key: cord- -pdvddowh authors: baltina, l. a.; fairushina, a. i.; baltina, l. a. title: synthesis of amino acid conjugates of glycyrrhizic acid using n-hydroxyphthalimide and n,n'-dicyclohexylcarbodiimide date: - - journal: russ j gen chem doi: . /s sha: doc_id: cord_uid: pdvddowh synthesis of amino acid conjugates of glycyrrhizic acid with the use of n-hydroxyphthalimide, n,n'-dicyclohexylcarbodiimide, and tert-butyl esters of l-amino acids (valine, isoleucine, phenylalanine, and methionine) was performed followed by deprotection with trifluoroacetic acid. the target amino acid conjugates were isolated by column chromatography on silica gel in – % yield. the structure of the prepared compounds was confirmed by ir and ( )c nmr spectroscopy. glycyrrhizic acid is the main triterpene glycoside of roots licorice (glycyrrhiza glabra l. and glycyrrhiza uralensis fisher) that belongs to the natural glycosides with promising properties for medicine for designing new immunomodulators and antiviral agents [ ] [ ] [ ] [ ] [ ] . a number of conjugates of glycyrrhizic acid with amino acids and dipeptides possesses meaningful anti-aids- activity [ , ] , and is of interest as anti-sars cov-agents [ ] and inhibitors of epstein-barr virus [ ] . it was found recently that some amino acid conjugates of glycyrrhizic acid possess significant virus inhibition activity towards pandemic influenza virus А/h n /pdm [ , ] . earlier we developed methods for the preparation of glycyrrhizic acid conjugates with alkyl and benzyl ( nitrobenzyl) esters of amino acids with the use of nhydroxybenzotriazole (hobt), n,n-dicyclohexylcarbodiimide (dcc), or n-hydroxysuccinimide (hosu) that allowed synthesis of the derivatives of glycyrrhizic acid with two or three amino acid residues or their esters depending on the reaction conditions [ , , , ] . n-hydroxyphthalimide (hopt) has been also applied as a nucleophilic reagent at the formation of the amide bond in the carbodiimide mediated synthesis of peptides [ ] ; but its use in the synthesis of amino acid conjugates of glycyrrhizic acid has not yet been reported. the goal of the present work was the synthesis of amino acid conjugates of glycyrrhizic acid with the use of hopt and dcc and tert-butyl esters of l-amino acids for activation of carboxy groups of the glycoside molecule. the condensation of glycyrrhizinic acid with amino acids was performed in two steps by conversion of it to activated ester using hopt and dcc in dioxane or tetrahydrofuran at the ratio : hopt : dcc = : ( - . ) : ( - . ). after separation of precipitated n,n-dicyclohexylurea (dccu), a solution of ester was brought into the reaction with amino components, l-amino acids tert-butyl esters hydrochlorides, in the presence of an excess of tertiary amine as a base (triethylamine) at room temperature ( - °c) for h (scheme ). carboxyl-protected conjugates , , , and were formed in - % yield; their physicochemical characteristics were the same as described before [ ] . deprotection of the products obtained was performed by treating with trifluoroacetic acid in methylene chloride at - °c. free amino acid con-doi: . /s jugates , , , and were isolated by column chromatography on silica gel in - % yields. the structure of the prepared compounds was confirmed by ir and c nmr spectroscopy and by the comparison of their properties with those of the model compounds prepared by the described methods [ , , ] . thus, in ir spectra of conjugates , , , and the absorption maxima of oh and nh groups ( - cm - ) and conh fragment ( - cm - ) were detected. in c nmr spectra of conjugates , , , and an additional signals of the carbon of cooh group of the amino acid moiety in a weak field ( - ppm), carbon atoms α-СНnh at - ppm, and another signals of amino acid residues were observed. the data of elemental analyses matched well the calculated values. experimental h and c nmr spectra were recorded on a bruker АМХ- spectrometer operating at and . mhz. ir spectra were registered on an ir prestige- shimadzu spectrophotometer as mulls in mineral oil. optical rotation angles were measured on a perkin-elmer polarimeter (l = dm) at - °c (λ na = nm). thin layer chromatography was done on sorbfil plates (by plc "sorbpolimer") with the use of a solvent system СНСl -meoh-h o, : : ; spots visualization was done with % solution of h so in etoh with following heating at - °c for - min. column chromatography was performed on a silica gel (fraction - mm). the solvents were purified by the known methods [ ] . solvents were evaporated in a vacuum at - °c. the used glycyrrhizic acid was isolated from the roots of liquorice ural (glycyrrhiza uralensis fisher) of the siberian populations [ ] . the following chemicals were applied: n-hydroxybenzotriazole and n,n-dicyclohexylcarbodiimide by sigma aldrich, hydrochlorides of tert-butyl esters of l-amino acids by chemapol. general method for the synthesis of glycyrrhizic acid conjugates. n-hydroxyphthalimide ( mmol) and triethylamine ( - mmol) were added to the filtrate. the mixture was kept for h at - °c with occasional stirring. then the mixture was poured in cold % solution of nahco , the precipitate was filtered off, washed with water, dried, and reprecipitated from aqueous ethanol to provide carboxylprotected conjugates , , , and in - % yield. to remove the ester tert-butyl group the obtained products ( . - . g) were dissolved in a mixture of methylene chloride and trifluorocetic acid ( : , ml). the mixture was kept for h at - °c, then evaporated in a vacuum and purified by column chromatography on silica gel eluting with a mixture chloroformmethanol-water ( : : , : : , : : , : : , vol). the individual fractions (by tlc) were combined and evaporated. this work was financially supported by the russian science foundation (grant no. - - ). solodka: bioraznoobrazie, khimiya, primenenie v meditsine (liquoruce: biodiversity, chemistry, application in medicine). novosibirsk: akademicheskoe izdatel'stvo "geo osnovnye metody obrazovaniya peptidnykh svyazei (peptides. the main methods of the peptide bonds formation sputnik khimika (the chemist's companion) chemistry for sustainable development key: cord- -rjyipcfv authors: chernyshov, vladimir v.; yarovaya, olga i.; fadeev, dmitry s.; gatilov, yuriy v.; esaulkova, yana l.; muryleva, anna s.; sinegubova, katherina o.; zarubaev, vladimir v.; salakhutdinov, nariman f. title: single-stage synthesis of heterocyclic alkaloid-like compounds from (+)-camphoric acid and their antiviral activity date: - - journal: mol divers doi: . /s - - - sha: doc_id: cord_uid: rjyipcfv abstract: an effective technique for one-stage synthesis of new polycyclic nitrogen-containing compounds has been developed. the procedure involves refluxing mixtures of camphoric acid with aliphatic or aromatic diamine without catalysts. in cases where the starting amine has a low boiling point (less than °c), phenol is used as a solvent, as it is the most optimal one for obtaining products with good yields. it has been shown that the use of lewis acids as catalysts reduces the yield of the reaction products. a set of compounds have been synthesized, which can be attributed to synthetic analogues of alkaloids. in vitro screening for activity influenza virus a was carried out for the obtained compounds. the synthesized quinazoline-like agent has inhibitory activity against different strains of influenza viruses. graphical abstract: [image: see text] electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. influenza represents one of the most serious challenges to medical science and health care all over the world. it causes annual epidemics and from time to time pandemics, both resulting in significant increase in morbidity and mortality [ ] . due to fast replicative cycle, ability to reassort the fragments of segmented genome and lack of correcting activity of viral polymerase influenza virus can quickly select mutants that do not match the virus-inhibiting antibodies and can therefore escape from the immune response [ ] . several classes of chemically distinct compounds are currently used for treatment of influenza: amantadine and rimantadine (two blockers of m proton channel) [ , ] , four neuraminidase inhibitors (oseltamivir, zanamivir, peramivir and laninamivir) prevent budding of viral progeny [ ] , umifenovir (arbidol) is used against influenza in russia and china [ , ] , pyrazinecarboxamide derivative favipiravir (t- ) was approved for stockpiling for potential treatment of pandemic influenza [ , ] , and baloxavir marboxil (xofluza ® ) that has been recently approved by fda interferes with endonuclease activity of viral pa subunit of polymerase complex [ ] . the same features of influenza virus that cause the emergence of antibody-escape mutants lead also to the selection of drug resistance to direct-acting antivirals. indeed, all current isolates of influenza virus are resistant to adamantane derivatives so that who does not recommend their use for treatment of influenza anymore [ ] [ ] [ ] . in - almost total resistance of seasonal influenza a(h n ) viruses to oseltamivir was achieved. these strains were further replaced with oseltamivir-susceptible pandemic influenza viruses a(h n )pdm [ , ] . taken together, these facts suggest that novel anti-influenza drugs of alternative mechanism(s) of activity and viral target(s) are therefore of high priority for medicinal science and health care. the abundance, crystallinity and variety of transformations of (+)-camphor were interesting throughout the history of organic chemistry. our work is devoted to the investigation of chemical properties of (+)-camphoric acid (product of oxidation of (+)-camphor). camphoric acid is a cyclopentane derivative containing two carboxylic acid functional groups on the first and third carbon atoms. the c- atom has an additional methyl group, thereby converting camphoric acid into an enantiomeric ditopic organic linker with different coordination regimes. moreover, the coordination chemistry of camphoric acid specifically gives rise to many interesting chiral features applicable to both materials and life sciences, such as asymmetrical synthesis or crystallization, homochiral structural design, chiral induction, absolute helical control and ligand handedness [ ] . there are examples of the use of camphoric acid derivatives as ligands [ ] [ ] [ ] . the condensation of carboxylic acids with amines and anilines is known to be a classical way of preparing amides. this interaction is shown for a wide spectrum of various substrates [ ] . the methods for preparing -substituted benzimidazoles based on direct cyclocondensations of carboxylic acids with benzene- , -diamine are also well known, but almost all of them involve rigid conditions, high temperatures or acidic conditions [ ] [ ] [ ] . for example, many carboxylic acids are condensed with benzene- , -diamine at temperatures of about ° [ ] [ ] [ ] . thus, we consider it relevant to study the interaction of camphoric acid with various aliphatic and aromatic diamines. this study is important, because all nitrogen-containing heterocyclic compounds play a great role in medicinal and organic chemistry in whole. the present work is devoted to the synthesis of new polycyclic nitrogen-containing compounds from (+)-camphoric acid and aliphatic or aromatic diamines. the first investigated reaction was interaction between ethylenediamine and (+)-camphoric acid . as a result of refluxing eq. camphoric acid with eq. ethylenediamine in phenol, product was obtained (scheme ). this technique led to an almost quantitative conversion to after h with % yield. product was not observed, if refluxing the starting reagents was carried out without a solvent, apparently due to the low boiling point of the starting amine . also we used toluene, dmso and o-xylene as a solvent for this interaction. it should be noted that using toluene or xylene significantly increased the conversion time to h. the use of dmso as a solvent resulted in a double decrease in the desired product. thus, using phenol as a solvent proved to be the most optimal for the production of the tricyclic product . refluxing a tenfold excess of camphoric acid with diamine in i-proh gives us a mixture of cyclic amide of symmetrical structure and product ( : ). product was purified by column chromatography and isolated with % yield. further, the interaction of , -diaminopropane with camphoric acid was investigated. refluxing with in phenol for h led to compound with % yield (scheme ). unfortunately, we were unable to isolate the cyclic amide of a symmetric structure based on , -diaminopropane. product was obtained by refluxing with in an i-proh with % yield. this interaction allows us to obtain compound and presumably mixture of polycyclic compounds with a similar structure to the compound , which, unfortunately, are not received in a pure form. for compounds , and perchlorate of the compound ( ·hclo ) x-ray crystallographic analysis was carried out (fig. ) . then, we decided to investigate the possibility of obtaining compounds of a similar structure from aromatic amines. thus, o-phenylenediamine was chosen as the next object of our research. we show that refluxing of mixture of with without a solvent for h leads to the formation of a mixture of two benzimidazole derivatives a and b with a total yield of % (scheme ). in this case, we carried out a study of the conversion rate and the ratio of the final products, by replacing camphoric acid with its anhydride, and also by adding mol% of lewis acid to the reaction mixture, for example, anhydrous zinc chloride. it is shown that the use of the lewis acid increases the conversion time from to h and decreases the total yield of the reaction products to % and the quantity of the major isomer a in the final mixture. using camphoric acid anhydride as a starting reagent slightly reduces the overall yield of the reaction products. compounds a and b have been isolated after the column chromatography with a yield of % and %, scheme interaction of camphoric acid with aliphatic diamines respectively. the structure of compound a has been confirmed by x-ray crystallographic analysis (fig. ) . the benzimidazole derivative a was previously described [ ] . the procedure involved refluxing the mixture of reagents with mol% of boric acid in toluene for h, and a dean-stark trap was used for the azeotropic removal of h o. we have also shown the possibility of synthesizing compound a by single-stage synthesis without solvent and catalysts, which significantly reduced the reaction time. then, we chose naphthalene- , -diamine as parent aromatic diamine. refluxing double excess of with without a solvent for h led to the mixture of compounds a and b with a total yield of % (scheme ). compounds a and b have been isolated by column chromatography with a yield of % and %, respectively. we consider that the minor product in all the transformations above is obtained by the primary nucleophilic attack to a more sterically hindered carboxylic group. this assumption is confirmed by h, c and d nmr spectra (see supporting information). also, the interaction of o-aminobenzylamine with was studied. refluxing the mixture of and excess of without a solvent for h resulted in compound with % yield (scheme ). x-ray crystallographic analysis shows that, in this case, the main product is the compound formed as a result of the primary nucleophilic attack to a more sterically hindered carboxyl group (fig. ). synthesized polycyclic nitrogen-containing compounds , , a, b, a, b, can be referred to as synthetic analogues of natural quinazoline alkaloids. nowadays, natural and synthetic quinazolines attract considerable attention due to their diverse and sometimes very high biological activity [ ] . for example, the most common active metabolites of plants of the genus peganum are quinazoline alkaloids (such as vasicine a, desoxyvasicine b, vasicinone a, deoxyvasicinone b [ ] ). these natural compounds have a broad spectrum of native biological activity, in particular: anti-ad for a-b [ ] , anti-parasitic for a-b [ ] , insecticidal for a [ ] . at the same time, synthetic derivative of quinazoline diproqualone was previously used as an analgesic for osteoarthritis and rheumatoid arthritis [ ] (fig. ) . along with the pronounced biological activity, the compounds of the quinazoline series can be successfully used as chiral catalysts [ , ] . thus, the use of vasicine a as an organic catalyst for direct c-h arylation of unactivated arenes with aryl iodides/bromides without assistance of any transition metal catalyst has been described [ ] . vasicine a, a quinazoline alkaloid, from the leaves of adhatoda vasica, has been utilized as an efficient catalyst for metal-and base-free henry reaction of various aldehydes with nitro alkanes [ ] . the quinazoline structure possibly imparts rigidity to the ligand and hence consistently high scheme interaction of camphoric acid with aromatic diamines enantioselectivity [ ] . at present time, attention of numerous groups is being paid to the synthesis of analogues of natural alkaloids. synthetic and natural quinazoline alkaloids can exhibit pronounced antiviral properties [ , ] . the compounds synthesized in this work contain both the monoterpenic fragment and the n-heterocycle. we have previously shown that various derivatives of monoterpenoids, in particular compounds including a , , -trimethylbicyclo[ . . ]heptane scaffold and n-heterocyclic fragment, exhibit antiviral properties against the influenza virus [ , ] . in this regard, the obtained derivatives were screened for their inhibitory activity against influenza virus a h n . for each compound, the values of % cytotoxic dose (cc ), % virus-inhibiting dose (ic ) and selectivity index (si) were calculated. the results are shown in table . adamantane-and norbornanebased derivatives were used as reference compounds due to their close similarity to the compounds under investigation in having rigid cage fragments in their structures. it is worth noting that compounds , , b, b, are less cytotoxic than reference compounds. compounds , , b, are most effective in inhibiting the influenza virus a (h n ) and can be used for further studies this type of activity. we believe that aliphatic polycyclic compounds (with a similar structure to compounds , ) or compounds containing an additional aromatic cycle may exhibit potentially high antiviral activity, but in this case, we are particularly interested in the isomers with the hem-dimethyl bridge directed upwards. for compound , which showed the highest activity, we studied the antiviral activity against different strains of influenza virus (table ) . it has been shown that compound has inhibitory activity against different strains of influenza virus a. the compound synthesized has inhibitory activity against strain h n (comparable to reference compounds) and strain h n (exceeding that of reference compounds). unfortunately, the inhibitory activity of compound against strain h n is lower than that of the reference compounds. activity of compounds , , , a, a- in conclusion, a simple and effective method of singlestage synthesis of polycyclic nitrogen-containing heterocyclic compounds, synthetic analogues of natural alkaloids, is suggested for the first time. it was shown that the optimal technique of synthesis is refluxing mixtures of parent compounds in phenol or without solvent and catalysts. using this method, we synthesized a number of polycyclic amides containing in their structure both a heterocyclic fragment and a bicyclic fragment ( , , a, b, a, b, ) and two cyclic symmetrical amides ( , ) . the structures of all synthesized compounds were confirmed by a complete set of spectral data, including x-ray crystallographic analyses of crystalline products , , , a and . in vitro screening for inhibitory activity against influenza virus was carried out for the obtained compounds and compound , was shown, exhibits inhibitory activity against different strains of influenza virus a (h n , h n , h n ). compounds , , b and their derivatives, in turn, can be used in the further study of this type of activity. media centre influenza (seasonal) fact sheet constraints, drivers, and implications of influenza a virus reassortment multiscale simulation reveals a multifaceted mechanism of proton permeation through the influenza a m proton channel structural basis for proton conduction and inhibition by the influenza m protein antiviral treatments arbidol as a broad-spectrum antiviral: an update arbidol: a broad-spectrum antiviral compound that blocks viral fusion favipiravir as a potential countermeasure against neglected and emerging rna viruses favipiravir (t- ), a broad spectrum inhibitor of viral rna polymerase baloxavir marboxil investigators group baloxavir marboxil for uncomplicated influenza in adults and adolescents adamantane resistance among influenza a viruses isolated early during the - influenza season in the united states the origin and global emergence of adamantane resistant a/h n influenza viruses comparison of antiviral resistance across acute and chronic viral infections drug resistance in influenza a virus: the epidemiology and management chiral chemistry of metalcamphorate frameworks novel tridentate ligands derived from (+)-camphoric acid for enantioselective ethylation of aromatic aldehydes enantioselective alkylation of aromatic aldehydes with (+)-camphoric acid derived chiral , -diamine ligands synthesis of some new chiral bifunctional o-hydroxyarylphosphonodiamides and their application as ligands in ti(iv) complex catalyzed asymmetric silylcyanation of aromatic aldehydes boric acid-catalyzed direct condensation of carboxylic acids with benzene- , -diamine into benzimidazoles synthesis, reactivity and biological activity of benzimidazoles synthesis and biological evaluation of ′-[(benzimidazole- -yl)methyl]biphenyl- -sulfonamide derivatives as dual angiotensin ii/endothelin a receptor antagonists novel pyrazolo[ , -d]pyrimidine with -( h-benzimidazol- -yl)-phenylamine as broad spectrum anticancer agents: synthesis, cell based assay, topoisomerase inhibition, dna intercalation and bovine serum albumin studies efficient propylphosphonic anhydride ( ® t p) mediated synthesis of benzothiazoles, benzoxazoles and benzimidazoles simulating microwave chemistry in a resistance-heated autoclave made of semiconducting silicon carbide ceramic synthesis and tuberculostatic activity evaluation of novel benzazoles with alkyl quinazoline derivatives: synthesis and bioactivities chemistry, pharmacology and medicinal properties of peganum harmala l rapid and sensitive detection of the inhibitive activities of acetyl-and butyryl-cholinesterases inhibitors by uplc-esi-ms/ms alkaloids from the seeds of peganum harmala showing antiplasmodial and vasorelaxant activities toxicity and growth inhibitory activities of methanol extract and the β-carboline alkaloids of peganum harmala l. against two coleopteran stored-grain pests quinazolinone: an overview evidence for involvement of cationic intermediate in epoxidation of chiral allylic alcohols and unfunctionalised alkenes catalysed by mn iii (quinazolinone) complexes -aminoquinazolinones as chiral ligands in catalytic enantioselective diethylzinc and phenylacetylene addition to aldehydes vasicine catalyzed direct c-h arylation of unactivated arenes: organocatalytic application of an abundant alkaloid vasicine from adhatoda vasica as an organocatalyst for metal-free henry reaction and reductive heterocyclization of o-nitroacylbenzenes vasicine as tridentate ligand for enantioselective addition of diethylzinc to aldehydes synthesis, antiviral activity and cytotoxicity evaluation of schiff bases of some -phenyl quinazoline- ( )h-ones antiviral alkaloids produced by the mangrove-derived fungus cladosporium sp. pjx- synthesis and in vitro study of novel borneol derivatives as potent inhibitors of the influenza a virus synthesis of camphecene derivatives using click chemistry methodology and study of their antiviral activity acknowledgements this work was supported by the russian science foundation ( - - a). the authors confirm that this article content has no conflict of interest. key: cord- -r y oo authors: clark, david a.; munshi, upender k. title: feeding associated neonatal necrotizing enterocolitis (primary nec) is an inflammatory bowel disease date: - - journal: pathophysiology doi: . /j.pathophys. . . sha: doc_id: cord_uid: r y oo neonatal necrotizing enterocolitis which develops after feeding preterm infants is characterized by severe intestinal inflammation and profound systemic metabolic acidosis. the fermentation of undigested dietary carbohydrate by colonic flora yields gases (co( ) and h( )) and short chain organic acids. these organic acids can disrupt the intestinal mucosa and initiate inflammation driven predominantly by resident mast cells and by granulocytes which are recruited from blood. a systemic acidosis ensues derived from intestinal acids, not classic lactic acidosis produced from anaerobic metabolism. the systemic acidosis further compromises inflamed bowel leading to bowel necrosis. neonatal necrotizing enterocolitis (nec) is a devastating illness. well over , articles from over , individual authors have been written in the past years (google scholar search -june ). the incidence of nec in the united states is approximately - % of pre-term very low birth weight babies (birth weight < g), which translates into at least cases of nec annually [ ] [ ] [ ] [ ] . it is one of the most common causes of death in premature babies surviving respiratory distress. signs and symptoms associated with nec are noted in table . there are radiographic diagnostic criteria for more advanced disease [ ] [ ] [ ] . pneumatosis intestinalis, with or without hepatic portal venous gas, is considered as pathognomonic of nec. free peritoneal air resulting from bowel wall perforation or a sentinel loop due to extensive bowel wall necrosis indicates an advanced stage of nec. laboratory investigations reveal elevation of acute phase reactants similar to neonatal sepsis syndrome, thrombocytopenia and metabolic acidosis, all in the context of inflamed and necrotic intestine from a disrupted intestinal mucosa [ ] [ ] [ ] [ ] . nec is not a specific diagnosis but a constellation of signs and symptoms with several proposed etiologies. spontaneous intestinal perforation (sip) presenting as pneumoperitoneum has been recognized as a distinct entity from nec. sip often * corresponding author. e-mail address: clarkd@mail.amc.edu (d.a. clark). presents early without pneumatosis or portal venous gas [ ] . in a sick preterm infant the distinction may not be resolved until surgical resection or autopsy. for the purpose of this review we will broadly classify nec into two main categories: ( ) primary nec which typically occurs in an apparently stable preterm infant who is feeding enterally with no recognizable prior triggering event and ( ) secondary nec which afflicts a preterm or term infant, who may or may not be feeding. these babies almost always have a recognizable triggering condition [ , ] . primary nec (lack of any inciting trigger) occurs beyond the first week of postnatal life and accounts for the majority of nec ( - % of cases) in neonatal intensive care units [ ] . secondary nec, a subset of which has been termed spontaneous intestinal perforation (sip) comprises about - % of all cases each preceded by a proximate cause [ ] [ ] [ ] . examples of secondary nec (sip) include term infants with congenital cyanotic heart disease, polycythemia, post-exchange transfusion, hypoxemic-ischemic insult with multiple organ failure and preterm infants with transfusion associated nec [ , ] . there is no national database for reporting the various forms of nec. once nec is clinically apparent, the clinical features of both categories look similar with varying degrees of severity. in secondary nec, earlier recognition and avoidance of inciting events as well as careful monitoring of the infants who have them may either prevent nec or result in occult blood - % positive blood culture elevated c-reactive protein early detection and prompt management of nec, potentially improving clinical outcomes. there is no such strategy applicable to primary nec since it sets in without warning events in an otherwise stable preterm infant who is either on full or substantial amount of enteral feeding. many factors such as osmolality of formula, timing and advancement of feedings, and primary gastrointestinal infection have been proposed as the inciting event but none have proven significant. exclusive breast feeding has been shown to lower the incidence [ , ] . there are several excellent articles describing potential and detailed subsets of pathophysiology that should be considered [ , ] . this manuscript focuses on the broad view of the initiation and propagation of primary nec. nec is the end result of a process and is not caused by the same pathophysiology in every premature infant. vascular (ischemia reperfusion) injury and infection are infrequent causes of nec and both are considered as secondary nec. primary nec is an inflammatory process without a clear understanding of its genesis. both lead to intestinal necrosis but there remain specific subtleties differentiating them [ , ] . without regard to the cause of severely damaged intestine, the treatment is the same, including cessation of feedings, gastric decompression, parenteral nutrition, supportive care, correction of acidosis, hypotension and blood loss, broad-spectrum antibiotics, and transfusion as necessary. serial examinations are performed and abdominal x-rays are taken in an attempt to detect intestinal perforation early. a perforation may be managed by peritoneal drainage or a laparotomy to resect necrotic bowel, commonly leaving a diverting intestinal stoma. unfortunate complications include short bowel syndrome, strictures, adhesions, entero-colonic fistulas, peritoneal abscesses and various complications of prolonged parenteral nutrition. the intestine of the low birth weight premature infant is immature in many of its functional aspects [ ] . there is decreased digestion and absorption of many essential nutrients. there is limited ability to both secrete enzymes as well as decreased intestinal motility. undigested carbohydrate is substrate for intestinal flora which produce organic acids and gases. the liver which metabolizes a number of compounds absorbed from the intestine does so with variable degrees of success. the preterm infant's immature intestinal barrier is less efficient at controlling toxins and organisms, specifically bacteria and viruses that are able to induce mucosal disruption. these organisms may initiate a local inflammatory response releasing mediators that are toxins. these should be removed by the liver, to prevent a severe systemic response. table delineates the characteristics of primary and secondary nec [ , , ] . the unfed infant commonly has multiple organ involvement before the bowel damage which is limited to a single site, commonly within the small bowel [ ] . if the infant has been fed, the onset is much later (beyond days) with over % of cases occurring after a month of age. the affected site is the distal ileum and proximal colon where undigested substrate (carbohydrate) first meets a significant load of bacteria. the intestine is the first organ affected and then there is subsequent systemic illness. a key difference between the two (table ) is that the acidosis of secondary nec is lactic acidosis whereas the acidosis of the late onset form (primary nec) is a mixed metabolic acidosis comprising more than one short chain fatty acid or organic acid [ , ] . clinical studies are hampered, because many key data elements are not considered or reported, including the prenatal history, time of rupture of the membranes, prenatal steroids or antibiotics, the mode of delivery, post natal steroids, the management of respiratory distress, the microbiology of the individual baby as well as of the environment of the nicu. most studies are unfunded and are commonly retrospective as a secondary or tertiary analysis. many important variables are missing, including events preceding the onset of nec in the individual baby. because there has been no well-defined etiology, investigator bias adds to the confusion. in general, the prevailing basic theory is that the preterm infant is born with multiple limitations of an immature intestine including malabsorption, immune dysregulation, a poorly regulated intestinal circulatory system, poor intestinal motility, and mucosal barrier defects [ , , , ] . animal models of nec have had limited utility. there is no premature primate nec model. the rat and mouse models of nec have been called into question, given that they depend on a combination of significant hypoxia combined with cold stress, neither which are likely to be sustained insults in the appropriate care of the premature baby. associations that are critical include prematurity with an inverse relationship, increasing incidence of nec as gestational age decreases [ , , , , ] . the feeding association is important as carbohydrate metabolized by the bacteria is the source of organic acids in the intestinal lumen, and the gases of pneumatosis intestinalis and portal venous tree. timing is important because subclinical cases may recover within several days once cautious feeding and supportive care have been initiated. a concept which is no longer relevant is the diving seal reflex. it is a mature reflex not found in seal pups. osmolarity is not an issue since the gastrointestinal tract is designed to dilute and concentrate nutrients. the stomach which by initial exposure is the most susceptible organ to hyperosmolarity is rarely involved. the classic theory does not correlate well with the epidemiology. about % of patients afflicted with nec are preterm, less than weeks gestation, and have been enterally fed, many for several weeks [ , , ] . most of the babies have gained weight demonstrating maturing intestinal function which then becomes impaired. the majority of the babies have not had an apparent hypoxic-ischemic insult and one quarter of the cases of nec occur beyond a month of age, suggesting perinatal events have little to no significance in primary nec. therefore, the etiology of nec falls into two broad categories; secondary nec encompassing infection, approximately %, and ischemia reperfusion injury - %. primary nec describes a neonatal form of inflammatory bowel disease which accounts for - % of all the clinical cases of nec. ischemia-reperfusion injury has been reported. however, the majority of the babies that have ischemic risk factors do not develop nec. most babies who develop primary nec have no identifiable ischemic insult [ ] . there are also many babies who do not develop nec even though they have obvious, severe ischemic insult. there is no correlation with nec of premature babies and acute tubular necrosis, perinatal asphyxia, and intraventricular hemorrhage. animal models of ischemia have been the primary nec model since the s. in a sheep model, event reducing the hematocrit to % and giving a hypoxic insult of % oxygen for min, oxygen consumption was not compromised as there was increased tissue o extraction and therefore no intestinal damage [ ] . in a canine model of nec, two hours of hypoxia and resuscitation, if the dogs were sacrificed within h there was intestinal vascular congestion and hemorrhage [ ] . if the animals were allowed three days before sacrifice there was full recovery of the intestine with normal histology. the intestine has the ability recover if there is not a continued insult. regarding infection, there are many reported cases of neonatal appendicitis, hirschsprung's disease, colonic obstruction, diverticulitis and other forms of localized inflammation that have some of the characteristics of necrotizing enterocolitis but with a clear underling etiology. in each of these examples enteric bacteria play a primary role [ , ] . at birth newborns are sterile but there is rapid colonization of skin, umbilical cord, oropharynx, and intestinal tract. once the intestine is colonized with as many as million to billion bacteria/gram of stool, portal bacteremia is relatively common [ ] . an efficient liver prevents that threat from becoming systemic. a number of organisms have been reported as associated with necrotizing enterocolitis [ ] . these include viruses: coronavirus, rotavirus, enterovirus, and parvovirus b [ , ] . the viruses cause disease by direct infection. the associations with bacteria have been more spotty and include, e. coli, klebsiella species, enterobacter species, and staphylococcus species. bacteria have several means by which to damage the intestine, including toxin production [ ] . bacteria also ferment carbohydrates of any variety with lactose being the most commonly malabsorbed carbohydrate in milk (breast milk or formula). the fermentation process of bacteria produces several gases, carbon dioxide and hydrogen. carbon dioxide is soluble and rapidly dissolved. hydrogen is the gas in the bowel wall of pneumatosis intestinalis as well as the portal gas a series of organic acids (short chain fatty acids) are produced in the fermentation process including lactic acid, butyric acid, propionic acid, acetic acid, isobutyric acid, and formic acid [ ] . the speed of fermentation is important. generating acid quickly initiates local intestinal inflammation and skip lesions. the acids generated are the source of the systemic mixed metabolic acidosis. the most commonly used medications in the neonatal intensive care unit are ampicillin and gentamicin [ , ] . carbonero et al. reported that klebsiella species and other gram negative rods exposed to low dose ampicillin increase their genomic ␤-lactamase becoming more resistant as well as simultaneously increasing the beta-galactosidase activity [ ] . therefore, as antibiotic resistances increases carbohydrate fermentation also increases rapidly. this sets the stage for high concentrations of multiple organic acids within the intestine that cause mucosal inflammation, leading to systemic acidosis. the concept of intraluminal organic acids inducing colitis is well described in that organic acids, acetic, butyric acid and propionic acid have been used in animal models to induce inflammatory colonic disease [ , [ ] [ ] [ ] [ ] . modeled after the concept that helicobacter sp. causes gastric ulcers, recently there has been a concerted effort to determine a specific pathogen responsible for causing the intestinal inflammation of nec. several studies have shown there is a change in the intestinal flora in children who develop necrotizing enterocolitis in that the proteobacteria constitute as much as % of the organisms in the stool, in cases within three days preceding the clinical diagnosis of nec [ ] . included in this group are the previously mentioned gram negative rods e. coli, klebsialla sp. and others. despite the systematic search, no consistent pathogen has been identified by culture techniques or by genomic analysis. the organism colonizes the gi tract while the baby is being fed sterile liquid formula or breast milk. the hypothetical organism would have to be one that preferentially affects the distal ileum and proximal colon, the primary sites of disease in preterm babies. no other intestinal or colonic pathogen has shown any similar pattern. there is no seasonal pattern as is commonly found with intestinal infections. toxins have rarely been identified in preterm nec. although an important etiology for gi disease in older children they are not an issue in newborns in that toxin receptors are not yet developed. the common intestinal flora found in all babies and in all intensive care nurseries may be the culprits in a subtle way, not by their invasive characteristics nor their ability to produce toxins, but simply by their ability to ferment carbohydrate and produce organic acids more quickly than the capacity of the preterm infants' defense mechanisms, the mucosa, and the portal system and liver can accommodate. to initiate intestinal damage a mechanism to disrupt the mucosal barrier is a central concept. only three of the many mechanisms of disruption of the intestinal mucosal barrier in animal models [ , ] seem to be of any significance. these are ischemia reperfusion injury, bacterial toxins, and organic acids. since there is minimal likelihood of bacterial toxins or ischemic reperfusion injury, the organic acids should be the focus. inflammation likely begins with carbohydrate (lactose) malabsorption and leads to bacterial fermentation and the generation of multiple organic acids, short chain fatty acids which disrupt the mucosal barrier. a local activation of the proinflammatory defense response along with poor motility and stasis initiates intestinal inflammation and potentially necrosis. a number of cells are responsible for the inflammation including cells which initiate and exacerbate the process. these are listed in table . of particular note here is the mast cell [ ] [ ] [ ] [ ] [ ] . the mast cell has not been well studied since it is consumed in the inflammatory process and is no longer present in necrotic tissue. the mast cell has over different mediators, some rapidly eluted, some from preformed granules, and others secondary or newly generated. mediators reported associated with nec (table ) include leukotrienes c , d , interleukin- , oxygen radicals, platelet activating factor and thromboxane all of which can be found in an activated mast cell [ ] [ ] [ ] [ ] . of the original list of systemic and gastrointestinal signs and symptoms of feeding-associated primary nec the one critical factor is mixed metabolic acidosis. with hypoxic ischemic injury the lactic acidosis is transient and readily managed. the mixed metabolic acidosis of nec is sustained which often takes many hours to fully resolve. using tandem mass spectroscopy, we have examined the whole blood organic acid profile of babies in the nicu with severe metabolic acidosis (fig. ) . babies (n = ) with hypoxic ischemia injury, and conditions other than nec have lactic acid as the primary circulating metabolite causing the acidosis. babies with nec (n = ) and severe metabolic acidosis had propionic acid as the primary organic acid. propionic acid is one of the important metabolic fermentation products of members of the proteobacteria specifically e. coli, and klebsiella sp. [ , ] . endogenous sources of propionic acid result from catabolism of a small number group of amino acids. protein catabolism is unlikely in preterm infants who are growing after having been fed successfully. the primary source of the propionic acid producing metabolic acidosis is the intestinal bacteria which continue to generate acid as long as substrate is available. this explains the location of nec as distal and proximal colon where the most rapid fermentation occurs. fermentation more distal is less likely since the substrate is consumed especially in the context of poor motility, inflamed distal ileum and proximal colon. based on newborn screening program data from new york state (ny state doh -personal communication) preterm infants < weeks gestation often have limited capacity to clear propionic acid. this organic acid is metabolized only by the liver. other organic acids such as lactic and short chain fatty acidic with fewer than carbons (c ) are metabolized by the krebs cycle present in the majority of tissues. in newborn screening programs propionic acid is a common false/positive in babies who are fed. the propionic acid is elevated prior to weeks gestation and resolves without true disease. this suggests a maturational defect of the liver to metabolize propionic acid. the enzyme (propionyl coa carboxylase) converts propionic acid to methylmalonyl-coa and a vitamin b dependent enzyme rearranges it to succinyl coa the precursor of succinic acid, which is within the krebs cycle. prior to birth without intestinal colonization there is no generation of organic acids from the intestine and therefore no need to have the hepatic enzymes functional. based on these observations we have developed a piglet intraluminal model of nec which includes all of the key features, skip lesions, pneumatosis, portal gas, systemic acidosis, thrombocytopenia, neutropenia and others [ ] . the model is induced only by a rapid lactose fermenting gram negative rod incubated with preterm formula. there is no hypoxic or ischemic insult [ ] . this model may explain the utility of probiotic administration to reduce the risk of nec [ ] [ ] [ ] . in summary, the initiation of nec is linked to rapidly fermenting bacteria producing a series of organic acids that cause local intestinal inflammation. intact sustained blood flow delivers acids along with hydrogen (portal gas) by the portal system to the liver. the liver metabolizes most organic acids efficiently in the term infant but in the preterm baby the liver is overwhelmed leading to systemic acidosis specifically propionic acidemia. the systemic mixed metabolic acidosis, led by propionic acid, exacerbates the damage to already inflamed intestine resulting in intestinal damage (primary nec). epidemiology of necrotizing enterocolitis necrotizing enterocolitis in very low birth weight infants: biodemographic and clinical correlates roberton's textbook of neonatology necrotizing enterocolitis necrotizing enterocolitis in term neonates: data from a multihospital health-care system intraluminal pathogenesis of necrotizing enterocolitis the canadian neonatal network, incidence and timing of presentation of necrotizing enterocolitis in preterm infants necrotizing enterocolitis in full-term infants: case-control study and review of the literature breast milk and neonatal necrotizing enterocolitis role of human milk in extremely low birth weight infants' risk of necrotizing enterocolitis or death mapping the new world of necrotizing enterocolitis (nec) what causes necrotizing enerocolitis and how can it be prevented development of gastrointestinal function: risk factors for necrotizing enterocolitis epidemiology of neonatal necrotizing enterocolitis: a population based study necrotizing enterocolitis in the asphyxiated full-term infant gastrointestinal blood flow and oxygen consumption in the newborn lamb: effect of chronic anemia and acute hypoxia pathogenesis of early necrotizing enterocolitis in the hypoxic neonatal dog diversity of the human intestinal microbial flora nosocomial necrotizing enterocolitis outbreaks: epidemiology and control measures rotavirus-associated necrotizing enterocolitis: an insight into a potentially preventable disease? intestinal lesions containing coronavirus-like particles in neonatal necrotizing enterocolitis: an untrastructural analysis reported medication use in the neonatal intensive care unit: data from a large national data set antibiotic exposure in the newborn intensive care unit and the risk of necrotizing enterocolitis bacterial pathogenicity determinant associated with necrotizing enterocolitis acute intestinal injury induced by acetic acid and casein: prevention by intraluminal misoprostol the role of bacterial infection in the pathogenesis of inflammatory bowel disease host-mediated inflammation disrupts the intestinal microbiota and promotes the overgrowth of enterobacteriacae fecal microbiota in premature infants prior to necrotizing enterocolitis profile and sites of eicosanoid release in experimental necrotizing enterocolitis hemodynamic and permeability characteristics of acute, experimental necrotizing entercolitis the essential role of mast cells in orchestrating inflammation histamine is a transient marker of small intestinal injury induced by luminal acetic acid and casein the human mast cell: an overview human mast cells, bacteria, and intestinal immunity eosinophilia in very low birth weight neonates a systematic review of serologic tests in the diagnosis of necrotizing enterocolitis a complete piglet model of neonatal necrotizing enterocolitis pediatric academic societies probiotic bacteria and intestinal epithelial barrier function probiotics in the prevention of necrotizing enterocolitis probiotics reduce the risk of necrotizing enterocolitis in preterm infants: a meta-analysis key: cord- -mubjm l authors: weng, jing-ru; lin, chen-sheng; lai, hsueh-chou; lin, yu-ping; wang, ching-ying; tsai, yu-chi; wu, kun-chang; huang, su-hua; lin, cheng-wen title: antiviral activity of sambucus formosananakai ethanol extract and related phenolic acid constituents against human coronavirus nl date: - - journal: virus res doi: . /j.virusres. . sha: doc_id: cord_uid: mubjm l human coronavirus nl (hcov-nl ), one of the main circulating hcovs worldwide, causes respiratory tract illnesses like runny nose, cough, bronchiolitis and pneumonia. recently, a severe respiratory illness outbreak of hcov-nl has been reported in a long-term care facility. sambucus formosananakai, a species of elderberry, is a traditional medicinal herb with anti-inflammatory and antiviral potential. the study investigated the antiviral activity of sambucus formosananakai stem ethanol extract and some phenolic acid constituents against hcov-nl . the extract was less cytotoxic and concentration-dependently increased anti-hcov-nl activities, including cytopathicity, sub-g fraction, virus yield (ic = . μg/ml), plaque formation (ic = . μg/ml) and virus attachment (ic = . μg/ml). among the phenolic acid constituents in sambucus formosananakai extract, caffeic acid, chlorogenic acid and gallic acid sustained the anti-hcov-nl activity that was ranked in the following order of virus yield reduction: caffeic acid (ic( ) = . μm) > chlorogenic acid (ic( ) = . μm) > coumaric acid (ic( ) = . μm). caffeic acid significantly inhibited the replication of hcov-nl in a cell-type independent manner, and specifically blocked virus attachment (ic( ) = . μm). therefore, the results revealed that sambucus formosana nakai stem ethanol extract displayed the strong anti-hcov-nl potential; caffeic acid could be the vital component with anti-hcov-nl activity. the finding could be helpful for developing antivirals against hcov-nl . human coronavirus (hcov) nl , the member of the genus alphacoronavirus in the coronaviridae family, is one of common human coronaviruses (li and lin, ; huang et al., ) . hcov-nl genome is a positive-strand rna with near . kb nucleotides containing ′ untranslated regions (utr), orf a/b, spike(s), orf , envelope(e), membrane(m), nucleoprotein (n), and ′utr (geng et al., ) . orf a/b encodes overlapping replicase a and ab via the − ribosomal frameshift at the nucleotide , . the papain-like (plpro) and c-like ( clpro) proteases embedded within replicase a and ab process cis-and trans-cleavage activity to divide replicase a and ab into nonstructural proteins (nsps) that modulate in viral rna replication. among common human coronaviruses like hcov- e, hcov-hku , and hcov-oc , hcov-nl is one of main circulating hcovs in the fall and winter worldwide, causing mild upper respiratory tract illnesses like runny nose, cough and sore throat in young children, young adults and elderly (cui et al., ; dijkman et al., ) . interestingly, hcov-nl has been frequently detected than other hcovs, influenza viruses, and rhinovirus in the specimens from the young adults with acute respiratory infection (cough and body aches or chills or fever/feverishness) (davis et al., ) . importantly, hcov-nl is also associated with lower respiratory tract illnesses, such as pneumonia and bronchiolitis in young children and elderly (huang https://doi.org/ . (huang https://doi.org/ . /j.virusres. received june ; received in revised form september ; accepted september et al., ) . hcov-nl infection is the high prevalence ( . %) in hospitalized patients with pneumonia in winter. recently, a severe respiratory illness outbreak in a long-term care facility in louisiana has been reported to be associated with the hcov-nl infection in winter (hand et al., ) . among cases aged from to , patients with pneumonia have to be hospitalized and patients are dead. moreover, screening children with acute undifferentiated febrile illness in rural haiti indicates that hcov-nl is identified in the blood samples from four patients aged from to years who have no respiratory symptom, but two cases have headache and the others exhibit influenza virus causing abdominal symptoms (beau de rochars et al., ) . therefore, hcov-nl is the notable pathogen as the etiology of mild and severe respiratory diseases, even acute undifferentiated febrile illness. sambucus formosananakai, also known by sambucus chinensis lindl and sambucus javanica, is a species of elderberry, belongs to family adoxaceae, and grows in subtropical and tropical asian areas, including taiwan, china, japan, cambodia, india etc. (lin and tome, ; yang and chiu, ; hong et al., ) . sambucus formosananakai is a traditional medicinal herb in taiwan for reducing inflammation, enhancing circulation, and treating rheumatoid and low back pain, neuritis, dermatitis, and infection diseases (yang and chiu, ) . the chemical components of s.ambucus formosananakai include sambuculin a, oleanolic acid, α-amyrin and β-amyrin, β-sitosterol, ursolic acid, pomolic acid, lupeol palmitate, glycyrrhetic acid, phenolic acid constituents, and flavonoids (chen et al., (chen et al., , liao et al., ; lin and tome, ; yang and lin, ; zhang et al., ) . in addition, phenolic acid constituents of s.ambucus formosananakai, including caffeic acid, caffeotannic acid, chlorogenic acid, coumaric acid, ferulic acid, and gallic acid, have been identified as the members of the most important active components with anti-inflammatory, anti-tumor and anti-hepatotoxic activities (chen et al., ; liao et al., ; yang and lin, ; zhang et al., ) . active components of sambucus formosananakai are similar to those of other sambucus species, including sambucus nigra l. and sambucus lanceolata, which process antioxidant, antiradical, antiviral, antimicrobial, and anti-inflammatory activities (barak et al., ; krawitz et al., ; mandrone et al., ; pinto et al., ; pliszka, ; porter and bode, ; turek and cisowski, ) . especially, the extract of sambucus nigra l. exerts the antiviral activity against influenza a and b viruses, human immunodeficiency virus, and the herpes simplex virus type (krawitz et al., ; serkedjieva et al., ; zakay-rones et al., ; amoros et al., ; mahmood et al., ; roschek et al., ) . sambucus nigraphenolic acids like caffeic acid show the highly inhibitory effect on the in vitro replication of influenza a virus (porter and bode, ; utsunomiya et al., ) . since sambucus formosananakai contains such antiviral active components in the extract of sambucus nigra l., the antiviral activity of sambucus formosananakai is worthy to further investigate. the study explored the anti-hcov-nl activity of sambucus formosananakai stem ethanol extract and some markers of its phenolic acid constituents, like caffeic acid, chlorogenic acid, coumaric acid, ferulic acid, gallic acid. the study indicated the inhibitory activity of sambucus formosananakai extract and its phenolic acid constituents on hcov-nl induced cytopathic effect, virus yield, and the early stage of hcov-nl replication in concentration-dependent and cell-type independent manners. hcov-nl provided by dr. lia van der hoek (academic medical center, the netherlands) was amplified in llc-mk cells, as described in our prior study . llc-mk cells were cultured in the minimum essential medium (mem) containing mm l-glutamine, μg/ml penicillin, μg/ml streptomycin, and % fetal bovine serum (fbs) at ℃ in a ％ co incubator. llc-mk cells were further used to perform antiviral assays and examine antiviral mechanism. human airway calu- cells were cultured in mem supplemented % fbs and antibiotics mentioned above, maintained at the - % confluent, and then also used to confirm the antiviral activity of indicated phenolic acid constituents. dried stems of sambucus formosana nakai (supplemental fig. a) were purchased from the medicinal herb pharmacy in taichung, and identified as described in flora of taiwan (yang and chiu, ) . dried stem slices (supplemental fig. b) were soaked in % ethanol with the sonication for h; the stem ethanol extract was filtered by whatman no. paper, then lyophilized in an iwaki fdr- p freeze dryer, as described in our previous study (wang et al., ) . the powder of sambucus formosana nakai stem ethanol extract was kept in the sterile bottle; the stock solution of mg/ml in dimethyl sulfoxide was prepared and stored in − °c freezer and the test solutions of the stem extract were diluted by the media. the phenolic acid constituents such caffeic acid, chlorogenic acid, coumaric acid, ferulic acid, and gallic acid, were purchased from sigma-aldrich company. llc-mk cells ( × cells/well) were cultured in the -well plates overnight, quintuplicate treated with sambucus formosana nakai stem ethanol extract or its phenolic acid constituents (caffeic acid, chlorogenic acid, coumaric acid, ferulic acid, and gallic acid) for days, and then incubated with . mg/ml -( , -dimethylthiazol- -yl)- , diphenyltetrazolium bromide (mtt) for additional h. after the cell lysis by isopropanol, yielding absorbance (od - ) at the wavelength of nm and nm was assessed using a micro-elisa reader. cell viability (%) and cytotoxic concentration showing % toxic effect (cc ) were subsequently determined, as previously described (wang et al., ) . llc-mk cells ( × cells/well) were cultured in the -well plates overnight, and then infected by × pfu (plaque formation unit) hcov-nl , representing as a moi (multiplicity of infection) of . to significantly induce viral cytopathic effect (cpe) in llc-mk . the cells were simultaneously added with hcov nl , and treated with sambucus formosana nakai stem ethanol extract at the concentrations of , , , and μg/ml or the phenolic acid constituents at the concentrations of , , , and μm, respectively. after the -h incubation at ℃ and ％ co , hcov nl -induced cytopathic effect (cpe) with cell swelling, rounding, vacuoles, and eventual detachment was photographed using microscope, in which vacuoles in cpe from hcov nl -infected llc-mk cells were more predominant at °c (lednicky et al., ) . in the cell cycle assay, the cells were harvested h post infection, stained with propidium iodide, and then examined using flow cytometry, as described in our prior study . to determine the production of progeny virions (virus yield assay), the cultured media were collected for determined the virus titers using plaque assay. llc-mk cell monolayer in the well of -well plates was added with μl of serial -fold dilutions of above cultured media. after a -h incubation, the cell monolayer was overlaid by the medium containing . % agarose ( ml per well) for -day incubation at °c in a co incubator to allow the plaque formation. finally, the cell monolayer was stained with the solution of naphthol blue-black dye and plaque number calculated. fifty percent ( %) inhibitory concentration (ic ) for virus yield reduction by the stem ethanol extract and the phenolic acid constituents was determined. in the plaque formation inhibition assays, llc-mk cell monolayer cultured in -well plates were infected with pfu of hcov-nl that were the maximum number of plaques counted in the well of -well plates. after the addition with hcov nl , the cells were immediately treated with the ethanol extract at the concentrations of , , , and μg/ml, caffeic acid and chlorogenic acid at the concentrations of , , , and μm, respectively. after a -h incubation at °c in a % co incubator, the mixtures were removed from the wells; the cell monolayer was cultured with the medium containing . % agarose and performed by the plaque assay mentioned above. in the virucidal assay, hcov-nl ( × pfu) was added into in the eppendorf tube and directly treated with the ethanol extract, caffeic acid, or chlorogenic acid for h at °c. for minimizing the antiviral effect of indicated agents in the cells, μl (near pfu hcov-nl ) of the -fold dilution from the mixtures of virus and the extract or phenolic acids was added to the mk cell monolayer in the -well plate to determining the residual viral infectivity using the plaque assay described above. virucidal activity was calculated based on the comparison of the residual infectivity of hcov-nl in the treated groups with the un-treated group. in the hcov-nl attachment assay, llc-mk cell monolayer in -well plate was placed at °c for h, infected with hcov-nl ( pfu), and then immediately added with the ethanol extract, caffeic acid or chlorogenic acid. after an additional -h incubation at °c, the mixture of virus and the extract or phenolic acids was removed from the well; the cell monolayer was overlaid with mem medium containing . % agarose at °c in a % co incubator, and then executed by plaque assay, as mentioned above. the attachment inhibition was analyzed according to the ratio of the plaque number in treated groups to the un-treated group. to measure the inhibitory activity of caffeic acid on the replication of hcov-nl in human airway epithelial cells, calu- cells ( × cells/well) were cultured in the -well plates overnight, and then infected by × pfu hcov-nl (moi = . ). after the addition of hcov nl , the cells were simultaneously treated with caffeic acid at the concentrations of , , and μm. after the -h incubation at ℃ in a ％ co incubator, swelling, rounding, and eventual de-attachment in hcov-nl -induced cpe were more predominant at °c (lednicky et al., ) , and then the images were recorded by microscope. subsequently, the cells were fixed with % paraformaldehyde solution in pbs for min, incubated with the quench buffer ( mm nh ci) for min, permeabilized and blocked using the cell perforation and blocking solution containing % albumin bovine (affymetrix) plus triton x- (thermofisher) for h at °c, and then reacted with hcov-nl -immunized sera in % bsa ( : ) overnight at °c and secondary antibody alexa fluor anti-mouse igg in % bsa ( : ) for h at °c (thermofisher). after staining with ′, diamidino- -phenylindole (dapi, thermofisher) min at room temperature, the images of mock, infected, and infected/treated cells were photographed using fluorescent microscopy. the infectivity was represented as the ratio of red fluorescent signals (hcov-nl -positive cells) to blue fluorescent signals (total nucleuses) was calculated by image j. the p value was calculated based anova using spss program and student t-test with the data from three independent experiments. when the p value was less than . , the result of the assay was statistically significant. sambucus formosana nakai stem ethanol extract had a low cytotoxicity with a cc value of . μg/ml to llc-mk cells (supplemental fig. , table ). subsequently, anti-hcov-nl ability of the stem ethanol extract was assessed with cytopathicity, cell cycle and virus yield assays (figs. and ) . sambucus formosana nakai extract concentration-dependently reduced cytopathicity in hcov-nl infected cells (fig. a) , in which vacuolation in infected cells at °c appeared more predominantly, as described in the prior report (lednicky et al., ) . the extract also significantly decreased the sub-g fraction in infected cells (fig. b) . in addition, sambucus for-mosananakai extract inhibited the in vitro production of progeny hcov-nl by concentration-dependent manners. the plaque assay indicated that the ic value of sambucus formosananakai extract on the virus yield was . μg/ml (fig. ) . remarkably, the results demonstrated that sambucus formosananakai stem ethanol extract served the significantly antiviral activity against hcov-nl . to discover the inhibitory action of sambucus formosananakai extract on stages of hcov-nl replication, plaque formation, virucidal activity, and virus attachment assays were performed using the plaque assays (fig. , table ). sambucus formosananakai extract meaningfully inhibited the plaque formation with an ic value of . μg/ml (fig. a) . in the virucidal assay, sambucus formosananakai extract at , , and μg/ml had a slight virucidal activity with lower than % inhibition to interfere with the hcov-nl particle infectivity compared to the mock control (fig. b ). in the virus attachment assay, sambucus formosananakai extract concentration-dependently reduced the hcov-nl attachment onto llc-mk cell monolayer in -well plates incubated at °c, which result demonstrated that the ethanol extract had a influentially inhibitory effect on hcov-nl attachment with an ic value of . μg/ml (fig. c) . the results demonstrated that sambucus formosananakai extract specifically suppressed the viral plaque formation and virus attachment during hcov-nl replication. the phenolic acid constituents were rich in the extract of sambucus nakai extract were less cytotoxic to llc-mk cells, in which cc values of caffeic acid, chlorogenic acid, and gallic acid were greater than μm (supplemental fig. , table ). in the inhibitory assay of hcov-nl induced cytopathic effect, caffeic acid, chlorogenic acid, and gallic acid, but not coumaric acid and ferulic acid, diminished the cytopathicity in the infected cells (fig. a) . the antiviral activity of the phenolic acid constituents in the in vitro production of hcov-nl was ranked in the following order of virus yield reduction: caffeic acid (ic = . μm) > chlorogenic acid (ic = . μm) > coumaric acid (ic = . μm) (fig. b , table ). the results verified that the phenolic acid constituents, like caffeic acid, chlorogenic acid, and gallic acid exhibited the prominent antiviral activity against hcov-nl , as potential anti-hcov-nl components in the sambucus for-mosananakai extract. to examine the antiviral mechanism of caffeic acid and chlorogenic acid against hcov-nl , the assays of plaque formation, virucidal activity and virus attachment were subsequently performed (fig. , table ). caffeic acid had a stronger inhibitory activity on the plaque formation than chlorogenic acid, in which the ic values on the plaque formation were . μm for caffeic acid and . μm for chlorogenic acid, respectively (fig. ) . caffeic acid, but not chlorogenic acid, concentration-dependently served the virucidal activity (ic = . μm) and powerfully reduced hcov-nl attachment to fig. . effects of sambucus formosana nakai extract on plaque formation, virucidal activity and virus attachment. mk- cell monolayer was infected with hcov-nl , simultaneously treated with the extract for h, and then covered with the agarose overlay medium. after -day incubation at °c in a co incubator, plaques were determined after crystal violet staining. the inhibitory activity of the extract on the plaque formation was according to on the ratio of loss plaque number to mock-treated group (a). in the virucidal assay, the extract was mixed with hcov-nl ( pfu), then incubated at °c for h. the extract/virus mixture was diluted by -fold dilution and examined for the residual infectivity by plaque assay (b). in the attachment assay, hcov-nl was mixed with the extract, then immediately added onto mk cell monolayer for h at °c. after washing, the cell monolayer was overlaid with . % agarose medium for days at °c in co incubator. attachment inhibition was determined based on the residual plaques (c). *, p value < . ; **, p value < . compared with mock-treated cells. fig. . inhibitory effects of the phenolic acid constituents on viral cytopathicity and virus yield in hcov-nl infected cells. hcov-nl at moi of . was added to llc-mk- cell culture and then immediately treated with the phenolic acid constituents. virus-induced cytopathic effect was photographed h postinfection by microscopy (a). supernatant of hcov-nl -infected/treated cells was harvested hpi; virus yield in supernatant was determined plaque assay. the rate of virus yield reduction was calculated based on the ratio of loss particle number to mock-treated group (b). ca, caffeic acid; ch, chlorogenic acid; co, coumaric acid; fe, ferulic acid; ga, gallic acid. **, p value < . , ***, p value < . compared with mock-treated infected cells. the cell surface (ic of . μm), respectively (fig. ). in addition, infectivity inhibition assay with human airway epithelia calu- cells was performed when hcov-nl -infected cells with a moi of . were treated with and μm caffeic acid after a -h incubation at °c. microscopic photography indicated that swelling and rounding were observed in hcov-nl -infected calu- cells (fig. a, top) , as described in the previous study (lednicky et al., ) . caffeic acid at and μm significantly lessening viral cpe (fig. a, top) . immunofluorescent staining demonstrated that caffeic acid concentration-dependently reduced hcov-nl infectivity determined according to the ratio of hcov-nl -positive cells to total cells stained with dapi (fig. a, middle and bottom) . the inhibitory assay with immunofluorescent staining on hcov-nl infectivity indicated that caffeic acid had a potent antiviral activity against the replication of hcov-nl in calu- cells (ic = . ± . μm) (fig. b) . the results revealed that caffeic acid displayed the potent anti-hcov-nl activity in a cell-type independent manner, specifically inhibiting on the attachment stage of hcov-nl replication. this study was the first report on the antiviral efficacy of sambucus formosananakai extract and its related phenolic acid constituents against hcov-nl . sambucus formosananakai extract exhibited the low cytotoxicity and markedly decreased cytopathic effect and sub-g arrest in hcov-nl -infected cells, in which was associated with the virus yield reduction in a concentration-dependent manner (figs. and , table ). moreover, sambucus formosananakai extract showed the potent antiviral activity against hcov-nl with the ic values in low microg/ml ranges, such ic of . μg/ml for virus yield, ic of fig. . effects of caffeic acid and chlorogenic acid on plaque formation, virucidal activity and virus attachment. mk- cell monolayer was infected with hcov-nl , simultaneously treated with the caffeic acid or chlorogenic acid for h, and then covered with the agarose overlay medium for -day at °c in a co incubator. after crystal violet staining, the inhibitory activity of caffeic acid and chlorogenic acid on the plaque formation was according to on the ratio of loss plaque number to mock-treated group (a). in the virucidal assay, the caffeic acid or chlorogenic acid was mixed with hcov-nl ( pfu), then incubated at °c for h. the -fold dilution of the compound/virus mixture was examined for the residual infectivity by plaque assay (b). in the attachment assay, mk cell monolayer was infected with hcov-nl ( pfu), immediately treated with the caffeic acid or chlorogenic acid for h at °c, washed, and overlaid with . % agarose medium for days at °c in co incubator. attachment inhibition was determined based on the residual plaques (c). ca. caffeic acid; ch, chlorogenic acid. *, p value < . ;**, p value < . ; ***, p value < . compared with un-treated infected cells. fig. . inhibition of hcov-nl infectivity in human airway epithelial cells by caffeic acid. calu- cells were infected with hcov-nl at a moi of . and immediately treated with caffeic acid for h at °c. images of virus-induced cpe effect were photographed by a light microscope (a, top). in addition, mock, infected, and infected/treated cells were performed using immunofluorescence staining anti-hcov-nl immunized sera and secondary antibody alexa fluor antimouse igg (a, middle); total cells were stained with dapi (a, bottom). infectivity was determined according to the ratio of hcov-nl -positive cells to total cells (b). **, p value < . ; ***, p value < . compared with un-treated infected cells. . μg/ml for plaque formation, and ic of . μg/ml for virus attachment. the results were consistent with the previous reports in that sambucus spp., such sambucus nigra l. served the antiviral properties against influenza a and b viruses, and the herpes simplex virus type (krawitz et al., ; serkedjieva et al., ; zakay-rones et al., ) . in addition, sambucus juice was the key recipe for the "virus blocking factor" that processed the in-vitro antiviral activity against many respiratory viral infections, including influenza a h n , rhinovirus b subtype , respiratory syncytial virus, parainfluenzavirus subtype , and adenovirus c subtype (fal et al., ) . sambucus nigra l. has been recognized as was a potentially safer alternative to treat upper respiratory symptoms, such common cold and influenza (hawkins et al., ) . thus, sambucus formosananakai might be the alternative medicinal herb for caring the respiratory viral infections, especially coronavirus-associated common cold. among six phenolic acid constituents in the sambucus formosananakai extract, caffeic acid had the highest anti-hcov-nl potency with the inhibitory effect on the virus yields (ic = . μm), plaque formation (ic = . μm), and virus attachment (ic = . μm) (figs. and , table ). caffeic acid has also been demonstrated the antiviral activity against hcov-nl in a cell-type independent manner (fig. ). in addition, chlorogenic acid and gallic acid exhibited antiviral activity against hcov-nl (figs. and , table ). the ic value on the inhibition of virus yield was . μm for chlorogenic acid, and . μm for gallic acid, respectively. the phenolic acid constituents have been identified as the major components in the sambucus formosananakai extract and sambucus australis by lc-ms/ms analyses (benevides bahiense et al., ; zhang et al., ) , therefore caffeic acid, chlorogenic acid and gallic acid might be the key components with anti-hcov-nl activity in the sambucus formosananakai extract. caffeic acid has been reported to process the antiviral activity against hepatitis b and c viruses, influenza a virus, and herpes simplex virus (wang et al., ; shen et al., ; tanida et al., ; utsunomiya et al., ; ikeda et al., ; langland et al., ) . caffeic acid reduced the production of hepatitis c virus in vitro via the initial stage of viral replication (tanida et al., ) . the antiviral mechanism of caffeic acid against hepatitis c virus was also associated with the activation of p -mediated keap /nrf signaling pathway for the ho- -dependent production of interferon α, which significantly suppressed the replication of hepatitis c virus (shen et al., ) . caffeic acid markedly decreased the virus yield and cytopathic effect in influenza a virus-infected cells, particular during the period within h post-infection, suggesting that caffeic acid works at the early stages of influenza a virus infection (utsunomiya et al., ) . in addition, caffeic acid had no virucidal effect, but significantly affected the production of progeny herpes simplex virus and the binding attachment to the heparan sulfate proteoglycans on cell surface (ikeda et al., ; langland et al., ) . interestingly, hcov-nl has been demonstrated to utilize the heparan sulfate proteoglycans as the co-receptor for attachment to target cells (milewska et al., ) . caffeic acid has also been identified to exhibit a high inhibitory effect on the enzymatic activity of angiotensin converting enzyme (ace) (chiou et al., ) ; docking studies revealed that caffeic acid also presented the good docking score with the hydrogen bond interactions with the residues in the activity site of ace (zozimus divya lobo et al., ) . thus, the inhibitory mechanism of caffeic acid on hcov-nl attachment and infection to cells might be due to that caffeic acid directly target and interfere the binding interaction of hcov-nl with heparan sulfate proteoglycans (co-receptor) and ace (receptor) on cell surface. chlorogenic acid and gallic acid have also been demonstrated to suppress the in vitro and in vivo replication of influenza a virus, enterovirus and hepatitis b and c viruses (ding et al., ; wang et al., ; you et al., ; govea-salas et al., ) . chlorogenic acid specifically inhibited the neuraminidase activity of influenza a viruses h n and h n that was the crucial mechanism of chlorogenic acid for blocking the release of progeny virions from infected cells (ding et al., ) . gallic acid served the antioxidant capacity that linked with down-regulating the genomic rna and proteins expression of hepatitis c virus (govea-salas et al., ) . therefore, the phenolic acid constituents of sambucus formosana nakai extract, like caffeic acid, chlorogenic acid and gallic acid, play the key antiviral components against hcov-nl and the other viruses. the study demonstrated the potent anti-hcov-nl- activity of sambucus formosananakai extract through the significant reduction of virus yield, plaque formation and virus attachment. caffeic acid, chlorogenic acid and gallic acid were reported as the phenolic acid constituents in the sambucus formosananakai extract, exhibiting the antiviral capacity with reducing the production of progeny hcov-nl particles in vitro. moreover, caffeic acid plays the important component with antiviral activity, as suggested to influence the binding of hcov-nl to the co-receptors (such heparan sulfate proteoglycans) and receptor (ace ). like sambucus nigra l., sambucus formosananakai might process the antiviral features against the broad spectrum of human respiratory coronaviruses, as useful for developing the antiviral agents in the future. all authors declare no potential conflict of interest. synergistic effect of flavones and flavonols against herpes simplex virus 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and genetic characterization of human coronavirus nl in primary human renal proximal tubular epithelial cells obtained from a commercial supplier, and confirmation of its replication in two different types of human primary kidney cells chlorogenic acid inhibits the replication and viability of enterovirus in vitro human coronaviruses: clinical features and phylogenetic analysis study on the chemical constituents of sumbucus chinensis lindl antihepatotoxic principles of sambucus formosana inhibition of hiv infection by flavanoids polyphenols pattern and correlation with antioxidant activities of berries extracts from four different populations of sicilian sambucus nigra l human coronavirus nl utilizes heparan sulfate proteoglycans for attachment to target cells polyphenolic profile and antioxidant activities of madeiran elderberry (sambucus lanceolata) as affected by simulated in vitro digestion polyphenolic content, antiradical activity, stability and microbiological quality of elderberry a review of the antiviral properties of black elder (sambucus nigra l.) products elderberry flavonoids bind to and prevent h n infection in vitro antiviral activity of the infusion (shs- ) from flowers of sambucus nigra l., aerial parts of hypericum perforatum l., and roots of saponaria officinalis l. against influenza and herpes simplex viruses caffeic acid inhibits hcv replication via induction of ifnα antiviralresponse through p -mediated keap /nrf signaling pathway inhibitory effects of caffeic acid, a coffee-related organic acid, on the propagation of hepatitis c virus free and chemically bonded phenolic acids in barks of viburnum opulus l. and sambucus nigra l inhibition by caffeic acid of the influenza a virus multiplication in vitro antiviral ability of kalanchoe gracilis leaf extract against enterovirus and coxsackievirus a epigallocatechin- -gallate inhibits the early stages of japanese encephalitis virus infection anti-hepatitis b virus activity of chlorogenic acid, quinic acid and caffeic acid in vivo and in vitro caprifoliaceae. flora of taiwan study on the chemical constituents of sumbucus chinensis lindl anti-pandemic influenza a (h n ) virus potential of catechin and gallic acid inhibition of several strains of influenza virus in vitro and reduction of symptoms by an elderberry extract (sambucus nigra l.) during an outbreak of influenza b panama simultaneous determination of four components in the herbs of sumbucus chinensis lindl. by high performance liquid chromatography identification of potent angiotensin converting enzyme inhibitors through virtual screening and structure-based pharmacophore design this study was supported by china medical university under the featured areas research center program within the framework of the higher education sprout project by the ministry of education, taiwan (chm - - and cmrc-chm- ). this project was also funded by grants from the ministry of science and technology, taiwan (most - -b- - -my , most - -b- - -my ), china medical university (cmu -bc- , cmu -asia- , cmu -asia- , and cmu -s- ). supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.virusres. . . key: cord- -q f el authors: farhadi, tayebeh; hashemian, seyed mohammadreza title: computer-aided design of amino acid-based therapeutics: a review date: - - journal: drug des devel ther doi: . /dddt.s sha: doc_id: cord_uid: q f el during the last two decades, the pharmaceutical industry has progressed from detecting small molecules to designing biologic-based therapeutics. amino acid-based drugs are a group of biologic-based therapeutics that can effectively combat the diseases caused by drug resistance or molecular deficiency. computational techniques play a key role to design and develop the amino acid-based therapeutics such as proteins, peptides and peptidomimetics. in this study, it was attempted to discuss the various elements for computational design of amino acid-based therapeutics. protein design seeks to identify the properties of amino acid sequences that fold to predetermined structures with desirable structural and functional characteristics. peptide drugs occupy a middle space between proteins and small molecules and it is hoped that they can target “undruggable” intracellular protein–protein interactions. peptidomimetics, the compounds that mimic the biologic characteristics of peptides, present refined pharmacokinetic properties compared to the original peptides. here, the elaborated techniques that are developed to characterize the amino acid sequences consistent with a specific structure and allow protein design are discussed. moreover, the key principles and recent advances in currently introduced computational techniques for rational peptide design are spotlighted. the most advanced computational techniques developed to design novel peptidomimetics are also summarized. different diseases may be caused by pathogens or malfunctioning organs, and using therapeutic agents to heal them has an old recorded history. small molecules are conventional therapeutic candidates that can be easily synthesized and administered. however, many of these small molecules are not specific to their targets and may lead to side effects. moreover, a number of diseases are caused due to deficiency in a specific protein or enzyme. thus, they can be treated using biologically based therapies that are able to recognize a specific target within crowded cells. under the biologic conditions, some macromolecules such as proteins and peptides are optimized to recognize specific targets. therefore, they can override the shortcomings of small molecules. recently, pharmaceutical scientists have shown interest in engineering amino acid-based therapeutics such as proteins, peptides and peptidomimetics. [ ] [ ] [ ] theoretical and experimental techniques can predict the structure and folding of amino acid sequences and provide an insight into how structure and function are encoded in the sequence. such predictions may be valuable to interpret genomic information and many life processes. moreover, engineering of novel proteins or redesigning the existing proteins has opened the ways to achieve novel biologic macromolecules with desirable therapeutic functions. protein sequences comprise tens to thousands of amino acids. besides, the backbone and side chain degrees of freedom lead to a large number of configurations for a single amino acid sequence. protein design techniques give minimal frustration through precise identification of sequences and their characteristics. [ ] [ ] [ ] [ ] considering energy landscape theory, the adequately minimal frustration in natural proteins occurs when their native state is adequately low in energy. the de novo design of a sequence is difficult because there are huge numbers of possible sequences: n for n-residue proteins with only natural amino acids. peptide design should incorporate computational approaches. it can benefit from searching the more advanced fields used for small molecules and protein design. however, the straightforward adoption of computational approaches employed to small-molecule and protein design has not be accepted as a reasonable solution to the peptide design problem. [ ] [ ] [ ] in the peptide drug design, the conformational space accessible to peptides challenges the small-molecule computational approaches. besides, the necessity for nonstandard amino acids and various cyclization chemistries challenges the available tools for protein modeling. furthermore, the aggregation of peptide drugs during production or storage can be an unavoidable problem in the peptide design procedure. rational design of a peptide ligand is also challenging because of the elusive affinity and intrinsic flexibility of peptides. peptide-focused in silico methods have been increasingly developed to make testable predictions and refine design hypotheses. consequently, the peptide-focused approaches decrease the chemical spaces of theoretical peptides to more acceptable focused "drug-like" spaces and reduce the problems associated with aggregation and flexibility. , for the discussions that follow, peptides can be defined as relatively small ( - residues) polymers of amino acids. in physiological conditions, several problems such as degradation by specific or nonspecific peptidases may limit the clinical application of natural peptides. moreover, the promiscuity of peptides for their receptors emerges from high degrees of conformational flexibility that can cause undesirable side effects. besides, some properties of therapeutic peptides, such as high molecular mass and low chemical stability, can result in a weak pharmacokinetic profile. therefore, peptidomimetic design can be a valuable solution to circumvent some of undesirable properties of therapeutic peptides. , in the biologic environment, peptidomimetics can mimic the biologic activity of parent peptides with the advantages of improving both pharmacokinetic and pharmacodynamic properties including bioavailability, selectivity, efficacy and stability. a wide range of peptidomimetics have been introduced, such as those isolated as natural products, synthesized from novel scaffolds, designed based on x-ray crystallographic data and predicted to mimic the biologic manner of natural peptides. using hierarchical strategies, it is possible to change a peptide into mimic derivatives with lower undesirable properties of the origin peptide. over the past years, computational methods have been developed to discover peptidomimetics. in a part of this review, novel computational methods introduced for peptidomimetic design have been summarized. peptidomimetics can be categorized as follows: peptide backbone mimetics (type ), functional mimetics (type ) and topographical mimetics (type ). the first generation of peptidomimetics (type ) mimics the local topography of amide bond. it includes amide bond isosteres, pyrrolinones or short fragments of secondary structure, such as beta-turns. such mimetics generally match the peptide backbone atom-for-atom, and comprise chemical groups that also mimic the functionality of the natural side chains of amino acids. a number of prosperous instances of type peptidomimetics have been reported. the second type of peptidomimetics is described as functional mimetics or type mimetics, which include small, non-peptide compounds that are able to identify the biologic targets of their parent peptide. at first, they were assumed to be conservative structural analogs of parent peptides. however, using site-directed mutagenesis, their binding sites to biologic targets were investigated. the results indicated that type peptidomimetics routinely bind to protein sites that are different from those selected by the original peptide. therefore, type mimetics maintain the ability to interfere with the peptide-protein interaction process without the necessity to mimic the structure of the natural peptide. type peptidomimetics reveal the best conception of peptidomimetics. they consist of the necessary chemical groups that act as topographical mimetics and contain novel chemical scaffolds that are unrelated to natural peptides. here, theoretical and computational techniques to design proteins, peptides and peptidomimetics are reviewed. however, the current review does not deeply highlight the computational aspects of amino acid-based therapeutic design, but only discusses the methods used to design the mentioned therapeutics. figure summarizes the key concepts presented in this study. as some examples, the structures of aldesleukin, leuprolide and spaglumic acid, important amino acid-based therapeutics approved by the us food and drug administration (fda), are shown in figure a computer-aided design of amino acid-based therapeutics figure a ) and leuprolide (pdb id: yy ; figure b ) were obtained from the protein data bank (pdb; http://www. rcsb.org/) and visualized by pymol tool. the structure of spaglumic acid was retrieved (in mol format) from pub-chem database (https://pubchem.ncbi.nlm.nih.gov/) with the pubchem id ( figure c ) and visualized using pymol. aldesleukin, a lymphokine, is a recombinant protein used to treat adults with metastatic renal cell carcinoma (https://www.drugbank.ca/drugs/db ). leuprolide, a synthetic nine-residue peptide analog of gonadotropin releasing hormone, is used to treat advanced prostate cancer (https://www.drugbank.ca/drugs/db ). spaglumic acid is used in allergic conditions such as allergic conjunctivitis. the drug belongs to a class of peptidomimetics known as hybrid peptides. hybrid peptides contain at least two dissimilar types of amino acids (alpha, beta, gamma or delta) linked to each other via a peptide bond (https://www.drugbank.ca/ drugs/db ). in the current study, all fda-approved therapeutics (in ) were retrieved from drugbank (https://www.drugbank. ca/biotech_drugs) and an analysis was conducted to compare their percentages. protein-based therapies, gene or nucleic acid-based therapies, vaccines, allergenics and cell transplant therapies made up . %, . %, . %, . % and . % of total approved therapeutics, respectively. small-molecule drugs made up . % of the approved therapeutics ( figure ). computational designing of proteins can be classified as follows: ) template-based designing in which three-dimensional ( d) farhadi and hashemian structure of a predefined template is adapted to design a sequence and ) de novo designing in which the amino acids' arrangement is changed to generate both sequence and d structure of a completely novel protein. the problem of predicting the fold of an unknown sequence could be solved by utilizing templates. since the fold is unaltered, the backbone atoms are directly located on this framework. moreover, to generate a functional protein, the side chains that can effectively stabilize the structure are added to the backbone. , routine concerns and methods for template-based protein design are reviewed below. selecting the template (scaffold) protein the template (also named as scaffold protein) contains a group of backbone atom coordinates. the coordinates can be retrieved from an available x-ray crystal structure or cautiously from a nuclear magnetic resonance (nmr) structure. computer-aided design of amino acid-based therapeutics fixing the backbone decreases the computational complication, but it may inhibit the main chain modifications to adjust sequence alternation. backbone flexibility can generate designed functionalities over the protein's normal function. the backbone flexibility is introduced through incorporating other closely associated conformations to an existing structure. [ ] [ ] [ ] recently, new functionalities were effectively introduced into the tim-barrel topology. this fold has been detected as one of the most shared structures in distinct protein superfamilies. sequence search and characterization in a design procedure, a protein sequence is selected such that it meets the energetic and geometric constraints established by the chosen fold. sequence search techniques sample different sequences and estimate their energies to gain the one owing the minimum energy. in order to identify the sequences subject to an objective function or a specific energy, a diverse strategies including optimization and probabilistic approaches have been developed. optimization processes may recognize candidate sequences using stochastic or deterministic methods. probabilistic approaches focus on characterizing the sequence space probabilistically. deterministic methods: to achieve a sequence folded into a global minimum energy conformation, deterministic methods search the whole sequence space and identify the global optima. , these methods include dead-end elimination (dee), self-consistent mean field, graph decomposition and linear programming. stochastic algorithms search the sequence space in an exploratory manner. these algorithms include monte carlo algorithms (simulated annealing), graph search methods and genetic algorithms. some of the most commonly used methods are discussed below. dee has been considered as a thorough search algorithm. to find and remove sequence-rotameric positions that are not portions of the global minimum energy conformation, dee compares two amino acid rotamers and removes the one with greater interaction energy. interaction energies are computed for each rotamer of the test amino acid, along with all rotamers of every other amino acid. the situation is repetitively examined for total amino acid states as well as their rotamers until it no longer holds true. , expanding the sequence length increases the combinatorial complication of dee exponentially. therefore, to design sequences of amino acids or larger, application of dee may be restricted. details of the theorem are explained elsewhere. , stochastic search algorithms: as mentioned before, deterministic approaches are perfect to design proteins with small sizes, but show the applied disadvantages with extension of sequence size. stochastic or heuristic methods are valuable to design large proteins. the most widely used method for protein design includes monte carlo sampling. , monte carlo method samples positions of complicated proteins in a way related to a selected probability distribution such as boltzmann distribution. boltzmann distribution specially weighs low-energy configurations. the monte carlo algorithm performs iterative series of calculations. at the primary step of each search, a partially accidental test sequence is generated, and its energy is calculated via a physical potential. during the primary step, both rotamer state and amino acid identity are adjusted and an efficient temperature controls the probable energy alterations. in the next step, named simulated annealing, the temperature gradually decreases and permits favorable sampling of lowerenergy configurations. multiple independent calculations are carried out to converge the system to a global minimum. , for more explanation about the theorems and details of the formulation of the probability distribution and weights, readers are referred to study previous reports. , probabilistic approach: probabilistic approaches are frequently employed when thorough information is not accessible for protein design. in a probabilistic approach, sitespecific amino acid probabilities may be utilized, rather than particular sequences. the procedure is partially motivated by the uncertainties to find sequences consistent with a specific structure. briefly, the backbone atoms are fixed or greatly constrained, side chain conformations are discretely handled, energy functions are estimated and solvation is handled by simple models. however, in order to offer valuable sequence information for design experiments and to find structurally significant amino acids, probabilistic techniques leverage structural characteristics of interatomic interactions. generally, monte carlo methods give a probabilistic sampling of sequences. , in addition, an entropy-based formalism has been defined to predict amino acid probabilities for a certain backbone structure. , the method employs concepts from statistical thermodynamics to assess the sitespecific probabilities. to address the whole space of existing compositions, the theory is not restricted by the computational enumeration and sampling. large protein structures with . variable residues can be supplied simply. sampling sequence space to generate conformations the chemical variability of a sequence and the number of various amino acids permitted at each position are defined as "degrees of freedom for each amino acid". moreover, each of the natural residues search the whole sequence space. drug design, development and therapy : submit your manuscript | www.dovepress.com to decrease the degrees of freedom for each amino acid and searching the sequence space, diverse approaches such as hydrophobic patterning have been proposed. monomers can be used to probe a protein structure and improve its function, other than the naturally occurring amino acids. sampling of side chain conformational space to form conformations side chain conformations are typically consistent with the energy minima of molecular potentials and can be obtained from a structural database. rotamer statuses are related to the repeatedly detected values of dihedral angles in the side chain of each amino acid. for example, the simplest amino acids including alanine and glycine have only one rotamer status, while the bigger amino acids have . diverse rotamer statuses. a variety of rotamer libraries including backbonedependent, secondary structure-dependent and backboneindependent libraries have been developed for protein design. , by using a rotamer library, one can discretize a meaningful state space to decrease the computational difficulty. rotamer libraries can be extended beyond the natural amino acids. the effective rotamers can model cofactors, ligands, water and posttranslational modifications. for example, to improve the modeling of protein-protein interactions and model water within proteins interiors, the structurally definite water molecules can be inserted as a solvated rotamer library. energy functions have been employed to quantify sequencestructure compatibilities. they include linear associations of hydrogen bonds made by backbone atoms, repulsion among atoms, hydrophobic attraction among non-polar groups and electrostatic interactions among sequential neighbors. the sequence of a protein is selected so that it can adjust the energetic and geometric constraints enforced by the favorite fold. constraints typically contain several intramolecular interactions such as van der waals, hydrophobic, polar and electrostatic interactions, as well as hydrogen bonds. generally, by using a scoring function, it is possible that energetic contributions of the mentioned parameters are taken into account. , , de novo design: designing the sequence and d structure through assembly of proteins fragments , or secondarystructure elements, , novel structures can be modeled de novo. in the design procedures, the backbone coordinates are generally constrained. summary and important findings of some proteins designed using computational approach including a retroaldol enzyme, the kemp elimination enzyme, a novel βαβ protein, a redesigned procarboxypeptidase, a novel α/β protein structure and the top are shown in table . peptide design methods have been categorized as ligand-and target-based design methods. in the ligand-based designing procedure, information derived from peptides is used to design novel therapeutic peptides. in the target-based method, information derived from target proteins is specifically utilized. typically, a hybrid approach including both ligand-and target-based design is utilized. ligand-based peptide design the ligand-based design has been classified as follows: ) sequence-based, ) property-based and ) conformationbased design. sequence-based approach uses the information of conserved regions and analyzes the multiple sequence alignments. this method is directed by the hypothesis that conserved regions are functionally and structurally significant. computational tools allow the ligand-based peptide design, although they lag behind bioinformatics strategies developed for protein designing. recently, using a method based on a pam matrix, the relationship between a series of collagen peptides and antiangiogenic activity including proliferation, migration and adhesion was analyzed. the pam matrix captured information of mutation rates among all pairs of amino acids. based on the results, regions at the c and n termini of the peptides were detected to be significant for an ideal activity and suggested as two distinct binding sites. the approach showed the potential worth of the sequence-based peptide design. in another report, a computational platform called sarvision was developed to support sequence-based design. sarvision signifies an important step for peptide sequence/activity relationship (sar) analysis. moreover, it pools the improved visualization abilities with advanced sequence/activity analysis. compared to small molecules, property-based design methods for peptides are in the early stages of development. in a recent study, the Δg decomposition per residue and the physicochemical characteristics of amino acids, such as hydrophilicity, hydrophobicity and volume, were used computer-aided design of amino acid-based therapeutics to model peptide binding to targets of interest. , finally, a model was built to estimate peptide Δg values for binding to the class i major histocompatibility complex (mhc) protein hla-a* . furthermore, in a wide range of studies, antimicrobial peptides were successfully analyzed by using the property-based approach. for example, a machinelearning method was employed to design novel antimicrobial peptides. the victory of the property-based methods with antimicrobial peptides may be explained by the fact that the desired biologic activity of membrane disruption is relatively nonspecific. in the case of conformation-based peptide design, computational techniques were developed to predict the conformational ensembles or structure of peptides and analyze the sars. , pep-fold is an online tool used to predict the d structures of peptides of length - residues. a remarkable suggestion from the data is that pep-fold seems to solve the conformational sampling problem. , in order to search conformational spaces of a peptide, long timescale molecular dynamic simulations have been employed. , besides, quantum mechanical calculations are promising to address the scoring deficiency in the peptide conformational examination. apparently, to affect the peptide design processes positively, improving the major theoretical and technical issues is necessary before such computationally sophisticated and costly procedures. conformation of a peptide may be modeled to generate a d pharmacophore hypothesis. a certain pharmacophore hypothesis is useful to determine the adme/tox activities or particular potencies of a peptide. for example, screening of a peptide library was jointed to generate a pharmacophore hypothesis to identify potent agonists of melanocortin- receptor isoforms. a combinatorial tetrapeptide library was screened, and sar and ligand-derived pharmacophore templates were generated. the pharmacophore hypothesis was proposed to allow continuous attempts in the rational design of melanocortin receptor molecules. target-based peptide design compared to ligand-based peptide design, target-based design appears to be in a more improved level. targetbased design is initiated with the computer-aided survey of a ligand-bound or unbound protein target to recognize its potential binding sites, prospective specificity surfaces and other pharmacologic activity elements. the phase is generally followed by an in silico design phase where computational methods perform, refine and evaluate peptide design ideas. some recently developed computational methods for targetbased peptide design are reviewed below. recently, an increase in the number of protein-peptide d structures deposited in the pdb has assisted to search the molecular mechanism and structural basis of peptide recognition and binding. information of crystal structures of protein-peptide complexes can improve our knowledge of the farhadi and hashemian chemical forces involved in the binding and special modes of binding. dynamic data of the complexes can be partially extracted from the solution nmr structures deposited in the pdb. to record the structures and functions of various protein-peptide complexes, the experimentally resolved structure data were gathered, annotated and analyzed, and several distinctive databases such as pepx, pepbind and peptiddb were generated. the pepx database, derived from the pdb, comprises unique protein-peptide interface collections. the pepbind database contains , proteinpeptide complex structures from the pdb. peptiddb is a curated database of protein-peptide complexes. the abundance of the structural information specifically on monomeric proteins could be gathered to design proteinpeptide interactions with no requirement for their sequence homology. protein-peptide docking precise docking of a highly flexible peptide is a major challenge. traditional docking protocols, such as autodock, vina , and moe-dock, developed for docking of small molecules, were also used to dock a peptide to a protein receptor. however, comparative studies revealed that these techniques would face failure if the docked peptides were . residues long. therefore, development of peptide-focused docking protocols is very important. other protein-protein docking tools such as z-dock and hex have been used for the computational peptide design in some studies. below, details of recently developed peptide-focused docking approaches are discussed. first, heuristic evolution procedures were applied to search the large conformational space of linear peptides before the binding. however, these procedures were not efficient and their use was limited. then, a scheme based on conformational sampling became common in the peptide docking. besides, several illustrative approaches were proposed to balance between the accuracy and efficacy of the flexible peptide docking. in this aspect, docscheme, dynadock and pepspec were integrated to online userfriendly interfaces and introduced. recently, pepcrawler and flexpepdock were developed as the peptide docking tools. it is reported that flexpepdock has sub-angstrom accuracy in reproducing the crystal structures of protein-peptide complexes. all of the flexpepdock-based methods assume previous information about the peptide-binding site. anchordock, a recently described algorithm, allows powerful blind docking calculations through relaxing the constraint. the program predicts anchoring origins on a protein surface. following recognition of the anchoring origins, an assumed peptide conformation is refined using an anchor-constrained molecular dynamic process. haddock, a well-known protein-protein docking tool, has been recently expanded to run the flexible peptide-protein docking. to handle a docking procedure, haddock uses ambiguous interaction restraints based on the experimental information about intermolecular interactions. this rigid body peptide docking is followed through a flexiblesimulated annealing process. the novel haddock strategy initiates docking computations from an ensemble of three dissimilar peptide conformations (eg, α-helix, extended and polyproline-ii) that are high informative inputs. cabs-dock is a recently introduced protein-peptide docking tool and runs a primary docking procedure whose outcomes can be refined by other tools such as flexpepdock. in the primary phase of the procedure, random conformations of a peptide are predicted and located around the protein target of interest. the process is followed by replica exchange monte carlo dynamics. subsequently, models are selected for the last optimization using the modeller tool to gain accurate scoring and ranking poses. , galaxypepdock was developed to use experimentally resolved protein-peptide structures for running the template-based docking pooled by flexible energy-based optimization. atomistic simulation atomistic monte carlo and molecular dynamics simulations are accurate, but they are meticulous techniques to investigate peptide-protein binding interactions. these techniques can also detect the thermodynamic profile and trajectory included in protein-peptide identification. these methods predict the association among conformations of a peptide in solution or protein. in a study, in order to describe the binding of a decapeptide to the cognate sh receptor, a long-term molecular dynamic simulation was used and a two-state model was built. in the first step, a relatively quick diffusion phase, nonspecific encounter complexes were generated and stabilized by using electrostatic energy. the secondary step was a slow modification phase, in which the water molecules were emptied out from the space between the peptide ligand and the receptor. in another report, by using monte carlo method, the mentioned two-state model was verified to trace some oligopeptide routes for binding to various pdz (post synaptic density protein, drosophila disc large tumor suppressor, and zonula occludens- protein) domains. drug design, development and therapy : submit your manuscript | www.dovepress.com computer-aided design of amino acid-based therapeutics the affinity of bh peptides to bcl- protein was investigated, and results showed the higher affinity of bound peptides occurred when the corresponding peptides were in a lower degree of disorder in unbound states and vice versa. these results showed that the highly structured peptides could increase their affinity through reducing the entropic loss associated with the binding. overall, in addition to the electrostatic and hydrophobic forces, protein-peptide interactions can be affected by the entropic effect and conformational flexibility that could be willingly examined with atomistic simulations. very recently, using a fast molecular dynamics simulation, the energetic and dynamic features of protein-peptide interactions were studied. in most cases, the native binding sites and native-like postures of protein-peptide complexes were recapitulated. additional investigation showed that insertion of motility and flexibility in the simulation could meaningfully advance the correctness of protein-peptide binding prediction. peptide affinity prediction most features of computational peptide design are based on the accuracy and efficacy of affinity prediction. hence, the fast and reliable prediction of peptide-protein affinity is significant for rational peptide design. in this aspect, two categories of prediction algorithms including sequence-and structure-based approaches were developed. the sequencebased method uses the information derived from primary polypeptide sequences to approximate and evaluate the standards of the binding affinity. the structure-based process takes the information derived from d structures of proteinpeptide complexes to predict the binding affinity. at the sequence level, the quantitative structure-activity relationships (qsars) have been widely utilized to forecast the binding affinity of peptides and conclude the biologic function. to model the statistical correlation between sequence patterns and biologic activities of experimentally assessed peptides, machine-learning methods such as partial least squares (pls), artificial neural networks (ann) and support vector machine (svm) have been used. the obtained correlations have been used to infer experimentally undetermined peptides. the relationship between the biologic activity and molecular structure is an important issue in biology and biochemistry. qsar is a well-established method employed in pharmaceutical chemistry and has become a standard tool for drug discovery. however, the predictive capacity of qsar techniques is generally weaker than statistics-based approaches. therefore, a combination of the qsar method with a statistic-based technique may bring out the best in each other and can be a trend in future developments of drug discovery. at the structural level, numerous reports on affinity prediction have addressed the mhc-binding peptides. plentiful mhc-peptide complex structure records have been deposited in the pdb. the significance of domain-peptide recognition has been recently illustrated in the metabolic pathway and cell signaling. to predict the protein-peptide binding potency, a number of strict theories were suggested based on the potential free energy perturbation. the theories computed the alteration of free energies upon the interaction between phosphor-tyrosine-tetra-peptide (pyeei) and human lck sh domain. furthermore, to obtain a deep insight into the structural and energetic aspects of peptide recognition by the sh domain, a number of molecular modeling experiments such as homology modeling, molecular docking and mechanism dynamics were used. peptide array strategies confirmed that some peptide candidates may be potent binders of the abl sh domain. very recently, an approach including quantum mechanics/molecular mechanics, semiempirical poisson-boltzmann/surface area and empirical conformational free energy analysis was developed to quantitatively illustrate the energetic contributions involved in the affinity losing of pdz domain and oppa protein to their peptide ligands. , de novo peptide design recently, in order to de novo target-based peptide design, two remarkable methodologies including the vital method and an approach developed by bhattacherjee and wallin were introduced. the vital method pools verterbi algorithm with autodock to design peptides for the binding sites of a target. the "bhattacherjee and wallin" approach explores both peptide sequence and conformational space around a protein target at the same time. this approach was tested on three dissimilar peptide-protein domains to assess its ability. a brief list of the existing computational resources employed in peptide design is presented in table . in recent years, some computational methods have been proposed to design peptidomimetics. these methods can be classified based on their specificity to translate peptides to peptidomimetics. to select the best method, drug design, development and therapy : submit your manuscript | www.dovepress.com farhadi and hashemian awareness about the structure of peptide-protein complexes is important. , herein, recently introduced methods for computer-aided design of peptidomimetics are presented. growmol is a combinatorial algorithm employed in the peptidomimetics design. growmol searches a variety of probable ligands for the binding sites of a target protein and produces molecules with the chemical and steric complementarity for the d structure of binding sites. this method was used to generate peptidomimetic inhibitors of thermolysin, hiv protease and pepsin. by using the x-ray crystal structures of pepstatin-pepsin complexes, growmol predicted therapeutic peptidomimetics against the aspartic proteases. the algorithm created some cyclic inhibitors bridging the side chains of cysteine residues in the pl and p inhibitor subsites. the binding modes were checked using x-ray crystallography. , ludi is another interesting software referring to the de novo methodology. by using natural and non-natural amino acids as building blocks, the software designed peptidomimetics against renin, thermolysin and elastase. conformational flexibility of each novel peptidomimetic was searched through sampling the multiple conformers of each amino acid. peptide-driven pharmacophoric hypothesis is the most perceptive computational technique discovered in the peptidomimetics design. the method is especially useful when the x-ray structures of protein-protein complexes exist. the main idea is to adapt the hot spot concept into the associated pharmacophoric feature concept. with a pharmacophorebased virtual screening process, this strategy can determine novel type mimetics. in fact, the side chains of each amino acid can be simply categorized based on the conventional pharmacophoric characteristics, such as hydrogen bond donors and acceptors, aromatic ring and charged and hydrophobic centers. for example, in a report, pharmacophore model directed synthesis of the non-peptide analogs of a cationic antimicrobial peptide identified an anti-staphylococcal activity. to make a pharmacophore hypothesis, a model of rna iii-inhibiting peptide (rip), a well-known heptapeptide inhibitor of the staphylococcal pathogenesis, was utilized. through the virtual screening of , commercially available small molecules based on the rip-based pharmacophore, hamamelitannin was discovered as a non-peptide mimetic of rip. hamamelitannin is a tannin derivate extracted from hamamelis virginiana. , in another study, two rounds of in silico screening were performed to discover potential peptidomimetics able to mimic a cyclic peptide (cyclo- [cpfvktqlc] ) that is known to bind the anb integrin receptor. at the end of the process, the most potent representatives were at least , times better than the original cyclopeptide (around mm). in a prosperous instance, virtual screening was done by using multi-conformational forms of a large commercial library. a target-based pharmacophoric model mapped the cd -binding site on hiv- gp . the pharmacophore hypothesis was made based on a homology model of the protein cavity. in a cell-based assay, two of the top scoring molecules were detected as micromolar inhibitors of hiv- replication. computer-aided design of amino acid-based therapeutics the pharmacophore-based screening was used to find the novel alzheimer's therapeutics as mimetics of neurotrophins. the therapeutic utilization of neurotrophins might be restricted because of several deficiencies such as its reduced central nervous system penetration, decreased stability and potency to enhance neuronal death through interaction with the p ntr receptor. the mimetism of particular nerve growth factor domains could inhibit neuronal death. peptidomimetics of the loop and loop domains of nerve growth factor can prevent neuronal death induced by p ntr-dependent and trk-related signaling. in another study, a full-computational pharmacophorebased approach assessed the fda-approved drugs as valuable candidates to inhibit protein-protein interactions. peptide structures were designated in terms of pharmacophores and searched against the fda-approved drugs to detect same molecules. the top ranking drug matches contained several nuclear receptor ligands and matched allosterically to the binding site on the target protein. the top ranking drug matches were docked to the peptide-binding site. the majority of the top-ranking matches presented a negative free energy change upon binding that was comparable to the standard peptide. geometry similarity method geometry similarity methods create a geometric similarity between non-peptide templates and peptide patches. in a study, the supermimic tool was developed to recognize peptide mimetics. in the program, a complex library of peptidomimetics composed of several protein structure libraries has been deposited. moreover, supermimic includes the d-peptides, synthetic components (reported as betaturn or gamma-turn mimetics) and peptidomimetic ligands obtained from the pdb. in the program, the searching process allows scanning a library of small molecules that mimic the tertiary structure of a query peptide followed by scanning of a protein library where a query for small molecule can adopt into the backbone. , sequence-based method recently, a method has been developed to rank peptide compound matches that are limited to short linear motifs in proteins and compounds with amino acid substituents. the algorithm allows mapping the side chain-like substituents on every compound of a large chemical library. the complete molecule can be signified by a short sequence, and each fragment in the molecule can be represented as a distinct letter abbreviation. a cross-search between the pubchem database (about . million molecules) and a non-redundant collection of , peptides obtained from pdb demonstrated that the algorithm can be useful for high-throughput measurements. to recognize a true positive, the method explored identified protein motifs against the national cancer institute developmental therapeutic program compound database. in another study, the similarity of amino acid motifs to compounds web server was developed to ease screening of identified motif structures against bioactive compound databases. the methodology was reported to be efficient since the compound databases were preprocessed to maximize the accessible data, and the necessary input data was minimal. in similarity of amino acid motifs to compounds, motif matching can be full or partial that may decrease or enhance the number of potential mimetics, respectively. using a novel search algorithm, the web service can perform a fast screening of known or putative motifs against ready compound libraries. the classified results can be examined by linking to appropriate databases. , fragment-based method replacement with partial ligand alternatives through computational enrichment is a fragment-based approach. by using structures of peptide-bound proteins as design anchors, the program can computationally find a non-peptide mimetic for specific determinants of known peptide ligands. hybrid peptide-driven shape and pharmacophoric method development and application of strategies for pharmacophore modeling indicate that the medicinal chemistry community has broadly accepted the intuitive nature of the pharmacophore concept. besides, shape complementarity has been identified as a significant element in the molecular identification between ligands and their targets. in virtual screening efforts, using the pharmacophore-and shape-based techniques distinctly may increase the rate of false-positive results. therefore, incorporating both pharmacophore-and shape-matching techniques into one program can potentially diminish the rate of false positives. recently, to discover novel peptidomimetics, a weboriented virtual screening tool named pepmmsmimic was developed to pool the conventional pharmacophore matching with shape complementarity. a library of million conformers were extracted from . million commercially available chemicals and gathered in the mmsinc database. the database was used as a skeleton to develop farhadi and hashemian pepmmsmimic. in the pepmmsmimic interface, the d structure of a protein-bound peptide is used as an input. then, chemical structures able to mimic the pharmacophore and shape similarity of the original peptide are proposed to involve in the protein-protein recognition. a list of in silico methods used to design potential peptidomimetics along with their strengths and weaknesses is presented in table . overall, design and development of therapeutics are tedious, expensive and time-consuming procedures. therefore, using modern approaches including computer-aided design methods can lessen the examination phase, price and failure of therapeutics discovery. computational methods used to design amino acid-based therapeutics can increase the range of available biotherapeutics. benefiting from the dramatic advance in bioinformatics, computational tools can be used to find and develop therapeutic proteins, peptides and peptidomimetics. , moreover, using the computational tools decrease the cost of therapeutics development, from concept to market, by up to %. however, in the computational protein designing, there are some challenges such as our inadequate knowledge of folding and physical forces that stabilize protein structures. moreover, sequences and local structures have many degrees of freedom that can complicate the sequence search. therefore, there is a requirement for effective methods to find sequences related to a particular structure and measure essential protein folding criteria. overall, in silico design of amino acid-based therapeutics includes many challenges that should be removed to improve the overall performance of the design processes. for example, although structure determination of all disease-related proteins through crystallography and nmr is a laborious task, it is necessary to gather much structural information of peptide-protein interactions. besides, development of vigorous algorithms to calculate protein-protein binding energies is essential. the estimation of binding constant between two macromolecules with an appropriate speedaccuracy tradeoff needs millisecond scale molecular dynamics. moreover, understanding of both protein-protein and protein-peptidomimetics recognition processes in a molecular level can be improved using higher accurate force fields such as quantum mechanical polarizable force. in recent years, there are growing examples on the approval of monoclonal antibodies (therapeutic antibodies) by the fda for treatment of various diseases. this important area of amino acid-based therapeutics has been covered in more depth elsewhere. , for more explanation about the theorems and details of antibody informatics for drug discovery as well as the computer-aided antibody design, readers are referred to study previous reports. , the authors report no conflicts of interest in this work. computer-aided design of amino acid-based therapeutics what is the future of targeted therapy in rheumatology: biologics or small molecules protein therapeutics: a summary and pharmacological classification in silico methods for design of biological therapeutics constructing novel chimeric dna vaccine against salmonella enterica based on sopb and groel proteins: an in silico approach in silico phylogenetic analysis of vibrio cholera isolates based on three housekeeping genes designing of complex multi-epitope peptide vaccine based on omps of klebsiella pneumoniae: an in silico approach theoretical and computational protein design annu evaluation of in silico protein secondary structure prediction methods by employing statistical techniques inhibition of mycobacterial cyp enzyme by sesamin and β-sitosterol: an in silico and in vitro study theory of protein folding: the energy landscape perspective toward an outline of the topography of a realistic protein-folding funnel artificial diiron enzymes with a de novo designed four-helix bundle structure computer-enabled peptide drug design: principles, methods, applications and future directions docking small peptides remains a great challenge: an assessment using autodock vina empirical estimation of local dielectric constants: toward atomistic design of collagen mimetic peptides recent work in the development and application of protein-peptide docking rational design of peptide drugs: avoiding aggregation computational peptidology: a new and promising approach to therapeutic peptide design strategies employed in the design and optimization of synthetic antimicrobial peptide amphiphiles with enhanced therapeutic potentials multifaceted roles of disulfide bonds. peptides as therapeutics peptidomimetics, a synthetic tool of drug discovery an in silico pipeline for the design of peptidomimetic proteinprotein interaction inhibitors (order no. ) natural products as sources of new drugs over the last years diversity-oriented synthesis of macrocyclic peptidomimetics structure-based design, synthesis, and biological evaluation of peptidomimetic sars-cov clpro inhibitors advances in amino acid mimetics and peptidomimetics a hierarchical approach to peptidomimetic design mimicking peptides… in silico peptidomimetic design rational design for peptide drugs peptidomimetics as a cutting edge tool for advanced healthcare an unusual functional group interaction and its potential to reproduce steric and electrostatic features of the transition states of peptidolysis low molecular weight, non-peptide fibrinogen receptor antagonists neurotrophin small molecule mimetics: candidate therapeutic agents for neurological disorders design of peptides, proteins, and peptidomimetics in chi space molecular technology. designing proteins and peptides molecular engineering: an approach to the development of general capabilities for molecular manipulation x-ray versus nmr structures as templates for computational protein design high-resolution protein design with backbone freedom prediction of protein-protein interface sequence diversity using flexible backbone computational protein design backbone flexibility in computational protein design de novo computational design of retro-aldol enzymes one fold with many functions: the evolutionary relationships between tim barrel families based on their sequences, structures and functions search and sampling in structural bioinformatics the dead-end elimination theorem and its use in protein side-chain positioning application of a self-consistent mean field theory to predict protein sidechains conformation and estimate their conformational entropy design of protein-interaction specificity gives selective bzip-binding peptides computational methods for protein design and protein sequence variability: biased monte carlo and replica exchange exploring the conformational space of protein side chains using dead-end elimination and the a* algorithm side-chain and backbone flexibility in protein core design dead-end elimination with a polarizable force field repacks pcna structures improved prediction of protein side-chain conformations with scwrl trading accuracy for speed: a quantitative comparison of search algorithms in protein sequence design using self-consistent fields to bias monte carlo methods with applications to designing and sampling protein sequences computational design and characterization of a monomeric helical dinuclear metalloprotein statistical theory of combinatorial libraries of folding proteins: energetic discrimination of a target structure achieving stability and conformational specificity in designed proteins via binary patterning photophysics of a fluorescent non-natural amino acid: p-cyanophenylalanine an expanded eukaryotic genetic code a "solvated rotamer" approach to modeling watermediated hydrogen bonds at protein-protein interfaces rotamer libraries in the st century improved side-chain prediction accuracy using an ab initio potential energy function and a very large rotamer library potential energy functions for protein design de novo design of foldable proteins with smooth folding funnel: automated negative design and experimental verification assembly of protein tertiary structures from fragments with similar local sequences using simulated annealing and bayesian scoring functions structure by design: from single proteins and their building blocks to nanostructures computational de novo design and characterization of a four-helix bundle protein that selectively binds a nonbiological cofactor using α-helical coiled coils to design nanostructured metalloporphyrin arrays kemp elimination catalysts by computational enzyme design de novo design of a βαβ motif high-resolution structural and thermodynamic analysis of extreme stabilization of human procarboxypeptidase by computational protein design design of a novel globular protein fold with atomic-level accuracy novel peptide-specific quantitative structure activity relationship (qsar) analysis applied to collagen iv peptides with antiangiogenic activity development of an informatics platform for therapeutic protein and peptide analytics two-level qsar network ( l-qsar) for peptide inhibitor design based on amino acid properties and sequence positions recent development of peptide drugs and advance on theory and methodology of peptide inhibitor design predicting the affinity of epitope-peptides with class i mhc molecule hla-a* : an application of amino acid-based peptide prediction a brief overview of antimicrobial peptides containing unnatural amino acids and ligand-based approaches for peptide ligands machine learning assisted design of highly active peptides for drug discovery pep-fold: an updated de novo structure prediction server for both linear and disulfide bonded cyclic peptides in silico predictions of d structures of linear and cyclic peptides with natural and nonproteinogenic residues long-timescale molecular dynamics simulations of protein structure and function how fastfolding proteins fold bond distances in polypeptide backbones depend on the local conformation identification of tetrapeptides from a mixture based positional scanning library that can restore nm full agonist function of the l p, i t, i s, a v, c y, and c r human melanocortin- polymorphic receptors (hmc rs) the protein data bank protein design with fragment databases pepbind: a comprehensive database and computational tool for analysis of protein-peptide interactions the structural basis of peptide-protein binding strategies protein-peptide interactions adopt the same structural motifs as monomeric protein folds highly flexible protein-peptide docking using cabs-dock computer-aided design of amino acid-based therapeutics virtual screening for potential inhibitors of ctx-m- protein of klebsiella pneumoniae in silico panning for a non-competitive peptide inhibitor comparative evaluation of eight docking tools for docking and virtual screening accuracy in silico designing of peptide inhibitors against pregnane x receptor: the novel candidates to control drug metabolism computation of the binding of fully flexible peptides to proteins with flexible side chains a flexible docking procedure for the exploration of peptide binding selectivity to known structures and homology models of pdz domains dynadock: a new molecular dynamics-based algorithm for protein-peptide docking including receptor flexibility structure-based prediction of proteinpeptide specificity in rosetta pepcrawler: a fast rrt-based algorithm for high-resolution refinement and binding-affinity estimation of peptide inhibitors rosetta flexpepdock ab-initio: simultaneous folding, docking and refinement of peptides onto their receptors sub-angstrom modeling of complexes between flexible peptides and globular proteins anchordock: blind and flexible anchordriven peptide docking a unified conformational selection and induced fit approach to proteinpeptide docking cabs-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site galaxypepdock: a proteinpeptide docking tool based on interaction similarity and energy optimization predicting peptide structures in native proteins from physical simulations of fragments mechanism of fast peptide recognition by sh domains binding free energy landscape of domain peptide interactions molecular dynamics simulations of pro-apoptotic bh peptide helices in aqueous medium: relationship between helix stability and their binding affinities to the anti-apoptotic protein bcl-xl structural and dynamic determinants of protein-peptide recognition quantitative sequenceactivity model (qsam): applying qsar strategy to model and predict bioactivity and function of peptides, proteins and nucleic acids recent advances in qsar and their applications in predicting the activities of chemical molecules, peptides and proteins for drug design comprehensive comparison of eight statistical modelling methods used in quantitative structure retention relationship studies for liquid chromatographic retention times of peptides generated by protease digestion of the escherichia coli proteome prediction of mhc-peptide binding: a systematic and comprehensive overview domain mediated protein interaction prediction: from genome to network calculation of absolute protein-ligand binding free energy from computer simulations prediction of binding affinities between the human amphiphysin- sh domain and its peptide ligands using homology modeling, molecular dynamics and molecular field analysis characterization of domain-peptide interaction interface: a generic structure-based model to decipher the binding specificity of sh domains why oppa protein can bind sequence-independent peptides? a combination of qm/mm, pb/sa, and structure-based qsar analyses characterization of pdz domain-peptide interactions using an integrated protocol of qm/mm, pb/sa, and cfea analyses computational design of peptide ligands exploring protein-peptide binding specificity through computational peptide screening multiple highly diverse structures complementary to enzyme binding sites: results of extensive application of a de novo design method incorporating combinatorial growth transformation of peptides into non-peptides. synthesis of computer-generated enzyme inhibitors towards the automatic design of synthetically accessible protein ligands: peptides, amides and peptidomimetics structure-based pharmacophores for virtual screening antimicrobial activity of small β-peptidomimetics based on the pharmacophore model of short cationic antimicrobial peptides small molecule inhibitors of hantavirus infection a dynamic target-based pharmacophoric model mapping the cd binding site on hiv- gp to identify new inhibitors of gp -cd protein-protein interactions alzheimer's therapeutics approved drug mimics of short peptide ligands from protein interaction motifs supermimic-fitting peptide mimetics into protein structures identification of potential small molecule peptidomimetics similar to motifs in proteins drug design, development and therapy web server to identify similarity of amino acid motifs to compounds (saamco) replace: a strategy for iterative design of cyclin-binding groove inhibitors swimming into peptidomimetic chemical space using pepmmsmimic mmsinc: a large-scale chemoinformatics database computational drug discovery developability assessment as an early de-risking tool for biopharmaceutical development antibody informatics for drug discovery computer-aided antibody design tumorhope: a database of tumor homing peptides drug-permeability and transporter assays in caco- and mdck cell lines pepx: a structural database of non-redundant protein-peptide complexes rosetta flexpepdock web server -high resolution modeling of peptide-protein interactions pdock: a new technique for rapid and accurate docking of peptide ligands to major histocompatibility complexes predicting peptide binding sites on protein surfaces by clustering chemical interactions protein-peptide complex prediction through fragment interaction patterns pep-sitefinder: a tool for the blind identification of peptide binding sites on protein surfaces vital: viterbi algorithm for de novo peptide design drug design, development and therapy : submit your manuscript | www.dovepress.com submit your manuscript here: http://www.dovepress.com/drug-design-development-and-therapy-journal drug design, development and therapy is an international, peerreviewed open-access journal that spans the spectrum of drug design and development through to clinical applications. clinical outcomes, patient safety, and programs for the development and effective, safe, and sustained use of medicines are the features of the journal, which has also been accepted for indexing on pubmed central. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. visit http://www.dovepress.com/testimonials.php to read real quotes from published authors. key: cord- -rx z orm authors: patra, amlan kumar title: an overview of antimicrobial properties of different classes of phytochemicals date: - - journal: dietary phytochemicals and microbes doi: . / - - - - _ sha: doc_id: cord_uid: rx z orm plants produce a great diversity of phytochemicals, the beneficial properties of which have been used by humans for centuries since the advent of human civilization. with the discovery of effective and potent antimicrobial compounds, these synthetic antimicrobial compounds are widely used to prevent and cure microbial diseases. however, the development of antibiotic resistant strains of bacteria, reduced efficacy and safety of antimicrobials and the search of new antimicrobials against emerging incurable diseases by conventional antimicrobial agents have revived to explore phytochemicals as an alternative to synthetic antimicrobial compounds. although numerous studies have been conducted in vitro and in vivo in the recent years on the efficacy of plant phytochemicals as antimicrobial agents, this chapter provides an overview of the antimicrobial properties of some major group of phytochemicals, namely, different phenolic compounds, alkaloids, saponins, iridoids and secoiridoids, polyacetylenes, glucosinolates, terpenoids, sulfinate, limonoids (tetranortepenoids) and anthranoids against pathogenic bacteria, fungi, viruses and commensal bacteria in the intestinal tracts of humans and animals. this chapter also discusses their antimicrobial mechanisms of action, the efficiency of different groups of phytochemicals against multiple-drug resistant bacteria, the effect of active dietary phytometabolites on the beneficial and pathogenic microbes of the gastrointestinal tracts and the outcomes of combination of phytofactors and drugs interactions. plants contain a wide array of phytochemicals , which have traditionally been utilized for centuries in folk medicines or ethnomedicines. the earliest information on the medicinal use of plants comes from china in bc (greathead ) , from india (in rigveda and atharvaveda) in bc (ramawat et al. ) , from mesopotamia in bc (newman et al. ) , and also from egypt in about bc (davidson and naidu ) . the natural medicines were widely used until the fi rst half of the twentieth century, when a shift towards synthetic medicines that were more effective, patentable and highly profi table, occurred (tyler ) . however, there have been increasing interests towards use of natural chemicals in medicinal purposes in recent years. these ethnomedicines are encouraging for both the public and national health care institutions as alternatives to synthetic drugs due to relatively lower incidences of adverse reactions compared to modern conventional pharmaceuticals along with their reduced cost (nair et al. ) . recently, the growing occurrences of multi-drug resistant strains of bacteria and the appearance of strains with decreased susceptibility to antibiotics have led to a resurgence of research interests in the discovery of novel antimicrobial agents from natural sources for therapeutic and preventive purposes against microbial diseases, food preservatives and feed additives in the animal industry. the ethnopharmacologists, botanists, microbiologists and natural-product chemists are constantly in search of medicinal effi cacy of plants and their phytochemicals , since the reported data so far available on plants are comparatively meager compared to the vast number of plant population. plants produce a great diversity of compounds. the structures of close to , compounds have already been elucidated and there are perhaps hundreds of thousands of such compounds in plants (pichersky and gang ) . only a few of these are part of 'primary' metabolic pathways (those common to all organisms). the rest are secondary metabolites or phytochemicals whose biosynthesis is restricted to selected plant groups (pichersky and gang ) . phytochemicals can be divided into many major classes depending upon the chemical structures, botanical origins, biosynthesis pathways or biological properties. the most phytochemical classifi cation scheme is based on chemical structures such as phenolics, alkaloids, saponins, terpenoids, limonoids, polyacetylenes and secoiridoids and so on. numerous studies have been conducted in vitro and in vivo in the recent years on the effi cacy of plant phytochemicals as antimicrobial agents. this paper presents the antimicrobial properties of some major group of phytochemicals against pathogenic bacteria, fungi and virus, and benefi cial microbes of the gastrointestinal tracts and their mechanism of action. phenolic compounds are a group of phytochemicals , which have a phenol structure, i.e. an aromatic benzene ring bearing at least one hydroxyl substituent (robbins ; vermerris and nicholson ) . phenolic compounds are commonly found an overview of antimicrobial properties of different classes of phytochemicals throughout the plant kingdom, where they protect the plants from microbial infections, ultraviolet radiation and chemical stressors. this large and diverse group of phytochemicals is classifi ed into many subclasses depending upon chemical structures and occurrence in plants. the commonly categorized subclasses of phenolic compounds are simple phenolics (resorcinol and phloroglucinol ), phenolic acids and aldehydes , coumarins , fl avonoids , chalcones , aurones , benzophenones , xanthones , stilbenes , benzoquinones , naphthaquinones , anthraquinones , betacyanins , lignans , and polyphenols (proanthocyanidin , galloyl, hexahydroxydiphenyl ester , hydroxy cinnamic acid , and phloroglucinol derivatives) (vermerris and nicholson ; handique and baruah ) . the detailed structures and chemistry of these phenolic compounds are presented elsewhere (vermerris and nicholson ) . foods containing phenolics are becoming an important part of diets due to their potential anti-oxidative properties. besides, these compounds have also potent anti-microbial properties. the phenolic acid and aldehyde group of phenolic compounds is characterized by the presence of a carboxylic acid or aldehyde group substituted on a phenol (table . ; vermerris and nicholson ) . the naturally occurring phenolic acids generally have two characteristic constitutive carbon frameworks: the hydroxycinnamic and hydroxybenzoic structures (robbins ) . majority of cinnamic and benzoic acid derivatives in plants are linked through ester, ether or acetal bonds to structural components, polyphenols , organic acids (quinic , maleic , tartaric and shikimic acid ), glucose and terpenes (robbins ) . chlorogenic acid is an ester of quinic acid and caffeic acid . some aldehyde analogues of phenols (e.g. vanillin ) are also grouped with phenolic acids (robbins ) . the numbers and positions of the hydroxyl and other groups on the aromatic ring can produce a large number of compounds in this subclass (robbins ; vermerris and nicholson ) . phenolic acids are present in a wide range of plants including in many common foods such as tea, coffee and berries. besides, phenolic acids and aldehydes could be formed by the intestinal microbial biotransformation of other phenolic compounds in the intestine, where they may infl uence intestinal microbiota. a number of simple phenols and phenolic acids possess antibacterial, antiviral and antifungal activities against a wide range of microbes, but at different concentrations. gallic acid and p -hydroxybenzoic acid reduced the viability of camplylobacter jejuni at concentrations as low as mg/l (ganan et al. ) . synaptic acid , vanillic acid , and caffeic acid were microbicidal at concentrations starting at mg/l. ferulic acid and cumaric acid were effective at a concentration of mg/l (ganan et al. ) . ozçelik et al. ( ) recently tested some phenolic acids such as gallic acid, caffeic acid, chlorogenic acid , and quinic acid for their in vitro antiviral, antibacterial, and antifungal activities. all these phenolic acids were inhibitory to herpes simplex virus type (hsv - ), whereas gallic acid, chlorogenic acid and quinic acid showed potent antiviral effect against parainfl uenza virus type at the therapeutic range of . - . mg/l. in general, antibacterial activity of phenolic acids is stronger against gram-positive bacteria than gram-negative bacteria (merkl et al. ; cueva et al. ) . the outer membrane of gram-negative bacteria provides them with a hydrophobic surface structure that is able to exclude certain hydrophilic molecules, making them inherently resistant to many antimicrobial agents including phenolic acids (alakomi et al. ; cueva et al. ) . gram-positive bacteria are enclosed in a plasma membrane covered by a thick peptidoglycan wall and lack an outer membrane (alakomi et al. ; cueva et al. ) . although, phenolic acids are effective against gram-negative bacteria, their antimicrobial effect is strain dependent (e.g. different strains of escherichia coli ; cueva et al. ) . phenolic compounds are usually poorly absorbed in the small intestine, and thus most of the dietary phenolics accumulate in the colon (clifford ; van duynhoven et al. ) . therefore, higher concentrations of phenolic acids may reach in the intestine than the concentrations in diets. phenolics may selectively suppress or stimulate (tzounis et al. ) . chlorogenic, quinic and gallic acids stimulated growth of lactobacillus collinoides relative to control cultures (no additive) up to concentrations of g/l of tomato broth media. in contrast, growth of lactobacillus brevis was little affected during early incubation, which has been suggested to be due to metabolism of these acids (stead ) . from structure-activity relationship , phenols having different alkyl chain length with hydroxyl groups could be important for antimicrobial actions (kubo et al. ) . p-hydroxybenzoic acid, protocatechuric , gentisic acid , vanillic acid , ferulic acid , caffeic acid and their methyl, ethyl, propyl and butyl esters were investigated for antibacterial action. it has been reported that the antimicrobial effect of phenolic acids derivatives increased with the increasing length of the alkyl chain (merkl et al. ) . the presence of hydroxyl groups on the phenol groups and oxidized status of phenol groups also determine the toxicity of microbes. the fl uidity of the cell membrane could be disturbed with increasing hydrophobic alkyl chains. the phenolic acids could enter the molecular structure of the membrane with the polar hydroxyl group oriented into the aqueous phase by hydrogen bonding and nonpolar carbon chain aligned into the lipid phase by dispersion forces (kubo et al. ) . thus, when the hydrophilic force exceeds hydrophobic one, the activity tends to disappear. also, the number and position of substitutions in the benzene ring of the phenolic acids and the saturated side-chain length infl uenced the bacteriocidal effects of phenolic acids against the different microorganisms, but in different ways against gram-positive and gram-negative bacteria (cueva et al. ) . for example, cueva et al. ( ) showed that for benzoic and phenylacetic acids, e. coli was inhibited in the following order of potency: non-substituted > -hydroxy- -methoxy-> -hydroxy-> -hydroxy-> , -dihydroxy-substituted acid. for phenylpropionic acids, the order differed slightly: nonsubstituted > -hydroxy-> -hydroxy-> , -dihydroxysubstituted acid. however, the potency of phenolic acids was in different order for lactobacillus spp. for benzoic acids, the order of potency was: -hydroxy-> -hydroxy-> non-substituted > -hydroxy- -methoxy-> , -dihydroxy-substituted acids, except for lactobacillus coryniformis cect ( -hydroxy-> non-substituted > -hydroxy > -hydroxy- methoxy-substituted acids). for phenylacetic acids, growth inhibition of lactobacilli was on the order of non-substituted > -hydroxy-> -hydroxy-> , -dihydroxysubstituted acids. for phenylpropionic acids, growth inhibition was as follows: nonsubstituted > -hydroxy-> -hydroxy > , -dihydroxy-substituted acids, except for lactobacillus fermentum cect ( -hydroxy > non-substituted > -hydroxyand , -dihydroxy-substituted acids) and lactobacillus plantarum lch (nonsubstituted > -hydroxy-> -hydroxy-> , -dihydroxy-substituted acids). coumarins are naturally found in many families of plants (apiaceae, asteraceae, fabiaceae, rosaceae, rubiaceae, rutaceae and solanaceae) and microorganisms, and approximately , coumarins have been isolated from these sources (weinmann ; smyth et al. ) . coumarins can be classifi ed into fi ve groups depending upon the structure, i.e. coumarins with substituents in benzene ring, coumarins with substituents in pyrone ring, furocoumarins, pyranocoumarins , and coumarin dimmers ( fig. . ; smyth et al. ) . coumarins exhibit a broad diversity for antimicrobial activity. o-acetylcolumbianetin, edultin, cniforin a, columbianadin and imperatorin isolated from the fruits of cnidium monnieri (l.) cuss exerted a little to no appreciable growth-inhibition of gram-positive and gram-negative bacteria (ng et al. ) . an amino-coumarin - -amino- -methylcoumarin showed broad-spectrum antibacterial and antifungal activities (liu et al. ) . melliou et al. ( ) studied the antibacterial activity of pyranocoumarins using an agar disc diffusion method. seselin, xanthyletin, -hydroxyseselin, and -hydroxyalloxanthyletin had no antibacterial effects. coumarin derivatives such as -methoxyseselin and its brominated derivatives, alloxanthoxyletine, the acetylated derivatives, and dipetalolactone were active against all the tested bacteria. a seselin derivative, -bromo- -benzoyloxyseselin showed moderate activity, while three coumarins containing acetoxy groups in pyrano ring were only active against the two gram-positive bacteria. a new coumarin -cajanuslactone isolated from pigeon pea leaves showed anti-bacterial activity against staphylococcus aureus (atcc ), and the minimum inhibitory concentration (mic) and the minimum bactericidal concentration (mbc) were . and . mg/ml, respectively . some seselin derivatives, including derivatives of -methoxyseselin, were found to be potent against human immunodefi ciency virus (hiv ) (xie et al. ) . it has been suggested that the presence of oxygenated substituents in the ether or ester form usually enhances the antibacterial activity, while the presence of free hydroxyl group reduces the activity (melliou et al. ) . this fact could be at least partially attributed to the reduced lipophilicity of the hydroxyl derivatives, which hinders the penetration through the bacterial cell wall (melliou et al. flavonoids are one of the largest groups of secondary metabolites that are distributed in various plant species. they have signifi cant antioxidant properties, which are benefi cial for health. these polyphenolic compounds are constructed basically with an a and c ring of benzo- -pyran- -quinone and a b ring. the main classes of fl avonoids ( fig. . ) (having a hydroxyl group at the -position), e.g. kaempferol , quercetin , galangin , datiscetin , morin , robinetin , isorhamnetin , tamarixetin , quercetagetin and myricetin ; ( ) fl avanones ( - bond saturated), e.g. hesperetin , taxifolin, eriodictyol and naringenin ; ( ) flavan- -ol, e.g. catechin and epicatechin; ( ) isoflavone , e.g. genistein , daidzein and coumestrol ; ( ) anthocyanidins : cyanidin , delphinidin , pelargonidin and peonidin (crozier et al. ) . the majority of fl avonoids commonly remain conjugated with sugars as glycosides . numerous fl avonoid derivatives showed antiviral activity against a wide range of viruses such as hsv , hiv , coxsackie b virus, coronavirus, cytomegalovirus, poliomyelitis virus, rhinovirus, rotavirus, poliovirus, sindbis virus, and rabies virus (de bruyne et al. ; evers et al. ; nowakowska ) . ozçelik et al. ( ) investigated the effects of quercitin, apigenin, genistein , naringin, silymarin and silibinin against hsv - and pi- virus. all fl avonoids inhibited hsv - activity, but only genistein inhibited parainfl uenza type- (pi- ) activity. of the three fl avonoids (baicalin, rutin and naringin) examined by ng et al. , baicalin was found to be the most potent in inhibiting the growth of s. aureus : of the strains tested were inhibited at mg/l. however, no inhibitory activity of rutin and naringin against s. aureus was observed at mg/l. at this concentration, naringin and baicalin inhibited two strains and rutin inhibited one strain of the eight p. aeruginosa strains tested. the fl avonoids compounds display different mode of antiviral action. for instance, baicalein probably block human cytomegalovirus infection at entry level while the primary mechanism of action for genistein may be to block immediateearly protein functioning off human cytomegalovirus (evers et al. ) . both these fl avoinoids did not inhibit the virus replication (evers et al. ) . puupponen-pimia et al. ( ) investigated falovonoid compounds (apigenin, (+)-catechin , chlorogenic acid , cyanidin chloride, delphinidin chloride, isoquercitrin, kaempferol , cyanidin- -glucoside (kuromanin), luteolin, myricetin , pelargonidin chloride, quercetin dehydrate and rutin trihydrate), and phenolic acids (caffeic acid , -coumaric acid, ferulic acid , trans-cinnamic acid) on gram-positive lactic acid bacteria of intestines, gram-negative e. coli cm and salmonella . myrecetin strongly inhibited the growth of lactobacillus as well as e. coli , but did not affect salmonella . luteolin was weakly inhibitory to gram-positive lactic acid bacteria but not to gram-negative bacteria. the anthocyanidins pelargonidin, delphinidin and cyanidin, as well as cyanidin- -glucoside, only inhibited growth of e. coli and had no effect on other bacterial strains (puupponen-pimia et al. ) . however, phenolic acids did not inhibit lactic acid bacteria, but inhibited gram-negative e. coli and salmonella sp. hatano et al. ( ) discussed that some prenylated fl avonoids such as licoricidin (an isofl avan) effectively suppressed the antibiotic resistance of methicillin-resistant s. aureus (mrsa ) compared to other fl avonoids. the addition of m g/ml of licoricidin shifted the mic of oxacillin from - to - m g/ml, and m g/ml of licoricidin reduced it to less than . m g/ml. the requirement for dimethylallyl or equivalent substituents suggests the importance of affi nity for the bacterial cell membrane. an overview of antimicrobial properties of different classes of phytochemicals phenolic acids show greater antimicrobial potency than their corresponding fl avonoids precursors such as the monomers (+)-catechin and (−)-epicatechin (ganan et al. ; cueva et al. ) . therefore, microbial transformations of dietary fl avonoid compounds in the intestine could lead to more potent microbialinhibitory compounds (phenolic acids ) and could reach greater concentrations in the intestine. this may selectively infl uence intestinal bacteria species, and therefore could affect the diversity and metabolic activity of the intestinal microbiota, including the transformation of phenolics in the gut (cueva et al. ) . epigallocatechin gallate exerted strong antibacterial growth against gram-positive bacteria than against gram-negative bacteria (yoda et al. ; engels et al. ) . it has been stated that gram-positive bacteria absorb more epigallocatechin gallate into their peptidoglycan cell wall and aggregate its presence, while gram-negative bacteria do not aggregate and absorb less epigallocatechin gallate (ikigai et al. ; engels et al. ) because of the repulsive negative charge of lipopolysaccharides on the surfaces of gram-negative bacteria. the binding of epigallocatechin gallate to peptidoglycan disrupts its function in osmotic protection, cell division, and cell wall biosynthesis (yoda et al. ) . detailed information of antimicrobial activities of fl avonoids has been discussed elsewhere in this book (chap. ). some phenolic acids (ellagic and gallic acids) or fl avonoids (fl avan- -ol, fl avan- - -diol or fl avan- -ol) in plants are esterifi ed or polymerized into dimeric, oligomeric or polymeric compounds. most abundantly present polyphenolic compounds in plants are tannins , which are usually of two types: hydrolysable tannins (ht) and condensed tannins (ct ). the ht are complex molecules with a polyol as a central core such as glucose, glucitol, quinic acids, quercitol and shikimic acid that is partially or totally esterifi ed with a phenolic group, i.e. gallic acid ( , , -trihydroxy benzoic acid ; gallotannins) or gallic acid dimmer hexahydroxydiphenic acid (ellagitannins) (haslam ) . the ct (proanthocyanidins) are mainly polymers of the fl avan- -ols (epi)catechin and (epi)gallocatechin units, which are linked by c -c and c -c interfl avonoid linkages (ferreira et al. ; hagerman and butler ) . the polyphenols also exert a wide range of antibacterial and antifungal activities. ellagitannin extracts inhibited a range of pathogenic organisms including vibrio cholerae , shigella dysenteriae and campylobacter spp . (silva et al. ; puupponen-pimia et al. ) . puupponen-pimia et al. ( ) reported that berry extracts exhibit selective inhibitory properties against intestinal bacteria such as staphylococcus , salmonella , listeria and lactobacillus strains, and the selective inhibitory actions varied with berry extracts. in general, pathogenic staphylococcus and salmonella were sensitive to various berry extracts and ellagitannins fractions, while pathogenic listeria and benefi cial lactobacillus were not inhibited. rauha et al. ( ) studied antimicrobial effects of some berry extracts against food spoilage and poisoning bacteria. the widest antibacterial activity was present in berries belonging to the genus rubus (cloudberry and raspberry) that are rich in ellagitannins. ellagic acid has been reported to exhibit a dose-dependent inhibitory effect (ic = mm) on helicobacter pylori isolated from peptic ulcer patients (chung ) . tannins isolated from dichrostachys cinerea roots exerted antimicrobial effects against s. aureus , e. coli , shiegella spp. and p. aeruginosa with mic of the tannins ranging between . and . mg/ml, while the mbc ranging between . and . mg/ml (banso and adeyemo ) . gallotannins extracted from the mango seed kernel inhibited the growth of gram-positive food spoilage bacteria and decreased the growth of gram-negative e. coli , but did not affect lactic acid bacteria (engels et al. ) . the antibacterial properties of cranberry juice with inhibition of e. coli adherence to mucosal surfaces by cranberry juice is reported to be associated with the presence of proanthocyanidins (howell et al. ) . many polyphenols have antiviral activities against different types of viruses (de bruyne et al. ; cheng et al. ) . it has been suggested that prodelphinidin b- ¢ -o -gallate (a proanthocyanidin gallate isolated from green tea leaf) showed anti-hsv - properties with the mechanism of inhibiting the attachment and penetration between cells and viruses possibly through the instability of viral glycoproteins (cheng et al. ) . the structure and functional groups of the polyphenol compounds may determine the effectiveness of the antiviral activities (de bruyne et al. ) . the content of small-molecular phenolic compounds have greater infl uence on the antibacterial activity of extracts than tannins (nazaruk et al. ) . thus, polyphenols could be cleaved by bacterial enzymes to form a number of phenolic acids in the intestine, where they may infl uence the microbial populations (bock and ternes ) . engels et al. ( ) recently studied the effects of gallotannins with different galloyl units from mango seed kernel on various gram-positive and gram-negative bacteria. gallotannins showed antibacterial activities with mics ranging from . g/l for s. aureus to . g/l for pediococcus acidilactici . they also observed that degree of galloylation did not affect the growth of bacteria. it has been suggested that the antibacterial activities of gallotannins are due to their strong affi nity for iron and the inactivation of membrane-bound proteins (engels et al. ) . it has also been shown that gallotannins changed the morphology of bacillus subtilis , which has been hypothesized due to inhibition of cell division by binding of gallotannins to the cell wall or inhibition of enzymes involved in cell separation (engels et al. ) . naphthoquinones are widely distributed in plants, fungi, and some animals. lapachol, plumbagone, juglone and lawsone are naturally occurring naphthoquinones an overview of antimicrobial properties of different classes of phytochemicals of plant origin that have antimicrobial effects against various pathogenic bacteria and fungi. adeniyi et al. ( ) reported that two dimeric naphthoquinones, diospyrin and isodiospyrin , isolated from the root of diospyros piscatoria (gurke), a common ingredient in several folk medicines, exhibited a broad spectrum of antibacterial activity against s. pyogenes and s. pneumoniae (mics of diospyrin ranged from . to m g/ml) salmonella choleraesuis serotype typhi ( s. typhi ) and mycobacterium chelonae (mics of diospyrin were between and m g/ml). isodiospyrin was more active than its racemic isomer diospyrin (mics against gram-positive bacteria ranged from . to m g/ml, while those against pseudomonas aeruginosa and s. typhi ranged from to m g/ml). another naphthoquinones, lapachol and b -lapachone, found in species of tabebuia, had relevant effects against candida albicans, candida tropicalis, and cryptococcus neoformans, and were more active than the reference standard, ketoconazole. lapachone showed strong antimicrobial activity than lapachol against the fungi (guiraud et al. ) . methanol extract from the dried inner bark of tabebuia impetiginosa exhibited potent antibacterial activity against h. pylori which contained lapachol and anthraquinones (park et al. ) . alkaloids have been defi ned as n-heterocyclic basic metabolites, although the defi nition does not clearly separate from other n-containing compounds. alkaloids have been classifi ed in many ways depending upon biogenic precursors or carbon skeleton characteristics. they have a great structural diversity compared with other classes of phytochemicals . alkaloids are generally known according to their carbon skeleton structures. pyridine (e.g. piperine), piperidine , quinoline , indole , pyrrolidine , quinazoline , isoquinoline , glyoxaline , lupinane , tropan , phenanthridine , imidazoline , alkaloidal amines and terpenoid types of alkaloids are commonly found in plants (hegnauer ) . alkaloid fractions isolated from strychnos potatorum l.f. (loganiaceae) seeds, which were of indole type, were tested for their antimicrobial properties against some pathogenic gram-positive, gram-negative and acid-fast bacteria and fungi. these fractions had shown considerable antimicrobial activity against both bacteria and fungi at the tested concentrations ( and m g/ml). further, the growth of proteus vulgaris , s. aureus , salmonella typhimurium, vibrio cholerae , mycobacterium tuberculosis, aspergillus niger and c. albicans were signifi cantly inhibited (mallikharjuna and seetharam ) . similarly, two benzophenanthridine alkaloids , dihydrochelerythrine and dihydrosanguinarinealkaloid constituents of bocconia arborea showed considerable antimicrobial activity against gram-positive and gram-negative bacteria and c. albicans ( navarro and delgado ) . sensitivity of dna and rna viruses to alkaloids may differ. ozçelik et al. ( ) investigated various alkaloids namely yohimbine and vincamine (indole -type), scopolamine and atropine (tropane-type), colchicine (tropolone-type), allantoin (imidazolidine-type), trigonelline (pyridine-type) as well as octopamine, synephrine, and capsaicin (exocyclic amine-type) for their antiviral activities against dna virus herpes simplex (hsv - ) type and rna virus parainfl uenza type- (pi- ). all the alkaloids were effective against hsv - at . - . mg/l, but atropin and octopamine showed potent antiviral activities against pi- at . - . mg/l (ozçelik et al. ) . antibacterial alkaloids from chelidonium majus linn, i.e. benzo [c] phenanthridine -type alkaloids, -hydroxydihydrosanguinarine, -hydroxydihydrochelerythrine were potently active against mrsa strains with mics/mbcs ranged from . to . and . to . m g/ml, respectively (zuo et al. ) . there are two rich sources of organosulphur compounds from plants; ( ) alliaceae family containing alliin -alliinase system and ( ) cruciferae ( brassicacae ) family e.g. brassica juncea , wasabia japonica (wasabi), armoracia rusticana (horseradish) and brassica oleracea (caulifl ower) containing glucosinolate-myrosinase (mithen ) . a number of sulphur-containing compounds can be derived from these plants through the action of myrosinase and alliinase enzymes. the primary sulphur-containing constituents in alliums spp. (e.g. a. sativum (garlic), a. cepa (onion), a. porrum (leek)) and brassica spp. (e.g. cabbage, kale, caulifl ower and turnip) are s -alk(en)yl-l-cysteine sulphoxides and g -glutamyl-s -alk(en)yl-lcysteine sulphoxides (block et al. ; ross and milner ; fig. . ) . the content of s-alk(en)yl-l-cysteine sulphoxides in garlic may range from . % to . % of fresh weight with s -allyl-l-cysteine sulphoxide (alliin ) being the largest contributor. by the action of alliinase enzyme present inside the cells, these compounds are converted into thiosulfi nate (a functional group consisting of the linkage r-s(=o)-s-r'), which are then spontaneously and enzymatically converted into a large array of volatile compounds, e.g. diallyl disulphide, diallyl trisulphide, allyl methyl disulphide and dipropyl and disulphide (mithen ) . antimicrobial activities of garlic and onion against a wide range of grampositive and gram-negative bacteria, virus and fungi are known for many years (ankri and mirelman ) . the antifungal activities of garlic oils appear to be more than the antibacterial activity (avato et al. ) . extracts of garlic exhibit the most potent antibacterial activity, followed by onion, and brassica including cabbage (kyung and lee ) . the principal antimicrobial compounds of allium and brassica are allicin ( s -allyl-l-propene thiosulfi nate ) and methyl methanethiosulfinate, respectively (kyung and lee ) . these compounds are derived from s -allyl and s -methyl derivatives of l-cysteine sulfoxide , respectively. avato et al. ( ) tested different mixtures of garlic distilled oils containing diallyl disulfi de (dds) and diallyl trisulfi de (dts), ranging from % to % and % to %, respectively, against yeasts ( c. albicans, c. tropicalis and b. capitatus ), gram-positive bacteria ( s. aureus and b. subtilis ) and gram-negative bacteria ( p. aeruginosa and e. coli ). incubation of garlic extracts made up of % dds and % dts did not show growth inhibition against all the tested microorganisms, whereas garlic oils with higher quantities of dds showed signifi cant inhibitory activity, increasing with the increase of dds amount, thus implicating the dds as the active antimicrobial agent (avato et al. ) . it has been reported that allicin (mic, m g/ml; mbc, m g/ml) was more potent than dds (mic range, - m g/ml; mbc range, - m g/ml), its corresponding sulfi de, but of a strength similar to that of diallyl tetrasulfide (mic range, - m g/ml; mbc range, - m g/ml) against h. pylori (o'gara et al. ) . kyung and fleming ( ) investigated the different s-compounds found in cabbages on the growth of bacteria and fungi. s -methyl-l-cysteine sulfoxide, sinigrin , and dimethyl sulfi de at ppm did not inhibit the growth of any of the bacteria and yeasts. dimethyl disulfi de at ppm retarded the growth of some bacteria, but was not bactericidal to any of the test microorganisms. dimethyl trisulfi de , methyl methanethiosulfi nate and methyl methanethiosulfonate had mics of ppm, between and ppm, and between and ppm, respectively for bacteria, and ppm, between and ppm and between and ppm for yeasts, respectively (kyung and fleming ) . there are numerous reports showing the effectiveness of garlic or allicin as antimicrobial agents in comparison to antibiotics (fujisawa et al. ; cai et al. ) . also, allicin with antibiotics may synergistically augment the antimicrobial actions (cai et al. ; an et al. ) . besides, thiosulfi nates and their derivatives show promising activity against multidrug resistant bacteria including mrsa (ankri and mirelman ; fujisawa et al. ) . the main mode of action of thiosulfi nate derivatives have been proposed to be due to its chemical reaction with the thiol groups of various enzymes (ankri and mirelman ) and thus antimicrobial properties of allicin may be abolished by cysteine, coenzyme a and glutathione (fujisawa et al. ) . antimicrobial activity of the diallyl sulfi des has been reported to increase with the number of sulfur atoms (o'gara et al. ) . glucosinolates are the sulphur-containing metabolites found in large number of edible plants. over glucosinolates are present in families of dicotyledonous angiosperms, most of which are clustered within the brassicaceae and capparaceae (fahey et al. ) . allyl (sinigrin ) and -butenyl (gluconapin) glucosinolate are found in brown mustard, p -hydroxybenzyl glucosinolate in white mustard, allyl and other glucosinolate in horseradish and wasabi, methylthiopropyl in cabbage and -hydroxy -butenyl glucosinolate in rapeseed ( fig. . ; fahey et al. ; mithen ) . the antibacterial and antifungal properties of glucosinolates are known for a long time (fahey et al. ) . intact glucosinolates do not show antimicrobial action, but the hydrolysis products of glucosinolates are active against various microorganisms (manici et al. ; tierens et al. ) . aires et al. ( a ) observed that the in vitro growth inhibition and the sensitivities of the individual bacteria are infl uenced by the structure of glucosinolates and their hydrolysis products. the most effective glucosinolate hydrolysis products were the isothiocyanates ; sulforaphane and benzyl isothiocyanate were the strongest inhibitory against the growth of human pathogenic bacteria. regarding action of glucosinolates products on the type of bacteria, -methyl sulfi nyl butylisothiocyanate exhibited antibacterial activity against a larger range of bacteria. indole- -carbinol had some inhibitory effects against the gram-positive bacteria, but had no effect against the gram-negative bacteria. indole- -acetonitrile had some inhibitory activity against the gram-negative bacteria. glucosinolates, nitriles and amines were ineffective at the doses up to m mol (aires et al. b ) . saavedra et al. ( ) evaluated the in vitro antibacterial actions of different classes of common dietary phytochemicals , i.e. simple phenolics -tyrosol, gallic acid, caffeic acid , ferulic acid , and chlorogenic acid ; chalcone -phloridzin; fl avan- -ol -(−) epicatechin; secoiridoid -oleuropein glucoside; glucosinolate hydrolysis products -allyl isothiocyanate, benzyl isothiocyanate and -phenylethyl isothiocyanate) against four pathogenic microbes. all of the isothiocyanates had signifi cant antimicrobial activities, while the phenolics were much less effi cient. no antimicrobial activity was observed with phloridzin. allyl isothiocyanate from cabbage had an mic between and ppm for bacteria and between and ppm for yeasts (kyung and fleming ) . iridoids is a group of cyclic monoterpenoids having iridane skeleton (cis- -oxabicycle-( . . )-nonane), which mostly remain as glycosides ( fig. . ; perez et al. ) . secoiridoids derive from iridoids by the elimination of the link - to yield the basic structure (perez et al. ) . this group of phytochemicals is found in a number of folk medicinal plants and many of them possess signifi cant biological and pharmacological activities (dinda et al. ) . a number of iridoids and secoiridoids (nepetalactones from serbian nepeta species , nestorović three iridoids, phloyoside , phlomiol, and pulchelloside , isolated from the rhizomes of the iranian fl ora eremostachys laciniata ( lamiaceae ) had low to moderate levels of antibacterial activity (mic = . - . mg/ml) against fi ve bacterial strains, bacillus cereus , citrobactor freundii , proteus mirabilis , p. aeruginosa , s. aureus (modaressi et al. ) . out of these three compounds, pulchelloside showed highest antibacterial activity against b. cereus , penicillin-resistant e. coli , p. mirabilis and s. aureus with an mic value of . mg/ml. nestorović et al. ( ) investigated the nepetalactones content in the methanol extracts of the shoot cultures of three endemic serbian nepeta species: nepeta rtanjensis , n. sibirica and n. nervosa , and evaluated the antimicrobial activity of these extracts against eight bacterial strains e. coli , p. aeruginosa , s. typhimurium , listeria monocytogenes , enterobacter cloacae (human isolate), b. cereus (clinical isolate), micrococcus fl avus and s. aureus , and eight fungal species: aspargillus fl avus , aspargillus fumigatus , aspargillus niger , fusarium sporotrichoides , fulvia fulvum, penicillium funiculosum, p. ochrochloron and trichoderma viride . trans, cisnepetalactone was present in shoots of n. rtanjensis , while cis,trans -nepetalactone stereoisomer was present in n. sibirica . no nepetalactone was observed in shoots of n. nervosa . all these extracts had signifi cant antibacterial and antifungal activities against all the tested species. n. rtanjensis extract showed the strongest antibacterial activity with mic of m g/ml. n. nervosa and n. sibirica extracts showed antibacterial activities with mic of - and m g/ml, respectively. similarly, n. rtanjensis, n. nervosa and n. sibirica extracts showed mic of - , - and - m g/ml, respectively. the presence of trans-nepetalactone in n. rtanjensis extract was probably responsible for strongest activity against bacteria and fungi, while cis-nepetalactone in n. sibirica extract showed higher antibacterial and antifungal activity than that of n. nervosa extract. (geng et al. a (geng et al. , b, . iridoid aglycone moieties, but not its glycosides , exhibit the antiviral activities. zhang et al. ( ) studied an anti-hepatitis c virus pseudoparticles (hcvpp) entry essay on both aqueous and methanol extracts of the fl owering tops of lamium album . iridoid glucoside lamalbid isolated from the methanol extract was inactive against hcvpp, whereas its aglycone, and epimers named lamiridosins a and b present as major constituents in the aqueous extract signifi cantly inhibited in vitro hcv entry (ic value of . m m). these were nontoxic to the hep g . cells at a concentration of m g/ml. they also demonstrated that the parent iridoid glycosides did not show anti-hcv entry activity, but the aglycones of shanzhiside methyl ester, loganin, loganic acid, verbenalin, eurostoside and picroside ii exhibited signifi cant anti-hcv entry and anti-infectivity activities. chemically, saponins are a group of high molecular-weight glycosides , in which saccharide chain units ( - residues) are linked to a triterpene (triterpene saponins ) or steroidal (steroid saponins) aglycone moiety, i.e. sapogenin (fig. . ) . they occur in a wide variety of plants with triterpene saponins (in soybean, alfalfa, quillaja, and guar), and are more widely distributed in nature than steroidal (in yucca, tomato, and oats) saponins ( hostettmann and marston ) . the steroidal saponins may possess furostanol or spirostanol (e.g. smilagenin and sarsapogenin) moiety. the saccharide chains are commonly attached at the c position (monodesmosidic), but some sapogenins contain two saccharide chains (bidesmosidic) attached at the c and c (via c ) position (vincken et al. ) . a large number of saponins could be possible depending upon the modifi cations of the ring structure of aglycone moieties and number of sugars added to it, and in turn producing different biological properties. many plant extracts containing saponins from various plants and purifi ed saponins show antimicrobial activities at different concentrations (sen et al. ; avato et al. ) . however, the types of saponins exhibit different spectra of antimicrobial effects. oleanolic acid isolated from the root bark of newbouldia laevis have broad-spectrum antimicrobial activity against gram-positive, gramnegative bacterial species and three candida species (kuete et al. ) . b -sitosterol- -o -b -d-glucopyranoside isolated from this plant also showed antibacterial effects on three gram-positive, six gram-negative bacterial species and three candida species. a saponin fraction from the stem of y. schidigera exhibited potent growthinhibitory activity with mic ranging from . to m g/ml against certain food-deteriorating yeasts ( c. albicans ), fi lm-forming yeasts ( debaryomyces hansenii, pichia nakazawae, zygosaccharomyces rouxii ), dermatophytic yeasts ( candida famata, hansenula anomala, pichia carsonii ), and against brewer's yeast ( saccharomyces cerevisiae ) (miyakoshi et al. ) . different saponins, i.e. tigogenin from tribulus terrestris , dioscin from the rhizomes of smilacina atropurpurea , minutosides from bulb of allium leucanthum were very active against different fungal strains such as c. albicans , c. glabrata and cryptococcus neoformans (zhang et al. a, b ; barile et al. ) . saponins appear to have stronger activities against fungi, and act by disrupting the membrane integrity of fungal cells. different extraction procedures and storage may affect the antimicrobial action of saponins probably due to chemical transformation of saponins (guclu-ustundag and mazza ) . commercially produced quillaja ( quillaja saponaria ) and yucca ( yucca schidigera ) saponins showed different antibacterial activities against e. coli , suggesting that saponins from various commercial sources differ in their biological activities (sen et al. ) . in this study, commercial saponin-rich quillaja and yucca extracts exhibited antibacterial activity against s. aureus and e. coli at different concentrations. the antimicrobial activity of saponins may also be modifi ed by the ph of media. the tea saponins exhibited greater antimicrobial activities against gram-positive s. aureus (mic < . vs. > . ), gram-negative e. coli (mic . vs. > . ) and c. albicans (mic < . vs. > . ) at low ph than high ph . (li et al. ) . some saponins , in general, exhibit stronger antimicrobial activity against gram-positive bacteria than against gram-negative bacteria (avato et al. ) . saponins fraction from soapnut pericarps ( sapindus mukurossi , tanaka et al. ( ) and guar ( cyamopsis tetragonoloba , hassan et al. a, b ) showed greater antibacterial activity against gram-positive bacteria than against gram-negative bacteria. conversely, saponins isolated from orchid tree ( bauhinia variegata l.) bark exhibited greater antibacterial activity for gram-negative bacteria than gram-positive bacteria at concentrations ranging from . to mg/ml (morrissey and osbourn ) . the relationships between saponin structures and antimicrobial activity are strongly noted. the structure of sapogenin moiety, chain length and composition of sugars infl uences the antimicrobial activities. the y. schidigera saponin fraction possessing a trisaccharide chain without any oxygen functionalities at c- and/or c- of the aglycone exhibited potent anti-yeast activity, while saponins with b-oh or -keto groups showed very weak or no activity. low activity was observed for saponins with a disaccharide chain and no activity was observed for the aglycones obtained after acid hydrolysis (miyakoshi et al. ) . yang et al. ( a ) noted that no activity was observed in the hecogenin saponins when its sugar moiety was less than four monosaccharide units. pentaglycoside was more active than tetraglycoside and shows extended antifungal spectrum against a. fumigatus . in the diosgenin saponin series, saponins with only triglycosides are active against c. albicans and c. glabrata , while the diosgenin saponins with monoglycoside and diglycoside did not show any activity. again, within the group of tigogenin saponins, their antifungal capacity was slightly infl uenced by the composition of the sugar moiety. the replacement of a glucosyl unit with a xylosyl unit showed enhanced activity against a. fumigatus . avato et al. ( ) suggested that the sugar moiety is not important for the antimicrobial effi cacy from their study since antibacterial activity increased from the saponin extracts to the sapogenin samples. terpenoid compounds derive from a basic structure of c isoprene units. they are classifi ed according to the number of isoprene unit involved for their synthesis, i.e. monoterpenoid (c ), sesquiterpenoids (c ), diterpenoids (c ), sesterterpenoids (c ) and triterpenoids (c ). they can be acyclic (myrcene and geraniol), monocyclic (cymene and carvacrol), bicyclic (pinene) and tricyclic with different groups (alcohol, phenol, and aldehyde). the most commonly occurring essential oils (eo) are included in two chemical groups (fig. . ) : terpenoids (monoterpenoids and sesquiterpenoids) and phenylpropanoids, which are synthesized through mevalonate and shikimic acid metabolic pathways, respectively (gershenzon and croteau ; calsamiglia et al. ) . among these two classes, terpenoids are the more diversifi ed group of plant bioactives abundantly found in many herbs and spices (gershenzon and croteau ) . within terpenoids, the most important components of eo of the majority of plants belong to the monoterpenoids and sesquiterpenoids (gershenzon and croteau ; calsamiglia et al. ) . phenylpropanoids have a side chain of three carbons bound to an aromatic ring of c (calsamiglia et al. ) . phenylpropanoids are less abundant compounds of eo compared with terpenoid family, but some plants contain in signifi cant proportions. the eo are a group of secondary plant metabolites obtained from volatile fractions of plants by steam distillation process (gershenzon and croteau ) . the eo are used traditionally by humans, for many centuries, which provide characteristic fl avor and aroma specifi c to many plants, and are used as antimicrobial agents and preservatives. the eo have diverse chemical composition, nature and biological properties. the eo can be obtained from fl owers, petals, leaves, stems, fruits, roots and barks and the concentrations of eo in these parts depends upon the stage of growth, environmental conditions (hart et al. ) . a number of eo are known for their strong anti-microbial activities against many pathogenic and non-pathogenic bacteria and fungi. curcumin and its derivatives, the phenylpropanoids, are the principal compounds in rhizome of curcuma longa (turmeric), which exhibit antibacterial properties against different bacteria and fungi. essential oil fractions of turmeric inhibited the growth of pathogenic gram-positive ( s. aureus and staphylococcus epidermidis ) and gram-negative ( e. coli , p. aeruginosa and s. typhimurium ) bacteria (singh et al. ) . the eo fraction was more effective against gram-positive compared to gram-negative strains, and was comparable to standard antibiotics gentamycin, ampicillin, doxycycline and erythromycin in these strains (singh et al. ) . a recent study by de et al. ( ) demonstrated that curcumin inhibited the growth of different clinical isolates of h. pylori with mics ranging from to m g/ml. the gingerols, another phynylpropanoids from zingiber offi cinalis (zinger), possess antifungal and antibacterial properties (park et al. ) . ginger extract containing gingerol inhibited the growth of h. pylori with mics ranging from . to . m g/ml (mahady et al. ) . constituents of eo differ in their antimicrobial activity against bacteria and fungi. investigating the antimicrobial properties ( bacterial species and fungi) of fi ve eo constituents (cineole, citral, geraniol, linalool and menthol), pattnaik et al. ( ) showed that linalool had the most antibacterial activity and inhibited bacteria, followed by cineole, geraniol (each of which inhibited bacteria), menthol and citral aromatic compounds, which inhibited and bacteria, respectively. however, the antifungal activities of these eo constituents did not follow the pattern of antibacterial activities. citral and geraniol oils were the most effective against fungi (inhibiting all fungi), followed by linalool (inhibiting fungi), cineole and menthol (each of which inhibited fungi) compounds (pattnaik et al. ) . it has been suggested that the ph of eo in culture media may modify antimicrobial properties. for example, anise oil had higher antifungal activity at ph . than at . , while the oil of cedrus deudorawas was most active at ph (janssen et al. ) . the structure and stereochemistry of the essential oils have profound infl uences on the antimicrobial activities. alkenyl substituents incorporated into nonphenolic ring structures of essential oils such limonene showed increased antibacterial activities compared with alkyl substituents such as p -cymene with alkylation showing more inhibitory effect on gram-negative bacteria (dorman and deans ) . from stereochemistry of eo, it has been reported that a -isomers such as a -pinene are less active relative to b -isomers such as geraniol and nerol; cis -isomers are inactive contrary to trans -isomers; compounds with methyl-isopropyl cyclohexane rings are the most active; or unsaturation of the cyclohexane ring further increases the antibacterial activity, e.g. terpinolene, terpineol and terpineolene (hinou et al. ; dorman and deans ) . however, griffi n et al. ( ) reported that the specifi city and level of antimicrobial activity of terpenoids were not always characterized by the functional groups, but were associated with hydrogen-bonding parameters, and for gram-negative organisms a combination of hydrogen-bonding parameters and molecular size parameters. the antimicrobial properties of eo from different sources have been discussed in details elsewhere (chap. ). chemically, limonoids are unique secondary metabolites, characterized by a tetranortriterpenoid skeleton with a furan ring ( fig. . ) . they are commonly isolated from citrous and maliaceae plants (hallur et al. ; rahman et al. ; vikram et al. ) . besides their health promoting effects, various limonoids have been shown to possess antibacterial, antifungal and antiviral effects (govindachari et al. ; battinelli et al. ; atawodi and atawodi ) . various limonoid compounds such as mahmoodin , azadirone , epoxyazadiradione, nimbin , gedunin, azadiradione , deacetylnimbin and -hydroxyazadiradione, isolated from various parts of azadirachta indica (meliaceae family) have been reported to have antimicrobial activities (siddiqui et al. ; govindachari et al. ; atawodi and atawodi ) . rahman et al. ( ) tested two limonoids isolated from the seeds of swietenia mahagoni ( meliaceae family), swietenolide and -hydroxy- -o-tigloylswietenolide against various multiple-drug-resistant bacterial strains including gram-positive ( s. aureus , s. pneumoniae and haemophilus influenzae ) and gram-negative ( e. coli , klebsiella pneumoniae, salmonella typhi, and salmonella paratyphi ) strains. the most potent activity of swietenolide was observed against h. infl uenzae , s. typhi , and s. paratyphi , whereas -hydroxy- -o-tigloylswietenolide was most active against s. pneumoniae, s. typhi, and s. paratyphi . the lowest activity was observed against k. pneumoniae for both compounds. the limonoids compounds may exhibit antibacterial properties against pathogenic bacteria by disrupting the quorum sensing system and biofi lm production. vikram et al. ( ) demonstrated limonin, nomilin, obacunone, deacetyl nomilin and limonin -o-b -d-glucopyranoside purifi ed from seeds of grapefruits to possess the anti-quorum sensing activity and inhibitory effect on biofi lm formation of pathogenic e. coli o :h with obacunone exhibiting strong antagonistic activity. limonoids also have signifi cant antiviral activity. limonin and nomilin showed inhibitory effects on hiv - replication in peripheral blood mononuclear cells and monocytes/macrophages, which was not cytotoxic at the active concentrations (battinelli et al. ) . the antiviral activity was not much infl uenced by structural differences by limonin and nomilin in this study (battinelli et al. parida et al. ( ) demonstrated in an in vivo study that azadirachtin obtained from a. indica inhibited dengue virus type- replication as confi rmed by the absence of dengue-related clinical symptoms in sucking mice and absence of virus specifi c bp amplicon. more than polyacetylene compounds have been characterized from plants, which are mainly prominent in the asteraceae, apiaceae and campanulaceae including many medicinal plants from various parts of the world (hudson ) . food plants of the apiaceae plant family such as carrots, celery, parsley, fennel and parsnip contain a group of bioactive aliphatic c -polyacetylenes including falcarinol, falcarindiol, panaxydiol, and polyacetylene -o-methylfalcarindiol (zidorn et al. ; christensen and brandt ) . avato et al. ( ) investigated the different polyacetylene compounds from the aerial organs of bellis perennis l. of the major constituents, methyl deca- , -diynoate and deca- , -diynoic acid, and their structural analogues, i.e. deca- , -diyne, dimethyl octa- , -diyne- , -dioate and deca- , -diyne- , -dioic acid, deca- , -diynoic acid and deca- , -diyne- , -dioic acid showed antimicrobial activity against gram-positive and gram-negative bacteria, respectively. polyacetylene carboxylic acids, ( e ), -octadecadiene- , -diynoic acid ( , -dihydrooropheic acid, and the known -octadecene- , , -triynoic acid (oropheic acid, isolated from the stem bark of mitrephora celebica demonstrated signifi cant activity against mrsa and mycobacterium smegmatis (zgoda et al. ) . similarly, pentayne diol, a polyacetylene which was isolated from bidens pilosa (a traditional medicinal herbs) showed highly potent and extensive inhibitory activities against several gram-positive and gram-negative pathogenic bacterial species, including mrsa , and vancomycin-resistant enterococcus faecalis and c. albicans (tobinaga et al. ) . in a recent fi nding, a polyacetylene compound from carlina acaulis , i.e. carlina oxide exhibited strong antibacterial activity against two mrsa strains, streptococcus pyogenes, p. aeruginosa, c. albicans , and c. glabrata with less toxicity to human hela cells (herrmann et al. ) . anthranoid compounds are widely distributed in various plants particularly in aloe , cassia, rheum , cassia and frangula , which are traditionally used in ethnomedicine for laxative and cathartic action (paneitz and westendorf ) . naturally occurring anthranoids can be chemically described as dihydroxyanthraquinones, -dianthrones and -anthrones, often present in plants as glycones (table . ; paneitz and westendorf ) . different anthranoids such as aloe-emodin, rhein, emodin, physcion and chrysophanol occur in rheum species. anthranoids have shown antimicrobial properties in different studies. the anthranoid compounds from the rhizome of rheum emodi exhibited antibacterial and antifungal activities (babu et al. ) . the antimicrobial effects of the three anthraquinones on s . aureus found to be in the order of rhein > emodin > , -dihydroxyanthraquinone (wu et al. ) . similarly, wang et al. ( ) demonstrated that the sequence of antimicrobial activity against bifi dobacterium adolescentis of the fi ve hydroxyanthraquinones was rhein > emodin > aloe-emodin > chrysophanol > physician. they also suggested the infl uence of substituent groups on phenyl ring in hydroxyanthraquinones against b. adolescentis activity might be related with the polarity and the sequence was carboxyl > hydroxyl > hydroxylmethyl > methyl and methoxyl. prenylated anthranoids from leaves of harungana madagascariensis have shown to inhibit bacillus megaterum (kouam et al. ) . additionally, the effect of emodin with antibiotics (ampicillin and oxacillin) was found to be synergistic or partially synergistic against mrsa, where emodin reduced the mics of the antibiotics (lee et al. ) . however, some of the anthranoids have potent mutagenic effect (paneitz and westendorf ) , which is required to consider when evaluating the antimicrobial properties of these compounds. there is considerable evidence that a number of phytochemicals have potential to become useful antimicrobial agents that could be employed as preventative or treatment therapies against microbial and viral diseases. although, there are some encouraging effects in vivo to inhibit pathogenic microbes without affecting benefi cial bacteria in the gastrointestinal tracts, more studies would be required for the safety and effi cacy of these phytochemicals to establish whether they could offer therapeutic benefi ts over conventional therapies. besides, the combination of some antimicrobial drugs and phytochemicals may act as better antimicrobial agents than antimicrobial drugs alone. for example, the application of dual combinations demonstrated synergy between streptomycin and gallic acid, ferulic acid , chlorogenic acid , allylisothiocyanate and -phenyle thylisothiocyanate against the gram-negative bacteria. moreover, they can act synergistically with less effi cient antibiotics to control bacterial growth (saavedra et al. ) . , -dihydroxyphenylacetic acid and -hydroxyphenylacetic acid increased the susceptibility of s . enterica subsp. enterica serovar typhimurium strains for novobiocin. in addition, organic acids present in berries, such as malic acid, sorbic acid, and benzoic acid , were shown to be effi cient permeabilizers of salmonella as shown by an increase in the -n-phenylnaphthylamine uptake assay and by lipopolrsaccharide release (alakomi et al. ) . cinnamon essential oil and its major component (trans-cinnamaldehyde) enhanced the antibacterial activity of clindamycin against a toxicogenic strain of clostridium diffi cile ) . in addition, the enhancement activity of different essential oils ( mentha longifolia l. and mentha spicata l.) and different monoterpenes (piperitone, carvone and menthone) on the antibacterial activity of nitrofurantoin has been reported (rafi i and shahverdi ; shahverdi et al. ) . the antibacterial activity of cefi xime, cephotaxime, vancomycin and tetracycline was also increased by curcumin (moghaddam et al. ) . allicin has a synergistic effect with amphotericin b against c. albicans via enhancing the phospholipid peroxidation reaction in vitro and in vivo , which suggests that allicin could reduce the amphotericin b dose to lessen side effects ) . due to the growing use of phytochemicals and other dietary phytochemical-rich supplements, it is required to understand whether problems might arise from using these preparations in combination with conventional drugs. there is lack of comprehensive studies that can establish the consequences of phytochemicals-drug interactions. however, all these evidence also suggest that intake of phytochemicals rich foods could be considered in future research while antimicrobial agents are applied to the body. plant genomes contain , - , genes, and about - % of these genes encode enzymes for secondary metabolism (bevan et al. ; somerville and somerville ) . the genome of a plant species encodes only a small fraction of all the enzymes that are required to synthesize the entire set of secondary metabolites found throughout the plant kingdom (pichersky and gang ) . identifi cation of particular genes for target phytochemicals and the genetic engineering techniques could allow expressing the biosynthetic pathways of some phytochemical synthesis in organisms such as e. coli , b. subtilis or s. cerevisiae . for example, miyahisa et al. ( ) reported that introduction of four genes for a phenylalanine ammonia-lyase, cinnamate/coumarate:coa ligase, chalcone synthase, and chalcone isomerase, in addition to the acetyl-coa carboxylase, in e. coli cells resulted in effi cient production of ( s)-naringenin from tyrosine and ( s)-pinocembrin from phenylalanine. finally, the possibility of using phytochemicals as antimicrobial compounds would be a paradigm shift towards the potential health benefi ts and safety overcoming the problem of microbial resistance to drugs. antibacterial activity of diospyrin, isodiospyrin and bisisodiospyrin from the root of diospyros piscatoria (gurke) (ebenaceae) initial in vitro evaluations of the antibacterial activities of glucosinolate enzymatic hydrolysis products against plant pathogenic bacteria the antimicrobial effects of glucosinolates and their respective enzymatic hydrolysis products on bacteria isolated from the human intestinal tract weakening of salmonella with selected microbial 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synthetic coumarins plant functional genomics the effect of chlorogenic, gallic and quinic acids on the growth of spoilage strains of lactobacillus collinoides and lactobacillus brevis study of the role of antimicrobial glucosinolate-derived isothiocyanates in resistance of arabidopsis to microbial pathogens isolation and identifi cation of a potent antimalarial and antibacterial polyacetylene from bidens pilosa phytomedicines: back to the future flavanol monomer-induced changes to the human fecal microfl ora metabolic fate of polyphenols in the human superorganism saponins, classifi cation and occurrence in the plant kingdom grapefruit bioactive limonoids modulate e. coli o :h ttss and biofi lm microcalorimetric assay on the antimicrobial property of fi ve hydroxyanthraquinone derivatives in rhubarb ( rheum palmatum l.) to bifi dobacterium adolescentis history of the development and application of coumarin and coumarin-related compounds antimicrobial properties and toxicity of anthraquinones by microcalorimetric bioassay anti-aids agents. . synthesis and structureactivity relationships of ( ¢ r, ¢ r)-(+)-cis-khellactone derivatives as novel potent anti-hiv agents antifungal activity of c- steroidal saponins five new iridoids from patrinia rupestris different susceptibilities of staphylococcus and gram-negative rods to epigallocatechin gallate polyacetylene carboxylic acids from mitrephora celebica antifungal activities and action mechanisms of compounds from tribulus terrestris l atropurosides a-g, new steroidal saponins from smilacina atropurpurea lamiridosins, hepatitis c virus entry inhibitors from lamium album polyacetylenes from the apiaceae vegetables carrot, celery, fennel, parsley, and parsnip and their cytotoxic activities antibacterial alkaloids from chelidonium majus linn (papaveraceae) against clinical isolates of methicillin-resistant staphylococcus aureus key: cord- - fcc i i authors: hassani, abdelkader; azarian, mohammad mahdi sabaghpour; ibrahim, wisam nabeel; hussain, siti aslina title: preparation, characterization and therapeutic properties of gum arabic-stabilized gallic acid nanoparticles date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: fcc i i gallic acid (ga) is a natural phenolic compound with therapeutic effects that are often challenged by its rapid metabolism and clearance. therefore, ga was encapsulated using gum arabic into nanoparticles to increase its bioavailability. the formulated nanoparticles (ganps) were characterized for physicochemical properties and size and were then evaluated for antioxidant and antihypertensive effects using various established in vitro assays, including , -diphenyl- -picrylhydrazyl (dpph), nitric oxide scavenging (no), β-carotene bleaching and angiotensin-converting enzyme (ace) inhibitory assays. the ganps were further evaluated for the in vitro cytotoxicity, cell uptake and cell migration in four types of human cancer cell lines including (mcf- , mda-mb ) breast adenocarcinoma, hepg hepatocellular cancer, ht- colorectal adenocarcinoma, and mcf- a breast epithelial cell lines. the ganps demonstrated potent antioxidant effects and have shown promising anti-cancer properties in a dose-dependent manner with a predilection toward hepg and mcf cancer cells. the uptake of ganps was successful in the majority of cancer cells with a propensity to accumulate in the nuclear region of the cells. the hepg and mcf cancer cells also had a significantly higher percentage of apoptosis and were more sensitive to gallic acid nanoparticle treatment in the cell migration assay. this study is the first to confirm the synergistic effects of gum arabic in the encapsulation of gallic acid by increasing the selectivity towards cancer cells and enhancing the antioxidant properties. the formulated nanoparticles also had remarkably low toxicity in normal cells. based on these findings, ganps may have promising therapeutic applications towards the development of more effective treatments with a probable targeting precision in cancer cells. . powder x-ray diffraction patterns of xrd analysis of (a) the none capsulated gallic acid, (b) gum arabic, (c) physical mixture of ganps, (d) the nanoparticles of gallic acid prepared using the freeze-drying technique. evaluation of the antioxidant properties. the free radical scavenging activity of the free and nano encapsulated gallic acid was assessed in vitro by the dpph assay. dpph is a stable organic free radical used to estimate the antioxidant activity of various compounds. trolox served as a positive control due to its capacity to dissolve in the aqueous system. in this assay, the color of the dpph changed from deep violet to a pale or colorless solution in the presence of ganps nanoparticles. as illustrated in fig. , the percentage of dpph scavenging activity of ganps was . %, . %, and . % for the concentrations of , , and µg/ml respectively; whereas for free gallic acid the scavinging activity was . %, . %, and . %, respectively for the same concentrations (p < . ). this confirms the retention of the complete function of gallic acid after nanoencapsulation in gum arabic nanoparticles. trolox revealed strong scavenging activity of . % against dpph at similar concentrations. the antioxidant evaluation of free and nano encapsulated gallic acid was also determined in murine macrophage cell line (raw . ) . initially, the cell viability of raw . cells in response to treatments was evaluated using the mtt reduction assay in the presence or absence of lps. as shown in fig. , the lps induction had shown a relative toxic effect on the raw . cells upon treatment with ga and ganps. both treatment forms had negligible cytotoxicity activity against raw . cells in comparison to the untreated lps-stimulated cells. treatment of raw . cells with ga and ganps caused a considerable inhibition of nitric oxide (no) production as depicted in fig. . nonetheless, the nano encapsulated ga exhibited potent antioxidant activity, when compared to the free ga. the antihypertensive activity of ganps. the in vitro antihypertensive activity of free gallic acid and ganps was evaluated by measuring the ace inhibitory activity of the enzyme following the method outlined by cushman and cheung . this assay is based on the conversion of hippuryl-histidyl-leucine to hippuric acid. the in vitro antihypertensive capacity of free ga and ganps was investigated by measuring its absorbance fig. . according to the results, significant cytotoxicity was elicited among the hepg , mcf , mda-mb , and ht cell lines after treatment with ic concentrations of free and nano encapsulated gallic acid as shown in fig. . there was negligible cytotoxicity among the mcf- a cells especially at concentrations equivalent to the ic concentrations used in cancer cells (fig. ) and also in comparison with the untreated mcf- a cells. interestingly, the hepg and mcf cells were more sensitive to the treatments than the ht and mda-mb cells with a significantly higher selectivity with ic of . and . µg/ml for free ga and ganps respectively as shown in fig. . the ic value of ganps was approximately half that of free gallic acid in hepg cells as shown in fig. . as demonstrated, the ht and mda-mb cells were less sensitive to the treatments with an ic concentration of . and . µg/ml respectively. the ganps demonstrated variable patterns of cellular uptake in hepg , mda-mb , ht , and mcf cancer cell lines. as shown in fig. , the ga/c nps labeled with c green fluorescent dye were successfully uptake by the majority of the cancer cell types with condensation in the nuclear region as shown in fig. a . in section b, the empty nanoparticles labeled with c didn't have any of the cells stained with pi indicating non of . inhibition (%) of free ga and gallic acid nanoparticles by β-carotene bleaching assay with statistical analysis using a one-way anova test. data shown are mean value ± sd (n = , *p-value ≤ . , **p-value ≤ . ). figure . ace inhibition (%) for free gallic acid and ganps after , , and min. data shown are mean value ± sd (n = , t test, *p-value ≤ . , **p-value ≤ . ). . therefore, with more dead cells due to ga/c nps, it will be challenging to have the real background color of c nanoparticles. the cell uptake study findings were consistent with the cytotoxicity study of cancer cells using the mtt reduction assay. the ga/c nps showed higher cellular uptake and toxicity in hepg and mcf cells by internalizing in the nucleus and cytoplasm of cancer cells. the mda-mb and ht cells had a lower fluorescence intensity of pi attributed to the lower toxicity of ga/c nps. www.nature.com/scientificreports/ the migration assay was carried out to assess the effect of ganps and free ga with the ic value of concentrations on mcf , mda-mb- , hepg , and ht . the pictures were captured using an inverted light microscope at h and h and the cell migration was calculated as percentages as illustrated in fig. . the findings of this study had shown that the ganps treatment significantly retarded the migration of hepg and mcf cells compared with mda-mb and ht cancer cells (fig. ). ganps had also demonstrated potent anti-proliferative properties in the wound zone. the ganps were prepared by the freeze-drying method with slight modifications including the high-pressure of homogenization at the pressure of , bar for cycles to formulate smaller particles. deionized water was used in the preparation to improve the dispersion of nanoparticles during the sonication process. the x-ray diffraction technique was carried out to investigate the amalgamation between gallic acid and gum arabic as shown in fig. . the physical mixture pattern as shown in the figure demonstrated ga peaks with a reduction of intensity. however, the diffraction peaks of the ganps were completely diffused due to the amorphous state and the lack of crystallinity of the nanostructure indicating the formation of a new state in the ganps. these changes were significantly related to the interaction between ga and gum arabic. furthermore, the lack of a clear peak in the x-ray diffraction of ga might be the cause for the appearance of non-crystalline large peaks. hence, the absence of crystallinity of pure compounds indicates the formation of a new phase, specifically by the conversion to the amorphous state. in the tem, the prepared nanoparticles had spherical elliptical shapes with the size distribution of individual particles ranging from to nm as shown in fig. . according to the zeta sizer, the nanoparticles size measurement was in a higher range between to nm in aqueous solution probably due to the dry phase of tem analysis. as demonstrated, the produced droplets had a small size to increase the surface area and the bioavailability of ga incorporated into gum arabic nanoparticles. the use of ga is limited by its fast metabolism, low bioavailability, and poor absorption. these pharmacokinetic challenges reduce the concentration of drug reached (cmax) and therefore lead to its rapid elimination . therefore, the encapsulation of ga into gum arabic nanoparticles was justified to effectively enhance its bioavailability and improve the therapeutic effects in cancer tissue by enhancing the uptake via transcellular or paracellular mechanisms . increasing the dispersion of the small nanoparticles aimed to increase the reactive surface area of gallic acid in the formulated nanoparticles . the release profile of gallic acid is depicted in fig. where the ganps released a burst of gallic acid with a remarkably higher percentage of . % at ph . compared to . % at ph . after , min. this release pattern may be attributed to the weak bonds between gallic acid and gum arabic that are prone to break faster in acidic ph. this feature might offer a therapeutic privilege in cancer tissue by enhancing the release of gallic acid among cancer cells. these cells are continuously exposed to rapid shifts in the acid-base balance due to the limited blood supply exposing them more to acidic ph . there was also a slow release of ga from ganps within , min at ph . and ph . . the release of ga was relatively slow at this phase compared with the initial release pattern. this pattern of release is probably due to the diminution of ga content in gum arabic www.nature.com/scientificreports/ nanoparticles. hence, there was a plateau stage that lasted till , min at ph . and ph . , in which about of . % and . % of ga was released from ganps at ph level of . and ph . , respectively. the release behavior of ga from ganps was dependent on the negative charge of phosphate anion which gave a higher affinity for ion exchange with ga as an encapsulated agent. at ph . , solubility decreased with an increase in particle binding properties due to the ionization properties of gum arabic. these findings are consistent with other studies in which the gum arabic polymer tended to disturb nps at ph close to and higher than . . antioxidants play a crucial role in the protection against the attack of free radicals thus helping to prevent cardiovascular and cancer diseases which represent the most common causes of mortality. oxidative stress significantly contributes to the etiology of these diseases through multiple pathways. therefore, it is vital to keep the physiological balance between antioxidants and free radicals through the administration of potent antioxidants. therefore, several in vitro assays were used in this study to confirm the antioxidant properties of ganps. the dpph test was based on the reduction of the purple-colored stable free radical dpph to the yellow diphenylpicrylhydrazine in the presence of free gallic acid and ganps. due to the existence of hydrogen-donating www.nature.com/scientificreports/ properties of ganps, the phenolic compound has been shown to quench oxygen derived from free radicals by donating an electron or hydrogen atom to the free radicals in various systems from in vitro studies , . trolox was used as a positive control to calculate the percentage of dpph inhibition. interestingly, ganps nanoparticles and gallic acid exhibited an in vitro scavenging activity in a dose-dependent manner at the concentrations gradient of - µg/ml in dpph as depicted in fig. . this property of nanostructured gallic acid was reported in the literature in which the underlying mechanism of dpph radical scavenging activity was attributed to the single electron transfert (set) and hydrogen atom transfer (hat) . set refers to a rearrangement of structure with a loss of a proton. usually, this mechanism produces quinone derivatives, resulting in the formation of a double bond with a change of proton signals. the gallic acid is stabilized after the reaction as a radical. in contrast, hat is attributed to the loss of proton with the stabilization action of nearby groups on the charge of the molecule. www.nature.com/scientificreports/ the quick proton exchange is considered as a contraction of the proton signals and it doesn't indicate any change in the chemical structure of the molecules . although the shown antioxidant effect of ga was lower than that of trolox, the formulated nanoparticles form had significantly enhanced these effects compared with free gallic acid due to the dpph scavenging properties of gallic acid combined with the hydroxyl units of gum arabic. from the data in fig. , it is apparent that the dpph test has examined the impact of free radical on test compounds. therefore, the strong absorption at nm in visible spectroscopy was influenced by the odd electron of dpph. the odd electron was paired in the presence of hydrogen of a free radical scavenger, indicating the capacity of gallic acid nanoparticles to scavenge free radicals without prior enzymatic activity . the antioxidant properties of gum arabic may be attributed to its hydroxyl groups, polypeptide, and its highly branched structure which may improve the stability and antioxidant properties of gallic acid within the nanoparticles . gum arabic is, therefore, a suitable encapsulant capable of forming stable nanoparticles for safe delivery and can be used as a potent antioxidant in future formulations. the in vitro cytotoxicity effect of the free ga and ganps in raw . cells was evaluated using mtt assay as demonstrated in fig. . the mtt results which reflect the number of viable cells confirmed that both ganps and ga did not have a significant cytotoxic effect on the raw . cells. in these cells, another antioxidant test was performed known as the nitric oxide inhibition assay which is important for evaluating the antioxidant and anti-inflammatory properties of potential agents. the assay is based on the production of nitrite metabolite from nitric oxide and its quantification using griess reagent . nitric oxide is an important molecule in the regulation of numerous physiological mechanisms such as blood pressure and pathological conditions such as inflammation, shock and neurotoxicity . due to the scavenging capacities of antioxidants, these agents are used to treat those deleterious disorders. the cells were challenged with lps which induces the expression of nitric oxide synthase protein. this enzyme in turn will enhance the production of nitric oxide from sodium nitroprusside and oxygen that are also induced by the oxidative challenge of lps. the production of peroxynitrite anion, a strong oxidant is based on the interactions between superoxide radical and no , . the results of the nitric oxide inhibition assay confirmed significant antioxidant effects in raw . cells. the free and nano encapsulated gallic acid at various concentrations accordingly inhibited the production of nitrite radical in the culture medium in a dose-dependent manner. as depicted in fig. , the hydroxyl radical scavenging activity of ganps was times higher than that of ga at concentrations ranging between . and µg/ml with ic values of . µg/ml and . µg/ml, respectively (p < . ). this result also confirmed that gallic acid maintained its antioxidant properties that were consistent with other reports . gallic acid was reported to have cyclooxygenase (cox- ) and nitric oxide synthase (inos) inhibitory properties in lpsinduced raw . cells modulating the pro-inflammatory cytokines such as il- , ifn-γ, and tnf-α . the ganps nanoparticles exhibited strong inhibition on no production at concentrations ranging from . to µg/ml. gum arabic was also reported to enhance the free radical scavenging properties of therapeutic agents by neutralizing and absorbing free radicals . nitric oxide produced as a proinflammatory mediator during the immunopathological phenomenon may cause chronic inflammation that is circumvented by macrophages. these cells were enhanced by gum arabic to exhibit antioxidant and hepatoprotective properties . the β-carotene bleaching assay is a quick and simple test based on the competition between ganps and β-carotene to neutralize the hydroperoxide-derived free radicals produced by the oxidation of linoleic acid. as shown in fig. , a concentration-dependent inhibition of β-carotene bleaching was recorded due to the presence of phenolic groups in gallic acid. the highest reducing power was recorded at the concentration of µg/ml, which exhibited approximately . % of antioxidant activity (p < . ). although both ga and ganps showed potent antioxidant activity; however, the nano encapsulated gallic acid appeared to be significantly more effective than free gallic acid. these findings may be attributed to the phenolic groups in gallic acid and the functional groups of gum arabic used as the coating material . oxidative stress is largely implicated in the pathogenesis of hypertension disease; where it is responsible for the activation of several mechanisms leading to vasoconstriction . therefore, the in vitro antihypertensive activity of the free and nano encapsulated gallic acid was assessed by measuring the ace-inhibitory capacity using a uv-visible spectrophotometer at nm. the inhibition of ace function is also one of the widely used therapeutic options in the hypertension disease as this enzyme is involved in the renin-angiotensin axis of blood pressure regulation which is largely involved in the pathogenesis of hypertension . the ace that is renowned now being the target of the recent coronavirus infection is the catalyst converting angiotensin i to angiotensin ii in lung and kidneys . this study is the first evidence confirming the ace inhibitory activity of ganps in addition to its antioxidant activity providing a promising treatment for hypertension disease. as shown in fig. , the ace-inhibitory capacities of gallic acid and ganps were time-dependent in which the ace inhibitory activity of ganps and gallic acid at min was . % and . %, respectively using µl of µg/ml treatment solution. the use of gum arabic polymer as nanocarrier improved the antihypertensive capacity of ganps that explains the high percentage of ace inhibition compared to that of free gallic acid. such antihypertensive effects were reported in animal models of hypertension disease using free gallic acid and gum arabic treatments. several phenolic natural products including gallic acid are known to have ace inhibitory activity but with a poorly understood mechanism . the enhanced antihypertensive property ganps could be imputed to the high amount of gallic acid released from the nanoparticles. the evaluation of cell viability is a common assay to determine the in vitro cytotoxicity of various biomaterials. the mtt assay is largely known as a rapid quantitative and colorimetric assay to evaluate cell viability for different purposes. the assay depends on the reduction of mtt salt by mitochondrial dehydrogenases inside the viable cells. upon reduction, the yellow tetrazolium salt changes to the corresponding purple formazan. in this study hepatocellular, colorectal, and breast adenocarcinoma cell lines were used in addition to normal epithelial www.nature.com/scientificreports/ cells. figure demonstrates the cytotoxic effect of ga, ganps in these cell lines after h of exposure. it has been observed that both ga and ganps exerted potent anti-proliferative effects in cancer cells with minimal toxicity in normal epithelial cells as shown in section a of fig. . as depicted, the ic values were significantly low in all the cancer cells compared with epithelial cells. these results were consistent with previous reports confirming the antineoplastic effects of gallic acid where it significantly increased apoptosis and apoptotic markers in esophageal and melanoma cancer cells by interfering with akt/mtor pathway with no apparent toxicity in normal cells , . interestingly as shown in fig. , hepatocellular cancer (hepg ) and breast cancer (mcf- ) cells appeared to be more sensitive to gallic acid nanoparticles than the mda-mb breast, and ht colorectal cancer cells. ganps at the concentration of . µg/ml caused a reduction to . % of viable hepg cells n compared to untreated cells (p < . ). the nanoparticles appeared to be more potent in hepg cells where a lower percentage of cell viability ( . %) was recorded at ( µg/ml) concentration compared to . % of free gallic acid. these findings could be attributed to the effect of gum arabic. in one study, gum arabic was found to have a targeting specificity towards hepatic cells by its galactose groups that attach with asialoglycoprotein receptors (asgrp) of liver cells in which the nanocarriers stimulated receptor-mediated endocytosis of the therapeutic agents . thus, the chain in gum arabic reacted as natural targeting ligands for asialoglycoprotein receptors (asgrp) on hepatocytes, which have improved the cytotoxicity effect of gallic acid nanoparticles. consequently, the high cytotoxicity effect of ganps against hepg cells might be attributed to the galactose units of gum arabic polymer. ganps had also demonstrated enhanced cytotoxicity in mcf breast cancer cells compared to mda-mb breast cancer cells. this significant potency in mcf cancer cells may be attributed to the modulation of estrogen receptor function as reported in several studies [ ] [ ] [ ] . these receptors are present in mcf cells and are potent targets approached in the treatment of breast cancer; however, the mda-mb cells lack these receptors. therefore ganps have a better selective index toward hepatic cancer cells and mcf breast cancer cells probably due to the chemical structure of gum arabic polymer and the selectivity toward hepatic and breast cancer cell receptors as demonstrated in fig. . the aforementioned toxicity findings were consistent with the cellular uptake study. in this assay, coumarin- (c ) was selected for the observation of ganps penetration to the cells with propidium iodide (pi) counterstain that is characterized by its penetration into nonliving cells. as shown in fig. , there was a significant increase in the uptake of pi among hepg and mcf cells compared with other cancer cells that may be attributed to the higher uptake of ganps leading to more apoptosis. the high fluorescence of pi as shown in fig. masked the green color of c due to the förster resonance energy transfer phenomena. as mentioned earlier these cells were more sensitive to ganps due to the chemical structure of gum arabic as confirmed in another report where the use of gum arabic assisted the delivery of curcumin and c into hepatocellular carcinoma cells . the enhanced uptake of pi was consistent with several reports of the proapoptotic and cell cycle arrest properties of gallic acid in cancer cells , , , . in these studies, the mechanism of action was revealed in the form of enhancement of caspase, endonuclease dependant pathways, extrinsic apoptotic pathways, and disruption of p kip /skp complexes that are crucial in regulating cell cycle procession in addition to modulating the level of il- . these findings confirm the therapeutic properties and the efficient cell internalization of gallic acid nanoparticles. thus the use of gum arabic as a carrier permit facile passage of gallic acid through the cell membrane as well as increase its solubility. ganps treatment was further evaluated for its effect on the migration of hepg , ht , mcf , and mda-mb cancer cells as a measure of invasiveness and metastasis . the treatment of cells using ganps has revealed a significant reduction in the percentage of migration of hepg and mcf cells. these findings are consistent with the reported antimetastatic properties of gallic acid in gastric, cervical, melanoma, and oral cancer cells that are attributed to the interference of gallic acid with nf-kappab activity, matrix metalloproteinases, ras-erk and the pi k/akt signaling pathways [ ] [ ] [ ] [ ] . this study is the first to report the augmenting property of gum arabic in encapsulating gallic acid improving the therapeutic outcomes of several in vitro assays. the potent antioxidant properties of gallic acid were significantly enhanced by gum arabic coating attributed to the chemical properties of both compounds; this study also revealed several antineoplastic properties offered by the nanoformulation. the ganps enhanced the selective uptake in hepatic cells enhanced the cytotoxicity in breast cancer (+ estrogen receptor) and confirmed its proapoptotic effects. therefore ganps formulation is a potential candidate for the prevention and treatment of hepatic and breast cancers. extrapolation of the in vitro cytotoxicity effects of ganps to in vivo cytotoxicity effects demands further investigation in light of its application as a cancer chemotherapeutic agent. materials. gum arabic polysaccharide was bought from ennasr company (sudan). gallic acid (ga) used in this study was purchased from sinar scientific company (malaysia). dexamethasone, trolox, , -diphenyl- -picrylhydrazyl (dpph), hippuryl-histidyl-leucine sulfonamide, angiotensin-converting enzyme, n-( -naphtyl) ethylenediamine dihydrochloride, sodium nitrite, linoleic acid, dulbecco's modified eagle's medium (dmem), fetal bovine serum (fbs) and streptomycin were purchased from sigma-aldrich company (malaysia). tween , b-carotene, quercetin, and α-tocopherol were purchased from r&m company (china). hepg , mcf , mda-mb , ht , mcf- a, and raw . cells were obtained from atcc (american type culture collection). throughout all experiments, deionized water was used. | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ preparation of gallic aid nanoparticles. the ganps were synthesized using the freeze-drying technique with few modifications. the aqueous solution containing gallic acid (ga) and gum arabic in : molar ratio was obtained by dissolving . g of ga in ml deionized water containing . g/ml of gum arabic. the mixture was kept under mild agitation at room temperature for h. the final suspension was subjected to a high-pressure homogenizer at a pressure of , bar for cycles and was then frozen at − °c. the final product was freeze-dried for h at − °c. in the confocal microscope study of cell uptake and apoptosis, the ganps labeled with c was prepared using a modified protocol of the same freeze-drying technique as described in some reports . an aqueous solution containing gallic acid (ga) and gum arabic in : molar ratio was obtained by dissolving . g of ga and . mg of coumarin- in ml of ethanol then mixed with . g of gum arabic dissolved in ml of dmso. the mixture was kept under mild agitation at room temperature for h. the final product was then freeze-dried for h at − °c. preparation of the physical mixture. the physical mixture of gallic acid (ga) and gum arabic was performed in a : ratio as lyophilized complex. ga and gum arabic were admixed into homogeneous powder using pestle and mortar. characterization of ganps nanoparticles. x-ray diffraction (xrd). the ganps, gum arabic, and ga powder samples were investigated using shimadzu refractometer, xrd (tokyo, japan). powder x-ray diffraction (xrd) patterns of ganps, gum arabic, and ga were recorded using cuk α incident beam, λ = . Å, and voltage of kv. analysis of samples was performed at θ = °- ° and a scan speed of ° per minute. size and zeta potential. the zeta potential and size of ganps were characterized using a zeta sizer (malvern nano-zs-zs, zeeman) with dynamic size. www.nature.com/scientificreports/ deionized water were added to the flask with vigorous shaking for min. blank emulsion, devoid of β-carotene was prepared. aliquots ( µl) of β-carotene emulsion were transferred into the -well plate containing µl of ga and ganps at various concentrations and incubated in the dark at °c. different concentrations of α-tocopherol were used as a positive control reflecting nearly complete inhibition of β-carotene bleaching. the absorbance measurements were recorded immediately at nm at min intervals for min. the antioxidant activity (aa) was assessed using the following equation: where a , a t , and a c , a tc are the absorbances of sample at t = , t = min and absorbance of control at t = , t = min. the anti-hypertensive assay using angiotensin-converting enzyme (ace) inhibition assay. the in vitro ace inhibition activity of gallic acid ga in gum arabic nanoparticles was assessed in vitro based on the conversion of hippuryl-histidyl-leucine to hippuric acid in the presence of ace enzyme . one hundred microliters of gallic acid ga and ganps were mixed with ace ( µl, ph . ) and incubated at °c for min. next, . mm of hippuryl-histidyl-leucine ( µl) was added to the mixture and incubated for , , and min. the enzymatic reaction was halted after the addition of µl of mol/l hci. hence, ml of ethyl acetate was added to remove the resultant hippuric acid. the solvent was evaporated at °c and re-dissolved in ml of n of nacl. the concentration of hippuric acid was evaluated by measuring the absorbance at nm. a blank devoid of ga and ganps was prepared at background subtraction. cell culture and cytotoxicity assay. a list of cancer cell lines selected to demonstrate the antineoplastic properties using the mtt assay which is based on the enzymatic reduction of tetrazolium salt -( , -dimethylthiazol- -yl)- , diphenyltetrazolium bromide (mtt) by active living cells. the cancer cell lines were all of an epithelial type representing a list of common cancer types including colorectal adenocarcinoma (ht ), hepatocellular cancer (hepg ), breast adenocarcinoma (mda-mb and mcf ), and mcf- a breast epithelial cell lines. all the cell lines were free of mycoplasma infection and the number of passages used in the experiments ranged between ( ) ( ) ( ) . the cells were maintained in dmem supplemented with % of penicillin-streptomycin and % of fetal bovine serum (fbs). the cells were harvested using trypsin-edta solution and examined using an inverted microscope (olympus ck ). a hemocytometer was used to determine cell density. standard solutions of ga and ganps were prepared and dissolved to concentrations ranging between . - µg/ml. this assay was performed as described in our previous reports to assess cytotoxicity activity of treatments in the selected cell lines . two hundred microliters of cell suspension with a density of × cells/ ml were placed into each well of -well plate and later incubated at % co and °c for h. after incubation time, the medium was replenished and the cells were treated with ga and ganps at concentrations ranging between . and µg/ml for cancer cells and . - , µg/ml for normal cell lines to determine the ic values. chemotherapeutic controls were also used with different concentration gradient from . to µg/ml ( -fluorouracil for ht , tamoxifen for hepg cells, and doxorubicin for mcf , mcf- a, and mda-mb . the last row of the -well plate was left for the control experiment under similar conditions. after h of postincubation, µl of mg/ml of mtt solution was added to each well, in which the plate was covered with aluminum foil and incubated for h. the medium was removed and replaced with µl dmso to dissolve the remaining purple formazan precipitate. the absorbance was recorded using an elisa reader at nm. this colorimetric assay focused on the reduction of mtt to purple formazan by the mitochondrial enzymes of the metabolically active cancer cells . the ic was calculated from the concentration of drug that inhibits % of cell growth. the experiment also included the mcf- a normal breast cell lines in the mtt assay to calculate the selective index for each of the cancer cell lines. all experiments were carried out in triplicate and the outcomes are presented in standard deviation and mean values. cellular uptake of ganps. the study also included a qualitative assessment of cell uptake in hepg , mcf , mda-mb , ht cancer cell lines based on the use of coumarin (c ) and propidium iodide (pi) fluorescent dyes. the use of both dyes in this experiment was adapted from win et al. . c is widely used for tracking of nanoencapsulation emitting green light at nm . while pi fluorescence dye was used as a counterstain selective to nonviable cells and cells with compromised cell membranes where it binds with base pairs of double-stranded dna giving the unique red fluorescence emission while sparing viable cells with intact cell membranes . the experiment started by culturing the cancer cells on glass coverslips with a cell suspension of cells/ cm in complete media inside the co incubator at °c for h allowing cell adherence to the coverslip. the cells were then subjected to the ic values of ga/c nps in serum-free medium for h. consequently, the cells were incubated with pbs buffer containing pi ( µg/ml) for min at room temperature after washing with pbs buffer; then washed again with pbs buffer followed by fixation with % ethanol in the freezer (− °c) for min. the coverslips were then examined using the confocal microscope with excitation wavelength nm under multichannel mode. scratch migration assay. the in vitro cell migration assay was performed by scratch assay to assess cell mobility following the protocol adapted from kovarikova et al. . the hepg , mcf , mda-mb , and ht cells were seeded in a -well plate with the cell density of ( × ). upon reaching % of confluence level, the scratch was carried out using a yellow pipette tip with an average diameter of mm. the cells were then washed www.nature.com/scientificreports/ times with pbs and treated with ganps at concentrations of . - µg/ml and were further incubated for h. based on the dino eye application connected to the inverted microscope, a marker line was used to select the position of the picture captured. various pictures were captured at h and h after the treatment at a magnification of ×. the experiment was performed in triplicate. hence, the percentage of migration of the aforementioned cell lines was calculated using the following formula: statistical analysis. t tests and one-way anova test followed by the holm-sidak multiple comparisons were performed using graphpad prism version . . (san diego, california usa) to evaluate the differences among the treatment groups. a difference at p < . was considered significant. received: march ; accepted: august scientific reports | ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports/ hallmarks of cancer: the next generation reactive oxygen and nitrogen species in carcinogenesis: implications of oxidative stress on the progression and development of several cancer types oxidative stress: harms and benefits for human health synthesis of zno nanoparticles and evaluation of antioxidant and cytotoxic activity blood radicals: reactive nitrogen species, reactive oxygen species, transition metal ions, and the vascular system pharmacological effects of gallic acid in health and diseases: a mechanistic review gallic acid ameliorates hyperglycemia and improves hepatic carbohydrate metabolism in rats fed a high-fructose diet gallic acid attenuated lps-induced neuroinflammation: protein aggregation and necroptosis pharmacokinetics of gallic acid and its relative bioavailability from tea in healthy humans bioavailability of gallic acid and catechins from grape seed polyphenol extract is improved by repeated dosing in rats: implications for treatment in alzheimer's disease enhanced retention and cellular uptake of nanoparticles in tumors by controlling their aggregation behavior a nanoliposome-based photoactivable drug delivery system for enhanced cancer therapy and overcoming treatment resistance nanocarriers as an emerging platform for cancer therapy controlled release and angiotensin-converting enzyme inhibition properties of an antihypertensive drug based on a perindopril erbumine-layered double hydroxide nanocomposite employment of enhanced permeability and retention effect (epr): nanoparticle-based precision tools for targeting of therapeutic and diagnostic agent in cancer effect of acacia gum on blood pressure in rats with adenine-induced chronic renal failure synthesis and antioxidant properties of gum arabic-stabilized selenium nanoparticles spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung application of förster resonance energy transfer (fret) technique to elucidate intracellular and in vivo biofate of nanomedicines nanoencapsulation of gallic acid and evaluation of its cytotoxicity and antioxidant activity colloidal astaxanthin: preparation, characterisation and bioavailability evaluation polysaccharide-based nanoparticles by chitosan and gum arabic polyelectrolyte complexation as carriers for curcumin. food hydrocoll the chemistry, physiology and pathology of ph in cancer dpph radical scavenging activity of ten natural p-terphenyl derivatives obtained from three edible mushrooms indigenous to china encapsulation of gallic acid/cyclodextrin inclusion complex in electrospun polylactic acid nanofibers: release behavior and antioxidant activity of gallic acid gallic acid suppresses inflammation in dextran sodium sulfate-induced colitis in mice: possible mechanisms a h nmr investigation of the interaction between phenolic acids found in mango (manguifera indica cv ataulfo) and papaya (carica papaya cv maradol) and , -diphenyl- -picrylhydrazyl (dpph) free radicals novel calcified gum arabic porous nano-composite scaffold for bone tissue regeneration inhibition of nitric oxide production in lipopolysaccharide-activated raw . macrophages by jeju plant extracts nitric oxide signaling, metabolism and toxicity in nitrogen-fixing symbiosis % of migration of cells = (distance of scratch at h − distance of scratch at h) (distance of scratch at h) × bacterial lps-mediated acute inflammation-induced spermatogenic failure in rats: role of stress response proteins and mitochondrial dysfunction antioxidant and anti-inflammatory activities of broccoli florets in lps-stimulated raw cells anti-inflammatory potential of ellagic acid, gallic acid and punicalagin a&b isolated from punica granatum antioxidant and anti-inflammatory activities of freeze-dried grapefruit phenolics as affected by gum arabic and bamboo fibre addition and microwave pretreatment protective effect of arabic gum against acetaminophen-induced hepatotoxicity in mice synthesis of antioxidant polymers by grafting of gallic acid and catechin on gelatin effect of gum arabic on quality and antioxidant properties of papaya fruit during cold storage oxidative stress and hypertensive diseases the vasoprotective axes of the renin-angiotensin system: physiological relevance and therapeutic implications in cardiovascular, hypertensive and kidney diseases a review of ace inhibitors and arbs in black patients with hypertension gallic acid reduces blood pressure and attenuates oxidative stress and cardiac hypertrophy in spontaneously hypertensive rats inhibitory effect of trichlormethiazide on development of doc hypertension in rats discovery of new angiotensin converting enzyme (ace) inhibitors from medicinal plants to treat hypertension using an in vitro assay anticancer effects of gallic acid isolated from indonesian herbal medicine, phaleria macrocarpa (scheff.) boerl, on human cancer cell lines gallic acid induces apoptosis in a .s human melanoma cells through caspase-dependent and -independent pathways asialoglycoprotein receptor (asgpr) as target autoantigen in liver autoimmunity: lost and found comparison of cytotoxic effects of pentagalloylglucose, gallic acid, and its derivatives against human cancer mcf- and mda mb- cells gallic acid based steroidal phenstatin analogues for selective targeting of breast cancer cells through inhibiting tubulin polymerization preparation of fe o magnetic nanoparticles coated with gallic acid for drug delivery gum arabic-curcumin conjugate micelles with enhanced loading for curcumin delivery to hepatocarcinoma cells gallic acid induces g /m phase arrest of breast cancer cell mcf- through stabilization of p (kip ) attributed to disruption of p (kip )/skp complex 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activity of orotic acid-loaded gum arabic nanoparticles the inhibitory activity of cocoa phenolic extract against pro-inflammatory mediators secretion induced by lipopolysaccharide in raw . cells essential oils as antioxidants: their evaluation by dpph, abts, frap, cuprac, and β-carotene bleaching methods review of angiotensin-converting enzyme inhibitory assay: rapid method in drug discovery of herbal plants effect of shrna mediated silencing of yb- protein on the expression of matrix collagenases in malignant melanoma cell in vitro formulation, characterization and biological activity screening of sodium alginate-gum arabic nanoparticles loaded with curcumin effects of particle size and surface coating on cellular uptake of polymeric nanoparticles for oral delivery of anticancer drugs cellular uptake of coumarin- as a model drug loaded in solid lipid nanoparticles hpc viability measurement: trypan blue versus acridine orange and propidium iodide methods for studying tumor cell migration and invasiveness the publication of this article was funded by the qatar national library. the authors would also like to acknowledge universiti putra malaysia for awarding the funds toconduct this research under project number gp-ips/ / . the authors declare no competing interests. correspondence and requests for materials should be addressed to w.n.i. or s.a.h.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- - tionxun authors: fenner, frank; mcauslan, b.r.; mims, c.a.; sambrook, j.; white, david o. title: the nature and classification of animal viruses date: - - journal: the biology of animal viruses doi: . /b - - - - . - sha: doc_id: cord_uid: tionxun nan virology began as a branch of c h apt er pathology, the study of disease. at the end of the nineteenth century, when the microbial the nature and classification etiology of many infectious disof animal viruses eases had been established, pathologists recognized that there since been discovered, but two still apply: (a) unlike even the smallest microorganisms (chlamydiae), viruses contain no functional ribosomes or other cellular organelles, and (b) in rna viruses the whole of the genetic information is encoded in rna, a situation unique in biology. other distinctions apply to some but not all viruses, e.g., the isolated nucleic acid of viruses of several genera is infectious (i.e., the virus can be generated intracellularly from a single molecule of nucleic acid), and viruses of most genera contain either no virus-coded enzymes, or one or more enzymes that belong to particular classes (neuraminidases and nucleic acid polymerases). it is impossible to define viruses satisfactorily in a sentence or even a paragraph, bearing in mind both their intracellular states and the extracellular particles or virions. virions consist of a genome of either dna or rna enclosed within a protective coat of protein molecules, some of which may be associated with carbohydrates or lipids of cellular origin. in the vegetative state and as "provirus" (see chapter ), viruses may be reduced to their constituent genomes, and the simplest "viruses" may be transmitted from one host to another as naked molecules of nucleic acid, possibly associated with certain cellular components. at the other extreme, the largest animal viruses, e.g., the poxviruses and the leukoviruses, are relatively complex. lwoff's concept that "viruses are viruses" has had important theoretical and practical consequences; on the one hand, it emphasized their similarities irrespective of the nature of the host (animal, plant or bacterium), and, on the other hand, it led to the possibility of freeing viruses from the rules of bacteriological nomenclature. however, the operational division of viruses made according to type of host continues to be used by the majority of virologists most of the time, and it is significant that the international committee on nomenclature of viruses (icnv), although dedicated to a universal classification, operates through subcommittees on bacterial, invertebrate, plant, and vertebrate viruses (wildy, ). the simpler viruses consist of nucleic acid and a few polypeptides specified by it. more complex viruses usually also contain lipids and carbohydrates; in the great majority of viral genera these chemical components are not specified by the viral genome but are derived from the cells in which the viruses multiply. in exceptional situations, cellular nucleic acids or polypeptides may be built into viral particles. viruses contain only a single species of nucleic acid, which may be dna or rna. viral nucleic acid may be single-or double-stranded, the viral genome may consist of one or several molecules of nucleic acid, and if the genome consists of a single molecule this may be linear or have a circular configuration. as yet, no animal viral nucleic acid has been found to be methylated, or to contain novel bases of the type encountered in bacterial viruses or mammalian transfer rna's, but some virions contain oligonucleotides rich in adenylate, of unknown function. the base composition of dna from animal viruses covers a far wider range than that of the vertebrates, for the guanine plus cytosine (g+c) content of different viruses varies from to %, compared with to % for all chordates. indeed, the g+c content of the dna of viruses of one genus (herpesvirus) ranges from to %. the molecular weights of the dna's of different animal viruses varies from just over to about million daltons; the range of molecular weights of viral rna's is much less, from just over to about million daltons. the nucleic acid can be extracted from viral particles with detergents or phenol. the released molecules are often fragile but the isolated nucleic acid of viruses belonging to certain genera is infectious. in other cases, the isolated nucleic acid is not infectious even though it contains all the necessary genetic information, for its transcription depends upon a virion-associated transcriptase without which multiplication cannot proceed. all dna viruses have genomes that consist of a single molecule of nucleic acid, but the genomes of many rna viruses consist of several different molecules, which are probably loosely linked together in the virion. in viruses whose genome consists of single-stranded nucleic acid, the viral nucleic acid is either the "positive" strand (in rna viruses, equivalent to messenger rna) or the "negative" (complementary) strand. preparations of some viruses with genomes of single-stranded dna consist of particles that contain either the positive or the complementary strand. viral preparations often contain some particles with an atypical content of nucleic acid. host-cell dna is found in some papovaviruses, and what appear to be cellular ribosomes in some arenaviruses. several copies of the complete viral genome may be enclosed within a single particle (as in paramyxoviruses) or viral particles may be formed that contain no nucleic acid ("empty" particles) or that have an incomplete genome, lacking part of the nucleic acid that is needed for infectivity. terminal redundancy occurs in the dna of some vertebrate viruses, but most sequences are unique. the largest viral genomes contain several hundred genes, while the smallest carry only sufficient information to code for about half a dozen proteins, most of which are structural proteins of the virion. the major constituent of the virion is protein, whose primary role is to provide the viral nucleic acid with a protective coat. as predicted by crick and watson ( ) , from a consideration of the limited amount of genetic information carried by viruses, the protein shells of the simpler viruses consist of repeating protein subunits. sometimes the viral protein comprises only one sort of polypeptide chain, although, more commonly, there are two or three different polypeptides. the proteins on the surface of the virion have a special affinity for complementary receptors present on the surface of susceptible cells. they also contain the antigenic determinants that are responsible for the production of protective antibodies by the infected animal. viral polypeptides are quite large, with molecular weights in the range , - , daltons. the smaller polypeptides are often but not always internal, the larger ones often but not always external. there are no distinctive features about the amino acid composition of the structural polypeptides of the virion, except that those intimately associated with viral nucleic acid in the "core" of some icosahedral viruses are often relatively rich in arginine. viral envelopes usually originate from the cellular plasma membrane from which the original cellular proteins have been totally displaced by viral peplomers and a viral "membrane protein" (see fig. - ) . the peplomers consist of repeating units of one or two glycoproteins, the polypeptide moiety of which is virus-specified while the carbohydrate is added by cellular transferases. in many enveloped viruses, the inside of the viral envelope is lined by a viral protein called the membrane or matrix protein. not all structural viral proteins are primary gene products, since with many viruses the viral mrna is translated into a large polypeptide that is enzymatically cleaved to yield two or more smaller virion proteins. cleavage is often one of the terminal events in the assembly of the virion and it can occur in situ after most of the proteins are already in place. although most virion polypeptides have a structural role some have enzymatic activity. many viruses contain a few molecules of an internal protein that functions as a transcriptase, one of the two kinds of peplomers in the envelope of myxoviruses has neuraminidase activity, and a variety of other enzymes are found in the virions of the larger, more complex viruses. in addition to polypeptides that occur as part of the virion, a large part of the viral genome (most of it, with the large dna viruses) codes for polypeptides that have a functional role during viral multiplication but are not incorporated into viral particles. few of these "nonstructural viral proteins" have been characterized. except for the large and complex poxviruses, which constitute a special case, lipid and carbohydrate are found only in viral envelopes and are always of cellular origin. the lipids of viral envelopes are characteristic of the cell of origin, though minor differences between the viral envelope and the normal plasma membrane may be demonstrable. about to % of the lipid is phospholipid and most of the remainder ( - %) is cholesterol. some of the viral carbohydrate occurs in the envelope as glycolipid characteristic of the cell of origin, but most of it is part of the glycoprotein peplomers that project from the viral envelope. during the years that followed the introduction of negative staining for the electron microscopic study of viruses (brenner and home, ), a general the structure of animal viruses picture was obtained of the structure of representatives of most of the groups of animal viruses that were known at the time (review: home and wildy, ). three structural classes were distinguished: isometric particles, which were usually "naked" but in some groups were enclosed within a lipoprotein envelope; long tubular nucleoprotein structures, always (with viruses of vertebrates) surrounded by a lipoprotein envelope; and in a few groups, a more complex structure. accepting a number of new terms defined by lwoff et al. ( a) , caspar and his colleagues analyzed the principles underlying the structure of simple viruses (review: caspar, ). their basic concepts remain valid, but subsequent work has rendered some of the original definitions ambiguous; where necessary these have been modified. virion (plural virions) is used as a synonym for "virus particle." the protein coat of an isometric particle or the elongated protein tube of viruses with helical symmetry is called the capsid. it may be "naked," or it may be enclosed within a lipoprotein envelope (peplos) which is derived from cellular membranes as the virus matures by budding. where the capsids directly enclose the viral nucleic acid, as is usual with tubular capsids but less common with isometric capsids, the complex is called the nucleocapsid. with most isometric particles and in all complex virions, the capsid encloses another protein structure containing the viral genome, called the core. capsids consist of repeating units of one or a small number of protein molecules. three levels of complexity can be distinguished. chemical units, the ultimate gene products, are single polypeptides that may themselves constitute the structural units, or several polypeptides may form homo-or heteropolymers which constitute the structural units. the structural units, or groups of them, may be visualized in the electron micrographs as morphological units. morphological units that form part of a capsid are called capsomers; those projecting from the envelope are the peplomers (sometimes called "spikes," an unsatisfactory term since they are never pointed and may, indeed, have knob-shaped ends). the chemical units are sometimes held together by disulfide bonds to form the structural units, hence the practice of using reducing agents in polyacrylamide gel electrophoresis when analyzing viral proteins to determine their constituent polypeptides. the structural units are held together to form the capsid by noncovalent bonds, which may be polar (salt and hydrogen bonds) or nonpolar (van der waals and hydrophobic bonds). the capsids of some viruses are readily disrupted in molar calcium or sodium chloride, suggesting electrovalent bonds between the structural units; others are unaffected by salt and can only be disrupted by detergents, suggesting that they are hydrophobically bonded. it has been found that the isometric virus particles that have been adequately studied by x-ray diffraction and electron microscopy have capsids in which the capsomers are arranged with icosahedral symmetry. according to caspar and (b) . the capsids consist of morphological suhunits called capsomers, which are in turn composed of structural suhunits that consist of one or more chemical suhunits (polypeptide chains). many icosahedral viruses have a "core" (not illustrated), which consists of protein(s) directly associated with the nucleic acid, inside the icosahedral capsid. in viruses of type b the envelope is a complex structure consisting of an inner virus-specified protein shell (membrane protein, made up of structural suhunits), a lipid layer derived from cellular lipids, and one or more types of morphological subunits (peplomers), each of which consists of one or more virus-specified glycoproteins (modified from caspar et ah, ) . klug ( ) , this occurs because the icosahedron is that polyhedron with cubic symmetry which, if constructed of identical subunits, would least distort the subunits or the bonds between them. a n icosahedron ( fig. - ) has equilateral triangular faces, vertices, where the corners of triangles meet, and edges, where the sides of adjacent pairs of triangles meet. it shows twofold symmetry about an axis through the center of each edge ( fig. - a) , threefold symmetry w h e n rotated around an axis through the center of each triangular face ( fig. - b) , and fivefold symmetry about an axis through each vertex ( fig. - c ). each triangular face may be thought of as containing, and being defined by, three asymmetric units (i.e., units that have no regular symmetry axes themselves) so that a minimum of sixty asymmetric units are required to construct an icosahedron. the triangular faces of an icosahedron can be subdivided into smaller identical equilateral triangles, to form a solid called an icosadeltahedron. only certain subdivisions are possible; the number of new triangles per facet is called the triangulation number (t), and t = h + hk + k , where h and k are any pair of integers. when h = k (t - , , , etc.), or when either h or k = (t = , , , , etc.), the triangles are arranged symmetrically on the underlying icosahedral face, but with other values for h and k (e.g., h = , k = and t = ) they are in a skew arrangement. a complete description then requires determination of the hand of the structure (right-dextro or left-zevo). the hand of the icosahedral shells of some papilloma viruses has been investigated by klug and finch ( ) and finch and klug ( ) , who concluded that the human papilloma virus (human wart virus) had a t = d icosahedral surface lattice whereas rabbit papilloma virus had a t = l lattice. in an icosadeltahedron with a triangulation number of , each icosahedral face has and the whole solid x = asymmetric units. these structural units may differ in shape and clustering so that the morphological units (capsomers) visible by electron microscopy may differ greatly in viruses with the same triangulation number. there are three basic types of clustering pattern: . the three units defining each triangular face may cluster at the center of the triangle, forming trimer capsomers ( fig. - d ). . the structural units may cluster at the vertices of the triangles, so that where five triangles meet at the vertices of the icosahedron there are pentamer capsomers, and where six triangles meet on faces of the icosadeltahedron there are hexamer capsomers ( fig. - e) . . pairs of structural units from adjacent triangles may cluster on the edges between the triangle to give dimer capsomers ( fig. - f ). the pattern seen on the surface of the virion need not reflect the way in which the structural units are bonded together, and gives no clue as to whether the structural units are constituted by single chemical units or are homo-or heteropolymers of the chemical units. however, the number of structural units in each capsomer can be guessed at from the arrangement and size of the capsomers ( fig. - ) . all known animal viruses whose genome is dna have isometric (or complex) capsids, as do all those whose genome is double-stranded rna and the viruses of two major families (picornaviridae and togaviridae) whose genome consists of a single molecule of single-stranded rna. tobacco mosaic virus occupies a unique position in virology. not only was it the agent whose "viral" nature was first appreciated (beijerinck, ), and the first virus to be crystallized (stanley, ), but more is known of its physical and chemical structure than any other virus (reviews: caspar, ; kaper, ). the virus particles are nonenveloped straight rods, which consist of repeating polypeptide (chemical) units, which are the structural units and also, without clustering, constitute the capsomers. these protein molecules are arranged in a helical manner so that except at the ends of the particles every capsomer is in a structurally equivalent position in relation to the long axis of the rods. many plant viruses and a few bacteriophages have similar nonenveloped tubular virions, their capsomers being arranged in helices whose pitch is characteristic for the virus group. such viruses are structurally defined by their length and width, the pitch of the helix, and the number of capsomers in each turn of the helix. tubular nucleocapsids are found in many groups of viruses of vertebrates, but only among those whose genome consists of single-stranded rna. none of these occurs as "naked" virions; the flexuous helical tubes are always inside lipoprotein envelopes. the diameters of the nucleocapsids of several viruses have been measured, but in only a few cases is the length or the pitch of the helix known. the best studied example, the nucleocapsid of sendai virus, a paramyxovirus, is a helix about /xm long and nm wide, with a pitch of . nm (finch and gibbs, ) . there are about hourglass-shaped structural units in the nucleocapsid, with either eleven or thirteen units per turn of the helix. the structural units are single polypeptides with a molecular weight of about , daltons, arranged with their long axes at an angle of about ° to the long axis of the nucleocapsid, which therefore has a herringbone appearance in electron the structure of animal viruses micrographs (see plate - ). the contact surface between adjacent turns of the basic helix is conical, so that contact is maintained even when the nucleocapsid is sharply flexed, and the viral rna is thus protected. unlike cells, which contain several different species of nucleic acid that subserve different functions, the only nucleic acid in viruses, apart from small amounts of host frna in leukoviruses, is their genome. it may consist of either dna or rna, it may be single-or double-stranded, it may be linear or cyclic, and the genome may consist of one or several molecules of nucleic acid. although the only detailed studies have been made on a few plant and bacterial viruses (review: tikchonenko, ), it is clear that the interaction between the viral nucleic acid and the capsomers is different in nucleocapsids with helical and icosahedral symmetry. in tobacco mosaic virus, there is a maximum regular interaction between the single strand of viral rna and the protein subunits which form a protective coat around it. a similar relationship probably exists in most animal viruses with tubular nucleocapsids, but in some viruses (e.g., influenza virus) the integrity of the tubular structure is destroyed by treatment with rnase but not by proteases, suggesting a different relationship of rna and protein. in icosahedral viruses, on the other hand, there can be no such regular relationship of the nucleic acid and each polypeptide subunit. in the simplest isometric viruses, the folding of the flexible single-stranded rna may have some regularity in relation to the capsomers and their constituent chemical subunits. x-ray diffraction studies of turnip yellow mosaic virus (klug et al., ), for example, show that a significant portion of the single rna chain is deeply embedded within the protein shell, large segments being intimately associated with the structural units, which as hexamers and pentamers make up the capsomers. the presence of the rna in and about these positions enhances the definition of capsomers seen in electron micrographs (finch and klug, ) . except for some togaviruses, even the simple isometric viruses of vertebrates have a more complex structure than this, since they contain several virus-coded poly pep tides. one or more of these poly pep tides are known to be "internal" to the capsid and it is thought that these rather than the capsomers interact with the viral rna. reovirus particles have two concentric protein shells, each consisting of well-defined morphological units. the proteins of the larger dna viruses are arranged in several layers, not all of which display symmetry. the internal proteins of many dna viruses are highly basic and are thought to be bonded to the viral nucleic acid, constituting a core within the isometric capsid. although occasionally used in a more general way to refer to the outer viral coats of some complex viruses like the poxviruses (mitchiner, ), we think that it is desirable to restrict the use of the term "envelope" to the outer lipoprotein coat of viruses that mature by budding through cellular membranes. enveloped viruses contain - % of lipid, all of which is found in the envelope. chemical analyses show that the lipid is derived from the cellular membranes through which the virus matures by budding, but all the polypeptides of viral envelopes are virus-specified. herpesvirus is the only virus of vertebrates that matures by budding through the nuclear membrane, and its envelope contains several virus-specified glycoproteins. all other enveloped viruses bud through cytoplasmic membranes, and contain one or more different polypeptides. the togaviridae have an isometric core to which a lipid layer is directly applied, and virus-specified glycoprotein peplomers project from this. all animal viruses with tubular nucleocapsids are enveloped, and in these the lipid layer from which glycoprotein peplomers project is probably applied to a protein shell (the membrane protein; see fig. - ), which may be relatively rigid, as in rhabdovirus, or readily distorted (as in the myxoviruses) so that in negatively stained electron micrographs the virions appear to be pleomorphic. viruses that have large genomes have a correspondingly complex structure. apart from the undetermined nature of the "cores" of many of the isometric viruses (e.g., herpesvirus and adenovirus), the virions of the two largest animal viruses (poxvirus and iridovirus) have highly complex structures, which are described in the appropriate sections of chapter . the rna viruses that have the largest (single-stranded) genomes, those of the leukovirus genus, also have a highly complex structure with an envelope enclosing an icosahedral capsid that, in turn, surrounds a tubular nucleocapsid. the aim of classification in biology is to make an ordered arrangement of a particular class of biological objects that will indicate their similarities and differences. adoption of a system of classification also involves consideration of the nomenclature of the objects to be classified. linnaeus introduced a latinized binomial nomenclature into biology years ago, and phylogenetic classifications of animals and plants based on the theory of evolution have since been introduced. international codes of nomenclature with rigid sets of rules, and judicial commissions to pass judgement on proposed names, have been set up for the naming of plants and of animals. an international code of nomenclature of bacteria and viruses was approved in and has since been revised (buchanan et ah, ). although they are primarily concerned with nomenclature, all these codes involve agreement upon a system of classification. codes are based on "acceptances," i.e., beliefs we would like to justify but are unable to prove, the principal one being that we are able to arrange living things in an orderly system that is indicative of both rank in a hierarchy and phylogenetic relationships (cowan, ) . classifications of animals and plants attempt to be scientific by deriving their taxa from a consideration of phylogenetic relatedness. more recently this approach has been reinforced by tests for genetic relatedness, i.e., the information content of the genetic material of the agents concerned. this has been tested by homology experiments with dna's ex- tracted from the cells of a variety of animals (mccarthy, ) , and it is to be expected that the phylogenetic and the molecular biological approaches will eventually be combined. the classification of bacteria into the same hierarchical pattern as that of plants and animals (phyla, subphyla, classes, orders, suborders, families, genera, and species) has led to a chaotic situation (cowan, ) . some bacterial taxonomists are looking to numerical methods, readily exploited with the aid of electronic computers, for the solution of their problems (sneath, ) . disadvantages of this approach are that the weighting of characters tends to be involuntary, and that pleiotropism may lead to some characters being scored more than once. most virologists believe that certain characters of viruses, such as the type, amount, and conformation of the viral nucleic acid, are taxonomically more important than characters like host range or pathogenic potential. molecular biology provides an alternative to phylogenetic relationships for making a scientific classification of microorganisms, viz., by the determination of genetic relatedness, using both the genetic material and the polypeptides that it specifies (mandel, ) . there are two groups of agents, the mycoplasmas and the viruses, for which detailed "official" classifications are still in the process of formation. because of their small genomes, they are particularly suitable for molecular taxonomy, i.e., classification based on the molecular weights and base ratios of their genomes, and on the results of nucleic acid hybridization experiments. applied to mycoplasmas, this approach has disproved claims that these microorganisms were derived from certain bacterial species (razin, ) . nucleic acid hybridization experiments have now been performed with many different viruses; detailed references to the results obtained will be given in chapter . in general, they have provided some useful data on relationships within genera and species, but not at higher taxonomic levels. with the methods used thus far many viruses now allocated to the same genus have shown little or no homology of their nucleic acids. indeed, nucleic acid hybridization may be too critical a method to be useful except for the comparison of closely related viruses, and less exacting tests for the similarity of viral genomes may be more pertinent when considering different viral species. bellett ( a,b) analyzed the data available in on the molecular weights and base ratios of the nucleic acids of different viruses. his results on the "clustering" of the viruses of vertebrates are consistent with the genera proposed by the icnv (wildy, ). however, newer knowledge about the fragmented nature of the genomes of some rna viruses and of the varied modes of their transcription and translation (baltimore, b) suggests that these data, where available, should be added to the parameters used by bellett. the differences between the molecular weights of the dna's of the dna viruses of vertebrates are such that sophisticated analysis is not needed to define the currently accepted families and genera. until about , little was known about viruses other than their pathogenic behavior. most early proposals for viral classification were confined to either plant or animal viruses and were based mainly upon the symptomatology of diseases caused by them, which tended to classify the host responses rather than the viruses. bawden ( ) made the pioneering suggestion that viral nomenclature and classification should be based upon properties of the virus particle. in the early 's bawden's approach was exploited by animal virologists (andrewes, ), and viruses were allocated to groups which were usually given latinized names constructed from a chosen prefix plus the word "virus." thus, myxovirus (andrewes et ah, ) group, ) , and adenovirus (pereira et ah, ) groups were described. in the meantime, a classification using quite different criteria had been established by epidemiologists. since they were so concerned with the transmission of infection, epidemiologists have used a classification based on the mode of transmission of disease; they have grouped viruses together as "respiratory viruses," "enteric viruses," or "arthropod-borne (arbo-) viruses." the last term, in particular, has been widely used, but it is generally agreed that this epidemiological classification, although useful is in no sense taxonomic. concurrently with these suggestions relating to the viruses of vertebrates, lwoff ( ) insisted upon the similarities between viruses, whatever their natural host, and the differences between viruses and all other biological entities. he was instrumental in arranging for the establishment of an international committee (anon., ; lwoff and tournier, ) to discuss nomenclature. its major proposal was to select "type species" upon which names for groups would be based. it also proposed a classification based on (a) the chemical nature of the nucleic acid, (b) the symmetry of the nucleocapsid (helical, cubical, or binal), (c) the presence or absence of an envelope, and (d) certain measurements: for helical viruses, the diameter of the nucleocapsid, for cubical viruses, the triangulation number and the number of capsomers. the official international committee on nomenclature of viruses (icnv), which was set up at the ninth international congress for microbiology in , adopted the physicochemical criteria of lwoff and tournier, but rejected the detailed hierarchical classification. the more important nomenclatural proposals accepted by icnv were: (a) an "effort should be made" toward a latinized binomial system of nomenclature, (b) the "law of priority" is unacceptable, (c) no taxon should be named from a person, and (d) anagrams, siglas, hybrids of names, and nonsense names should be prohibited. before describing the classification of animal viruses that we shall use throughout this book, it is appropriate to consider some of the problems of classification and nomenclature that have not yet been tackled by icnv. one of the most important is the level of taxa that should be used. so far, only three families of animal viruses have been accepted (see below), but it is clear that large and heterogeneous groups currently classed as genera (e.g., poxvirus, herpesvirus, paramyxovirus, leukovirus; wildy, ) should be regarded as families. indeed, it would not be unreasonable to regard all the currently accepted isolated "genera" as families, some of which (e.g., adenovirus) might at this a classification of animal viruses stage contain only a single genus. the conventional physicochemical criteria [(a) nucleic acid: type, strandedness, fragmentation, and molecular weight; (b) virion: shape, size, and symmetry] are suitable for classification at this level of family/genus, perhaps assisted by the serological cross-reactivity of "group" antigens where these have been recognized. at the other end of the nomenclatural spectrum, there is hopeless confusion in the ways in which the terms "species," "type," "subtype," and "strain" are used. for example, "types" of influenza virus exhibit no serological crossreactivity and their nucleic acids do not hybridize; they should be regarded at least as distinct species. on the other hand, many alphaviruses and flaviviruses with distinct names, which exhibit extensive serological cross-reactivity, should perhaps be regarded as types within the same species. serological cross-reactivity and nucleic acid hybridization tests are probably most useful for making comparisons at this "species" level. the icnv, working under the chairmanship of professor p. wildy, presented its first report at the tenth international congress for microbiology in mexico city in , and has published valuable basic data on forty-three viral groups encompassing viruses of bacteria, invertebrates, plants, and vertebrates (wildy, ) . in spite of the problems referred to above, we shall follow the classification set out in the report, amplifying it with proposals that have come forward since then, but maintaining accepted usages of the terms "type," "strain," etc. only two families were established by icnv in (wildy, ): papovaviridae and picornaviridae, and subsequently the family togaviridae was defined. most accepted "groups" of vertebrate viruses were given generic names. no species names were adopted by icnv, although "t> ~>e species" were designated for several of the genera. the family papovaviridae (sigla: pa = papilloma; po = poly oma; va = vacuolating agent, sv ) encompasses two genera, polyomavirus (poly = many; oma = tumor) and papillomavirus (papilla ^nipple; oma = tumor), which differ substantially in size and nucleic acid content of the virion (table - ) but share many other properties. an important property of many papovaviruses is their capacity of produce tumors. in nature, some produce single benign tumors (which may undergo malignant change) and are highly host specific ; others may cause primary malignant tumors within a short period of their inoculation into newborn rodents. the adenoviruses (adeno = gland) are nonenveloped icosahedral dna viruses which multiply in the nuclei of infected cells, where they may produce a crystalline array of particles. many serological types have been isolated from human sources. these have an antigen that is shared by all mammalian strains, but differs from the corresponding antigen of avian strains. allocation to the genus is made primarily on the basis of the characteristic size and symmetry of the virion as seen in electron micrographs (icosahedron with capsomers). most adenoviruses are associated with respiratory infection and many such infections are characterized by prolonged latency. some multiply in the intestinal tract and are recovered in feces. many adenoviruses, from both mammalian and avian sources, produce malignant tumors when inoculated into newborn hamsters. in the laboratory, stable hybrids have been produced between certain adenoviruses and the polyomavirus, sv (see chapter ). the herpesviruses (herpes = creeping) are readily recognized by their morphology. their icosahedral capsid is assembled in the nucleus and acquires an envelope as the virus matures by budding through the nuclear membrane. electron microscopic examination by negative staining of many previously unclassified viruses showed that several of them had large icosahedral capsids with capsomers enclosed within lipoprotein envelopes, similar to the type species, herpes simplex virus. when examined further, such viruses were found to be dna viruses that multiplied in the nucleus, and have now been included in the genus herpesvirus. table - shows some of the viruses now regarded as members of this genus; a more complete list is given by andrewes and pereira ( ). there is a group-specific antigen(s) associated with the nucleocapsids and demonstrable by immunodiffusion, and several type-specific antigens associated with the nucleocapsid and envelope. some type-specific antigens crossreact (e.g., herpes simplex viruses type and type and b virus). different herpesviruses cause a wide variety of types of infectious diseases, some localized and some generalized, often with a vesicular rash. a feature of many herpesvirus infections is prolonged latency associated with one or more episodes of recurrent clinical disease. this genus (irido = iridescent) was defined on the basis of several viruses of insects whose structure and nucleic acid content have been carefully studied . nature and classification of animal viruses (bellett, ; wrigley, ) . several dna viruses of vertebrates that are similar in morphology and certain other characteristics have been tentatively grouped with the genus iridovirus (table - ) . like poxviruses, but unlike other dna viruses, iridoviruses multiply in the cytoplasm. their dna consists of a single linear molecule, with a molecular weight of about - million daltons, and the virion is a large and complex nonenveloped icosahedron, with an outer shell composed of about capsomers. the vertebrate ''iridoviruses" may be enveloped. several enzymes are found within mature virions. the best studied of the vertebrate "iridoviruses" are some viruses of frogs, notably fv (review, granoff, ); the most important economically is african swine fever virus (review, hess, ). the poxviruses (pock = pustule) are the largest animal viruses, and contain a larger amount of dna ( - million daltons of double-stranded dna) than any other virus. the structure of the brick-shaped virion is complex, consisting of a biconcave dna-containing core surrounded by several membranes of viral origin. there is a poxvirus group antigen which is probably an internal component of the virion, and can be demonstrated by complement fixation or gel diffusion tests. several enzymes, including a transcriptase, are found within mature virions. multiplication occurs in the cytoplasm and the virions mature in cytoplasmic foci. occasionally, the virion may be released within a loose membrane derived from the cytoplasmic membrane. this is not essential for infectivity, and must be distinguished from the envelope of viruses that mature by budding through cellular membranes. the genus is divided into several subgenera (table - ) , and there are several poxviruses that have still to be classified. the properties outlined for the genus apply to all the subgenera, except that the virions of members of the subgenera b and c (see table - ), and swinepox virus, are narrower than those of other poxviruses, and virions of subgenus b (orf) have a distinctive surface structure. species within each subgenus show a high degree of serological cross-reactivity by neutralization as well as complement fixation tests. genetic recombination occurs within, but not between, subgenera; nongenetic reactivation (complementation) occurs between most poxviruses of vertebrates (chapter ). certain viruses that multiply in insects have many of the attributes of poxviruses and have been tentatively called entomopoxviruses (entomo = insect) (review: bergoin and dales, ). poxviruses cause diseases in man, domestic and wild mammals, and birds. these are sometimes associated with single or multiple benign tumors of the skin, but are more usually generalized infections, often with a widespread vesiculo-pustular rash. several poxviruses are transmitted in nature by arthropods acting as mechanical vectors. the viruses of rodents cause acute fulminating disease when inoculated into newborn hamsters (kilham, ) . the adenovirus-associated viruses are not known to cause any symptoms. the picornavirus group (sigla: pico = small; rna = ribonucleic acid), which includes a very large number of viruses, was accepted by icnv as a family, picornaviridae, with three genera: enterovirus (entero = intestine), rhinovirus (rhino = nose), and calicivirus (calici = cup). newman et al. ( ) believe that this subdivision of the family is unduly restrictive; on the basis of particle density, base composition of the viral rna's, and stability at various phs they differentiate cardioviruses (cardio = heart) from enterovirus, and foot-and-mouth disease virus and "equine rhinovirus" from the genus rhinovirus (table - primarily inhabitants of the intestines, and a large number of serotypes have been found in the feces of man and of various animals. the enteroviruses of man have been subdivided into three major subgroups : poliovirus, three serotypes; echovirus (acronym: echo = enteric cytopathogenic /luman orphan), thirty-four serotypes; and coxsackievirus (coxsackie = town in new york state), twenty-four serotypes of type a and six of type b. the polioviruses, which show some serological cross-reactivity, are distinguished by their capacity to paralyze humans. coxsackieviruses were originally defined in terms of their capacity to multiply in infant mice, but subsequently some echoviruses were found to do the same. it has been recommended that all future en-short descriptions of the major groups of rna viruses teroviruses that are discovered should be numbered sequentially from , irrespective of subgroups (rosen et al., ) . most infections with enteroviruses are inapparent, a few are associated with gastrointestinal disorders, and some may cause generalized infections with rash, central nervous system involvement, including poliomyelitis and aseptic meningitis, or specific damage to the heart. genus: rhinovirus [r/ : . - . / : s/s: v/ ]. the rhinoviruses resemble the enteroviruses in several characteristics but they are acid labile (ph ) and have a buoyant density (in cscl) of . - . g/cm . most have a low ceiling temperature of growth and are characteristically found in the upper respiratory tract of man and various animals. there are a large number of different serotypes of human rhinoviruses, and there are several serotypes of foot-and-mouth disease virus, which resemble rhinoviruses in some respects, but not in others (table - ) . most rhinoviruses cause mild localized infections of the upper respiratory tract, but foot-and-mouth disease virus causes a severe generalized disease with rash in cattle. during the last quarter century intensive world-wide efforts have been made to recover viruses which would multiply in both arthropods and vertebrates, and some different agents with these biological properties are now known. they have been called "arthropod-borne viruses," a name which was shortened to "arborviruses" and then (in order to avoid the connotation of "tree") to "arboviruses." the arboviruses have been defined, on epidemiological grounds (mode of transmission), as a group comparable to the "respiratory viruses." arboviruses are viruses which, in nature, can infect arthropods that ingest infected vertebrate blood, can multiply in the arthropod tissues, and can then be transmitted by bite to susceptible vertebrates (world health organ., ). for many years arboviruses have been recovered from vertebrate tissues and suspensions of arthropods by the intracerebral inoculation of mice, and advantage has been taken of certain chemical and physical properties found to be commonly associated with them to avoid confusion with murine picornaviruses. the property generally tested was sensitivity to lipid solvents. many arboviruses have lipoprotein envelopes and their infectivity is destroyed by these reagents (theiler, ; casals, ) . there was thus a tendency to equate sensitivity to lipid solvents with "arbovirus." during the last decade it has been recognized that the arbovirus group is quite heterogeneous in its physicochemical properties (see table - ). some members are not enveloped (orbivirus, nodamura virus), and those sensitive to lipid solvents belong to at least three major groups (togaviridae, rhabdovirus, and bunyamwera supergroup). this preamble has been necessary because in the past the term "arboviruses" has been regarded as applying particularly to viruses with the physicochemical properties of the group a and group b arboviruses. these viruses now form two genera (alphavirus and flavivirus) of the family togaviridae (toga = cloak). (table - ) and show serological cross-reactivity by the hemagglutinin-inhibition test. the arthropod vectors are mosquitoes, but some alphaviruses may be transmitted congenitally by vertebrates. in nature, they usually cause inapparent infections of birds, reptiles, or mammals, but some can cause generalized infections associated with encephalitis in man and in other mammals. genus: flavivirus [r/ : / - : s/s: v,l/ , di, ac]. this genus (flavi = yellow) comprises the group b arboviruses. all members show serological crossreactivity. the arthropod vectors may be ticks or mosquitoes, and some of them may be transmitted by the ingestion of contaminated milk. they differ from the alphaviruses in that budding usually occurs into cytoplasmic vacuoles rather than from the plasma membrane. most cause inapparent infections in mammals and less commonly in birds, but generalized infections of man may occur with visceral symptomatology (e.g., yellow fever), rashes (e.g., dengue), or encephalitis (e.g., japanese encephalitis). other possible members of the family togaviridae. on the basis of the physicochemical definition proposed, several other viruses that are not transmitted by arthropods should probably be included in this family. generic names have not yet been proposed for these viruses, which include rubella and equine arteritis viruses, and the two serologically related viruses of hog cholera and bovine mucosal disease. in early classifications, some members of two very different genera, now distinguished from each other as orthomyxovirus (ortho = correct; myxo = mucus) and paramyxovirus, were grouped together as myxovirus (andrewes et ah, ). the common properties were an rna genome, a tubular nucleocapsid, and a pleomorphic lipoprotein envelope that carried the properties of hemagglutination and enzymatic elution. the term "myxovirus" is now only used as a vernacular expression to encompass the viruses that have these properties {viz., influenza, mumps, newcastle disease, and parainfluenza viruses); it has no taxonomic status. type a influenza viruses have been recovered from a number of different species of animal (birds, horses, and swine) as well as man; types b and c are specifically human pathogens. they are an important cause of respiratory disease in man and other animals, and some of the avian influenza viruses may cause severe generalized infections. in contrast to the orthomyxoviruses, the paramyxoviruses (para = alongside; myxo = mucus) are enveloped viruses whose rna occurs as a single linear molecule with a molecular weight of about million daltons (table - ) . the tubular nucleocapsid has a diameter of nm and is about . μπι long. it is enclosed within a pleomorphic lipoprotein envelope nm or more in diameter; long filamentous forms with the same diameter also occur. three serologically related viruses, those of measles, distemper, and rinderpest, have been tentatively allocated to the paramyxovirus genus on the basis of the morphology of the virion and nucleocapsid; they do not have a neuraminidase; respiratory syncytial virus is different again. some paramyxoviruses cause localized infections of the respiratory tract and several produce severe generalized diseases; among the latter some are characteristically associated with skin rashes. the genus arenavirus (arena = sand) was defined in terms of the electron microscopic appearance of the virions in thin sections, and serological crossreactivity (rowe et al., a) . the pleomorphic enveloped virions are - nm in diameter (sometimes larger), and have closely spaced peplomers. the structure of the nucleocapsid is unknown, but in thin sections the interior of the particle is seen to contain a variable number of electron-dense granules - nm in diameter, hence the name. all members of the genus are associated with chronic inapparent infections of rodents; some cause acute generalized diseases in other hosts (e.g., lassa fever virus in man). b characteristics : single-stranded rna probably in several pieces, total molecular weight . million daltons; lipoprotein envelope - nm in diameter; multiply in cytoplasm; mature by budding from plasma membrane. all members share a group-specific antigen. envelope encloses "granules" - nm in diameter; some of these are cellular ribosomes. the bunyamwera "supergroup" of arboviruses (bunyamwera, a locality in africa) was established by casals (world health organ., ) to bring together a number of minor arbovirus groups linked by distant serological reactions between occasional "bridging" viruses. the subgroups of viruses included are shown in table - . all these viruses, numbering well over , are known or suspected to be arthropod-borne. morphologically, those that have been studied have enveloped roughly spherical virions - nm in diameter with a tubular nucleocapsid. several other arboviruses serologically unrelated to those of the "supergroup" have a similar morphology (table - ). their genome consists of single-stranded rna probably occurring in several pieces; its molecular weight has not been determined. the outstanding characteristic of the genus leukovirus (leuko = white) is that all members contain an rna-dependent dna polymerase ("reverse transcriptase"). the viruses contain three or four pieces of single-stranded rna, with a total molecular weight of to million dal tons, associated with a helical nucleocapsid, which is enclosed within a capsid with cubic symmetry. this is, in turn, enclosed within a lipoprotein envelope about nm in diameter, containing peplomers which confer the type specificity. leukoviruses mature by budding from the plasma membrane. genome is a linear molecule of single-stranded rna, - million daltons molecular weight, consisting of three to four linked pieces and probably associated with tubular nucleocapsid. structure of virion is complex, the nucleocapsid being enclosed within a capsid of cubic symmetry, which is enclosed in an envelope that carries type-specific antigens. virion also contains species-specific (e.g., feline or murine) and interspecies-specific (e.g., avian or rodent) antigens. as table - illustrates, the genus leukovirus accepted by icnv is clearly an inadequate taxon for the variety of viruses that now fulfill the physicochemical criteria set out above. the term "oncornaviruses" (nowinski et al., ) is also not suitable for the taxon as a whole or any subgroup of it, for not all the viruses conforming to the physicochemical specifications of the genus leukovirus are tumor viruses, many "rna tumor viruses" are not transforming (temin, ) , and in any case the leukemia-sarcoma viruses and the mammary tumor virus (both of which produce tumors) belong to different subgenera. in order not to prejudge a classification of the group, we shall adhere to the accepted generic name, but indicate four subgenera. subgenus a includes the "c-type particle" viruses, some of which cause leukemia-sarcoma and are currently being subjected to intensive study. the mammary tumor virus, which is the best known representative of the subgenus b, differs from viruses of subgenus a in morphology and maturation ("b-type particles") and shows no serological cross-reactivity with the murine viruses of subgenus a. subgenus c includes a group of serologically related viruses that cause slowly progressive diseases in sheep. they have all the physicochemical properties of leukoviruses. although they do not cause neoplastic disease, they will transform cells that are nonpermissive for viral growth (takemoto and stone, ). the viruses of subgenus d (foamy agents) include a number of viruses of monkeys, cats, and cattle, that have no known pathogenic potential but have been frequently isolated from tumors (as "passenger viruses") or healthy animals. they have a different morphology from other leukoviruses (clarke et al., ) and produce an intranuclear antigen as well as cytoplasmic antigens in infected cells (parks and todaro, ), but they contain a reverse transcriptase and are much more resistant to uv irradiation than other rna viruses. the rhabdoviruses (rhabdo = rod) are enveloped rna viruses with singlestranded rna of molecular weight million daltons. the rna is associated with a very regular double-helical nucleocapsid nm in diameter, enclosed within a bullet-shaped shell that measures about x nm (table - ) . several arboviruses belong to this genus, which also includes rabies virus and the virus of hemorrhagic septicemia of trout. it has been claimed that rabies virus can be adapted to multiply in drosophila melanogaster (plus and atanasiu, ). several viruses with a somewhat similar morphology cause diseases of insects and plants (table - , and see table ii of howatson, ), but it may well turn out that these resemblances are superficial. examination of the nature of their genomes and polypeptides is necessary before it can be confidently stated whether these viruses rightly belong to the genus rhabdovirus or even to an enlarged family that might be called rhabdoviridae. nature and classification of animal viruses the mammalian serotypes share a common antigen, which differs from the group antigen of the avian serotypes.clover wound tumor virus, which multiplies in plants and leafhoppers, resembles the reoviruses of vertebrates morphologically and chemically but does not cross-react with them serologically. bluetongue virus, an arbovirus, was found to resemble the reoviruses in some properties but not in others (review: howell and verwoerd, ). subsequently, a large number of similar viruses have been recognized (table - ) and the name "orbivirus" (orbis = ring) was suggested for them (borden et a\., ). reoviruses and orbiviruses may eventually be grouped together in the same family for which the name "diplornavirus" has been suggested (verwoerd, ) . apart from its "illegality" (according to icnv rules), the occurrence of other quite different viruses with genomes of double-stranded rna (like some of the viruses of fungi and insects) cautions against ready acceptance of this term.all members of the genus multiply in arthropods as well as vertebrates. some of them (bluetongue and colorado tick fever viruses) cause severe generalized diseases with viremia in some vertebrates. it is pleasing to note that several of the viruses listed as "unclassified" in the first edition of this book have now been allocated to genera (lymphocytic choriomeningitis of mice, arenavirus; mouse hepatitis virus, coronavirus; rubella virus, togaviridae; visna virus, leukovirus; and african swine fever virus, iridovirus). a few unclassified viruses remain that warrant special mention here, such as the human hepatitis viruses, the agents of the subacute spongiform encephalopathies (scrapie, etc.), lactic dehydrogenase elevating virus (ldv), and the marburg agent. experiments with human volunteers many years ago (neefe et al, ) and again more recently (krugman et al., ) have shown that the diseases commonly known as infective hepatitis and serum hepatitis are caused by two viruses that differ serologically, in their clinical expression, and in their usual routes of transmission. because both can be transmitted orally it is better to use noncommital names for them, and "serum hepatitis" is now termed hepatitis b; infective hepatitis, hepatitis a. study of these viruses has been greatly inhibited by the lack of susceptible laboratory animals (chimpanzees may get clinical hepatitis; marmosets and rhesus monkeys subclinical infection, while other laboratory animals are insusceptible), and the difficulty of obtaining reproducible cytopathic changes in cultured cells. the recognition in the sera of cases of serum hepatitis of lipoprotein particles of characteristic serological specificity, called "australia antigen/ hepatitis-associated antigen (haa), and now hepatitis b antigen (hb-ag), has led to a great expansion in studies on the incidence and pathogenesis of hepatitis b, but the actual virions have not yet been unequivocally demonstrated (see chapter ). serologically unrelated particles of similar morphology have been reported to occur in feces from patients with hepatitis a (cross etal, ). four diseases of similar nature, scrapie of sheep, transmissible encephalopathy of mink, and kuru and creutzfeld-jakob disease in man appear to be caused by similar agents, which differ from all known viruses by being nonimmunogenic. the causative agents are filtrable, highly heat-resistant, and highly resistant to ionizing radiation. it has been suggested that they may be small molecules of naked rna, protected by being closely associated with cellular membranes (diener, a), but a definitive description of these agents is still awaited. this virus, which occurs as an inapparent infection in many laboratory mice and as a contaminant of cells and viruses derived from or passaged through mice (review: notkins, ), shows some resemblances to the togaviruses. it appears to have an isometric core and a lipoprotein envelope, and its rna is infectious. however, the viral rna is large, perhaps million dal tons (darnell and plagemann, ). in germany, in , a small outbreak of a serious new disease occurred in laboratory workers who had handled the tissues of recently imported vervet monkeys (review: siegert, ). the causative agent grows in cultured cells and kills guinea pigs. studies with inhibitors suggest that it contains rna; of known viruses it most closely resembles rhabdoviruses in structure but is much larger and more pleomorphic (murphy et ah, b) . the foregoing account has shown how varied are the agents that we classify as viruses, for reasons based on their composition and their mode of intracellular replication. we can only speculate about their origins and relationships to each other, except in cases where the relationship is very close. it seems likely that different viruses belonging to any one genus, and in at least some cases, different genera allocated to a particular family, may be phylogenetically related. no use- . nature and classification of animal viruses fui suggestions can be made concerning the relationships between families or genera (except in some of the cases where genera have been allocated to the same family), a fact which underlines the undesirability at this stage of our knowledge of erecting any taxa at levels higher than the family.two suggestions have been made concerning the origin of viruses: (a) that they are the result of progressive parasitic degeneration of microorganisms (green, ) and (b) that they have developed from components of the cells of their hosts (andrewes, ; luria and darnell, ) , or are indeed still a permanent part of the host's genome (todaro and huebner, ) . with our present knowledge of the morphological and chemical complexity of the poxviruses, it is not difficult to envisage these agents as being the next degenerate step in the series: bacterium, rickettsia, chlamydia. although they resemble bacteria in most important respects, rickettsia and chlamydiae are, like viruses, obligate intracellular parasites lacking the metabolic equipment for independent multiplication.on the other hand, some dna viruses could well have arisen from episomes, by the acquisition of genetic information specifying a protein coat. even this may not be essential, if diener's ( b) observations on potato spindle tuber virus are confirmed and generalized. the two alternatives are not mutually exclusive; some viruses may have evolved from cellular organelles like chloroplasts or mitochondria, themselves probably derived from bacteria (swift and wolstenholme, ). it is difficult to see where most rna viruses could have originated except from cellular rna's.comparing the nearest neighbor nucleotide doublet frequencies of the nucleic acids of several large and small viruses, subak-sharpe ( ) noted that the patterns shown by small viruses with genomes of less than million daltons (two enteroviruses, three parvoviruses, two polyoma viruses, and two papilloma viruses), closely resembled the pattern of mammalian dna. on the other hand, the doublet frequency patterns of several viruses with large genomes (two herpesviruses and a poxvirus) differed strikingly from that of mammalian dna. the doublet patterns of three adenoviruses resembled each other and showed a slight resemblance to the pattern of mammalian dna, which could derive from some earlier natural fusion of genomes, like that recognized as a laboratory artifact with adenoviruses and sv (see chapter ). this evidence supports the notion that the small viruses may have originated from vertebrate cells whereas the herpesviruses, poxviruses, and probably the adenoviruses did not. these large viruses may have originated from the nucleic acid of cells of a different phylum, or as suggested earlier, by parasitic degeneration of microorganisms. key: cord- -dlqs ay authors: nan title: sequences and topology date: - - journal: curr opin struct biol doi: . / - x( ) -t sha: doc_id: cord_uid: dlqs ay nan . garrell j, modolell j: the drmm~hila locus, am antagonist of proneural geors that, lilac these genes, ~.ncodes a helix-loop-helix protein. ce/ , : - in crystals of ~or-nib-glu(oz~)-leu-mb-ala-leu-an~alm-lys(z)-alb.ome. pro~ natl acx*d sci usa , : - cloning and expre~inn of two distinct high-afl~nlty p~eptot~ ~geat¢ting with acidic and b~ic ~last growth imctogs. embo j . emboj emboj , : emboj - . he~gst l~ lf~w~m~ t, g~lwr~ i~. the ryb l gene in the l~lmion ye~tt sd~m~acctm~ pombe lfalcodin a gtp-bindin s protein belated to rho and ypt: structot~, expfemflon and identificetion of its human homulogue. embo j ~ , : embo j ~ , : - serotouln receptor that activates adenyiate cyclase domain • of lutropin/choriogonadotropin receptor expressed in t~ cells binds choringonadotropin with ltlgh /tmnlty cllgol~o onlal orsanization of adrenergic receptor genes molecoiat chagactegization of a rat ~ n-adrenerl#c receptor identificatlon of rpo , a vaccinia virus rna polymerase gene with structut~ similarity to a eucaryotic transcription elon~ation factor nucleotide sequence analysis of the l g~ne of vesicular stomafltia virus (new jersey serotype) --identification of conserved domai~l~ in l proteins of nonsegmented negative-strand rna viruses a novel u,man immunodeflclency virus type- protein, try, shares ~'quences with tat, ent~ and rev proteins phosphoprotein and nucleo~psid protein evolution of vesicular stomatitis virus new jersey identification of a conserved region common to cadherius and hl~u~llzat s~ a hema~u!tinin~ sequence and evolutionary relationships of african swine fever vh*es thymidine kinase all unusual stnlctul~ of a putative t cell oncugene whlch allows production of similar proteh~ from distinct messengeg rnas ~ ldentilfication of a rd protein factor which binds to the rolls sarcoma virus ltr enhancer --po~lble homology with the serum response factor genetic variation and multigene fannllles in aj~rics~t swine fever virus sequence of the genome rna of rubella vh'us --evidence for genetic rearrangement du~n~ tosavirus evolution derse i~ equine infectious anemia virus tat--insights into the structure, function, and evolution of lentivtrus tran.~activator proteins a colrpoeison of the genome organization of capripoxvirns with that of the orthopo~ ~golutionary orlffln of human and s imian lmtmo~odeflciency vir~jes a new supe~mlly of putative ntp-bindin domaan,t encoded by genomes of small dna and rna viruses envelope gene sequence of htlv- isolate mr- and its comlmrlann with other htlv-i isolates evolutionary relationship between luteovtruses and other rna plant viruses based on sequence motifs in their putative rna pulymet-am~ and nucleic acid hellcases isolation and sequence analysis of caenothabd/~s br/~w~e repetitive elements related to the ~ dqana transposon tcl jmolevo selective clo~ sequence analysis of the hum l sequence* which t~ in the rehttively recent l~tt jowcs m& sequences related to the matte tra~acmable element acin the genum zea. j m / evo/ evulutiotmtv pattern of the hemas~utinin gene of inflmmza-b viru~s ~ulated in japan ~ cocir~lating linesses in the same epidemic semon the dna binding subuult of nf-kaplm-b is identical to factor kbfl and homologous to the rel oncogene product sequencins analyses and com~ of pmrainfluenza virus type- a and type- b np protein genes. virok>gie complete sequence of the gcnomic gna of o'nyon -nyong virus and its use in the constroctton of alphavir~ phylogenetic trecs molecular clouln s of the rinderpest virus matrix gene --comparative sequence analysis with other paramyxorirm~. vi~logy cautd~an p~ ancestry of a human endogenous retrovirus ~ determination of an epitope of the diffuse systemic sclerosis marker antisen dna topoisomerase-l: sequence $ mllagity with retroviral ~ protein suggests a possible cause for autoimmunity in systemic sclerosis. pro natlacad s i u& , : ~ . mcgeoch dj: pgotein sequence cota~lxs show that the 'psuedoprotesses' encoded by poxviruses and certain retrovirus~ belong to the deoxyoridine triphtmphate family ~sk life: ~es of comme//na yellow mottle vlrus's complete dna sequence, genomlc discontinuities and transcript su est that it is a pararetrovlnm i~l~titis c vllrll~ sborl~ amino acid sequence similarity with pe~tivirutu~ and flavivirus~ as well as members of two plant vlgus superggoupo mo~mann ti~ homology of cy~kine synthesis inhibitory factor (el- ) to the epstein-barr virus gene bcrfi nucleotlde sequence analysis of sa-omvv, a vlsna-related ovine lentivirus --phylo~-netic history of lentivirmms single copy seqoences in g~qgo dna retmmable a repetitive hnman aetrotrmmposon-llke family. y mo/e~/ , : re¢otnblnation resulting in unusual features in the polyomavlrus genome isolated from a murine tumor cell line sequence anal~is of rice dwarf elxytoreovirus genome sewments s , s , and s -comparison with the equivalent wound tumor virus segments ho~tu~ ~ s is a ehylngcueticellly distinct human endogenous reteovtgal rlement with structural mad sequence homology to simian sarcoma virus (ssv). vi~ologie identification of a novel -kl)a cell surface receptor common to pm~cee~flc polypeptide molybdenum hydroxylas~ ~ the amino acid seqoence of chicken hepatic solfite oxidase frequency of a]mloglnai h |lm~tn haemoglobins ~ by c ~ t trmmitions in cpg dlnucleotid~ evidence for conservation of ferritin sequences amon plants and animctbt and for a transit peptlde in soybean a -kda llpo~ortin from human mononuclear cells appears to be identical with the placental inhibitor of blood coagulation distinct fercedoxins from rhodobacter-capsulstus -complete amino acid sequences and molecular evolution n~ptide sequence analysis and molecular cloning reveal two calcium pump isoforms in the human erythrocyte membgane cloning and characterization of a novel member of the cytochrome-p subfamily iva in rat prostate a directiy repeated sequence in the ~-globin promoter resulates transcription in murine efythroleukemla cells isolation and chamcterizatinn of the alkane-inducibie nadph-cytochrome-p- olf, idoreductsse gene from candida-tropicalls -identification of invarlant residues wlthin slmilmr amino acid sequences of direr'sent flavoproteins protein klnase-c inhibitor proteins -purification from sheep brain and sequence similarity to lipocortins and - - mci~ aveml~ b& sequence homology between purple acid phosphatases and phusphoprotein pho*phatsses --are phesphoprotcin phosphatatms metalloproteins collt~|nln~ oil~-bridged dinuclcar metal centers negative regulation of the human ~-globin ca~ne by transcriptional interference: role of an mu repetitive ~lement amino acid sequence of chicken catisequestrin deduced from c dna -comp~rison of caisequestrin and aspartactin caisequestrin, an intesccilular calciumbinding protein of skeletal muscle sarcoplssmic reticulm, is homolokous to ~, a putstive latminin-binding protein of the exteac¢llular matr~ bovsm~ ]prote~ c inhihl.gog with structugll and fun~ hotdoio~ou~ ]~-.gtl~ to hum~zn plum~ protein c inhibitor sequence of silkworm hemolymph antitrypsin deduced from its cdna nucleoude sequence --~on of its homology with ~.rplus. l b~cbem (tokyo) human mm~t cell tryptm~e multiple cdnas and genes reveal • multigene serlne protemje lmmlly howam> jc: msc ore. n k#on encoding protehm iteleted to the multidtog ite~letance family of tra~membt'mne tratmpofters m~, a tks~me-speclfi¢ b•tmment membrane protein, is a ia.minin.like protein commrvation of a cytoplasmic ~xy-termitml domain of couexin , a gap junctional protein, in mammal heart and brain the a~lba//a~ plasma membrane h+-a~ multigene ~ -genomle sequence and expression of • rd lsoform, f b/ /owra op#n of calliphora peripheral l~otoreceptors r - --homology with d~ rhl and po~tmnsi~domd processing evolution of rhodopsin supergene family --independent divergence of visual pibments in vertebrates and insects and po~ibly in mollusks ct~tpl¢ the g~ne~ amino acid ~'~m~me gene of sac~baromy~wcet~-v/~ae --nucleotide seque~tce, protein similarity with the other i~kers yeast amino acid petmme~mes, and nitrogen cataboht~ repreulon the -kda peroxlsomal membrane protein is a member of the mdr (p-glycoprotein)-related atp-bindin~g protein superfamlly a new clam of lym~o-real/vacuolar protein sorelng signais. l b~/chem complete amino acid sequence and homologies of human erythrocyte membrane protein band . . proc natl acad scd us a the primary structure of a halorhodopsin from n pbaraom~--structural, functional and evolutionary impnoations for bacterial rhod~ and haloghodopslns soluble lactose. blndln~ vertelmue lectlns: a ~ family the a regulatory subunit of the mltochondrlal fi-atpa~ complex is a heat shock protein. identification of two highly conserved amino acid sequences amon~ the ~x-subunits and molecular ~ sequence of h ilmlfl ~ l~ieat~ • novel gene family of integral membrane proteoglycans a protein with homology to the c-termimml relationaxip~ between/m~-nylate cycla~ and n•+,k+-ati~se lit pat pancgtmti¢ islets human na+ ,k+-ati~¢ genes ~ beta~ubunit gene family conmina at lcest one gene and one ~ evolution of the mltc cles~l genes of a new world lh'imate from ancestral homologues of human non-clessical genes the cdna sequence of mouse pllp- arid homololgy to hntman cd c~ll s~e antitpm and promot#ycen core unk proteins ~tjott of cdna encodin~ a hnman sperm membr~e protein related to a amyloid protebm ptwlflcstlo~ c~mu'actet~muon, and con with memb~ne carbonic anhydrase from human kidney hypermumbility of cpg dinucleoudes in the propcptide-enced/ng sequence of the human alb~tmi~ gene dystt'ophhl in electric or~n of to, pedo-~ homologous to that in h,ml~ muscle botste~ i~ homolosy of a yeast acun-binding protein to signal trmmductlon proteins slid myosin- the complete sequence of drosophila alpha-spectrin --conservation of structurml domahm between alplm-~ and alpl~t cttnin •~ettaatflon of a lqbrilisr collqwn gene ~ spruces reveais the etdy evolutionary appearance of two collqwn gene fmmilk~ the predicted amino acid sequence of ct-lnternexin is that of a novel neuronal lntegmedla~ ~ent protein otsen bl~ type xil collm~n. a larbe multidomnln molecule with partial homology to type ix cousllem / b/d aera amyioid protein in i~mni~l amyloidmfls (plmnlah type) ks homolollotm to gd$oilmb an ac, tht-i~h,.da~g protein. b/~bera b/q b~ res commun key ji~ ~ of a proline-rich cell wall protein gene ~ of soybesn. a ~ ana/ysis. j b/o/~em chicken liver evolutionary rehttinnships and impflcations for the resulation of phoophohpsse-a from snake venom to human secreted forms identification of a locality in snake venom a-ncurotoxins with a slsnlficant comlm*itinmd similarity to marine smdl ct-conotoxins: implications for evolution and structure activity al~ph[biml~ albmtm|nm ~s members of the albumin, alpha-l~toprotein. vitamin-d-binding protein mul~ flmily ~ni~on of the hnm~n llpoprotein lllmse gene and evolution of the llpase gene family e~'t~ion of cloned human reticulocyte - ipoxygenase and immunological evidence that -hpoxygetmses of different cell types are related identification of a protein alt~ inttaspecific evolution of a gene family coding for urinary proteins conservation between yeast and man of a protein a~ociated with i small nuclear rlbonucleoprotein stl~ctute and partial amino acid sequence of calf thymus dna topobmmaertt~-ii -coml~on with other type-h emmyme~ ol~nudeotide correlations between infector and hem genomes hint at evolutiotmry relationships. nu /e~ scot/~ik p& carotenoid desmurases fi, om ~ ~and nmoowo~craua are stru~ and l~n~'tinnally comerved and eonmin domains homolosons to flavoprotein dimdflde oxldoreductm~ deininger pi stt'uc~uee and vsrisbihty of recently inserted alu family members a novel neutrolphfl chemmtttactant generated duan an ln~ammmtory reaction in the l~mt peritoneal cal~lt~ tt~ t~t~o -l~tl'~t~tloil~ ~ amino acid seque~tce and structural relmtmmhip to interkukin-& b~ffx~m j the multlfimctinna -methylmllcyllc acid syn~ ge~e of ~~ ~ its ge~e structmm ieimive to tl~t of other po~lyketide symhase~. f.urj b/odaem mammalkm ublquitin carrier prmmtmh but not i~:i~k, ame ltdated to the -kda yeast , rad . bk~chem b/qohys res commun chambers gk: sequence. structure and evolution of the c.ene codin b for ~t-gi~erol- -phe~plmte ~rdrotfm~ in om,qt~ the cotaplete sequence of bogu/ktmm nenrotoxin type-#, and com~ with other clostrldhtl neugoto~hm if: a pamlly of cxam~fltutive c/bbp-llkc dna blndln~ proteins attenuate the il-l~t induced, ni~b mediated trans-activation of the ansiotemflnogen gene acute-phase response element different fort~ of ultmhithomx proteim generated by alternative spttcim~ are functionally equivalent evolution of collagen-iv genes from a -batm pair faton --a role for lntrmm ht gem~ evolution evolution of the insulin superfamlly tcetins are structoraily related sertoli cell proteim who~ ~on is tightly coupled to the iprtsence of germ cells ivarie r~ a bovine homolo s to the human myolletti c determination factor myf~ sequence conservation and ' proce~ing of transcripts proteiu sertne threonine phoephatmes -an expanding family coppes zl divergence of duplicate genes in three sciaenid species (perciformes) from the south co~t of uruguay coasfaneda m: rrs~j~o~a (mu-~--a~) repetitive dna seqmmce l~vointion in ~hically mstinct isolates. cor~ bnz~n physiol repetitive seq~ce involvement in the duplication and divergence of mouse lysozyme genes the structure of a subtermlnal nut/e/ a /ds res schoofs i~ h~ between amino acid sequenc~ of ~ v~'lt~tm'stte peptide hormones and peptides ~mlated fi-on~ invertebrate sources. corn# bm&.n mg~ol bun'nng s, ~us r& lqatelet gtycoprotetn nb-ma protein antssonim from snake venoms ---evidence for s fumlly of p~telet-~sgqpttlon lnhll~tol~ hikher plant orilgins and the whylogeny of gt~en allpte simihtrity between the t~ ~ sindln s proteins abf how big is the univet~ of e~otm worklwide diffegences in the ~ncideace of type ! diabetes are ammciated with amino acid variation at pos/tion of the hi~-dq ~ chain yeast general trtnscelptimt l~ctor gf! --sequence requirements for binding to dna mad evointhmky commrvttion. nudeg m/ds res concerted ]rv~ution of primate mplm smelllte dna. e'~kmce foe tm an~mt~ sequence sbm'ed by goal~ md human x ~e alpha ~ttdllte the nuchl~m~ sequence of etve ribommaal protein genea from the o/anene. of ~~ impacattom concem~ the mtytosene~ relationship bet~-en cyanelles and chloropluts wmslanoer l~ a new member of a secretory protein gene family in the dipteran c~t~onomot~ tentaus ~ a variant repeat stracture the ~r sequence ~ --die.inn on the x-chromosome and y-chromosome of a large set of closely related sequence~, most of wmda are i~eudogene~ ba~ttmo~e l~ cloning of the pso dna binding subutdt of nf-kapi~-b -homolo~" to gel and dortml l-~te two-monooxr~muse from m~ --clon~ nucleotide sequence, and primary structu~ homology within an enzyme family genetic hot~o~n~ty ~ acute and chronic acute forms of spinal muscular atrophy genetic variants of bovine ~-lactogiobulin --a novel wild.type ~-lacto#obulin w and ~ts primary sequence. b/or (~rn h tt e sey/er l~ltogh~ dna evolution in the olmcm species subgroup of drooophll~ f mot evot lovell-badge l~ a gene mapldng to the sex-determining gegion of the mouse y chromommae ~ a member of a novel ~ of zmbryonk~ly genes ~titmte , -dioxy~mm~ from p~.udomotm~ pustfi~mtion, characterization, ~md compm'tson of the f.mtymes from psemffmmm~m ta~o~k-ron/and aaammms~ spec~clties of the peptidyl prolyl cis-tratm isomeric activities of cydophmn and fk- bindh~ protein --evidence for the existence of a family of distinct enzymes. b~x/aem/ary mltochondrl~ dna evolution in primates -tt-atmltion gate has been extremely low in the lemug homeobox containing genes in the nematode ~enorbabd/f~ elk.gamin nucleic ac shdic add fateesses of ~ • voluttomu.y origins have serine active sites f~entlal arginlne residues dewact-rrer l~ the ltilm omal rna ~-quence of the s~t anemone anemom~s ssdcmta and its evolutionary intuition amomqg other eukaryotes inferred b'om s~l,.m.~ comlmrttmas of a heat shock g~ae in two nematorl~ the l~'/o multtgene family of ok~hag of cdna ~ for the ~ omin of human complement component ca~bi~una protein, seqaenoe homolo~ with thc a c~t~:~a~h proc natl acad s¢t usa highly conserved core domain and unique n terminus with presumptive regulatory moti~ in a hmman tata factor (l'lql~) [letter] identification cimractertzaflon of a novel member of the nerve growth fmctor/besln.dertved neurotrophic factor family ~ bind to s~dlfmme [eal(~-so )l~l-lcer ] and has a sequence homology with other pt'otelns that bind sulfated glycoconjut~tes anllllo acid seqmmce of clnnamomin, a new member of the elicitin family, and its comparison to cryptogein and capsicetn soluble and mtmo[~tle~ioc~ta~l h~ low-ml~n|ty adenomne binding protein (adenotin) --properties and homology with mtmmall~la and avian stress protelus. b~-/~om/stry edolatlon of complementary dna$ f~lcoding a cerebellum-enriched nuclear factor-i family that activates tt'anscription from the mouse m~.lin basic protein promoter ye~mt mltochondrlal dna polymet'ase is related to the family a dna polymerases nudeotide and deduced amino add sequence of a human cdna (nqo ) corresponding to a second membeg of the nad(p)h --quinone oxldoreductase gene family --extensive polymorphism at the nqo gene locus on chgomo~ome- . b/oc.heraistry ult~ sltnlltt'leles a~llolltll enzyme pterin binding sites as demonstrated by a monoeinnal amiidiotypic antibody blundell tl molecular anatomy: phylogenetic relationship* derived from three~limenslonal structure~ of proteins subfamily structure and evolution of the hnmtn . family of repetitive scquence~. f mot evo selmt~te mltochondrlal dna sequences are contiguous in htlmsa~ genol~ic dna l~t~lit~ within mmmm~lla~ sogl~tol deh~ --the prlmm'y structure of the human liver enzyme heterogeneous modifications of the l /alo ltrote~a of ibtegleuldn-~t cells are concentrated in a/,ti~hly r~qg~.titlv ~ amino-t~ vaults.ell rebofmcleoprotein structures are msl~ conserved among higher and lower e~tes rnas le~d support to the monophyletic nature of the ~erla lmmunoloslcal ~lmllmtties ~etween cytosolic and partictdate tissue trans#utamilsc. febs lat mans~ti x#tope m~w~zed by a protective m~aodonm antibody is identical to the sta~e-specific embryonic antlgen-l. proc naa acad sa o~ the murg gene of t-brucei contains multiple dom.l.m of extensive editinil and is hofaoin~m~ to a subultit of nadh dehy~ neparm-bindl~ nenrotrophtc x~tor (hbnf) and mk, member's of z new i~mily of homolosous~ developmentally l~ted proteitm pugmattion and strucrmml ~on of pttcentel nad + .mtked -hydroxyproma#andm dehydtoffmase ~ the primary structure reveals the enzyme to belon to the short-alcohol l)ehydrogena~ l~mlly. b/ochemistry structores and homologies of carbohydrate ~pho~ system ep~l~[ln, a ~o~a-gmjoclated mudn, is generated by a polymorphlc gene encodin splice variants with alternative amino termini a new member of the leucine zipper class of proteins that binds to the hia drct promoter. sc/ence attalysi~ of cdna for human ~ ajudgyrin i~dicltes a repeated structure with homology to tissue-differentiation a~td cell-cycle control protein the b subunlt of a rat hetefomeric ocaat-binding transcription factor shoes a striking sequence identity with the yeast hap transcription factor homology to mouse s-if and sequence similarity to yeast pt~ stgucttu'e and evolution of the small nuclear rna multigene family in primates: gene amplification under nat-¢wal selectinn? ident~catinn of an additional member of the proteln.tyrushle-phosp~ family --l*vidence f~ alternative spliclog in the tyrmine phosphzmme domain a ~le am~o acid difference dis~ishes the human and the rat sequences of statlmaln, a ubiquitous intracehular pho~phoproteln ~ with cell item comp~ison of the seve~le~ gene* of drosop~ffa t~'ff~ end ma ~ muty, an adenine ~ active on g-a mislmirs, has homology to ~t evolution of largesubunit iutna structuge --the ~cation of imvetbe~t d dommin amon mmjor phyiolpmetic groups discrepancy in diveqlenoe of the mltodtondrlal and nuclear genomes of m sensor/and y~ j mot evot ~ adenylate deamll~t~. a mt~flige~e fam~ in p..m~,n, and rats isolmion and structure of ceerol#m, itna,~le hat~ peptmes, from the smm~m, ~ mo~ comp a~a rmm~ i~ vmotocin ge~ of the teleom f.,xott intro~ botany. ~ hot~ ot'l~mization. b~hemioy the adb gene areal share features of sequence structure and nudeast~protected sites. m /cell bto/ the amino-acid sequence of multip/e lectins of the #.corn barnacle m~us-lgo~ and its homology with .animal ]~'tllls. bioclx'm btqobys acta amino add ~.-quence of mtmkey erythrocyte glycophorba mk. its amino acid ~'qu~'~icc ]f][~ a stri~tl~ homology with that of human glycophorin a flsp~r p& drtmophila proliferating cell nuclear antigen. structural and functional homology with its mammalian coonterpart phylogeny of n|trogen*me s~queac~ in ][~mnkla and other nlteogen-fixing ml~m$ vertebrate prot~mlne c~ne evolution. . sequence alignments and gene structure florin l~ a major styl~ matrix polypeptid~ (sp ) is a member of the f~thogenesia-reiated proteins superciass complete amino acid sequence of rat kidney ornithine aminoteat~fet-~e --identity with ijver omithine aminotransferme. l bnxl;em (tokyo) rlbonuclease p --function and variation. j b/o/~bem the primary strum of glycoprotein-m from bovine adrenal medullary granules --sequence similarity with bnmmn serum protein- , and rat sertoli cell giycoprotein- compm'ative ~quence/umlysis of m~mmantan f'a~or ix protaotegs the amino acid sequence of the b nman l~ia polymet'a~-h -kda subunit hrpb is highly cotmerved among eukaryotes phylogenetic conservation of atylsulfatases --cdna cloturing and expre~ion of hnman aryisul~t~e-b. j b/o/cbem c.oll/l~'vlltion and diversity in fatnllies of coated vemcle adaptlns cllaracterizaflon of petel porcine bone sialoproteins, soca'~ted phosphopgotein ! (sppi, osteopontin), bone siaioprotein, and a .kda glycoprotetn ~ demonstration that the -kda glycoprotein is derived from the carboxyl terminus of sppi characterization of matteuccin, the . s storag~ prote~ of the ostcich fern -evolutionary iteiatinnshlp to angiosperm seed storage ~ a new mmber of the glutamine-rlch protein gene family is characterized by the absence of internal lgepe~ts and the androgen control of its expression in the subm*ndlbuiar gland of pad novel insect n~ with homology to peptides of the vea'te~ tachykinin family identircation of a novel platelet-derived neutrophli-chcmaotgctic po~ with structural homology to piatelet-factor- a novel repeated dna sequoncc located in the intergenic regions of ba~tceial chromosomes. nuc eic.,k:ids res the proianlin storage protellx¢ of cere~ seeds ~ structure and evolution functional analysis of the '-terminal part of the balbiani ring gene by hlterspecies sequence comparison dr= mammaban ~yl phosphate symhetase (cp*) --cdna sequence and evolution of the cl m domain of the syrian hamster multifunctional protein cad mammalian dihydroorotase --nudeotide sequence, peptide sequences, and evolution of the imhydroorotsse domain of the multifunctinnal protein cad a receptor for tumor necrosis factor defines an unusual family of cellular and viral proteins the control of flower morphogenesis in a~..ffd~um majusthe protein shows homoinff~ to transcription factors an element of symmetry in ytmst tata-box binding protein transcription factor-lid --consequence of an ancestra/ duplication? c-type natciuretic peptide (cnp): a new member of nateinretic peptide family identified in porcine brain evolution of antioxidant m~: ediol-dependent petoxidm~.s and thiol~ ~umong ptocaryotes towards the evolution of ribozymes alkyl hydroperoxide reductase from sa/mone/ta ~ur/um --sequence and homology to thinredoxin reductase and other fiavoprotein disuliide oxidoreducmses fc: nonuniform evolution of duplicated, developmentally controlled c~azrion genes in a sillumoth the fission yeast cutl + gene regulates spindle pole body duplication and has homolosy to the buddin structural homology b~ween the hnmmn fur gene product mad the sub---like protea~ encoded by ye~t/~x . nuc~ a¢/ds res nudeotide sequences and novel steuctut~ features of hnm=. and cimm~ lighter ~# primary stt~t~ and expression of a nuclear-coded subunit of complex-n n~ to protetm specified by the chtoropiast genome. b/ chera bnfhys r~ commun a novel gene member of the human giycophorin-a and glycophorin-b genc fatuily -molecular cloning and expression the x-chromosome of monotremes shares a highly conserved region with the eutherlan and marsupial x-o~romosomes despite the absence of x-chromosome ittactt~tion c~lract~tion and or~= nl~tion of dna sequences adjacent to the evidence for a new fmily of evolutionarily conserved homeobox genes elellatltlll and albolabrin purified peptides from viper venoms --homologies with the rgds domain of flbrinogen and yon willebrand pactor measurement of $~tiv~-site homology between potato and l~bbit muscle alpha-glum phosphoryiases through use of a iane~r free energy relationship white ~ weiss ~ the neuroflbromatosis typed gene encodes a protein related to gap the dna damage-inducible gcne-dinl of saocbarom q~ewcet~#.s/ae encodes a regulatory subunit of elbonucleotide reductase and is identical to gnr fhlgegprinting of ne~lr-homogeneous dna hgase-i and ligase-h from eh,m~n cells --similarity of their amp-binding domains control of m na st~mlity in • chnoc~qg.~um, by 'inverted ltepeats: effects of stem and loop mutations on degradation ofxtmba mlna/n vt~ nuc/e~ ac alternative messenger rna structures of the ciil-gene of bacteriophage ~. determine the rate of its tt'ansbttion initiation alternative mrna structures of the cm genc of bacta~ophage ~ detc:'mine the rate of its translation initiation. j mo/b~ / a model fog iina editing in klnetopiastid mltochondrla --guide rna molecules transcribed from max/circle dna provide the edited information elements and coding sequences. j mol bio , : - . chang c-y, ~ d-a, mohandas til chung b-c: stt~ctut~e, ~-quence, chromo~maal location, and evolution of the human fercedoxin gene family. dna cell b/o/ , : - key: cord- -wydc wyd authors: siebert, agnieszka; wysocka, magdalena; krawczyk, beata; cholewiński, grzegorz; rachoń, janusz title: synthesis and antimicrobial activity of amino acid and peptide derivatives of mycophenolic acid date: - - journal: eur j med chem doi: . /j.ejmech. . . sha: doc_id: cord_uid: wydc wyd the series of novel amino acid and peptide mycophenolic acid (mpa) derivatives was obtained as potential antibacterial agents. coupling of mpa with respective amines was optimized with condensing reagents such as edci/dmap and t p/tea. amino acid analogs were received both as methyl esters and also with the free carboxylic group. the biological activity of the products was tested on five references bacterial strains: klebsiella pneumoniae atcc (esbl), escherichia coli atcc , pseudomonas aeruginosa atcc , staphylococcus aureus mrsa atcc , staphylococcus aureus mssa atcc . peptide derivatives proved to be the most versatile ones, their mic values relative to most strains was lower than mpa alone. it has been noted that the activity of amino acid derivatives depends on the configuration at the chiral center in the amino acid unit and methyl esters indicated better antimicrobial activity than analogs with free carboxylic group. mycophenolic acid (mpa) possesses interesting scope of biological properties including antimicrobial, antifungal, antiviral, immunosuppressive and antitumor features. mpa (fig. ) was discovered in by italian physician bartolomeo gosio, who collected the fungus from damaged maize and called it penicillium glaucum [ e ]. it was found that the fungus exhibited antimicrobial activity against anthrax bacterium [ ] . although mpa occurred to be the first antibiotic isolated in pure and crystalline form, further studies were not continued for a long time [ ] . in the s, the biochemical causes of immunodeficiency were investigated in children, and inosine -monophosphate dehydrogenase (impdh) was established as an enzyme responsible for the undesired immune response in autoimmune diseases. studies towards a suitable inhibitor were started, followed by a successful clinical trial with the compound under the trade name cell cept (fig. ) , which was approved for use in kidney transplantation by the us food and drug administration in [ ] . mpa is a competitive and reversible inhibitor of inosine- -monophosphate dehydrogenase (impdh), predominantly isoforms ii, which is present in tumor cells and in activated lymphocytes [ , ] . mpa is an active substance of immunosuppressive drugs for the prevention of acute and chronic rejection of allogenic organ transplants. so far, there are clinically applied two derivatives of mpa: mycophenolate mofetil (mmf) known as cellcept produced by roche and mycophenolate sodium (mps) (fig. ) under the trade name myfortic manufactured by novartis [ e ]. mpa possesses significant antimicrobial properties e.g. against anthrax bacterium [ ] , botrytis cinerea [ ] ), antifungal like against rhizoctonia solani [ ] , antiviral for instance against dengue virus [ ] , avian reoviruses (arv) [ ] , coronaviruses (mers_cov) [ ] and rotavirus [ ] . mycophenolic acid exhibits also a wide spectrum of anticancer properties [ e ] . recent studies in showed that the combination of rapamycin with mpa significantly improves clinical symptoms (erythema, edema, rash and rash) at atopic dermatitis, also reduced epidermal exfoliation, edema and cellulite [ ] . on the other hand, multidrug resistance is also a significant problem in treatment of bacterial infections, and structural modifications of mpa could provide promising antimicrobial agents. taking into account such a large number of positive aspects of the mpa, we attempted to modify its structure towards microbiological activity. we optimized synthesis of several new amino acid and peptide mpa derivatives by conjugation with various condensation reagents. new synthesized compounds have been tested on five reference strains: klebsiella pneumoniae atcc (esbl), escherichia coli atcc , pseudomonas aeruginosa atcc , staphylococcus aureus mrsa atcc , staphylococcus aureus mssa atcc . according to research in molecular modeling, polar group at the end of the side chain of mpa interacts with ser of impdh and is one of the crucial factors for maintenance of its activity [ ] . in literature were reported amide mpa analogs bearing glycine [ e ] alanine [ ] valine, glutamic acid, leucine and phenylalanine [ ] moieties as potential anticancer and immunosupressive agents. in order to obtain an amide bond between the mpa molecule and the amino acid, a very large amount of condensing agents was checked, for example: -ethoxy- -ethoxycarbonyl- , dihydroquinoline (eedq) with pyridine, n,n -dicyclohexylcarbodiimide (dcc) with nmm o-(benzotriazol- -yl)-n,n,n ,n tetramethyluronium hexafluorophosphate (hbtu) with n-methylomorpholine (nmm) or method of mixed anhydrides (isobutyl chloroformate). unfortunately, none of the above methods occurred to be effective, since low conversion of substrates or difficulties with purification of target amides [ ] . edci ( -ethyl- -( dimethylaminopropyl)carbodiimide) was successfully used as a condensing reagent in the presence of dmap without any racemization suppressant in the synthesis of optically active amino acid derivatives, where amino group of chiral amino acid is acylated [ e ]. this method proved to be suitable for the synthesis of most of compounds e . amino acid mpa derivatives e were obtained by means of a condensation reagent -ethyl- -( -dimethylaminopropyl)carbodiimide (edci) in the presence of -(dimethylamino)pyridine (dmap) acting as a base in anhydrous n,ndimethylformamide (dmf) (scheme ). in case of derivatives of mpa with isoleucine or malonate moiety the best results were received using the t p/et n procedure, where triethylamine acts as a base and propanephosphonic acid anhydride (t p) is a condensing reagent (scheme ). the method using t p in the presence of tea provides amides in high yields with very low epimerization and is particularly well-suited for the coupling of racemization prone acids and amines, including relatively non-nucleophilic anilines, with easy reaction setup and product isolation [ , ] . finally, synthesis of compounds and [ ] was optimized using t p/tea method. the results of the synthesis of mpa analogs e are given in table . in order to investigate the influence of methyl ester on activity against chosen microorganisms, methyl esters were hydrolyzed with lithium hydroxide monohydrate to produce derivatives with free carboxylic group e (scheme ). the yields of the obtained analogs were presented in table . the next step was to obtain pentapeptide derivatives containing in their structure tuftsin and retro-tuftsin , , . the protected tuftsin and retro-tuftsin derivatives, and pentapeptides, were synthesized by the mixed anhydride method with isobutyl chloroformate and n-methylmorpholine (nmm) in anhydrous dmf [ e ]. tuftsin is known as an natural immunomodulator of a wide range of biological properties, occurring in human blood and other mammals, is able to stimulate certain white blood cells (monocytes, macrophages, and neutrophils). this tetrapeptide provides also antitumor, antimicrobial, anticoagulant and analgesic properties [ e ] . despite of such advantageous properties of tuftsin, the peptide is not stable, its half-life in blood is min. it is hydrolyzed by leucine aminopeptidase, carboxypeptidase b, proteinase and subtilisin to tripeptides such as h-lys-pro-arg-oh and h-thr-lys-pro-oh. the resulting compounds inhibit the activity of tuftsin [ ] , but tuftsin analogs with biologically active compounds such as mpa could improve its metabolic properties. another way to stabilize the tuftin molecule is to attach another amino acid to the ε-amino group of lysine, thus forming a pentapeptide, which could be also implemented in designed conjugates. in the course of optimization of the consecutive stages towards final peptides we applied condensation reagents such as edci, eedq, or t p. in the case of tuftsin analog the most effective one occurred to be t p procedure in the presence of tea (scheme ), whereas compounds bearing retro-tuftsin , were optimized with edci in the presence of dmap (scheme ). mycophenolic acid and peptides were coupled in anhydrous dmf at c to produce mpa derivatives , , in reasonable yields. the results of these experiments are shown in table . the fmoc protective group was removed with e % diethylamine solution towards target compounds , , . fmoc removal yields were within the range of e %. the present study evaluated the effect of the obtained compounds e , , , on five reference strains: klebsiella pneumoniae atcc (esbl), escherichia coli atcc , pseudomonas aeruginosa atcc , staphylococcus aureus mrsa atcc , staphylococcus aureus mssa atcc . selected compounds differ in the structure of the skeleton to be able to see if any of its components are of particular importance for antibacterial activity. the micro dilution method in the broth gave the possibility of determining the minimum concentration of microbial growth inhibitor (mic) within a narrow and precise concentration range. by carrying out a -well microplate study, the analysis of results may consist of a visual assessment of the degree of turbidity in a given well or a spectrophotometer (od ) [ , ] . when testing waterinsoluble products (compounds with arginine), incompletely dispersible, may interfere with reading of absorbance, it is advisable to use colored growth indicators of microorganisms. the activity of sparingly soluble compounds may be erroneous. in the presented work, a redox indicator -resazurin was used as a measure of the presence of live microorganisms present in tested compounds. resazurin is subjected to reduction of activity by active metabolic microorganisms by changing the color from blue (resazurin) to pink (resorufin) and the intensity of color correlates with the size of the population. it has been found in this and other works that the use of resazurin as an indicator of metabolic activity of bacteria increases the sensitivity of detection [ ] . for standard antibiotics and mpa for e. coli it was possible to determine mic (bold type in table ), for the remaining compounds for all tested bacteria mic was determined. inhibitory concentrations of tested compounds e , , , for selected microorganisms (mg/ml) is shown in table . the scope of the examined derivatives mpa was chosen individually. the methodological basis for further elaboration in the implementation and interpretation of minimal inhibitory concentrations is eucast, supplemented by the recommendations of the scientific societies, data from the publication and own experience [ ] . investigated compounds exerted a variety of effects depending on the species of bacteria. it was demonstrated that most of tested bacterial species (except for acinetobacter baumannii for which mic mpa was the same, and p. aeruginosa, where no activity was observed) were inhibited by compound (mic e mg/ml) as compared to mpa ( -! mg/ml). in case of compound the inhibition of k. pneumoniae was comparable to the control effect of ampicillin ( mg/ml). the mic for this compound is at mic of mpa. the mic for compound is lower than mic of mpa. for the remaining bacteria there is no clear activity (at the concentration limit). compounds , , , , , , exhibited the greatest activity against s. aureus, but the mssa strain was more sensitive than mrsa. in case of analog , for s. aureus mssa, the scheme . synthesis of amino acid derivatives of mpa e using the edci/dmap method. scheme . synthesis of amino acid derivatives of mpa , [ ] using the t p/tea method. table yields of obtained compounds e . compound is equal to mg/ml (concentration limit) and is higher than for there was no bacteriostatic effect of the tested compounds on pseudomonas aeruginosa. the remaining compounds e caused very poor growth inhibition (< % control) or no inhibitory effect on the growth of bacteria. the bactericidal effect was not found for any mpa derivative. compounds e exhibit poor activity against all groups of microorganisms, no susceptibility was observed. analyzing the results, it can be seen that the peptide derivatives of mpa , , have better activity than the parent compound itself. in case of strains k. pneumoniae atcc (esbl) and s. aureus mrsa atcc their values mic were lower ( e for k. pneumoniae atcc (esbl), e for s. aureus mrsa atcc ) than for the antibiotics themselves: kanamycin and ampicillin. a slightly better microbiological activity for s. aureus mrsa strains revealed derivatives , containing in his peptidic structure b-alanine moiety. there was no difference in effect between tuftsin skeleton and retro-tuftsin. microbiological activity of amino acid esters depends both on substituent and configuration in amino acid moiety. it can be clearly seen, that in case of threonine , and arginine , d enantiomer is definitely less active against all strains. although damino acids can enhance stability of the active substance due to its improved resistance against proteases [ ] , obtained results suggest that natural configuration of the tested compounds is privileged against investigated bacterial strains. deprotection of methyl esters e changed activity considerably. we observed a significant advantage of derivatives protected by methyl ester over those with a free carboxylic group. this is noticeable for all strains of bacteria, and could be explained by better cell membrane penetration due to lower polarity [ ] . we have received new mpa derivatives using edci and t p condensation reagents. the obtained compounds were tested on five different bacterial cell lines. by analyzing the results it can be concluded that mpa modifications such as , , , , , are promising for s. aureus, while , , , modifications are promising for k. pneumoniae. it was noted that the compounds with the free carboxyl group e exhibit poor activity against all groups of microorganisms. microbiological activity of amino acid esters depends both on substituent and configuration in amino acid moiety. it was found that derivatives of d configuration , were much less active than those of l configuration , . these results confirm that structure modifications of mpa derivatives can be designed towards antimicrobial properties. all reactions were performed in inert atmosphere with magnetic stirring. dmf was purified by distillation from benzene/water. the reactions were monitored by tlc on merck f silica gel precoated plates. the following solvent systems (by volume) were used for tlc development: ch cl :meoh:ch cooh ( : : . , v/v/ the value in bold type -mic, is the lowest concentration causing complete inhibition of growth or decreasing the number of bacterial population by over %; another -mic . value with the ">" sign -concentration inhibiting growth by e % relative to positive control. value with the sign ">>" -concentration causing very poor growth inhibition (less than % of control). "ni" -no inhibitory effect at the compound concentrations used or stimulation of growth. amino acid methyl ester hydrochloride e ( . mmol), mpa ( . mmol) and dmap ( . mmol) were dissolved in ml anhydrous dmf. then the solution cooled to c in an ice bath, then added ( . mmol) of edci (in case of , . mmol of t p in % dmf was used [ ] ). stirring was continued under nitrogen at c for h and then at room temperature. the progress of the reaction was controlled by tlc. after completion of the synthesis, the solvent was evaporated under reduced pressure using a vacuum pump. the product was purified by preparative thin layer chromatography. structures of synthesized derivatives e were established by spectroscopic methods ( h nmr, c nmr) and ms, hplc-ms. hydrolysis of the esters e was carried out by removing the methyl ester from the carboxylic group. methyl ester e ( . mmol) was dissolved in . ml of methanol. then, was added . g ( . mmol) of lithium hydroxide monohydrate dissolved in . ml of water. the mixture was stirred on a magnetic stirrer for h at room temperature. the solution was acidified with n hcl and extracted with ethyl acetate (etoac). the extract was dried with mgso , evaporated and purified by preparative thin layer chromatography with respective solvent system a-d to produce compounds e . . , . , . , . , . , . , . , . , . the procedure for the synthesis of compounds , and by the mixed anhydride method has been published previously [ e ]. the boc-protecting groups was removed by treatment with tfa. peptide ( . mmol), mpa ( . mmol) and tea ( . mmol) were dissolved in ml of anhydrous dmf. the solution was then cooled to c in an ice bath, followed by the addition of ( . mmol) of t p in % dmf. the reaction mixture was stirred in a spherical flask filled with inert gas (nitrogen) at c for h and then at room temperature for h. the progress of the reaction was controlled by tlc plates. after completion of the synthesis, the solvent was distilled off under reduced pressure using a vacuum pump. the product was purified using preparative thin-layer chromatography. compound ( . mmol) was treated with ml of chloroform followed by the addition of . e . ml of diethylamine (dea). the deprotection reaction was carried out at room temperature for e h and controlled by tlc. then, solvent was distilled off under reduced pressure using a vacuum pump. the product was purified using preparative thin-layer chromatography. peptide , ( . mmol), mpa ( . mmol) and dmap ( . mmol) were dissolved in ml of anhydrous dmf. the solution was then cooled to c in an ice bath, then of edci was added. the whole was stirred with magnetic stirrer in a spherical flask under inert gas (nitrogen) at c for h and then at room temperature for h. the progress of the reaction was controlled by tlc plates. after completion of the synthesis, the solvent was distilled off under reduced pressure using a vacuum pump. the product was purified using preparative thin-layer chromatography. compounds , ( . mmol) was treated with ml of chloroform followed by the addition of . e . ml of diethylamine (dea). the deprotection reaction was controlled by tlc plates. the solvent was distilled off under reduced pressure using a vacuum pump. the product was purified using preparative thin-layer chromatography. at the beginning of the experiment the effect of solvents dmso ((at concentrations ranging from . % to . %) on bacterial cells viability was investigated by measurement od and by plating on a solid medium. the bacterial growth after e h incubation was compared to control strain without dmso. no impact of dmso on bacterial cells was determined at concentration below . %. the research was done in triplicate. the stock solution for conjugates of mycophenolic acid was prepared in % dmso. serial dilutions were made with water and the growth medium to a final concentration of mg/l. the solutions were frozen and thawed only once and then discarded. the screening sensitive bacteria to mpa derivatives was performed with mics tests. mics were determined by the microdilution method in mueller hinton broth ii (mhb ii) on -well polystyrene microtiterplates according to the european committee on antimicrobial susceptibility testing (eucast) recommendations [ , ] . the cell bacterial cultures from the logarithmic growth phase were diluted in a medium at a ratio : ) and them suspension was diluted -fold in saline to received mcfarland . scale. on -well titration plates ml of mhb ii medium was applied. then, ml of the compound solution was added to each of the first wells in each column and serial double dilutions were made. in case of compounds , , , , and , , were in range from . mg/ml to . mg/ml in case of compound were in the range from . mg/ml to . mg/ml. two conventional antibiotics: ampicillin (sigma) or kanamycin (sigma) was used as a control. for ampicillin the range of concentration was from . to . mg/ml, for kanamycin from . to . mg/ml. finally, ml of suitable bacterial suspension was inoculated to each well to achieve cells/ml. inoculated plates were incubated at ± c for e h and mics were recorded. evaluation was performed spectrophotometrically (platereader af uv/vis and fluorescence microplate reader from eppendorf) od and visually. mics determined by spectrophotometric techniques were defined as the smallest concentration of the compound above which inhibition of bacterial cell growth was expressed as mic and mic ( % and %, respectively, of bacterial growth arrest). for confirmation results the resazurin ebased assay was also used. resazurin is an oxidation-reduction indicator used for the evaluation of cell growth [ , ] -pink or uncolored indicates growth and blue inhibition of growth. resinazurin ( ml of . % aqueous solution) was added to each well. samples were incubated at c for h (in the dark) and measurements of absorbance and visual evaluation of color according to control were made. the lowest concentration at which color change occurred is taken as the mic value. mbc value was not possible to determine in each case. mic values were determined in replicates in independent experiments. data were collected and statistically tested (anova parametric test and kruskal-willis nonparametric test). statistical work has been done using rstudio and using the microsoft excel in all calculations, assuming p < . as statistically significant. ricerche batteriologiche e chimiche sulle alterazoni del mais, riv. d'igiene sanita publica ann evaluation of mycophenolic acid production by penicillium bervicompactum mucl in batch and continuous submerged cultures synthesis and biological activity of mycophenolic acid-amino acid derivatives rediscovering mycophenolic acid: a review of its mechanism, side effects, and potential uses natural products: drug discovery and therapeutic medicine mycophenolate mofetil (rs- ): preclinical, clinical, and three-year experience in heart transplantation the structure of inosine '-monophosphate dehydrogenase and the disgn of novel inhibitors synthesis of the inosine '-monophosphate dehydrogenase (impdh) inhibitors mycophenolic acid trough level monitoring in solid organ transplant recipients treated with mycophenolate mofetil: association with clinical outcomes longitudinal evaluation of mycophenolic acid pharmacokinetics in pediatric kidney transplant recipients. the role of post-transplant clinical and therapeutic variables boraty nska, first polish forearm transplantation: report after months -chloro- , -dimethoxy- ( h)-isobenzofuranone and basidalin: antibiotic secondary metabolites from leucoagaricus carneifolia gillet (basidiomycetes) antagonism against rhizoctonia solani and fungitoxic metanolite production by some pennicillium isolates mycophenolic acid inhibits dengue virus infection by preventing replication mycophenolic acid inhibits avian reovirus replication thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of middle east respiratory syndrome coronavirus mycophenolic acid potently inhibits rotavirus infection with a high barier to sesistance development structureactivity relationships for inhibition of inosine monophosphate dehydrogenase and differentiation induction of k cells among the mycophenolic acid derivatives nad-based inhibitors with anticancer potential novel mycophenolic adeninę bis(phosphonate) analogues as potential differentiation agents against human leukemia effects of topically applied rapamycin and mycophenolic acid od tncbinduced atopic dermatitis-like lesions in nc/nga mice microbial modifications of mycophenolic acid preparation and antitumor properties of analogs and derivatives of mycophenolic acid antitumor activity of derivatives of mycophenolic acid total synthesis of lys tamandarin m: a potential affinity ligand total synthesis of the fumiquinazoline alkaloids: solidphase studies total synthesis of the quinazoline alkaloids (-)-fumiquinazoline g and (-)-fiscalin b general and scalable amide bond formation with epimerization-prone substrates using t p and pyridine an expedient route for the reduction of carboxylic acid to alcohols employing -propanephosphonic acid cyclic anhydride as acid activator the synthesis and structure of a potential immunosuppressant: n-mycophenoyl malonic acid dimethyl ester synthesis and cytotoxic activity of conjugates of muramyl and normuramyl dipeptides with batracylin derivatives biological activity of conjugates of muramyl dipeptides with batracylin derivatives synthesis and functional studies of tuftsin analogs containing isopeptide bond synthesis of conjugates of muramyl dipeptide and normuramyl dipeptide with retro-tuftsin (arg-pro-lys-throme) as potential immunostimulants synthesis of analogues of anthraquinones linked to tuftsin or retro-tuftsin residues as potential topoisomerase inhibitors recent developments in the synthesis and biological activity of muramylpeptides new conjugates of muramyl dipeptide and nor-muramyl dipeptide linked to tuftsin and retrotuftsin derivatives significantly influence their biological activity tuftsin: on the -year anniversary of victor najjar's discovery the peptide molecular links between the central nervous and the immune systems high-throughput assessment of bacterial growth inhibition by optical density measurements optimisation оf the microdilution method for detection of minimum inhibitory concentration values in selected bacteria resazurin-based -well plate microdilution method for the determination of minimum inhibitory concentration of biosurfactants the european committee on antimicrobial susceptibility testing, breakpoint tables for interpretation of mics and zone diameters inspiration from the mirror: d-amino acid containing peptides in biomedical approaches eucast expert rules in antimicrobial susceptibility testing investigation of the alamar blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity this work was financially supported by the gda nsk university of technology under grant ds/ . supplementary data related to this article can be found at https://doi.org/ . /j.ejmech. . . . key: cord- -awygbo y authors: vimr, eric r. title: unified theory of bacterial sialometabolism: how and why bacteria metabolize host sialic acids date: - - journal: isrn microbiol doi: . / / sha: doc_id: cord_uid: awygbo y sialic acids are structurally diverse nine-carbon ketosugars found mostly in humans and other animals as the terminal units on carbohydrate chains linked to proteins or lipids. the sialic acids function in cell-cell and cell-molecule interactions necessary for organismic development and homeostasis. they not only pose a barrier to microorganisms inhabiting or invading an animal mucosal surface, but also present a source of potential carbon, nitrogen, and cell wall metabolites necessary for bacterial colonization, persistence, growth, and, occasionally, disease. the explosion of microbial genomic sequencing projects reveals remarkable diversity in bacterial sialic acid metabolic potential. how bacteria exploit host sialic acids includes a surprisingly complex array of metabolic and regulatory capabilities that is just now entering a mature research stage. this paper attempts to describe the variety of bacterial sialometabolic systems by focusing on recent advances at the molecular and host-microbe-interaction levels. the hope is that this focus will provide a framework for further research that holds promise for better understanding of the metabolic interplay between bacterial growth and the host environment. an ability to modify or block this interplay has already yielded important new insights into potentially new therapeutic approaches for modifying or blocking bacterial colonization or infection. at least at some level common experience indicates to almost everyone that life is constrained by competition for limited resources. formally trained biologists understand this competition as central to evolution, the only fundamental theory in biology. for some microorganisms competitive success in colonizing a mammalian or avian host depends upon specialized metabolism that may support growth in only certain niches. for example, freter [ ] has summarized his own and the work of others by describing the mechanisms of association of bacteria with mucosal surfaces. ese mechanisms include "(a) chemotactic attraction of motile bacteria to the surface of the mucus [layer], (b) penetration and trapping within the mucus [layer], (c) adhesion to receptors…, (d) adhesion to epithelial cell surfaces, and (e) multiplication of the mucosa-associated bacteria. " e combined set of traits or phenotypes expressed by a given bacterium de�nes its potential "virulence factors" or relative colonization success [ , ] . in the current paper the �nal stage of the host-microbial interaction is exclusively focused upon multiplication of bacteria at mucosal surfaces. is focus further emphasizes escherichia coli as the predominant facultative anaerobe in animal hosts and its metabolic uses of host sialic acids for nutrition or surface decoration. e narrative approach is intended to support a uni�ed set of observations and hypotheses that could guide future research in the �eld designated microbial sialobiology [ ] . by understanding the metabolic use of a single group of prevalent mucosal sugars, the sialic acids, it may be possible to at least partially identify factors controlling which bacteria colonize only certain areas of the gastrointestinal tract or other mucosal surfaces. is goal is central to understanding microbial colonization in disease and health of humans and livestock [ ] . e gastrointestinal tract (git) is essentially an open tube containing a few valves located between mouth and anus and open to the environment at either end ( figure ). bacterial colonization begins during and aer birth and may continue to change or become restructured over time as in�uenced by complicated factors such as diet, overall health, and even geographic location. other mucosal or epithelial surfaces include the eyes and some sites not shown in figure like the nasopharynx, lungs, bladder, vagina, and urethra. some of these sites are normally sterile, for example, eyes, lungs, and bladder unless colonized during an ongoing infectious disease process. each of these extra-git sites expresses a variety of sialic acids that probably do not differ greatly from those found in the large intestine, though much less work has been done on this topic than on the mucus layer and epithelium of the animal large intestine. regardless of the relative disparity in detailed information between datasets, information about the large intestine should facilitate generalizations to all mucosal sites in healthy and diseased states where microbial involvement is known or suspected. note that listed in the legend to figure are bacteria that permanently colonize the large intestine. is group must be in constant competition thus separating the colon from normally sterile sites that usually remain uninfected or, when infected, it is usually by a single species resulting in either clearance by or death of the host. most information about the pathogenic and commensal gut bacteria comes from standard (sometimes referred to as classical) methods of microbial culture and measurements of nutrient use. more current methods such as high-throughput sequencing for identifying both cultivable and noncultivable bacteria as well as nuclear magnetic resonance spectroscopy or mass spectrometry for identifying hundreds of small molecules in complex samples are generating datasets for statistical analyses [ ] . however, when the exact identities or functions of important nutrients are unknown, or the metabolic pathways needed for their metabolism are not described, it is unclear how the more recent methods will offer many new insights until supported or refuted by direct experiments aer the necessary basic pathways have been elucidated. erefore, and at the risk of being repetitive, the goal of this current paper is to understand the metabolism of a remarkably distinct, chemically varied, and prevalent family of mucosal sugars that are known in some cases and hypothesized in others to in�uence in minor-to-major ways the capacity for bacterial niche specialization or disease potential. some of the ways this information could be applied to speci�c practical (therapeutic) uses have been described [ ] . anatomic variation between gits re�ects the digestive needs of a given animal species. carnivores such as cats and dogs, or human omnivores have a less developed cecum (the appendix) than monogastric herbivores, ruminants, or granivorous birds ( figure ). despite this and other anatomic differences most of the digestion and absorption of foodstuffs occurs in the animal small intestine such that carbohydrate, protein, and fat are all digested and mainly absorbed from this site before the undigested residuum empties into the colon [ ] . e many factors limiting or selecting for bacterial diversity in most anatomical compartments is in stark contrast to the large intestinal microbiota in its richness and depth of both permanent and occasional inhabitants ( figure ). table lists some of the bacterial species isolated from the healthy human intestine [ ] . e genus/species designations given to some bacteria have changed over time, and other species unique to nonhumans ( table ) expands the diversity of bacteria residing within animal gits. despite the enormous numbers of intestinal bacteria estimated at [ ] , and the wide species diversity of the colonic microbiota, two facts emerged from standard analyses of the major cultivable bacterial groups [ ] . first, e. coli is to times more prevalent than clostridia, streptococci, or lactobacilli and one million times more common than yeasts, while to times less prevalent than bacteroides in the normal human cecum or feces. second, e. coli is found in the rumen and abomasum of cows and crop of chickens as well as the stomachs and entire small intestines of pigs, chickens, cats, and humans living in tropical environments [ ] . a discussion of factors limiting e. coli to the terminal ileum and colon of healthy humans from temperate climates will not be attempted. neither will an attempt be made to either support or refute metagenomic analyses that suggest many more, uncultivable bacterial species may exist than those species already identi�ed by standard procedures (tables and ). however, and for the purposes of this paper, it is essential to note the guiding principle suggested by the above data. namely, bacteroides outcompetes e. coli by nutritionally exploiting residual foodstuffs not already absorbed by the host including carbohydrates that are undigestible by e. coli, while e. coli outcompetes all other enterics or other major bacterial groups by mechanism(s) unknown. is paper will address a hypothesis that could explain the evolutionary success of e. coli. failing that goal, the present paper will at least provide a coherent assessment of recent data explaining how bacteria metabolize a major group of host-derived metabolites. if the central hypothesis introduced above is correct, which nutrient(s) does e. coli exploit for survival and outcompeting most of its rivals? erefore, the conceit of this paper is that e. coli has evolved to efficiently exploit host-derived nutrients, and its success as the preeminent facultative large intestinal anaerobe is at least partly owed to an evolutionarily optimized use of host-derived sialic acids. anyone interested in exploring the genesis of this paper's central hypothesis should read the delightful treatise by koch [ ] . in this work koch argues, on the basis of biophysical data, at least a partial explanation for how e. coli became evolutionarily successful. indeed, it is in this author's opinion that koch's article is the single best of all possible articles on the subject of evolutionary success by suggesting that e. coli could be the most highly evolved species on the planet. the large intestine e sialic acids is a designation given to a group of over naturally occurring nine-carbon keto acids found mainly in animals of the deuterostome embryonic lineage (star�sh to humans). ese sugars are synthesized rarely by bacteria, and then mostly by pathogens that use sialic acids to masquerade as immunological self, not at all in plants or protostomes except for perhaps a few larval insect stages, and probably not by fungi though the jury remains out in this case [ ] . erefore, when speaking of sialic acid metabolism (sialometabolism) the process is limited to mostly bacterial species that exist as animal commensals or pathogens [ , ] . faillard [ ] covered the early history of sialic acids since their discovery in the s to the modern era beginning around . chemists conducted most research during the initial stage of sialic acid discovery. however, roland schauer was an early proponent during this time of a different or at least more expansive view of sialic acids, as he clearly recognized that their unique chemical structures and skewed phylogenetic distribution was likely to be signi�cant to diverse biological phenomena. his many insights helped lead to the modern �eld of sialobiology as a subset of glycobiology and ultimately to the current view of microbial sialobiology described in this paper. indeed, schauer was the �rst to show that a bacterium, clostridium perfringens, appeared to have some mechanism for metabolizing sialic acid in the bacterial growth medium [ ] . e most common sialic acid, -keto- -deoxy- -acetamido-d-glycero-d-galacto-nonulosonic acid, is abbreviated neu ac re�ecting the backbone neuraminic acid ring, the acetamido group at the carbon position , and the glycerol tail composed of carbons - ( figure (a) ). various chemical groups attached to the glycerol tail or ring carbon hydroxyl groups de�ne most neu ac derivatives. �y far the most common derivatives bear o-acetyl groups at carbon positions , , , or . ese additions are catalyzed by o-acetyl transferases in both bacteria and eukaryotes using acetylcoenzyme a as acetyl donor. o-acetylated sialic acids are abbreviated neu ( , , ) , ac or to re�ect the position(s) sequenced microbial genomes of some of the species found in the healthy human intestine with recent designations given in parentheses [ ] . genera were queried for similarity to acetyl xylan esterase [ ] . e lower the expected value ( ) is re�ects the likelihood that a match is not due to chance. b indicates the percentage of identical amino acids within the speci�ed alignment length. e number of identical amino acids/the alignment length is given in parentheses. of the acetyl ester(s). n-glycolylneuraminic acid (neu gc), synthesized by a hydroxylase that adds a hydroxyl group to the carbon- acetamido of neu ac (figure (a)) is the other major form of sialic acid in most animals other than humans. its absence in humans is due to a null mutation in the hydroxylase gene, indicating gene function was lost aer the split of the human ancestor from that of the great apes. ajit varki and his colleagues have speculated about the biological consequences of neu gc's absence in humans [ ] , but it is possible that the loss has no major consequence other than one less nutrient for bacteria to exploit in humans. less common derivatives include an oxidized form of neu ac, -(acetylamino)- , -dideoxy-dglycero-d-galacto-octanoic acid (adoa), a carbon position - lactone (neu ac , l), and a -deoxy anhydro form, neu ac en (figures (b)- (d), resp.). e structural diversity of the sialic acids is matched by their regio-distributional differences along the length of the large intestine [ , ] , revealing an increasing sialic acid gradient from ileum to rectum. figure also shows that in humans neu ac, neu ac , l, and various o-acetylated forms are the most prevalent sialic acid derivatives. by contrast, the mouse as expected produces neu gc [ ] , but a possibly lesser amount of the other derivatives found in humans ( figure ). except for neu gc it is unclear whether differences between humans and mouse colonic sialic acids re�ects true species diversity or artifacts of the sampling and analytical methods used for detection. if these differences were real, the mouse would be a poor model for investigating sialometabolism in humans. most sialic acid is linked to other sugars including other sialic acids and the di-, oligo-, or polysaccharides formed by these carbohydrate linkages are attached to lipids (forming glycolipids) or proteins (forming glycoproteins) comprising the group of molecules called glycoconjugates. sialic acids are frequently, when present in a sugar chain, the terminal sugar linked to subterminal carbohydrate units through glycoketosidic bonds between the carbon- hydroxyl of the terminal sugar and subterminal hydroxyls at various positions depending on the acceptor. in the git as well as other mucosal surfaces sialic acids are a major component of mucins bathing the epithelial surfaces and the glycoconjugates comprising the epithelial glycocalyx including the glycolipids and glycoproteins bound to epithelial cell bacteria isolated from nonhuman animal intestines with newer designations given in parentheses. asterisks indicate bacteria isolated mainly or exclusively from the rumen [ ] . expect values ( ) and maximum percentage identities are as described in table . surfaces. e types of glycoconjugates and their interactions in health and disease have been recently reviewed [ ] [ ] [ ] . bound sialic acids by de�nition are unavailable to bacteria unless �rst released by sialidases (neuraminidase, e.c. . . . ), which hydrolyze the linkages between terminal sialic acids and subterminal sugars. ese hydrolases are produced by the host (endogenously) and by some bacterial species (exogenously). bacterial sialidases come in a great variety of structures and may be multifunctional [ ] . e combined actions of endogenous and exogenous (bacterially derived) sialidases is thought to be necessary for any further microbial utilization of host sialic acids for either synthetic or catabolic purposes [ ] . e reader is directed to the original literature and reviews describing the molecular characterization of the bacterial sialidase superfamily [ ] [ ] [ ] [ ] [ ] [ ] , and a more recent review listing further examples of bacterial sialidases [ ] for additional background information. once sialic acids are released by hydrolysis they are available like most other sugars free in solution for transport into the cell and catabolic fermentation or oxidation. at the neu ac catabolic pathway went undiscovered or, indeed, not even thought of until no doubt re�ects the phylogenetic rarity of sialic acid and its commercial expense at the time as an available potential nutrient for experimental testing [ ] [ ] [ ] [ ] . figure shows the canonical e. coli sialocatabolic operon (in color) and upstream repressor gene nanr. induced catabolic genes, their encoded polypeptides, and functions where known are also depicted in the �gure. once transported into the cell by nant the lyase encoded by nana releases n-acetylmannosamine (mannac) and pyruvate; the latter enters the oxidative tca pathway of energy production. nank phosphorylates the ring form of mannac yielding the -phosphate derivative mannac- -p. nane converts mannac- -p to n-acetylglucosamine- phosphate (glcnac- -p), the inducer of the nag operon, with deacetylation and deamination by naga and nagb, respectively, completing neu ac dissimilation to fructose- -phosphate. neu ac thus serves as a carbon and nitrogen source, a source of energy, and a source of precursors for cell wall synthesis. with minor variation [ ] , the canonical biochemical pathway exists in all microorganisms known to catabolize neu ac. variations in gene organization are widespread in different species, but utilization of neu ac for growth depends on some form of nanatek and nagab. e function of yhch is unknown, but as discussed below it is hypothesized to function in dissimilation of sialic acids other than neu ac, since deleting yhch had no effect on growth of e. coli on neu ac [ ] . bacteria such as e. coli serotype k and some neisserial serotypes synthesize sialic acids de novo and assemble them into cell surface glycolipids [ ] . in some cases, the capsular polysaccharides synthesized by these bacteria mimic host molecules and thus fail to elicit a host immunological response [ ] . in other sialo-positive serotypes the polysaccharides have no host analogs due to linkage differences and so form the basis of effective vaccines against bacterial meningitis. in all animal models tested loss of capsular sialic acids results in attenuation, indicating the essential role of the capsule in pathogenesis. e ineluctable conclusion is that bacteria presenting sialic acids at their surfaces do so as a mechanism of avoiding host immune surveillance or to otherwise mask the bacterial surface making it less susceptible to host defense mechanisms both innate and acquired. however, antibodies to capsules if present as a result of prior infection, passive transfer, or vaccination protect against disease, at least in the short term. another group of bacteria displaying surface sialic acid though lacking the de novo metabolic pathway include neisseria gonorrhoeae, haemophilus in�uen�ae and other haemophilus spp., and pasteurella multocida. ese bacteria acquire sialic acids from the host using either surface sialytransferase or hybrid synthetic-catabolic pathways including sialic acid transporters and a truncated synthetic system using only the activating enzyme and a sialyltransferase [ ] . experimental evidence in a natural p. multocida host, the cow, and a mouse model of invasive disease indicated that sialic acid transport was essential to pathogenesis [ ] . using substantially the same approaches, sialic acid uptake was independently con�rmed to be essential in turkey pathogenesis, another natural host of this facultative pathogen [ ] . sialic acid human a mouse c f : relative distribution of sialic acids in the human and murine gut. sialic acid abundances were determined for the human git compartments highlighted in rectangles [ ] . mouse values are for the small and large intestine [ ] . a deletion of nana did not affect p. multocida pathogenicity [ ] , suggesting that catabolism of host sialic acids was not essential to virulence. similarly, nana was unessential for colonization of the mouse nasopharynx by h. in�uen�ae [ ] . e combined results described above unambiguously support an essential function of bacterial sialic acid decoration for evading immune responses but provided little indication that an ability to catabolize these sugars was important to either colonization or disease. however, as discussed below, studies in other bacteria strongly suggest that host sialic acid catabolism has at least a minor role in pathogenesis in different species, and possibly a major role in colonization. none of the bacteria to be discussed below either synthesize sialic acid de novo or use a truncated catabolic-synthetic pathway for surface decoration. erefore, the sole function of sialocatabolism in these bacteria must be growth at the expense of host mucosal sialic acids. a variety of studies have suggested sialocatabolism is important to colonization or �tness in animal hosts. however, none of the studies has been independently con�rmed, and some of the effects of deleting nana or the sialate uptake system have shown less than dramatic effects on �tness. for example, deleting the nana orthologs in vibrio cholerae suggested a minor (< -fold) decrease in competitive index when compared to wild type [ ] . single-infection experiments showed no signi�cant difference with wild type, except at one early sampling interval [ ] . a greater �tness effect ( - times less) was reported for a nana deletion in vibrio �ulni�cus, and a -fold increased ld aer intraperitoneal injection in mice [ ] . however, neither of the above studies rule out whether the effects were caused by an inability to metabolize sialic acids or toxicity resulting from intracellular sialic acid accumulation in the nana mutants [ ] . jeong et al. [ ] indicated there was no toxicity observed in vitro, but this statement was contradicted experimentally in a later study by some of the same authors [ ] , making the in vivo results suspect or at least requiring independent con�rmation before they can be taken seriously. furthermore, a nana mutation in an uropathogenic strain of e. coli had no �tness defect in mouse bladder or kidneys, consistent with the effect of peptide or amino acid utilization in these extraintestinal sites [ ] . however, some of the same authors later showed a -fold decreased �tness of an uropathogenic e. coli nana mutant during bacteremia [ ] . again it is unclear whether this effect is due to sialic acid toxicity or lack of its contribution to growth under in vivo conditions. much more work is needed before any conclusions can be drawn from these studies that do, however, at least point to either a nutritional or detoxifying effect of sialate aldolase (nana) in bacterial-host interactions. by constructing a double mutant defective in sialate uptake and aldolase, one can experimentally control for both the nutritional and antitoxicity functions of bacterial sialocatabolism. using an e. coli nanat double mutant in streptomycin-treated mice the mutant was - times less able than wild type to colonize the host, consistent with a previously reported potential role of sialocatabolism in mucin utilization [ , ] . interestingly, enteropathogenic e. coli o did not appear to use sialic acid for colonization, which was one of the sugars used by commensal e. coli, suggesting sugars not used by the resident population support colonization of the pathogenic strain. in a recent study e. coli passage through the mouse intestine selected for derivatives with increased metabolic efficiency, including genes controlled by the nanr sialorepressor [ ] . e problem with these otherwise elaborate studies [ ] [ ] [ ] [ ] is that the mice are treated with streptomycin to reduce the normal intestinal microbiota to allow a better chance of introduced strains to colonize. in terms of sialic acid utilization, this treatment means that all or most of the free sialic acid for growth must come from endogenous (host) sialidases, and any residual sialidase-positive bacteria remaining aer the drug treatment. is conclusion follows from repeated observations that e. coli lacks sialidase and must rely on other providers for free sialic acids in vivo. uncompromised studies are needed before any �rm conclusions can be drawn about the role of sialocatabolism in e. coli gut colonization. a seemingly more convincing study suggesting the role of sialocatabolism in streptococcus agalactiae (group b streptococci, gbs) was recently published [ ] . gbs are a leading cause of neonatal meningitis in human newborns and a common inhabitant of the vagina mucosal surface. except for glucose there are few obvious carbohydrates that gbs can use for energy production. because gbs lacks sialidase, any source of free sialic acid must come from endogenous or other microbial sialidase activities in the vagina. e authors showed that exogenous addition of sialic acid in a mouse model increased wild type growth in the vagina and had, as expected, no effect on a sialate transport-defective mutant [ ] . ese results add to the overall hypothesis of the current and earlier paper [ ] by suggesting that host-derived sialic acids are important to colonization and disease potential. a study similar to that described above for gbs [ ] was previously carried out in s. pneumoniae [ ] . by contrast to gbs, inactivating the s. pneumoniae sialate uptake system had only a -fold decreased �tness. however, the dramatic in vivo effects seen with gbs were only observed when exogenous sialic acid was injected into the animal model, which is problematic unless the results are compared to the expected general increase in all coresident species utilizing sialic acids in the nares, lungs, or vagina. in other words, the sialouptake defect in gbs had little or no effect on colonization in any of these sites relative to wild type unless exogenous sialic acid were coadministered, which is the expected result essentially making the mouse an unnecessary "furry test tube. " both streptococcal studies [ , ] also can be criticized on the basis of genomic comparisons of sialocatabolic loci in s. pneumoniae. figure shows the known or predicted sialocatabolic genes in three sequenced strains: d , one of the original avery isolates, atcc , and tigr . despite a few differences in overall gene arrangement the gene duplications or triplication of nan orthologs nana (lyase, blue), nane (epimerase, green), yhch (unknown, orange), yjhc (unknown, grey), and nank (kinase, purple) point to past recombination events in the streptococcal sialocatabolism regions of these strains ( figure ). of note from this analysis is the nana orthologs of strain d bear identical point mutations early in the sequence resulting in an inability to catabolize exogenous sialic acids. despite this defect d is as pathogenic for mice as tigr or other wild type streptococcal strains indicating that a natural sialocatabolicdefective mutant might be unaffected for colonization or disease potential. work is in progress in my laboratory to resolve the contradictory evidence, which includes one other study claiming s. pneumoniae d uses sialic acid derived from hog gastric mucin for growth [ ] . as discussed above, �. in�uen�ae and p. multocida catabolize sialic acids and sialic acid transport is essential for virulence while use of sialic acid as energy source is not [ ] [ ] [ ] [ ] . ese �ndings were con�rmed and extended in vivo for nontypeable �. in�uen�ae (nthi), an important agent of middle ear (otitis media) infections especially in children [ , ] . us, unlike e. coli and possibly gbs and s. pneumoniae, an ability to catabolize host-derived sialic acids might not necessarily correlate with colonization or pathogenesis. e regulatory mechanism controlling �. in�uen�ae sialic acid uptake and catabolism is similar to that described previously for e. coli [ , [ ] [ ] [ ] . however, the importance ascribed to this regulatory system [ ] [ ] [ ] has been recently challenged [ ] . regardless of the discrepancies, another area where host-derived sialic acid may be important to nthi is bio�lm formation under both in vivo and in vitro conditions [ ] [ ] [ ] [ ] . while these studies support a role for sialic acid in bio�lm formation in vitro, the entire concept of nthi bio�lms in the middle ear and by extension the role of host-derived sialic acids in otitis media has been challenged [ ] . e discrepancies between groups investigating substantially identical phenomena using similar methodologies warrants caution when extrapolating in vitro results to in vivo conditions. even in vivo results may be misleading when the relevance of the animal model might be �awed. �ther bacteria where bio�lms and sialic acids might be important to infection include pseudomonas aeruginosa, an environmental opportunist, and s. pneumoniae, an important cause of ear infections, meningitis, septicemia, and pneumonia in especially young, old or immunocompromised human beings. both microorganisms express sialidase(s) at their surfaces, although the p. aeruginosa enzyme seems to cleave sialic acid-like molecules (pseudaminic acids) found on a variety of bacterial species including p. aeruginosa [ ] , but not animals of the deuterostome lineage [ ] . furthermore, and unlike s. penumoniae, p. aeruginosa lacks the catabolic genes to transport or metabolize sialic acids. however, in both bacterial species sialidase seems to be required for bio�lm formation in vivo [ ] [ ] [ ] . competitive sialidase inhibitors that bind to the respective enzyme's active sites appeared to reduce bio�lm formation and in vivo �tness, suggesting that these inhibitors, normally prescribed for viral in�uenza infections, may be useful clinically for treating pseudomonad and streptococcal infections. similar to bio�lm formation in nthi, where hostderived sialic acid presumably in�uences bio�lm formation by incorporation into bacterial surface structures, the pseudomonad sialidase might modulate pseudaminic acid levels on bacterial surface structures thereby promoting or inhibiting bio�lm formation. e streptococcal situation is much more complicated, not least by con�icting evidence showing an effect of neu ac but not neu gc on bio�lm formation when contaminating amounts of neu ac in the neu gc used was probably in excess of the effective neu ac concentration [ ] . furthermore, s. pneumoniae expresses up to three sialidases each producing a different hydrolytic product [ ] . more work is obviously needed to con�rm the potentially exciting �ndings, especially when competitive sialidase inhibitors might form the basis of a useful therapeutic approach. for example, the major sialidase expressed by all strains of s. pneumoniae has been linked to phase-variation during infection and modi�cation of the leukocyte in�ammatory response [ ] [ ] [ ] , supporting the possibility of a general approach aimed at blocking sialidase activity. as indicated throughout the current paper sialic acids are present in free form at low amounts presumably resulting from the actions of endogenous sialidases. at least four forms of human sialidase have been identi�ed with one located at the plasma membrane [ ] . in principle any one of the endogenous sialidases could gain access to mucosal sialoglycoconjugates and release free sialic acid product. in complex microbial communities like those at mucosal surfaces, bacteria express a wide variety of sialidases that can either be excreted, surface-associated, intracellular, or periplasmically located. for example, mizan et al. [ ] showed that p. multocida uses its two different surface sialidases to grow on different sialoglycoconjugates by releasing free sialic acid for transport and catabolism by products of the sialocatabolic operon [ ] . however, the complexity of sialometabolism at mucosal surfaces is likely to be greater than a simple scavenging model might otherwise indicate. consider in addition to simple scavenging of free sialic acids (figure (a)) two models with distinctly different outcomes but both involving sialidases unique to the bacterial species. figure (b) shows an example of a "spitter" in which terminal sialic acid, as part of a glycoconjugate, is cleaved by a periplasmic sialidase but by a strain otherwise lacking all other sialocatabolic functions [ ] . e outcome is a sort of acid re�ux whereby the released sialic acid enters the extracellular milieu while the subterminal sugars are subsequently hydrolyzed by speci�c glycosidases, then transported and used for cell growth. clearly, this is a growth strategy that sacri�ces the sialic acid in turn to gain access to subterminal sugars on carbohydrate chains, underscoring the previous conclusion that the diversity of bacterial sialocatabolic pathways evolved in response to the apotheosis if not emergence of sialic acids in the deuterostome lineage [ ] . erefore, the only difference between a spitter and a "swallower" (figure (c)) is that the latter has a sialocatabolic system to exploit the full richness of carbohydrate substrates. by de�nition, swallowers like bacteroides spp [ , ] should not be in competition with e. coli unless their sialo uptake systems are so ine�cient that they allow sialic acid re�ux and consequent scavenging by e. coli or other sialidase-negative species. koch searches for alternative energy sources [ ] . e foraging system requires surface-associated glycosidases, outer membrane oligosaccharide transporter, and periplasmic glycosidases to release monosaccharides, inner membrane transporters, and the intracellular metabolic functions to produce energy from the imported sugars. in other words, b. thetaiotaomicron is an example of a spitter or a swallower ( figures (b) and (c), resp.), depending on its ability to metabolize sialic acid. while the above studies identify the nutritional use of enteral carbohydrates for bacterial nutrition, they contribute directly nothing to understanding the metabolism of speci�c mucosal sialoglycoconjugates. some investigators have demonstrated metabolism of sialylated mucins isolated from various mucosal surfaces. in one study mucins from germ-free rats were incubated with total cecal microbiota from conventionally raised rats. sialylated mucins were degraded more rapidly than the neutral or sulfated forms suggesting an overall optimized use of sialic acids by intestinal bacteria [ ] . although ocular �uid from many humans is sterile, some studies have shown that other people released sialic acids diffuse into the periplasm between the outer and inner membranes (om and im, resp.,) for transport by speci�c permease(s) into the cytoplasm. (b) e spitter mode of acquisition involves sialidase release but inability to further metabolize sialic acid. ese bacteria then sequentially release glcnac (open squares), galactose (gal, open circles), and n-acetylgalactosamine (galnac, bold circles) from the idealized oligosaccharide for subsequent dissimilatory pathways. note that the entire oligosaccharide chain may be degraded within the periplasm. (c) e swallower mode is identical to that of the spitter, except that swallowers catabolize the released sialic acid(s). note that the scavenger and spitter modes are available to gram-positive bacteria that lack a periplasmic space. maintain a commensal bacterial population without incident. ese commensals were shown to degrade sialylated ocular mucins indicating the primary carbon and energy sources for these bacteria are carbohydrates found at ocular mucosal sites [ ] . similarly, burnaugh and colleagues showed that in vitro growth of s. pneumoniae on human glycoconjugates relied on the sequential action of several different surfacebound glycosidases, including the major sialidase-a [ ] . however, a mutant defective for this sialidase was still able to colonize the mouse lung, suggesting either free sialic acid is not essential to the host-microbe interaction or that other sources of this sugar are to be found in the lung [ ] . alternatively, the contribution of the sialidase to disease might be host species-speci�c, underscoring the potential pitfall when extrapolating too freely between in vitro and in vivo results. probably the best commercially available source of chemically characterized sialomucin for experimental investigation is bovine submaxillary gland mucin (sgm). sensitive �uorometric methods exist to identify neu ac, all of its oacetylated derivatives, and neu gc or its derivatives [ ] . however, sgm cannot adequately represent the vast variety of carbohydrates detected in human mucins. for example, using electrospray ionization quadrupole time-of-�ight mass spectrometry, neutral, and acidic carbohydrate chains were detected from mucin oligosaccharides isolated from the ileum, cecum, transverse colon, sigmoid colon, and rectum [ ] . neutral oligosaccharides do not contain neu ac or sulfate residues while acidic chains included neu ac, sulfate, or both neu ac and sulfate [ ] . anthony cor�eld and his colleagues were the �rst to show that some mucosal bacteria synthesized sialidase, glycosulfatase, and sialate oacetyl esterase, supporting the idea that acidic sugars are a nutritional source for bacteria residing in the git [ ] . as these authors noted [ ] , because sulfated carbohydrates and o-acetylated sialic acids reduce glycosidase activity, bacteria evolved mechanisms to remove the modi�cations so that the "released" carbohydrates could become more readily available for nutrition. some in vivo experimental results support this conclusion. research with capnocytophaga canimorsus, a member of the bacteroidaceae family, underscores how bacteria feeding off mammalian cell surface glycoconguates gain competitive advantage [ ] . however, the authors failed to cite an earlier publication by michael malamy and his associates demonstrating essentially the same phenomenon with bacteroides fragilis [ ] . ese investigators showed that a b. fragilis sialidase-negative mutant could not compete against wild type when growing in tissue culture or a rat-pouch model of human abscess. bacterial growth in both models was equivalent until the time glucose was exhausted, suggesting that the wild type exploited sialoglycoconjugates that were unavailable to the mutant [ ] . both studies [ , ] focus on the need for increased attention to bacterial sialidase substrate speci�cities because the variety of sialic acids and their linkages to subterminal sugars is so diverse. for example, using a novel system of chemoselective labeling, parker et al. [ ] showed that the minor s. pneumoniae sialidase-c strongly preferred neu ac to neu gc. e paucity of neu gc in humans may in part explain why s. pneumoniae is such a successful human pathogen while not generally a problem in other animals. ese observations concerning substrate availability further suggest that animal models of human infectious diseases may not accurately report reliable information. e unavoidable conclusion is that testing therapeutics aimed at inhibiting sialometabolism could require human volunteers. while investigations of sialomucin and other sialoglycoconjugate substrates will continue to expand understanding of bacterial sialometabolism, it seems essential to have a uni�ed theory for at least one bacterium. is theory would include all known and putative sialocatabolic functions thus allowing directed approaches aimed at understanding metabolic pathways while facilitating extrapolation to other sialo-capable bacterial species. e. coli remains the best model organism for developing a uni�ed theory of sialometabolism. �� ��en�i��a�ion o� ��e e. coli sialoregulon e e. coli sialocatabolic system is regulated by repressor protein, nanr, whose structural gene is located immediately upstream of the nanatek-yhch operon (figure ) . nanr binds to a unique operator with three ggtata repeats separated by two or three nucleotides [ ] in addition to the canonical nan operon (with colored arrows having the same designations as given in figure ), other genes regulated by nanr include nanc (pink), nanm (teal), and nans (magenta), which is homologous to the axe genes shown for streptococci in figure . another coregulated operon is composed of a putative permease, yjhb (gold) and oxidoreductase, yjhc (grey). responds to exogenous sialic acid with nana induction up to -fold [ ] , indicating the important function of the lyase for both nutritional use of sialic acids and detoxi�cation [ , ] . except for the unknown function of yhch, the canonical nan operon is dedicated to catabolism of neu ac [ ] [ ] [ ] [ ] ] . however, when transcriptome analysis of a nanr mutant was compared to wild type, or when wild type bacteria were grown with neu ac or glycerol as sole carbon source [ ] , two additional nanr coregulated operons were identi�ed by their increased message production representing �ve additional genes ( figure ). both nancms and yjhbc include nanr operators upstream of the putative transcriptional start sites for each operon. e functions of three of the �ve coregulated genes is known or at least supported by some experimental evidence. e nancms operon is composed of genes encoding an outer membrane porin (nanc), sialate mutarotase (nanm), and sialate o-acetyl esterase (nans). e porin is not required for growth of e. coli on neu ac unless outer membrane porins ompf and ompc are absent [ ] . e recently solved crystal structure of nanc con�rms its similarity to porins with presumed selectivity for acidic oligonucleotides [ ] . e nanc -stranded -barrel tertiary structure de�nes an open pore with average radius of . Å lined by two strings of basic amino acid residues apposed across the pore. e alignment of basic residues is conserved within a family of diffusion channels that likely facilitates the entry of acidic oligosaccharides [ ] . e similarity of nanc to this family of diffusion channels was thought to indicate preferential uptake of sialooligomers [ ] . however, there is little indication that such oligomers would exist outside of polysialic acid in the central nervous system [ ] , nor any known periplasmic or intracellular e. coli sialidase that could convert oligomers to free sialic acids. a recent transcriptome analysis of e. coli indicated that nanc was induced when e. coli is growing in bio�lms [ ] . given that nanc is part of an operon controlled by nanr, it is difficult to see how induction could occur unless the operon was under control of some regulator other than nanr. interestingly, nanc was one of the genes identi�ed by a targeted mutagenesis approach in salmonella enterica serovar typhimurium strain atcc as having decreased �tness during competitive mouse infection [ ] . e combined results of crystallography, transcriptome, and animal studies strongly suggest that nanc is a porin that is important to host colonization and disease. at it is part of the sialoregulon further suggests it somehow facilitates utilization of host sialoglycoconjugates or at least their released sialic acids. like most sugars in solution, the pyranose neu ac ring continuously rotates by opening and closing between the thermodynamically more stable -anomer with axially directed hydroxyl at the carbon- position (figure (a) ) and the -anomeric form (< % of the total neu ac in an equilibrium solution) with hydroxyl directed equatorially. e mutarotation time to equilibrium starting from a pure solution of the -anomer is on the order of an hour, such that at equilibrium the mixture contains > % -anomer [ ] . by contrast to this equilibrium mixture, all neu ac or derivatives attached to glycoconjugates are in -glycoketosidic linkages [ ] . because mammalian and bacterial sialidases are retaining hydrolases, the -isomer is exclusively released from substrates aer enzyme cleavage. since spontaneous rotation is slow, and if as seems logical bacterial sialate transporters recognize the thermodynamically predominant sialate in solution, bacterial mutarotase encoded by nanm catalyzing the -to -isomeric sialate conversion may enhance competitive success at mucosal surfaces. us, nanm could increase the scavenging potential for sialates in an animal host where bacteria rely at least in part on sugars released by endogenous or exogenous sialidases for growth. is is an attractive idea with some supporting evidence [ ] . mutarotation from -to -neu ac is easy to entertain when the enzyme is located in the periplasm. however, some bacteria have more than one copy of nanm suggesting a cytoplasmic location for at least some neu ac mutarotases [ ] . a cytoplasmic location for mutarotase is problematic because the lyase encoded by nana requires -neu ac substrate (figure (a) ). it is conceivable that nana pulls the -anomeric form, presumably the form transported by nant, in the direction of the -isomer. however, at best, there would seem to be competition between cytoplasmic nanm and nana. erefore, because neu ac accumulation in the cytoplasm is potentially toxic [ ] , perhaps nanm functions primarily as a detoxifying enzyme in the event that -neu ac is the toxic form. in any case, nanm and its predicted orthologs are found in many but by no means all bacterial species with known or predicted canonical neu ac dissimilatory pathways (figure ), suggesting the mutarotase is not an essential component of sialocatabolism. indeed, an e. coli nanm mutant had at most a % reduction in growth rate relative to wild type under experimental conditions favoring overabundance of the -anomeric form [ ] , as might exist while bacteria scavenge neu ac in their natural hosts (figure (a) ). is relatively modest growth defect might be, however, a signi�cant factor helping to explain part of the overall puzzle why or how e. coli became the preeminent facultative anaerobe in the git. one obvious test would be to construct an e. coli nanm mutant and compare its �tness to wild type in an appropriate animal model. unfortunately, as discussed above, it is not entirely clear what an appropriate model would be unless the phenotypic effect in, say the mouse, were a dramatic one. e third and last gene of the nancms operon, nans, was previously thought to be a conditionally essential gene of e. coli for growth on glycerol as sole carbon source [ ] . however, steenbergen et al. [ ] published evidence that nans is a sialate o-acetyl esterase, indicating that the glycerol-growth defect previously reported [ ] was almost certainly caused by an uncharacterized secondary mutation in the test strain. in other words, growth of newly constructed nans mutants on neu , ac was eliminated while the mutant grew normally with glycerol [ ] . discerning the true function of nans was made possible by two key observations: a commercially available source of neu , ac and a bioinformatics survey of nans against the microbial genomic database which identi�ed weak similarity to an acetyl xylan esterase (axe) [ ] . because esterases frequently share conserved primary structural similarities including active site residues [ ] , it was logical that nans might be a sialate o-acetyl esterase because it mapped within a nanr-coregulated operon and was at least partly similar to axe [ ] . remarkably, when nans is screened against its close bacterial relatives none has a discernable copy of nans despite the presence of genetic information known to or to potentially encode and regulate the canonical neu ac dissimilatory pathway (figures and ). some of the species shown in figure that are related to e. coli include orthologs of nanc, namm, or yjhbc though none has a copy of nans regardless of whether the database is screened against nans or axe other than shigella dysenteriae (see below). by contrast to the absence of nans or axe orthologs in enteric bacteria closely related to e. coli, potential axe orthologs abound in git bacterial species (tables and ) , suggesting that an ability to metabolize o-acetylated sialic acids is a common phenotype of bacteria living on or at a mucosal surface [ ] . one drawback working with commercially available o-acetylated sialic acids is their relative lack of purity such that preparations of neu , ac or neu , ac contain impurities including neu ,( , )ac contaminants [ ] . clarke et al. [ ] recently reported the chemical synthesis of neu , ac , neu , ac , and neu ( , )ac derivatives in pure form. relatively straightforward chemical synthetic methods for preparing o-acetylated sialic acids should facilitate future research on these interesting and phylogenetically widespread neu ac derivatives. e identity of nans as an o-acetyl esterase was recently con�rmed by rangarajan et al. [ ] , who presented a crystal structure of the nans homolog from e. coli o :h . while there is nothing remarkable about the structure partial characterization of the nans active site residues suggested nans is the founding member of a subfamily of esterase [ ] . unlike e. coli with its three coregulated nan operons all known close relatives containing predicted nanr orthologs include only one or in the case of p. haloplanktis two nan operators (figure ). ese observations predict a general lack of coordinated nan expression in species related to e. coli, and that only e. coli is capable of metabolizing o-acetylated sialic acids within this related bacterial group. evidence that the latter conclusion is true came from an analysis of wild type s. enterica var typhimurium (s. typhimurium) grown on neu ac or neu , ac , while s. typhimurium wild type grew as expected with neu ac as sole carbon source, a result supported by the inability of a nana mutant to grow under the same condition, the wild type did not grow when the oacetylated sialic acid was provided as sole carbon source [ ] . is last result is consistent with the predicted absence of nans in s. typhimurium [ ] . e clear implication of these results is that with the possible exception of s. dysenteriae, the nan regions of species shown in figure lack the genetic information to encode esterase or the ability to metabolize o-acetylated sialic acids. s. dysenteriae, the causative agent of dysentery, includes a gene with an internal domain paralogous to nans and two predicted domains of unknown function at the n-or c-termini �anking the nans paralog ( figure ). interestingly, the nans paralog is located in the s. dysenteriae prophage that encodes shiga toxin. indeed, the prophage copy of nans immediately follows in the same transcriptional direction as the two genes encoding subunits of the holotoxin. one possibility for the close association of nans with toxin genes is that the epithelial toxin receptor somehow involves the need to convert o-acetylated sialic acid(s) to neu ac. other prophage copies of nans exist in e. coli o strains and other serotypes causing hemorrhagic disease (table ) . e prophage carrying shiga-like toxin in most ehec strains is similar to the s. dysenteriae phage as are the encoded toxin (stx or stx-like) subunits. as shown in table , some stx-positive bacteria are predicted to express a variable number of nans paralogs, where short refers just to the e. coli k- homolog (figure ), long to nans with n-and c-terminal domains, and partial to nans plus one or the other �anking domain. other strains of pathogenic e. coli from eaec, expec, and epec groups lack stx but may have multiple copies of nans that are invariably associated with prophage remnants. remarkably, one ehec appears to lack even the nanr-regulated copy of nans whereas other sequenced strains, like e. coli k- , lack stx, and nans paralogs (table ) and strains, atcc , se , and umnk lack any versions of nans. is bioinformatics survey beggars many questions warranting future investigation. do nans paralogs have o-acetyl esterase activity? if so, why are seemingly redundant copies of nans located in prophage or prophage remnants ? is expression of nans essential for dysentery or hemorrhagic diseases; if so, why do some strains lack even the otherwise common nans copy? indeed, one strain lacks even the canonical nanr-regulated nanatek-yhch operon (table ). in other words, so many e. coli strains have already been or are being sequenced that it is possible to �nd nearly every conceivable variant of nan organization. does this variation mean that some or all nan genes are nonessential to the e. coli lifestyle, or more likely that variants might be on their way to extinction or have partially different lifestyle than the majority of e. coli strains? what are the functions if any of the n-and c-terminal domains �anking nans paralogs? why is nans absent in some bacteria with otherwise intact nan systems ? finally, is e. coli nans really essential for the evolutionary success of this bacterium as a human and animal commensal, facultative, and sometimes frank pathogen? determining the answers to some of these questions will surely increase understanding of sialometabolism and have the potential to suggest new ways of manipulating mucosal bacterial physiology in general. as shown in tables and and figure , many bacterial git-inhabitants with predicted sialocatabolic systems include a copy of nans (axe). is �nding is consistent with a potentially important role of nans in supporting the commensal lifestyle involving utilization of host-derived sialic acids other than neu ac. for example, it is unclear why pneumococcal strains have distinct nan genetic organizations whereas all strains examined, like gbs, include one copy of nana in their genomes ( figure ). unpublished data from the author's laboratory has shown that the nans homologs in streptococci encode functional neu , ac o-acetyl esterases. e obvious experimental approach to extend these �ndings is to eliminate streptococcal esterase(s) and determine the effects on host colonization or disease. however, because the role of sialocatabolism in pneumococcal infection is suspect, the best candidate organism for the proposed studies is gbs, which seem to have a clearer dependency on sialocatabolism for colonization than pneumococci [ ] . compared to the at least partially characterized functions of nanatek and nancms, little is known about yhch or yjhbc except that these genes are coregulated by nanr in e. coli k- ( figure ). species closely related to e. coli have one or in the case of e. tarda, two yhch copies, whereas yjhb and yjhc are infrequently detected. e yhch ortholog in h. in��en�ae was puri�ed and its crystal structure solved [ ] . e resulting conjecture that it might function in catabolism of neu gc was not supported when an e. coli yhch null mutant was shown to grow as well as wild type on neu gc [ ] . however, solving the crystal structure of yhch does support a possible epimerase activity [ ] . despite the absence of positive data, the similarity of yhch to an epimerase, yjhb primary structure being similar to nant, and yjhc primary structure suggesting it is a possible oxidoreductase (figure ), strongly suggests that like nans, genes coregulated as part of the sialoregulon function in metabolism of sialates other than neu ac or o-acetylated sialates. note that s. pealeana and p. haloplanktis lack yhch and yjhb (figure ), suggesting that the spectrum of sialates metabolized by these bacteria might be less than for most e. coli strains. were a panel of all likely sialic acids present at mucosal surfaces available, it would be straightforward to determine all those derivatives of neu ac metabolized by e.coli but not by s. pealeana or p. haloplanktis. indeed, since some e. coli lack certain genes of the sialoregulon (table ) , these strains alone might suffice to determine the functions of yhch and yjhbc. erefore, instead of waiting for chemical methods that would probably be available only to a few laboratories, simply isolating all sialates from selected mucosae and exposing them to e. coli and relevant mutants or natural mutant phenocopies could facilitate identi�cation of all currently unknown gene functions, as long as the results are combined with simple chemical detection methods [ , ] . bacteria have evolved diverse sialate transport systems including symporters, abc-and trap-transporters [ , ] . nant is a proton symporter with instead of the usual membrane spanning domains [ , ] . by contrast, yjhb though similar to nant lacks the central hydrophilic domain found in nant [ ] . is domain is thought to be essential for uptake of neu ac, neu gc, and certain other sialates [ , , ] . erefore, the presumed sialate(s) transported by yjhb should be structurally distinct from more common sialates and might have speci�city for less common forms like adoa or neu ac , l (figures (b) and (c) , resp.). as shown in figure , neu ac , l seems to be a relatively common sialate in the large intestine, suggesting it could be a potentially important source of bacterial nutrition. chemical synthesis of sialyl lactones has been reported [ ] , suggesting simple experiments to determine its utilization by e. coli and possibly identify the function of yjhb. adoa is an oxidized form of neu ac that may serve as an essential hydroxyl free radical scavenger in tissues [ , ] . s. typhimurium is closely related to e. coli but has only one predicted operon regulated by nanr (figure ). however, immediately downstream of a duplicated copy of nane (mannac- -p to glcnac- -p epimerase) is a predicted sodium-solute symporter that was shown to complement an e. coli nant mutant for growth on neu ac in trans [ ] . is result suggests that s. typhimurium spends at least some of its time in an environment with at least physiological levels (c. -mm) of sodium, concentrations found commonly in all human or other animal hosts. e problem with the complementation study is no evidence was presented showing the sodium-solute sialate symporter (here designated nanv) in fact functions as such in s. typhimurium wt + sodium wt f : auxanographic analysis of neu ac utilization by s. typhimurium nant and nanv mutants. e indicated strains were grown in minimal medium with glycerol as sole carbon source and plated in top agar with no carbon source and with or without mm sodium chloride. black rectangles indicate areas where neu ac was added, with growth shown by the hazy zones or individual colonies. [ ] . figure shows the results of an auxanographic analysis of s. typhimurium strain wild type, nant, nanv and nantv double mutant growth on neu ac as sole carbon source. auxanography is a common procedure where bacteria suspended in so ( . %) agar are plated on top of . % bottom agar, both lacking at least one essential growth factor [ , ] . e analysis can be carried out qualitatively by sprinkling about mg of substrate at one point of the plate, or semiquantitatively by applying a precise amount either in a small liquid volume or onto a paper disk [ ] . e results as expected show growth of the wild type (wt) on neu ac and none by the nant mutant. however, whereas growth was observed for the wt on a plate where the top agar was supplemented with mm sodium, similar growth was observed for the nant mutant demonstrating the sodium-dependency of another sialate uptake system. e sodium-dependent phenotype of the nant mutant was lost when a nantv double mutant was plated in the presence or absence of sodium ( figure ). however, some few colonies observed in the double mutant with sodium suggest another sodium-sialate transporter remains to be identi�ed. it will be interesting to test this isogenic mutant series for �tness defects in animal models of salmonellosis. ese studies are in progress. methylated sialic acid (neu acme) it has been known since that e. coli uses neu gc as a sole carbon source [ ] . although as discussed above this sialic acid is not synthesized by humans it is found in most other animal hosts where it could serve an important nutritional source for bacterial colonization. because neu gc differs from neu ac by a single hydroxyl group at the carbon- position of neu ac (figure (a) ), there might be an enzyme that �rst removes the group before or aer transport of neu gc by nant. however, no such enzyme is known to exist in nature indicating that neu gc metabolism probably begins with cleavage by nana to release pyruvate and man-ngc. is activity of the sialate lyase has been demonstrated biochemically for the mammalian homolog of nana [ ] . since we know neu gc serves as a sole carbon source for e. coli and is cleaved by nana, how e. coli handles the resulting hydroxylated mannac derivative, manngc, should de�ne the pathway for catabolism of neu gc in e. coli and probably all other bacteria that catabolize this substrate. in a preliminary experiment from the author's laboratory the expected accumulation of neu gc by an e. coli nana mutant was con�rmed by previously described chemical methods [ ] , demonstrating that the hydroxyl, as expected, is stable aer uptake, that is, there is no dehydroxylase in the cell. figure shows that once neu g is cleaved by nana, the resulting manngc is likely to be phosphorylated by nank and epimerized by nane to yield n-glycolylglucosamine- -phosphate (glcngc- -p). if the naga deacetylase can remove the glycolyl group, yielding glcn- -p, then nagb would complete the pathway by converting glcn- -p to fructose- -p just as it does in the canonical neu ac pathway ( figure ). e remaining glycolic acid would then be a substrate for the glc system, which is induced by glycolate, yielding glyoxylate [ ] . glyoxylate can then either be condensed with acetyl coenzyme-a by malate synthase g, or two molecules acted upon by glyoxylate carboligase, which simultaneously decarboxylates the condensation product, tartronic semialdehyde, and reduces it to glycerate that is then phosphorylated to glycerate- -phosphate. ese reactions constitute what is known as the glycerate pathway [ ] . e e. coli pathway proposed for metabolism of neu gc in figure is straightforward to verify as it involves readily available methods of bacterial mutagenesis of known target genes. if neu gc is an important carbon source in vivo, a mutant defective in glycolate oxidase might have an interesting phenotype that should be easy to determine. a variant of the pathway proposed in figure has been speculated upon in mammalian cellular metabolism of neu gc [ ] . neu acme is sialic acid with a methyl group attached to the carbon- carboxylate group (figure (a) ). e. coli uses this sugar as sole carbon source despite the inability of nana to cleave methylated sialic acid [ ] . on the basis of pervious studies with nans [ ] , it is reasonable to conclude that an as yet unidenti�ed methyl esterase(s) exists in the periplasm of e. coli to convert the methylated sialate to neu ac. a pattern of metabolism similar to that of n-glycolyl or methyl group removal is envisioned for other sialates with lactyl or other simple chemical substitutions found in the git (figure ). e picture emerging from the admittedly still limited number of studies concerning the sialoregulon is that bacteria and especially e. coli have evolved metabolic functions to funnel the diversity of host-derived sialates to neu ac or other readily digestible forms of this sugar. is dataset suggests a simple model of sialocatabolism for at least some of the sialoregulon parts that should be universally true for all bacteria with homologous sialocatabolic functions. pathways in e. coli e model is subdivided into �ve parts specifying the various e. coli cellular compartments: extracellular space, outer membrane (om), periplasm or periplasmic space, inner membrane (im), and cytoplasm ( figure ). e various components of the sialocatabolon are then either substrates, porins allowing ingress of substrates to the periplasm where modi�ers convert sialate derivatives into forms transportable by nant or yjhb (permeases) located in the im, and further conversion cytoplasmically as needed to neu ac, followed by the actions of the canonical metabolic pathway ( figure ). sialates released from sialoglycoconjugates by endogenous or exogenous sialidase(s) are immediately available for passive diffusion into the periplasm through outer membrane pores (porins) ompc, ompf, and nanc with molecular weight exclusion sizes of about a disaccharide. ough nanc is unessential for growth on neu ac it might allow certain oligosaccharides with o-acetylated sialic acids to gain access to nans in the periplasm, which would release the acetyl group(s) thus facilitating conversion in the extracellular milieu to free sialic acids. is idea is easy to test because nans modi�es glycoketosidically linked o-acetylated sialic acids as well as the free sugars. erefore, a strain with a fully induced sialoregulon when exposed to sgm would allow one to determine if nanc facilitates oligosaccharide deacetylation. alternatively, nanc might simply facilitate ingress of one or more of the minor sialates not recognized well by the major porins. once in the periplasm indicating the alpha anomer while hatched diamonds represent the thermodynamically favored beta anomer). neu ac derivatives: acetyl (ac) groups indicated by their linkages to different neu ac carbon position (numbers in diamonds); neu , ac , neu , ac , neu , ac , neu , ac , neu , , ac , neu , , ac , and neu gc (diamond with oh) is neu ac with hydroxyl at position (see figure ), lt (lactyl), me (methy). bold diamond is neuraminic acid (neu, which lacks the carbon- acetamido group). triangle represents adoa while diamond with horizontal line represents neu ac , l (see figures (b) and (c) ). diamond with cross lines represents neu ac en (see figure (d)). other abbreviations are as given in the legend to figure . nanm, nans, and other unidenti�ed modifying enzymes facilitate conversion of sialates to form(s) recognized by nant and, possibly, yjhb. aer transport to the cytoplasm other reactions presumably occur that convert any remaining sialates to neu ac for subsequent cleavage to pyruvate and mannac by nana. e resulting mannac, pyruvate, and glycolate in the case of neu gc are then available for �nal conversion to carbon, nitrogen, and energy sources or cell wall precursors. sialocatabolism thus is capable of ful�lling all cellular metabolic needs, consistent with its widespread occurrence and diversity in mucosal bacteria. any new or original idea goes through at least three stages: �rst many say it is not true, then they say it is true but not interesting; �nally, it is deemed true and interesting but not new (paraphrased from "anonymous"). it is only when a new scienti�c �eld reaches the third ideation that it has any chance of attracting adherents and the subsequent funding necessary for expansion. e �eld of microbial sialobiology began in with discovery of a sialic acid catabolic system in e. coli [ , ] . at that time most microbiologists had no idea what sialic acids were, and when explained to them they said they are "just another sugar" of no particular interest or importance. ose few researchers who knew something about sialic acid structural and phylogenetic diversity either thought the sugars were either too unstable or otherwise inaccessible to have any special relevance to microbial growth and overall bacterial physiology, and so the �eld has had a long gestation. e current paper focused on advances in microbial sialobiology since the �eld was last reviewed in [ ] . major advances since then have been the expanding knowledge of the sialoregulon and tantalizing in vivo experiments supporting minor to de�nitive roles of sialometabolism in diverse host-microbe interactions. ese recent �ndings are quite separate from the well-known functions of host sialic acids as microbial or toxin adhesins or regulators of innate immunity, knowledge of which has had little success generating practical advances in biomedicine. by contrast, targeting e. coli k , certain neisserial serotypes, haemophilus spp, and p. multocida synthetic or hybrid catabolic systems of sialic acid surface decoration are already known to have therapeutic potential [ , , , , , , ] . on the basis of these practical advances and the basic theoretical and experimental underpinnings the pace of research in microbial sialobiology is likely to increase. erefore, the point of the current paper is to increase optimism by presenting a coherent uni�ed theory of bacterial sialometabolism. certainly some ideas will not withstand detailed scrutiny. indeed, for reasons described some of the in vivo studies are not even likely to be reproducible. all the likely failures should stimulate rather than impede attempts to sharpen experimental approaches. it is hoped that the basic scienti�c �ndings presented in this paper will stimulate the proper scrutiny and help guide the �eld during its mature stage. mechanisms of association of bacteria with mucosal surfaces control mechanisms of the large-intestinal micro-�ora and its in�uence on the host diversity of microbial sialic acid metabolism gut microbe-host metabolic interactions in health and disease: exploring host and gut microbiota connections could uncover the mechanisms of various diseases along with targets for drugs with which to treat them targeting microbial sialic 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sialic acid, is essential for vibrio �ulni�cus pathogenesis cooperative regulation of the vibrio �ulni�cus nan gene cluster by nanr protein, camp receptor protein, and n-acetylmannosamine -phosphate fitness of escherichia coli during urinary tract infection requires gluconeogenesis and the tca cycle dissemination and systemic colonization of uropathogenic escherichia coli in a murine model of bacteremia carbon nutrition of escherichia coli in the mouse intestine comparison of carbon nutrition for pathogenic and commensal escherichia coli strains in the mouse intestine genotype and phenotypes of an intestine-adapted escherichia coli k- mutant selected by animal passage for superior colonization exogenous sialic acid transport contributes to group b streptococcus infection of mucosal surfaces sialic acid transport contributes to pneumococcal colonization e ability to utilize mucin affects the regulation of virulence gene expression in streptococcus pneumoniae host-derived sialic acid is incorporated into haemophilus in�uen�ae lipopolysaccharide and is a major virulence factor in experimental otitis media sialic acid mediated transcriptional modulation of a highly conserved sialometabolism gene cluster in haemophilus in�uen�ae and its effect on virulence regulation of sialic acid transport and catabolism in haemophilus in�uen�ae sialic acid metabolism and regulation by haemophilus in�uen�ae: potential novel antimicrobial therapies sialic acid transport and catabolism are cooperatively regulated by siar and crp in nontypeable haemophilus in�uen�ae nontypeable haemophilus in�uen�ae strain produces a bio�lm containing n-acetylneuraminic acid that may mimic sialylated olinked glycans sialylation of lipooligosaccharides promotes bio�lm formation by nontypeable haemophilus in�uen�ae role of sialic acid and complex carbohydrate biosynthesis in bio�lm formation by nontypeable haemophilus in�uen�ae in the chinchilla middle ear identi�cation, structure, and characterization of an exopolysaccharide produced by histophilus somni during bio�lm formation haemophilus in�uen�ae bio�lms: hypothesis or fact? structural studies on the pseudomonas aeruginosa sialidase-like enzyme pa suggest substrate and mechanistic variations innovations in host and microbial sialic acid biosynthesis revealed by phylogenomic prediction of nonulosonic acid structure bacterial neuraminidase facilitates mucosal infection by participating in bio�lm production e nana neuraminidase of streptococcus pneumoniae is involved in bio�lm formation sialic acid: a preventable signal for pneumococcal bio�lm formation, colonization, and invasion of the host ree streptococcus pneumoniae sialidases: three different products phase variable desialylation of host proteins that bind to streptococcus pneumoniaein vivo and protect the airway interaction of pneumococcal phase variation, host and pressure/gas composition: virulence expression of nana, hyla, pspa and cbpa in simulated otitis media leukocyte in�ammatory responses provoked by pneumococcal sialidase human sialidase neu long and short are extrinsic proteins bound to outer mitochondrial membrane and the endoplasmic reticulum, respectively cloning and characterization of sialidases with - ′ and - ′ sialyl lactose speci�city from pasteurella multocida capnocytophaga canimorsus a human pathogen feeding at the surface of epithelial cells and phagocytes mucosal glycan foraging enhances �tness and transmission of a saccharolytic human gut bacterial symbiont selective growth of mucolytic bacteria including clostridium perfringens in a neonatal piglet model of total parenteral nutrition selective in vitro degradation of the sialylated fraction of germ-free rat mucins by the caecal �ora of the rat commensal ocular bacteria degrade mucins growth of streptococcus pneumoniae on human glycoconjugates is dependent upon the sequential activity of bacterial exoglycosidases deglycosylation of human glycoconjugates by the sequential activities of exoglycosidases expressed by streptococcus pneumoniae chromatographic analysis of the escherichia coli polysialic acid capsule structural diversity and speci�c distribution of o-glycans in normal human mucins along the intestinal tract e roles of enteric bacterial sialidase, sialate o-acetyl esterase and glycosulfatase in the degradation of human colonic mucin a role for bacteroides fragilis neuraminidase in bacterial growth in two model systems sialidase speci-�city determined by chemoselective modi�cation of complex sialylated glycans function and expression of an n-acetylneuraminic acidinducible outer membrane channel in escherichia coli nanc crystal structure, a model for outermembrane channels of the acidic sugar speci�c kdgm porin family temporal geneexpression in escherichia coli k- bio�lms analysis of pools of targeted salmonella deletion mutants identi�es novel genes affecting �tness during competitive infection in mice sialic acid recognition by vibrio cholerae neuraminidase chemical diversity in the sialic acids and related -keto acids: an evolutionary perspective sialic acid mutarotation is catalyzed by the escherichia coli -propeller protein yjht experimental and computational assessment of conditionally essential genes in escherichia coli yjhs (nans) is required for escherichia coli to grow on -o-acetylated nacetylneuraminic acid separate pathways for o acetylation of polymeric and monomeric sialic acids and identi�cation of sialyl o-acetyl esterase in escherichia coli k synthesis of the complete series of mono acetates of n-acetyl-d-neuraminic acid structural and enzymatic characterization of nans (yjhs), a -o-acetyl nacetylneuraminic acid esterase from escherichia coli o :h crystal structure of the bacterial yhch protein indicates a role in sialic acid catabolism a novel sialic acid utilization and uptake system in the periodontal pathogen tannerella forsythia derived structure of the putative sialic acid transporter from escherichia coli predicts a novel sugar permease domain chemoselective synthesis of sialic acid , -lactones sialic acid is an essential moiety of mucin as a hydroxyl radical scavenger characterization of the reaction between sialic acid (nacetylneuraminic acid) and hydrogen peroxide characterization of a novel sialic acid transporter of the sodium solute symporter (sss) family and in vivo comparison with known bacterial sialic acid transporters dipeptidyl carboxypeptidase-de�cient mutants of salmonella typhimurium oligopeptidase-de�cient mutants of salmonella typhimurium characterization of a sialate pyruvate-lyase in the cytosol of human erythrocytes glc locus of escherichia coli: characterization of genes encoding the subunits of glycolate oxidase and the glc regulator protein metabolism of vertebrate amino sugars with n-glycolyl groups: incorporation of n-glycolylhexosamines into mammalian glycans by feeding n-glycolylgalactosamine high-throughput identi�cation of chemical inhibitors of e. coli group capsule biogenesis as antivirulence agents acknowledgments e work from the author's laboratory was supported by grants from the national institutes of health institute of allergy and infectious disease. e author thanks eric sixmister for the helpful discussions. susan steenbergen assisted with editing and formatting the paper. kerry helms was responsible for the excellent artwork that increased the overall appearance of the paper. key: cord- -al gxjw authors: calder, philip c. title: n− fatty acids, inflammation, and immunity— relevance to postsurgical and critically iii patients date: journal: lipids doi: . /s - - -z sha: doc_id: cord_uid: al gxjw excessive or inappropriate inflammation and immunosuppression are components of the response to surgery, trauma, injury, and infection in some individuals and these can lead, progressively, to sepsis and septic shock. the hyperinflammation is characterized by the production of inflammatory cytokines, arachidonic acid-derived eicosanoids, and other inflammatory mediators, while the immunosuppression is characterized by impairment of antigen presentation and of t helper cell type- responses. long-chain n− fa from fish oil decrease the production of inflammatory cytokines and eicosanoids. they act both directly (by replacing arachidonic acid as an eicosanoid substrate and by inhibiting arachidonic acid metabolism) and indirectly (by altering the expression of inflammatory genes through effects on transcription factor activation). thus, long-chain n− fa are potentially useful anti-inflammatory agents and may be of benefit in patients at risk of developing sepsis. as such, an emerging application of n− fa is in surgical or critically ill patients where they may be added to parenteral or enteral formulas. parenteral or enteral nutrition including n− fa appears to preserve immune function better than standard formulas and appears to partly prevent some aspects of the inflammatory response. studies to date are suggestive of clinical benefits from these approaches, especially in postsurgical patients. the systemic inflammatory response syndrome is the name given to the uncontrolled inflammatory response to insult or injury involving excessive production of inflammatory cytokines such as tumor necrosis factor (tnf)-α, interleukin (il)- β, il- , and il- ( , ) . sepsis has been defined as "the systemic inflammatory response syndrome that occurs during infection" ( ) . sepsis is the leading cause of death in critically ill patients in western countries. using records from for state hospitals in the united states it was estimated that there were more than , cases of sepsis with a . % mortal-ity rate ( , deaths) and a total cost of almost us$ billion ( ) . death from septic shock is the result of multiple organ failures and represents the extreme end of a continuum of events of increasing severity and decreasing likelihood of survival ( , ; fig. ). the systemic inflammatory response syndrome, sepsis, and septic shock may together be termed as "septic syndromes." the involvement of inflammatory cytokines in septic syndromes has been long recognized and vervloet et al. ( ) wrote "these mediators [i.e., inflammatory cytokines] are largely, if not completely, responsible for the clinical signs and symptoms of the septic response to bacterial infection." in support of this idea, patients with sepsis were found to have markedly elevated circulating concentrations of tnf-α, tnf receptor , il- β, il- receptor antagonist (il- ra), and il- , and those patients with the highest concentrations were more likely to die ( ) ( ) ( ) ( ) . in addition, circulating white cells from septic patients exhibited high levels of activated nuclear factor kappa b (nfκb), a transcription factor that promotes the expression of numerous genes associated with inflammation, and again levels of activated nfκb were higher in those patients who went on to die ( ) . animal studies also support a role for inflammatory cytokines in the septic response. these studies have often used bacterial endotoxin (also called lipopolysaccharide) as a surrogate for infection, although endotoxin is a fragment of the gram-negative bacterial cell wall and not a viable organism. mice injected with endotoxin exhibit high circulating concentrations of tnf-α, il- β, il- , and il- , and survival of these animals can be improved by administering anti-cytokine antibodies ( , ) , cytokine receptor antagonists ( ) , or anti-inflammatory cytokines such as il- ( ) , or by knocking out the tnf-α receptor ( ) . despite this evidence, it is important to note that some studies report that many septic patients do not show detectable or elevated circulating concentrations of tnf-α or il- β ( ) ( ) ( ) ( ) . furthermore, it appears that inflammatory cytokines do play a beneficial role in sepsis. for example, in some animal models, blocking tnf-α increases mortality ( ) ( ) ( ) , while a tnf-α antagonist increased mortality in a clinical trial ( ) . thus, the situation regarding the pathological role of inflammatory cytokines in sepsis is unclear; it may be that a little is beneficial but that excess is harmful and that complete blocking negates the beneficial effects. another consideration is that there may be large between-individual differences in the generation of inflammatory cytokines, in the sensitivity to the harmful effects of these cytokines, and in the effects of blocking these cytokines. thus, there may be significant variation in the susceptibility of individuals to exhibit the systemic inflammatory response syndrome and to progress toward septic shock. this may partly relate to the extent and site of the initial injury, partly to the nature and site of the infection, if any, and partly to aspects of the patient's well-being prior to receiving the injury (e.g., nutritional state). it is now recognized that genetics may also play a role. in fact there are likely to be genetic variations in many aspects of the septic response to infection and injury. these most likely relate to adaptations of various population groups to withstand infection and injury in different ecological settings. in the context of this article, genetic variations in the propensity to produce inflammatory cytokines are of relevance. it is now recognized that there are single base variations in genes or in their promoter regions called single nucleotide polymorphisms or snps (pronounced "snips"). snps have been described for tnf-α, tnf-β, il- β, il- , il- , tnf receptors, il- receptors, il- ra, and for many other genes involved in the septic response ( ) . these snps are of functional significance since they partly determine the extent of expression of the gene once it is activated ( ) . thus tnf-α production by monocytes in response to endotoxin is higher in individuals who have a g rather than an a at - in the tnf-α gene promoter region ( ) . intriguingly, tnf-α production is also affected by a polymorphism in the tnf-β gene: tnf-α production by monocytes in response to endotoxin was higher if there was an a at + in the tnf-β gene than if there was a g ( ) . genotypes affecting tnf-α production appear to be of relevance with respect to sepsis mortality. for example, possession of a g at - in the tnf-α gene was found in % of patients with septic shock compared with % of controls, and among patients with septic shock this polymorphism was significantly more common among patients who died ( % vs. % among survivors) ( ) . in controlling for age, it was identified that, for the same clinical score, patients with a g at - of the tnf-α gene had a . -fold higher risk of death than those without a g ( ) . in another study, patients with sepsis who were homozygous for a at + in the tnfβ gene displayed significantly higher plasma tnf-α concentrations than heterozygotes or homozygotes for g, and they showed % mortality compared with % for heterozygotes and % for g homozygotes ( ) . in a more recent study, postoperative patients who were homozygous for a at + in the tnf-β gene had a . -fold higher risk of developing severe complications than heterozygotes ( ) . furthermore, among the patients who developed sepsis, those who were homozygous for a at + in the tnf-β gene were more likely to die ( vs. % for heterozygotes and % for homozygotes for g) ( ) . these findings raise the possibility of being able to identify patients at high risk of complications and mortality on the basis of genetic polymorphisms. although there has been much focus on the potential detrimental role of inflammatory cytokines in sepsis, other mediators including arachidonic acid-derived eicosanoids, reactive oxygen species, nitric oxide, and adhesion molecules are involved in the pathological processes that accompany critical illness. prostaglandin (pg) e is implicated in sepsis, burns, and critical illness ( , ) , while leukotriene (lt) b and oxidants released by neutrophils are involved in acute respiratory distress syndrome [see kollef and schuster ( ) ]. in addition to hyperinflammation, patients with sepsis also display immunosuppression ( ) ( ) ( ) . there are reports that septic patients have high circulating concentrations of the antiinflammatory cytokine il- and that these are strongly correlated with mortality ( , ) . note that this is contrary to the predicted effect of il- since this cytokine down-regulates tnf-α production and its early administration is protective in murine endotoxemia ( ) ( ) ( ) . however, the apparently harmful effect of il- may relate to the timing of its production. lymphocytes from patients with burns or trauma produce low levels of the t helper (th) -type cytokines [e.g., interferon (ifn)-γ] associated with host defense against bacteria and viruses but high levels of the th -and treg-type cytokines (il- , il- ) associated with inhibition of host defense against bacteria and viruses ( , ) . there also appears to be decreased monocyte expression of human leukocyte antigens (hla) ( ) ( ) ( ) ( ) , the proteins involved in antigen presentation to t cells, and this is associated with impaired ability of monocytes to stimulate t cells ( ) . interestingly, il- downregulates both th -type cytokine production and hla expression ( , ) , and this might be the origin of the harmful effect of this cytokine in septic patients. recent studies have revealed impaired proliferative or secretory functions of t cells from patients with sepsis, trauma, or burns ( , ) . the traditional view is that the immunosuppressed phase of septic syndromes lags behind the hyperinflammatory phase (fig. ) ; that is, initially sepsis is characterized by increased generation of inflammatory mediators (the systemic inflammatory response syndrome), but as it persists there is a shift toward an anti-inflammatory, immunosuppressed state sometimes called the compensatory anti-inflammatory response syndrome. however, some recent studies challenge this and suggest that the hyperinflammatory and immunosuppressed states coexist. some authors report that immunosuppression is present at the onset of sepsis ( , , ) , rather than being a later compensatory response. for example, tschaikowsky et al. ( ) identified that significantly decreased monocyte expression of hla-dr was evident at the onset of severe sepsis in postsurgical patients; in survivors there was some recovery of expression but in nonsurvivors there was a further decrease or even a permanent suppression of hla-dr expression. these authors identified that the timing of the peak of the systemic inflammatory reaction (identified as the time of maximum c-reactive protein concentration) coincided with the timing of the lowest monocyte expression of hla-dr. from this they concluded that decreases in monocyte hla-dr expression occur simultaneously with "signs of hyperinflammation" and as early as the onset of severe sepsis ( ) . thus, it appears that immune cells and cytokines have both detrimental and protective roles in patients as they move through the stages of sepsis. however, the traditional view that hyperinflammation precedes immunosuppression, as shown in figure , may be a simplification of the real situation, and this increases the challenge to finding interventions that might benefit high-risk patients. human immune and inflammatory cells are rich in polyunsaturated fa (pufa), especially arachidonic acid ( : n- ) [see calder ( ) ]. classically the influence of pufa on immunity and inflammation has been viewed as relating to their influence on eicosanoid generation ( ) ( ) ( ) ( ) . arachidonic acid is the principal substrate for cyclooxygenase (cox) and lipoxygenase (lox) enzymes giving rise to -series pg and thromboxanes (tx) or -hydroxyeicosatetraenoic acids (hete) and -series lt, respectively. these mediators have cell-and stimulus-specific sources and frequently have opposing effects (table ). for example, pge is produced mainly by monocytes, macrophages, and, to a lesser extent, neutrophils and inhibits the production of tnf-α and il- β [see miles et al. ( ) and references therein; , ] and il- ( , ) , while ltb is produced mainly by neutrophils, other granulocytes, and, to a lesser extent, monocytes and macrophages and increases the production of tnf-α and il- β [see rola-pleszczynski et al. ( ) and references therein]. thus, the overall physiological (or pathophysiological) outcome will depend upon the cells present, the nature of the stimulus, the timing of eicosanoid generation, the concentrations of different eicosanoids generated, and the sensitivity of target cells and tissues to the eicosanoids generated. recent studies have demonstrated that pge induces cox- in fibroblasts cells and so upregulates its own production ( ), induces production of il- by macrophages ( ) it is frequently considered that the effects of arachidonic acid are solely related to its role as an eicosanoid precursor. cell culture studies have shown that arachidonic acid acti- vates nfκb in a monocytic cell line ( ) , and induces tnf-α, il- α and il- β in osteoblasts ( ), il- in macrophages ( ) and osteoblasts ( ) , and cox- in fibroblasts ( ) , and it appears that these effects are exerted directly by arachidonic acid rather than by an eicosanoid metabolite. what is evident from these studies is that arachidonic acid may be able to regulate inflammatory mediator production in its own right and, if so, that it has effects that are sometimes the opposite of those of pge , for example, with respect to tnf-α production. a series of cell culture-based studies with human endothelial cells has suggested that another n- fa, linoleic acid ( : n- ), may also play a role in inflammation through activation of nfκb and increased production of tnf-α, il- , and other inflammatory mediators ( ) ( ) ( ) ( ) ( ) ( ) ( ) . increased consumption of long-chain n- pufa, usually as components of fish oil, by humans results in increased amounts of epa ( : n- ) and dha ( : n- ) in cells involved in immunity and inflammation [see calder ( ) ]. the incorporation of these fa from the diet into immune/inflammatory cells of humans is near-maximal within a few weeks ( , ) and occurs in a dose-dependent manner ( ) . incorporation of epa and dha into human cells is partly at the expense of arachidonic acid [see calder ( ) ], and the functional significance of this is that it decreases the amount of arachidonic acid available as a substrate for eicosanoid synthesis. thus, fish oil supplementation of the human diet has been shown to result in decreased production of pge ( - ), txb ( ), ltb and -hete ( , ) , and lte ( ) by inflammatory cells. however, the mechanism of the effect of long-chain n- fa on eicosanoid generation extends beyond simply decreasing the amount of arachidonic acid substrate. for example, epa competitively inhibits metabolism of arachidonic acid by cox ( - ) and -lox ( , ) . in vitro studies also report that dha can inhibit cox activity ( , ) but not that of -lox ( , ) . interestingly, however, both epa and dha suppressed cytokine-induction of cox- and -lox gene expression in cultured bovine chondrocytes and in human osteoarthritic cartilage explants ( , ) . by inhibiting cox and lox activities and by suppressing the up-regulation of the genes for these enzymes in response to inflammatory stimuli, long-chain n- fa act to oppose generation of eicosanoids from arachidonic acid. the final element of the effects of longchain n- fa on eicosanoid production is the ability of epa to act as a substrate for cox and lox enzymes, so giving rise to a different family of eicosanoids: the -series pg and tx, the series lt, and the hydroxyeicosapentaenoic acids (hepe). epa, which appears to be a good substrate for -lox ( , ) , is also a substrate for cox enzymes ( , ) . thus, fish oil supplementation of the human diet has been shown to result in increased production of ltb , lte , and -hepe by inflammatory cells ( ) ( ) ( ) , although generation of pge has been more difficult to demonstrate ( ) . the functional significance of this is that the mediators formed from epa are believed to be less potent than those formed from arachidonic acid. for example, ltb is -to -fold less potent as a neutrophil chemotactic agent than ltb ( , ) . recent studies have compared the effects of pge and pge on production of cytokines by cell lines and by human cells. bagga et al. ( ) reported that pge was a less potent inducer of cox- gene expression in fibroblasts and of il- production by macrophages. pge and pge had equivalent inhibitory effects upon production of tnf-α ( , ) and il- β ( ) by human mononuclear cells stimulated with endotoxin and upon production of ifn-γ production by mononuclear cells stimulated with mitogen ( , ) . however, il- production appeared to be less sensitive to pge than pge ( ) . studies using the isolated, perfused rabbit lung have identified contrasting effects of arachidonic acid-and epa-derived eicosanoids. infusion of escherichia coli hemolysin caused hypertension mediated by txb and increased vascular leakage mediated by -series lt ( ) . inclusion of arachidonic acid in the perfusate increased txb and -series lt generation, arterial pressure, and vascular leakage ( , ). in contrast, inclusion of epa in the perfusate decreased txb and -series lt generation, decreased arterial pressure and vascular leakage, and increased generation of txb and -series lt ( ) . perfusion with fish oil attenuated the hypertension induced by calcium ionophore ( ) . compared with soybean oil infusion, fish oil decreased the concentration of ltc by % and increased the concentration of ltc from barely detectable to very similar to that of ltc ( ) . in addition to long-chain n- fa modulating the generation of eicosanoids from arachidonic acid and to epa acting as substrate for the generation of alternative eicosanoids, recent studies have identified a novel group of mediators, termed e-series resolvins, formed from epa by cox- that appear to exert anti-inflammatory actions ( ) ( ) ( ) . in addition, dha-derived mediators termed d-series resolvins, docosatrienes, and neuroprotectins also produced by cox- have been identified, and these too appear to be anti-inflammatory ( ) ( ) ( ) . this is an exciting new area of n- fa and inflammatory mediators, and the implications for a variety of conditions may be of great importance. cell culture studies investigating the direct effects of arachidonic acid on inflammatory mediator production have also investigated effects of long-chain n- fa. epa did not activate nfκb in a monocytic cell line ( ), while epa and dha inhibited endotoxin-stimulated production of il- and il- by cultured human endothelial cells ( , ) . more recent studies showed that epa did not induce tnf-α, il- β, or il- α ( ) or il- ( ) in osteoblasts, and even countered the upregulating effect of arachidonic acid ( ) ; that epa and dha could totally abolish cytokine-induced up-regulation of tnf-α, il- α, and il- β in cultured bovine chondrocytes and in human osteoarthritic cartilage explants ( , ) ; and that epa or fish oil inhibited endotoxin-induced tnf-α production by monocytes ( ) ( ) ( ) ( ) . epa was also less potent than arachidonic acid in inducing cox- expression by fibroblasts and il- expression by macrophages ( ) . epa prevented nfκb activation by tnf-α in cultured pancreatic cells, an effect that involved decreased degradation of the in-hibitory subunit of nfκb (iκb), perhaps through decreased phosphorylation ( ) . similarly, epa or fish oil decreased endotoxin-induced activation of nfκb in human monocytes ( , , ) , and this was associated with decreased iκb phosphorylation ( , ) , perhaps due to decreased activation of mitogen-activated protein kinases ( ) . these observations suggest direct effects of long-chain n- fa on inflammatory gene expression via inhibition of activation of the transcription factor nfκb. animal feeding studies with fish oil support the observations made in cell culture with respect to the effects of long-chain n- fa on nfκb activation and inflammatory cytokine production. compared with feeding corn oil, fish oil lowered nfκb activation in endotoxin-activated murine spleen lymphocytes ( ) . feeding fish oil to mice decreased ex vivo production of tnf-α, il- β, and il- by endotoxin-stimulated macrophages and decreased circulating tnf-α, il- β, and il- concentrations in mice injected with endotoxin [sadeghi et al. ( ) and references therein]. several studies in humans involving supplementation of the diet with fish oil have demonstrated decreased production of tnf-α, il- β, and il- by endotoxin-stimulated monocytes or mononuclear cells (a mixture of lymphocytes and monocytes) ( ) ( ) ( ) ) . the study of caughey et al. ( ) reported a significant inverse correlation between the epa content of mononuclear cells and the ability of those cells to produce tnf-α and il- β in response to endotoxin. recent studies have confirmed the ability of dietary fish oil to decrease production of tnf-α ( ) and il- ( , ) by human mononuclear cells. furthermore, these studies provide for the first time information on the dose-response relationship between dietary intake of long-chain n- fa and production of these cytokines. it should be noted that there are also several studies that fail to show effects of dietary long-chain n- fa on production of inflammatory cytokines in humans [see calder ( ) for references]. it is not clear what the reason for this is, but the dose of n- fa used and other technical factors are likely to be contributing factors. one other factor that has recently been identified is polymorphisms in genes affecting cytokine production ( ) . it was found that the effect of dietary fish oil on cytokine production by human mononuclear cells was dependent on the nature of the - tnf-α and the + tnf-β polymorphisms. this study raises the possibility of being able to identify those who are more likely and those who are less likely to experience specific anti-inflammatory effects of fish oil. thus, examination of fa composition and of eicosanoid profiles, cell and tissue culture work, and animal and human feeding studies have revealed a range of anti-inflammatory actions of long-chain n- fa ( table ). these may be of benefit in sepsis, particularly during the "early" hyperinflammatory phase. the benefits of fish oil in animal models of experimental endotoxemia have been clearly demonstrated. for example, dietary fish oil or fish oil infused intravenously significantly enhanced survival of guinea pigs to intraperitoneal endotoxin compared with safflower oil ( , ) . dietary fish oil resulted in a decreased concentration of circulating postendotoxin eicosanoids (pge , txb , -keto-pgf α ) in rats and in decreased eicosanoid generation by alveolar macrophages ( , ) . furthermore, compared with dietary safflower oil, fish oil resulted in lower circulating tnf-α, il- β, and il- concentrations following endotoxin administration to mice ( ) . dietary fish oil also appears to decrease sensitivity to inflammatory cytokines ( , ) . fish oil decreased endotoxininduced metabolic perturbations in guinea pigs and rats ( , ) and improved heart and lung function and decreased lung edema in endotoxic rats ( , ( ) ( ) ( ) and pigs ( ) ( ) ( ) . in addition to effects on production of inflammatory eicosanoids and inflammatory cytokines, long-chain n- fa decreased generation of arachidonic acid-derived partial replacement of arachidonic acid in cell membrane phospholipids eicosanoids (many with inflammatory actions) inhibition of arachidonic acid metabolism by phospholipase a , cox, and -lox decreased induction of cox- , -lox, and -lox-activating protein however, it is the effects of lower amounts of long-chain n- fa that are of relevance to the patient setting. several studies in humans, typically providing long-chain n- fa as fish oil, and investigating aspects of cell-mediated immunity have been performed. phagocytic uptake of escherichia coli appears unaffected by dietary long-chain n- fa in humans ( ) ( ) ( ) ( ) . one study reported that fish oil decreased expression of hla-dp, -dq, and -dr on human monocytes ( ), suggesting impaired ability to present antigen, but there have been no studies attempting to confirm this finding. meydani et al. ( ) reported that fish oil providing . g epa plus dha per day decreased t-lymphocyte proliferation in older but not younger women. however, that study also reported increased oxidative stress in the older subjects ( ) , and it may be that the effect of n- fa was due to excessive lipid peroxidation. several other studies report no effect of various doses of longchain n- fa on lymphocyte proliferation ( , , ) , although there are studies reporting a decrease ( , ) . one recent study reported that long-chain n- fa caused a dosedependent increase in proliferation of t cells ( ) . it is noteworthy that the fish oil used was given in combination with an antioxidant mix. this might be important in terms of preventing excessive lipid peroxidation and so in determining the overall effect of n- fa. the study by meydani et al. ( ) also reported decreased production of il- in the older women, but this effect has not been confirmed by others in either older ( ) , young ( , ) , or mixed-age ( , ) subjects. a recent study reported a dose-dependent increase in ifn-γ production following n- fa supplementation as fish oil ( ) . that antioxidants were given in combination with fish oil may have been important in generating this finding. thus, the effects of long-chain n- fa on aspects of cellmediated immunity are rather unclear, although recent human studies suggest that adverse immune effects are not exerted at modest doses (see previous discussion for references) and that enhanced t-cell responses (proliferation and ifn-γ production) may occur at modest doses so long as antioxidants are also given ( ) . in terms of sepsis, the true test of immunocompetence occurs when live pathogens are administered. this is a different situation from using endotoxin that is not living and that therefore does not require a robust cell-mediated immune response to eliminate it. as indicated previously, it is clear that long-chain n- fa protect against the deleterious effects of endotoxin. however, the situation regarding live pathogens is much less clear. this is because animal studies, frequently using high intakes of n- fa, report opposing findings. infusion of fish oil into rats also receiving low-dose endotoxin decreased the number of viable bacteria in mesenteric lymph nodes and liver ( ) . fish oil did not decrease bacterial translocation across the gut, and so the authors concluded that fish oil must have improved bacterial killing. compared with linoleic acid-rich vegetable oils, fish oil fed to rats before exposure to live bacteria ( , ) resulted in increased survival, which was associated with decreased production of pge . more recently, infusion of fish oil after induction of sepsis by cecal ligation and puncture decreased mortality (and pge production) compared with vegetable oil ( ) . intragastric administration of fish oil into chow-fed rats before cecal ligation and puncture improved survival compared with saline or vegetable oil infusion ( ) . compared with vegetable oil feeding to mice, fish oil feeding increased survival to an intramuscular injection of klebsiella pneumoniae ( ) . the findings from these studies ( ) ( ) ( ) ( ) ( ) ( ) contrast with those reporting that fish oil feeding decreases the survival of mice to oral salmonella typhimurium ( ) and to intraperitoneal listeria monocytogenes ( ) , of guinea pigs to mycobacterium tuberculosis ( ) , and of neonatal rabbits to staphylococcus aureus ( ) . thus, animal studies do not provide a clear picture of the effect of high-dose fish oil on ability to survive an infectious challenge. there are few human studies that address exposure to long-chain n- fa and infection; most intervention studies performed to date have been too small and of too short duration to monitor infection as an outcome. however, it is worth noting that an epidemic of measles in greenland triggered by its introduction to a naive population by an infected danish sailor showed the same characteristics as previous epidemics in other naive populations ( ) . this suggests that the very n- fa-rich diet of the greenland inuits did not worsen their response to the virus and this could indicate that these fa do not increase infectious susceptibility in humans. surgery is typically accompanied by an inflammatory response that may be exaggerated in some patients, especially if the surgery is major. if the patient is exposed to pathogenic organisms and is unable to cope with these, then sepsis may develop. artificial nutrition is frequently used post-surgery and this may involve parenteral (i.e., intravenous) infusions, especially where the gastrointestinal tract is not fully functional (e.g., post-abdominal surgery). lipids are included in parenteral nutrition to provide an alternative source of calories to glucose and the lipid source used most frequently has been soybean oil, which is rich in the n- fa linoleic acid, although it also contains a proportion of α-linolenic acid ( : n- ). a meta-analysis of total parenteral nutrition suggested that inclusion of lipids might be detrimental (p = . for lipids vs. no lipids) ( ) , at least in very ill patients. it is not clear why this is, although a number of in vitro experiments have shown that soybean oil-based lipid emulsions can exert immunosuppressive effects [see calder et al. ( ) for references], which would clearly be detrimental in patients at risk of infection and sepsis. clinical trials provide conflicting evidence, some showing some immunosuppressive effects ( , ) and others not ( ) ( ) ( ) , at least in some patient groups. the concern about potential harm, the view of sepsis as a hyperinflammatory state followed by an immunosuppressed state (fig. ) , and the idea that n- fa might be "proinflammatory and immunosuppressive" has led to the development of alternative lipid emulsions for parenteral applications. emulsions using a mix of medium-chain triglycerides and soybean oil or based upon olive oil instead of soybean oil have been developed, but these will not be discussed here. however, of relevance to the present discussion is the development of emulsions that include fish oil as a partial replacement for soybean oil. several such emulsions have been tested in surgical patients. intravenous infusion of a lipid emulsion containing fish oil for d into patients who had undergone major abdominal surgery resulted in much higher ltc production by blood leukocytes stimulated ex vivo at d postoperation ( ) . in another study, patients who had undergone abdominal surgery received soybean oil or a mix of medium-chain triglycerides, soybean oil, and fish oil ( : : , by vol) for d post surgery ( ) . leukocytes from these patients produced more ltb and ltb isomers at postoperative days and . patients who had undergone major gastrointestinal surgery received a medium-chain triglyceride/soybean oil mix ( : , vol/vol) or a mix of medium-chain triglycerides, soybean oil, and fish oil ( : : , by vol) for d postsurgery ( ) . patients receiving fish oil got (days and ) and g (days , , and ) of long-chain n- fa per day. neutrophils from these patients produced less ltb and more ltb at postoperative days and . plasma tnf-α concentrations were lower in the fish oil group at day , while plasma il- concentrations were lower at day . the study did not report clinical outcomes. a more recent study infused a fish oil-rich formula on the day before abdominal surgery and on days to following abdominal surgery ( ) . on days and the patients also received standard total parenteral nutrition that included g of fat/d (n = ; n = in the control group). tnf-α production by endotoxin-stimulated whole blood tended to be lower at postoperative day in the fish oil group, but this was not significant. serum il- concentrations were significantly lower at days , , and in the fish oil group. monocyte expression of hla-dr was preserved in the fish oil group but declined at postsurgery days and in the control group. no differences in infection rates or mortality were observed. however, postoperative stay in intensive care tended to be shorter in the fish oil group ( . vs. . d) as did total hospital stay ( . vs. . days), although neither of these was a significant effect. postoperative stay on medical wards was significantly shorter in the fish oil group. another recent study compared the effects of lipid-free total parenteral nutrition or parenteral nutrition including % soybean oil or . % soybean oil plus . % fish oil for d after large bowel surgery ( ) . there were no differences between the groups with respect to the numbers of circulating lymphocytes, b cells, cd + cells, cd + cells, or natural killer cells before surgery or at days and postsurgery, although these were affected by surgery itself. there were no differences between groups with respect to t-lymphocyte proliferation, but il- production was increased in the fish oil group and the postsurgery decline in ifn-γ production was prevented by fish oil. these studies indicate that inclusion of fish oil in parenteral nutrition regimens for gastrointestinal surgical patients modulates generation of inflammatory eicosanoids ( ) ( ) ( ) and cytokines ( , ) and may help to counter the surgery-induced declines in antigen-presenting cell activity ( ) and t cell cytokine production ( ) . importantly, these studies do not reveal deleterious immunologic effects of fish oil infusion in these patients. furthermore, the only one of these fairly small studies to have examined hard end points like length of hospital stay suggests some clinical benefit from fish oil infusion in these patients ( ) . however, larger studies are required to evaluate the effects of this approach on complication rates, hospital stay, and mortality rate. a very recent report from a larger cohort of patients receiving parenteral nutrition postsurgery does indicate benefit of inclusion of fish oil in the regimen ( ) . patients received fish oil postoperatively (n = ) or controls received a : medium-chain triglyceride-soybean oil mix (n = ). there were no differences between the two groups with respect to the proportions of patients who died or developed wound infections or with respect to length of hospital stay. however, the proportion of patients who were readmitted to intensive care ( %) was significantly lower in the fish oil than in the control group ( %). a group of patients also received the fish oil-containing emulsion for d preoperatively (n = ). here there were a number of very significant benefits. this group showed a significantly decreased need for mechanical ventilation ( vs. % in the control group), a significantly shorter length of hospital stay ( vs. d) , significantly less need for readmission to intensive care ( vs. %), and a significantly lower mortality rate ( vs. %) ( ) . this study demonstrates a benefit from the inclusion of long-chain- fa in parenteral nutrition regimens used in abdominal surgery patients. however, it also demonstrates a much greater benefit if the fa are additionally provided before surgery, which, of course, is only possible in elective surgery. the greater benefit of preoperative infusion of longchain n- fa may relate to better incorporation of the fa into leukocytes and other tissues. enteral nutrition is an alternative form of artificial nutrition. it describes provision of nutrients directly into the gastrointestinal tract via a tube and is sometimes referred to as "tube feeding." enteral nutrition is used in patients with a functional gastrointestinal tract and is considered preferable to parenteral nutrition. the influence of enteral feeds including long-chain n- fa in their composition has been examined in surgical patients, generally in those who have undergone surgery to remove cancerous regions of the intestine. these studies have frequently used an enteral formula named impact ® (novartis, basel, switzerland), which contains arginine, long-chain n- fa, and nucleotides, each of which is lacking from control formulas. thus, any effects observed cannot be ascribed to a particular component of impact. the effect of impact on immunoinflammatory outcomes in surgical patients has been widely examined. daly et al. ( ) reported that impact results in time-dependent incorporation of epa into mononuclear cells and that this is associated with a timedependent decrease in pge production. studies have reported that impact increases phagocytosis by monocytes but not by neutrophils ( , ) , increases t-cell proliferation ( ) and cell-mediated immunity ( , ) , and decreases circulating concentrations of il- ( , ) . several of these studies report significantly improved clinical outcomes related to lower infection rate ( , , , ) and decreased length of hospital stay ( , , ) . studies of impact and similar enteral formulas investigating clinical outcomes in postsurgical patients have been subject to meta-analyses ( ) ( ) ( ) , which conclude that this approach to enteral nutrition significantly decreases infectious complications and length of hospital stay in elective surgery patients. it is possible that the modulation of inflammation and the improvements in immune function reported in these patients receiving impact contribute to the improved clinical outcomes. however, it is not possible to ascribe these benefits to long-chain n- fa. critically ill patients frequently require artificial support, depending upon the extent of organ damage or failure, and this will include nutritional support. the influence of enteral feeds including long-chain n- fa has been examined in critically ill patients; again, many of these studies have involved impact. a study in intensive care unit patients (a mix of trauma, sepsis, and major surgery patients) reported that impact resulted in higher t-cell proliferation at days and ( ), while a study of severe trauma patients reported greater hla-dr expression at day ( ) . these studies did not report improvements in clinical outcomes. studies of impact and similar enteral formulas investigating clinical outcomes in trauma and critically ill patients have been subject to metaanalysis ( ) ( ) ( ) . the most recent of these concluded that this approach to enteral nutrition decreases length of hospital stay but has no effect on infectious complications or mortality in critically ill patients ( ) . another trial performed in patients with moderate and severe acute respiratory distress syndrome used an enteral preparation that differed mainly in lipid source from the control ( ) . the control group of patients (n = ) received a formula in which the lipid source was % corn oil plus % soy lecithin. the experimental group (n = ) received a lipid source that was % canola oil, % medium-chain triglycerides, % borage oil, % fish oil, and % soy lecithin. the experimental formula also contained more vitamin c and vitamin e than the control and it contained β-carotene, taurine, and carnitine, which the control formula did not. patients receiving the experimental formula got about g of epa, g of dha, g of γ-linolenic acid, . g of vitamin c, iu of vitamin e, and . mg of β-carotene per day for d. by d the numbers of total leukocytes and of neutrophils in the alve-olar fluid declined significantly in the experimental group and were lower than in the control group. arterial oxygenation and gas exchange were improved in the experimental group. these patients had a significantly decreased requirement for supplemental oxygen, decreased time on ventilation support ( . vs. . d), and a shorter length of stay in intensive care ( . vs. . d) . total length of hospital stay tended to be shorter in the experimental group ( . vs. . d). significantly fewer patients in the experimental group developed new organ failure ( vs. %). the mortality rate was % in the experimental group and % in the control group, but this difference was not statistically significant. more recently, new data from this study have become available ( ) . patients receiving the experimental formula had significantly lower concentrations of il- in their alveolar fluid and tended to have lower concentrations of ltb and tnf-α. it is possible that the lower concentrations of ltb and il- , both of which are potent leukocyte chemoattractants, may have been responsible for the lower neutrophil infiltration reported in the experimental group, and indeed neutrophil counts were significantly associated with these concentrations ( ) . this study establishes that the experimental treatment decreases production of inflammatory mediators and infiltration of inflammatory leukocytes and that this can result in significant clinical improvement in extremely ill patients. because of the many differences in composition between the experimental and control formulas used it is not possible to ascribe the effects and benefits to any particular nutrient. however, the effects on ltb , il- and tnf-α concentrations are consistent with effects of long-chain n- fa reported elsewhere. recently, data from studies using parenteral nutrition with fish oil in sepsis patients have become available ( , ) . patients received a standard soybean oil-based emulsion or an emulsion containing fish oil for ( ) or ( ) d. blood leukocyte counts and serum c-reactive protein concentration tended to be lower, and production of ltb by stimulated neutrophils was significantly higher in patients receiving long-chain n- fa ( ). production of tnf-α, il- β, il- , il- , and il- by endotoxin-stimulated mononuclear cells did not increase during infusion of the fish oil-containing emulsion whereas production of the four proinflammatory cytokines was markedly elevated during the first d of soybean oil infusion ( ) . these studies establish that infusion of long-chain n- fa into patients with sepsis can modulate inflammatory mediator production and related inflammatory processes. however, the impact of this on hard clinical outcomes in these patients is not yet clear. in summary, long-chain n- pufa from fish oil decrease the production of inflammatory cytokines and eicosanoids. they act both directly, by replacing arachidonic acid as an eicosanoid substrate and by inhibiting arachidonic acid 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improves lymphocyte function during sepsis essential fatty acids influence survival in stress dietary fish oil supplementation in experimental gram negative infection and in cerebral malaria in mice fish oil decreases natural rresistance of mice to infection with salmonella typhimurium dietaey fish oil reduces survival and impairs bacterial clearance in c h/hen mice challenged with listeria monocytogenes influence of dietary (n- ) polyunsaturated fatty acids on leukotriene b and prostaglandin e synthesis and the time course of experimental tuberculosis in guinea pigs effect of dietary (n- ) and (n- ) fatty acids on in vivo pulmonary bacterial clearance by neonatal rabbits analysis of the incubation period for measles in the epidemic in greenland in using a variance components model total parenteral nutrition in the critically ill patient: a meta-analysis inhibition of lymphocyte proliferation in vitro by two lipid emulsions with different fatty acid compositions a prospective, 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perioperative administration of n- fatty acids impact of fish oil enriched total parenteral nutrition on dna synthesis, cytokine release and receptor expression by lymphocytes in the postoperative period perioperative administration of parenteral fish oil supplements in a routine clinical setting improves patient outcome after major abdominal surgery enteral nutrition during multimodality therapy in upper gastrointestinal cancer patients clinical outcome and immunology of postoperative arginine fatty acids, and nucleotide-enriched enteral feeding: a randomized prospective comparison with standard enteral and low calories/low fat iv solutions immune and nutritional effects of early enteral nutrition after major abdominal operations enteral nutrition with supplemental arginine, rna, and omega- fatty acids in patients after operation: immunologic, metabolic, and clinical outcome a prospective, randomised clinical trial on perioperative feeding with an arginine-, omega- fatty acid-, and rna-enriched enteral diet: effect on host response and nutritional status perioperative immunonutrition in patients undergoing cancer surgery enteral nutritional supplementation with key nutrients in patients with critical illness and cancer-a meta-analysis of randomized controlled clinical trials immunonutrition in the critically ill: a systematic review of clinical outcome should immunonutrition become routine in critically ill patients? a systematic review of the evidence effect of enteral nutrition on in vitro tests of immune function in icu patients: a preliminary report influence of arginine, omega- fatty acids and nucleotide-supplemented enteral support on systemic inflammatory response syndrome and multiple organ failure in patients after severe trauma and the enteral nutrition in ards study group ( ) effect of enteral feeding with eicosapentaenoic acid, γ-linolenic acid, and antioxidants in patients with acute respiratory distress syndrome enteral nutrition with eicosapentaenoic acid, gamma-linolenic acid, and antioxidants reduces alveolar inflammatory mediators and protein influx in patients with acute respiratory distress syndrome ) ω- vs, ω- lipid emulsions exert differential influence on neutrophils in septic shock patients: impact on plasma fatty acids and lipid mediator generation parenteral nutrition with fish oil modulates cytokine response in patients with sepsis key: cord- -pkj bjur authors: Çiçek, serhat sezai; wenzel-storjohann, arlette; girreser, ulrich; tasdemir, deniz title: biological activities of two major copaiba diterpenoids and their semi-synthetic derivatives date: - - journal: rev bras farmacogn doi: . /s - - -y sha: doc_id: cord_uid: pkj bjur the oleoresin of copaifera reticulata ducke, fabaceae, is a traditional brazilian remedy used for a wide range of applications. commonly named copaiba, the oleoresin has been found to exhibit strong antimicrobial effects in our previous study, which could be attributed to some of its diterpenoid constituents. in order to find new biological activities and to eventually enhance the before observed effects, (−)-polyalthic acid ( ) and kaurenoic acid ( ), together with eight prepared semi-synthetic derivatives ( a– c and a– e) were evaluated for their cytotoxic, antibacterial and antifungal properties. regarding the gram-positive bacteria enterococcus faecium and methicillin-resistant staphylococcus aureus, we found that both the exocylic methylene group and the carboxyl group were crucial for the activity against these two clinically relevant bacterial strains. investigation of the antifungal activity, in contrast, showed that the carboxyl group is unnecessary for the effect against the dermatophytes trichophyton rubrum and cryptococcus neoformans, indicated by low micromolar ic( ) values for both (−)-polyalthic acid diethylamide ( a) as well as (−)-polyalthic acid methyl ester ( b). apart from studying the biological activity, the structure of one semi-synthetic derivative, compound c, is being reported for the first time. during the course of the structure elucidation of the new compound, we discovered inconsistencies regarding the stereochemistry of polyalthic acid and its stereoisomers, which we clarified in the present work. [figure: see text] electronic supplementary material: the online version of this article ( . /s - - -y) contains supplementary material, which is available to authorized users. copaiba, or copaiba oil, is the name of an oleoresin obtained from selected species of the genus copaifera, fabaceae (leandro et al. ) . copaiba is widely used in the traditional medicine in brazil and other latin american countries for treatment of various diseases, such as skin and urinary tract infections, respiratory diseases, ulcers, rheumatism, herpes, tumours and tetanus disease (ohsaki et al. ) . due to its antibacterial, antihelminthic, trypanocidal and leishmanicidal applications, the oleoresin represents an important natural remedy for people without access to modern medicine and commercial drugs (ohsaki et al. ; leandro et al. ) . copaiba consists of sesquiterpenes and diterpenes, the latter usually being present as diterpene acids (leandro et al. ) . sesquiterpenes represent the main compounds that can count up to % of the oleoresin by weight, and show a rather stable metabolite pattern, with β-caryophyllene being the major constituent in most copaifera species (leandro et al. ). however, βbisabolene has been identified as the major sesquiterpene of copaifera reticulata ducke collected in the pará region (northern brazil) (pfeifer barbosa et al. ) . in contrast to the sesquiterpenes, copaiba diterpenoids show much higher interspecific variation and therefore attracted particular interest in recent years due to varying biological electronic supplementary material the online version of this article (https://doi.org/ . /s - - -y) contains supplementary material, which is available to authorized users. activities observed for the different copaifera species (leandro et al. ) . these activities comprise cytotoxic, antibacterial, antifungal and antiprotozoal properties as part of numerous in vitro and in vivo studies attempting to attribute the detected effect to certain compounds or compound classes. the cytotoxic potential of the copaiba oleoresin seems to mainly derive from its sesquiterpene constituents, as respective investigations on diterpenoids resulted in weak to moderate (but then non-selective) cytotoxic effects (cavalcanti et al. ; pfeifer barbosa et al. ) . the only exception is kolavenol, which showed antitumour effects against imc carcinoma in mice, determined by an increase of life span of the animals (ohsaki et al. ) . in contrast, the main sesquiterpenes showed selective moderate (β-bisabolol) to pronounced (β-caryophyllene and β-caryophyllene oxide) cytotoxic activities (kubo et al. ; yeo et al. ) . with respect to their higher concentration in the oleoresin, it is more likely that sesquiterpenes are responsible for the antitumour properties of copaiba (pfeifer barbosa et al. ) . on the other hand, copaiba diterpenoids showed good in vitro activities against causative agents of infectious parasitic diseases. copalic acid, β-hydroxycopalic acid and methyl copalate, for example, inhibited the growth of the amastigote forms of trypanosoma cruzi, whereas kaurenoic acid ( ) and some of its semi-synthetic derivatives were found active against its erythrocytic trypomastigote forms (vieira et al. ; izumi et al. ). (−)-polyalthic acid ( ), another major copaiba diterpenoid showed activity against t. brucei and amastigote forms of leishmania donovani (mizuno et al. ) . also here, semi-synthetic derivatives were prepared, of which some showed comparable to slightly higher activities than the natural product. however, neither (−)-polyalthic acid ( ) nor any of its derivatives was leishmanicidal against promastigote forms of l. donovani. moreover, copaiba diterpenoids revealed antibacterial activity against both gram-positive and gram-negative bacteria, with pronounced effects against bacillus subtilis and five different streptococcus species (tincusi et al. ; souza et al. ). in our previous study, we reported strong inhibitory effects against the two clinically relevant bacterial strains methicillin-resistant staphylococcus aureus (mrsa) and enterococcus faecium for three diterpene acids, of which one was kaurenoic acid ( ) (pfeifer barbosa et al. ). in the same study, antidermatophytic activity against two trichopyhton species was detected for (−)-polyalthic acid ( ), the major diterpenoid in the copaifera reticulata oleoresin, along with weak [(−)-polyalthic acid, ] to moderate (kaurenoic acid, ) cytotoxic effects against six cancer cell lines. these findings prompted us to prepare semi-synthetic derivatives of both compounds in an attempt to enhance their antimicrobial and cytotoxic properties and to get more insights into their structure-activity-relationships (sar). furthermore, additional screenings were performed to discover new lead compounds against both human and plant pathogens. (−)-polyalthic acid ( ) and kaurenoic acid ( ) were isolated from the oleoresin of copaifera reticulata ducke, fabaceae, as described in our previous studies (Çiçek et al. ; pfeifer barbosa et al. ) . n-chlorosuccinimide ( %) and triphenylphosphine (reagentplus, %), sulphuric acid (puriss., analytical grade) and lc-ms grade formic acid were purchased from sigma-aldrich co., st. louis, mo, usa. hydrochloric acid ( %, analytical grade) was obtained from honeywell, seelze, germany, while sodium hydroxide solution ( n) was purchased from carl roth gmbh, karlsruhe, germany. diethylamine (for synthesis), acetone (analytical grade), methanol (gradient grade or lc-ms grade) and water (lc-ms grade) as well as other analytical grade solvents used for purification were obtained from vwr international gmbh, darmstadt, germany. solid phase extraction (spe) columns (chromabond sb ml/ mg) were obtained from macherey-nagel gmbh & co. kg, düren, germany. deuterated methanol (lot p , . %), deuterated dmso (lot s , . %) and deuterated chloroform (lot q , . %) for nmr spectroscopy were obtained from eurisotop gmbh, saarbrücken, germany. conventional -mm sample tubes were purchased from rototec-spintec gmbh, griesheim, germany. semi-synthetic derivatives of compounds and were prepared in -ml v-vials (wheaton, millville, nj, usa) using a ret basic magnetic stirrer with integrated heater (ika-werke gmbh & co. kg, staufen, germany). lc-dad-elsd analysis was accomplished as stated in Çiçek et al. ( ) ; lc-ms and gc-ms analyses were conducted as described in pfeifer barbosa et al. ( ) . highresolution ms spectrum was recorded on microtof ii-high-performance tof-ms system (bruker®, billerica, ma, usa) equipped with an electrospray ionisation source. specific rotation of the compounds measured in methanol on a jasco p- polarimeter (jasco, pfungstadt, germany). nmr spectra were recorded using a bruker avance iii nmr spectrometer operating at mhz for the proton channel and mhz for the c channel with a mm pabbo broad band probe with a z gradient unit at k (bruker biospin gmbh, rheinstetten, germany). reference values were . ( h) and . ppm ( c) for methanol as well as . ( h) and . ppm ( c) for dmso, respectively. the compound ( μmol) and triphenylphosphine were dissolved in ml of dichloromethane, and the mixture was cooled in an ice bath. after adding μmol of n-chlorosuccinimide in small portions, the mixture was vigorously stirred for min at room temperature. diethylamine ( μmol) in μmol of pyridine was added, and the mixture was stirred for another min at room temperature. the mixture was subsequently concentrated under reduced pressure, and the by-products were removed by filtration and solid phase extraction using hexane as solvent to afford . mg of (−)-polyalthic acid-n,n-diethylamide ( a) and . mg kaurenoic acid-n,n-diethylamide ( a). the formation of ester was accomplished in the same manner except that diethylamine was substituted with methanol in the last reaction step. this reaction yielded . mg (−)-polyalthic acid methyl ester ( b) and . mg kaurenoic acid methyl ester ( b). the compound ( μmol) was dissolved under heating in a mixture of μl of water and μl of m sodium hydroxide. the solution was subsequently cooled to °c, and mg of ice was added before adding a cold solution of . mg potassium permanganate in μl of water over a period of min. the mixture was kept at to °c for another min before the precipitate was filtered off and washed with hot water. the filtrate was acidified with acetic acid, and the resulting precipitate was filtered of and washed with cold water and dried to afford . mg of ( s, s)- , -epoxy- , -dihydroxy- ( ), -entlabdadien- -oic acid [( s, ar, s, s, as)- -[ -(furan- yl)ethyl]- , a-dimethyl- -hydroxy- -hydroxymethyl- , , , , , a-hexahydro- h-naphthalene- -carboxylic acid] ( c) and . mg of α, -dihydroxy-ent-kauran- -oic acid ( c), respectively, after recrystallisation from a mixture of hexane and tert-butyl methyl ether. compound ( mg) was dissolved in a mixture of μl acetone, μl water and μl hydrochloric acid ( %) and kept at °c for h under stirring. the solution was then transferred to a beaker containing ml of water ,and the resulting precipitate was filtered and recrystallised from acetone to obtain . mg of α-hydroxy-ent-kauran- -oic acid ( d). compound ( mg) was dissolved in a mixture of μl methanol and μl sulphuric acid and kept at °c for h under stirring. the solution was subsequently cooled to − °c and the resulting precipitate was filtered and recrystallised from methanol to obtain . mg of α-methoxy-ent-kauran- -oic acid ( e). the samples were tested against the eskape panel of multidrug resistant bacterial human pathogens, including the grampositive bacteria enterococcus faecium (dsm ) and methillicin-resistant staphylococcus aureus (mrsa, dsm ), and the gram-negative bacteria klebsiella pneumoniae (dsm ), acinetobacter baumannii (dsm ), pseudomonas aeruginosa (dsm ) and escherichia coli (dsm ). furthermore the activity of the samples against four phytopathogenic bacteria, pseudomonas syringae (dsm ), xanthomonas campestris (dsm ), erwinia amylovora (dsm ) and ralstonia solanacearum (dsm ), and against two human pathogen yeasts, candida albicans (dsm ) and cryptococcus neoformans (dsm ), was carried out. all test strains were purchased from leibniz institute dsmz-german collection of microorganisms and cell cultures, braunschweig, germany. the bacteria were cultivated in tsb medium ( . % tryptic soy broth, . % nacl), except e. faecium which was cultivated in m medium ( % trypticase soy broth, . % yeast extract, ph . - . ) and r. solanacearum which was grown in m ( % glucose, . % peptone from soymeal, . % malt extract, . yeast extract). the cultivation of c. albicans took place in m / ( . % glucose, . % peptone from soymeal, . % malt extract, . % yeast extract) and for c. neoformans m was used as well. overnight cultures of the test organisms were prepared and diluted to an optical density ( nm) of . - . . to prepare the assay, the test samples ( mg/ml dmso stock solution) were dissolved in medium and transferred into a -well microtiter plate and μl of the cell suspension cultures was added to each well. the final assay concentration of the substances was μg/ml. the inoculated microplates were incubated for h at °c and rpm (e. faecium without shaking), or °c and rpm for h for the phytopathogen bacteria and c. neoformans, respectively. to detect the inhibitory effect of the substances μl of a resazurin solution ( . mg/ml phosphate-buffered saline) was added to the microplates and incubated again for - min before the fluorescence signal ( nm/ nm) was measured using the microplate reader (tecan infinite m ). for e. faecium the ph indicator bromocresol purple was used to determine the acidification caused by growing, and for r. solanacearum and c. neoformans the optical density at nm after incubation time was recorded using the microplate reader as well. the resulting values were compared with a positive control (chloramphenicol for the bacteria, except r. solanacearum where tetracycline was used, nystatin for c. albicans and amphotericin b for c. neoformans) and a negative control (no compound) on the same plate. for ic determination, a dilution series was prepared and the ic value was calculated by excel or graphpad prism as the concentration that shows % inhibition of viability on the basis of a negative control (no compound). measurements of cytotoxic activity and activity against dermatophytic fungi were performed as described in our previous study (pfeifer barbosa et al. ). preparation of semi-synthetic derivatives a- c and a- e semi-synthetic derivatives of (−)-polyalthic acid ( ) and kaurenoic acid ( ) were prepared on a small scale using -ml v-vials and applying between and mg of diterpene acid. derivatisations included the carboxyl group and the exocyclic methylene group, respectively. thus, n,n-diethyl amide ( a and a) and methyl ester ( b and b) as well as dihydroxy derivatives ( c and c) of both parent molecules were prepared as described in the experimental section. additionally, one monohydroxy derivative ( d) and one methoxy derivative ( e) of kaurenoic acid were prepared. all products were analysed by tlc using different mixtures of dichloromethane and ethanol as solvent and vanillin-sulphuric acid as spray reagent. amides and esters were additionally checked by gc-ms , whereas the other derivatives were analysed by uhplc-dad/elsd and uhplc-ms . for structure elucidation, one-and two-dimensional nmr experiments were performed. amidation and esterification of diterpenoids was accomplished using n-chlorosuccinimide and triphenylphosphine for conversion of the carboxylic acids and adding either n,n-diethylamine or methanol in pyridine to the reaction mixture as described in the protocols of frøyen ( frøyen ( , . the experiments were therefore downsized to μmol of reactants (instead of mmol) except for dichloromethane, of which ml (instead of ml) was used. purification was conducted as described in the protocols (concentration, filtration and washing with diethyl ether, silica gel column with ether as eluent). however, for the last purification step another silica gel column with n-hexane as eluent was preferred over crystallisation or distillation, respectively. all four derivatives were subsequently analysed by gc-ms showing the expected m/z ratios of ( a), ( a), ( b), and ( b) and by nmr spectroscopy comparing their spectra to literature data (narayanan and venkatasubramanian ; vieira et al. ; mizuno et al. ; santos et al. ). the methyl esters ( b and b) were additionally identified by comparing their mass spectra to the spectra available in the nist database. dihydroxy derivatives of compounds and were prepared with potassium permanganate in alkaline medium following a protocol for the hydroxylation of lambertianic acid (chernov et al. ) . lambertianic acid is an epimer of (+)-polyalthic acid at position c- and thus a stereoisomer of (−)-polyalthic acid ( ), showing different configurations at positions c- , c- , and c- . using scales of μmol of diterpenoid instead of mmol, the reaction yielded ( s, s)- , -epoxy- , -dihydroxy- ( ), -ent-labdadien- -oic acid ( c), a previously undescribed compound, and the known compound α, -dihydroxy-ent-kauran- -oic acid ( c). whereas the latter compound was identified by comparison of ms and nmr spectra to literature data (song et al. ) , structure elucidation of compound c is described in "structure elucidation and configuration of compound c". preparation of α-hydroxy-ent-kauran- -oic acid ( d), α-methoxy-ent-kauran- -oic acid ( e) was performed using acid-catalysed hydroxylation and methoxylation, respectively (cavalcanti et al. ). for these reactions, amounts of mg were applied instead of g and the resulting products were identified by comparison of their ms and nmr data to literature (chen et al. ; yaouba et al. ). as mentioned above, compound c was synthesised using a protocol (chernov et al. ) reported for the transformation of lambertianic acid, a stereoisomer of (−)-polyalthic acid ( ). the side chain at c- of lambertianic acid is βoriented leading to α-oriented hydroxylation at position c- , due to steric hindrance by the furanoethyl group. in contrast, (−)-polyalthic acid ( ) shows an α-oriented side chain at c- ; hence, the resulting hydroxy group at position c- had to have β-orientation and the hydroxymethylene group α-orientation instead. the α-orientation of the hydroxymethylene group was confirmed using noesy, where noe correlations were observed for the protons at c- on both the methylene group at c- and the c- methyl group. thus, the structure was identified as ( s, s)- , -epoxy- , -dihydroxy- ( ), -ent-labdadien- oic acid or (rel- s, ar, s, s, as)- -[ -(furan- -yl)ethyl]- , a-dimethyl- -hydroxy- -hydroxymethyl- , , , , , ahexahydro- h-naphthalene- -carboxylic acid, respectively (see "stereochemistry of polyalthic acid, daniellic acid and lambertianic acid"). same as (−)-polyalthic acid ( ) as described above, hydroxylation of (−)-polyalthic acid ( ) followed a protocol of chernov et al. ( ) , who used lambertianic acid as starting material. however, during the course of the structure determination of compound c and assignment of the absolute configuration, some inconsistencies have been observed with regard to the stereochemistry of polyalthic acid and its structural isomers, which we attempted to resolve hereafter. the first two of these four compounds to be reported were (−)-polyalthic acid ( ) and daniellic acid in . as the absolute configuration has not been determined yet, (−)polyalthic acid is correctly described as (rel- s, as, r, as)- -[ -(furan- -yl)ethyl]- , a-dimethyl- -methylenedecahydronaphthalene- -carboxylic acid. daniellic acid was isolated from daniellia oliveri (fabaceae, caesalpinioideae) (haeuser and ourisson ) , whereas (−)-polyalthic acid ( ) was obtained from polyalthia fragrans (annonaceae) and named polyalthic acid (gopinath et al. ) . both compounds were attributed to possess the "wrong" configuration corresponding to an ent-labdane scaffold and showing negative optical rotation values. both compounds are epimers, only differing in the orientation of the carboxyl group at position c- , which is axial for daniellic acid and equatorial for (−)polyalthic acid ( ). five years later, the first report for lambertianic acid, isolated from pinus lambertiana (pinaceae), was made (dauben and dauben and german ) . lambertianic acid was found to be the optical antipode of daniellic acid and thus to be the first furan diterpene with the so-called normal labdane configuration. the last of the four isomers was the optical antipode of (−)-polyalthic acid ( ), isolated from sequoia semperivirens, taxodiaceae (ohta and nawamaki ) . however, instead of choosing an original trivial name (e.g. semperiviric acid), the authors named the compound (+)-polyalthic acid, being (rel- r, ar, s, ar)- -[ -(furan- -yl)ethyl]- , a-dimethyl- -methylenedecahydronaphthalene- -carboxylic acid. thus, from then onwards, two forms of polyalthic acid existed in the literature. except for the last years, where (+)-and (−)-or ent-prefixes were used to differentiate between the two enantiomers (bardají et al. ; borges et al. ; carneiro et al. ; Çiçek et al. ; pfeifer barbosa et al. ; senedese et al. ) , only one publication in almost four decades adopted such prefixes (miyazawa et al. ) . the missing stereochemistry did not represent a problem as long as the studies were dealing with copaifera, because the ent-configuration is abundant in this genus (leandro et al. ). however, some wherefore, the identity of the right enantiomer remained unknown. thus, reporting the right configuration for polyalthic acid is absolutely necessary. another problem is that the introduction of (+)-polyalthic acid and the recent use of the ent-prefix for differentiation of the enantiomers seems to have caused confusions in some large databases, such as scifinder™ or reaxys™. for example, when searching for structure templates of polyalthic acid in the reaxys database, three suggestions are delivered, of which two display (−)-polyalthic acid and the other one shows (+)-polyalthic acid. however, by using the name entpolyalthic acid, also two structures are displayed, one for (−)-polyalthic acid and the second one for daniellic acid. for both compounds their semisystematic names are given along with twelve references per compound, thus giving equal priority to the correct and the incorrect structure. by looking for chemical structures in the scifinder database, the name polyalthic acid leads to the structure of (−)-polyalthic acid, which is historically correct, whereas the search for entpolyalthic acid gives the structure of daniellic acid and thus the wrong compound. alas, these inconsistencies or confusions, respectively, led to several publications showing the structure of daniellic acid instead of (−)-polyalthic acid (leandro et al. , Çiçek et al. , da silva et al. pfeifer barbosa et al. ) . the mentioned problems can be partly dealt with by using the prefixes of the respective optical rotations, leading to the exact structures for both (+)-and (−)polyalthic acid in the reaxys database and also to the correct structure for (+)-polyalthic acid with the scifinder database. only the name (−)-polyalthic acid does not yield any results with scifinder, but as the name polyalthic acid leads to the structure of (−)-polyalthic acid and the optical rotation can be retrieved from the experimental details, at least no misinformation is spread. we therefore suggest to use the prefix for the optical rotation only. in our previous study, we investigated the effect of copaiba diterpenoids against a- , hepg , ht- , hct- , a- and mda-mb- cancer cell lines as well as non-cancerous hacat cell lines resulting in weak to moderate non-selective cytotoxic effects with ic values of . to . μg/ml for (−)-polyalthic acid ( ) and . to . μg/ml for kaurenoic acid ( ) (pfeifer barbosa et al. ) . thus, the effect of derivatising either the carboxyl group or the exocyclic methylene group was the main aim of the present study. in a first screening at a concentration of μg/ml no effects for derivatives without the exocyclic methylene group ( c and c- e) were observed (table s ). compounds d and e were earlier tested on four different cancer cell lines (k , hl- , mda-mb and sf ) as well as on healthy peripheral blood mononuclear cells and compared with kaurenoic acid (cavalcanti et al. ). also here, the cytotoxic effect disappeared after derivatisation; wherefore, the authors hypothesised that the exocyclic methylene group is crucial for the cytotoxic effect. however, also derivatisation of the carboxyl group (compounds a, b, a and b) did not result in total growth inhibition at μg/ml, but rather affected healthy hacat cells at this concentration. because of the only low cytotoxic activity of compounds and found in our previous study and the not too promising results of the preliminary screening, we decided to focus on the antimicrobial effects of our compounds. due to the strong effects of (−)-polyalthic acid ( ) and especially kaurenoic acid ( ) against e. faecium and methicillinresistant staphylococcus aureus (mrsa) (pfeifer barbosa et al. ) , additional assays on klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa and escherichia coli as well as phytopathogens pseudomonas syringae, xanthomonas campestris, erwinia amylovora and ralstonia solanacearum were included in our investigations (table s ) . however, at a concentration of μg/ml, activity of compounds and as well as their derivatives against the additionally investigated organisms was rather low or nonexistent in contrast to the already known effects against e. faecium and mrsa. but also against the latter two strains derivatisations did not lead to an improved activity (table ) . here, only compound c showed (at least moderate) effects, with ic values of . μg/ml against e. faecium and . μg/ml against mrsa. in contrast, (−)-polyalthic acid ( ) and kaurenoic acid ( ) showed ic values of . and . μg/ml against e. faecium as well as . and . μg/ml against mrsa, respectively. thus, both the carboxyl group and the exocyclic methylene group seem to be pivotal for the activity against these two clinically relevant strains. (−)-polyalthic acid ( ) was the most potent compound against trichopyhton species dermatophytes in our previous study, with ic values of . μg/ml against t. rubrum and . μg/ml against t. mentagrophytes, respectively (pfeifer barbosa et al. ) , while kaurenoic acid ( ) only showed weak ( . μg/ml) to moderate effects ( . μg/ml) against these two strains ( table ) . derivatisation of the two natural products this time revealed a different picture, leading to one equally potent ( b) and another still moderately active compound ( a) against t. rubrum, with ic values of . μg/ml for (−)-polyalthic acid methyl ester ( b) and . μg/ml for (−)-polyalthic acid n,n-diethylamide ( a), respectively. notably, this effect was not observed against t. mentagrophytes, where the ic value was increased by the factor five. for kaurenoic acid ( ), a similar picture was observed. here, amidation ( a) and esterification ( b) yielded even more active compounds against t. rubrum, though on an overall higher level. still, also against t. mentagrophytes the activity was drastically reduced by derivatisation of the carboxyl group. of the remaining derivatives, only compound c showed activity, with an ic of . μg/ml against t. rubrum and . μg/ml against t. mentagrophytes. the compounds were furthermore investigated for their inhibitory potential against two yeasts, candida albicans and cryptococcus neoformans. while the activity against c. albicans was negligible (table s ) , the results against c. neoformans revealed interesting findings for compounds , a and b. all three compounds showed similar ic values, which were in the range of . to . μg/ml (table ) . however, below this concentrations the activity of (−)-polyalthic acid ( ) decreased rapidly in contrast to its two non-acidic derivatives ( a and b) (fig. ) . (−)-polyalthic acid ( ) and kaurenoic acid ( ) as well as their semi-synthetic derivatives ( a- c and a- e) were investigated for their cytotoxic, antibacterial and antifungal properties, with different outcomes. while the previously discussed importance of an exocyclic methylene group for the cytotoxic effect was also apparent in our study, we furthermore found that both the exocyclic methylene and the carboxylic acid group were necessary for the effect against e. faecium and mrsa. however, different results were obtained with regard to the antifungal activity of the two natural products and their derivatives. for t. mentagrophytes, the picture was somehow similar to the antibacterial assays, where the best antifungal activity was achieved when both an exocylic methylene group and a carboxylic acid were present. in contrast, the presence of an carboxyl group was not required for activity against c. neoformans and t. rubrum, as displayed by the similar ic values of , a and b. however, the low ic value of compound c clearly shows that at least the methylene group is crucial for the effect. besides gaining further insights into differential biological activities and some sars for copaiba diterpenoids, we present a novel semi-synthetic derivative of (−)-polyalthic acid. in the course of verifying the relative configuration of the new compound, we discovered ambiguities regarding the stereochemistry of polyalthic acid by two commonly used databases, which also led to incorrect chemical structures in several publications. we thus suggest to only use the terms (+)-and (−)- table antifungal effects of (−)-polyalthic acid ( ), kaurenoic acid ( ) and their derivatives. the ic values are in μg/ml. positive controls were clotrimazole (trichophyton rubrum and t. mentagrophytes) and amphotericin b (cryptococcus neoformans) t. rubrum t. mentagrophytes c. neoformans compound . ± . . ± . . ± . compound a . ± . . ± . . ± . compound b . ± . . ± . . ± . compound c polyalthic acid to differentiate between the two enantiomers in order to hopefully avoid such mistakes in future publications. copaifera reticulata oleoresin: chemical characterization and antibacterial properties against oral pathogens copaifera duckei oleoresin and its main nonvolatile terpenes: in vitro schistosomicidal properties development and validation of a rapid and reliable rp-hplc-pda method for the quantification of six diterpenes in copaifera duckei, copaifera reticulata and copaifera multijuga oleoresins kauren- -oic acid induces dna damage followed by apoptosis in human leukemia cells ent-kaurane diterpenoids from annona glabra synthetic transformations of higher terpenoids: xii.* transformation of lambertianic acid into , -epoxyabietane diterpenoids quantification of diterpene acids in copaiba oleoresin by uhplc-elsd and heteronuclear two-dimensional qnmr development of a validated ultra-high-performance liquid chromatography tandem mass spectrometry method for determination of acid 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cell lines acknowledgements the authors thank jana heumann for the measurements of the cell culture assays and fengjie li and claudia welsh for high-resolution ms and optical rotation measurements. author contribution ssc, aws and ug conducted the experiments; dt supervised the bioactivity testing; aws, ug and dt revised the manuscript; ssc designed the study and wrote the manuscript. all authors approved the final version of the manuscript.funding information open access funding provided by projekt deal. conflict of interest the authors declare that they have no conflict of interest. the authors declare that no experiments were performed on humans or animals for this study. the authors declare that no patient data appear in this article. the authors declare that no patient data appear in this article.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/ . /. key: cord- -ml luc t authors: sollner, johannes; grohmann, rainer; rapberger, ronald; perco, paul; lukas, arno; mayer, bernd title: analysis and prediction of protective continuous b-cell epitopes on pathogen proteins date: - - journal: immunome res doi: . / - - - sha: doc_id: cord_uid: ml luc t background: the application of peptide based diagnostics and therapeutics mimicking part of protein antigen is experiencing renewed interest. so far selection and design rationale for such peptides is usually driven by t-cell epitope prediction, available experimental and modelled d structure, b-cell epitope predictions such as hydrophilicity plots or experience. if no structure is available the rational selection of peptides for the production of functionally altering or neutralizing antibodies is practically impossible. specifically if many alternative antigens are available the reduction of required synthesized peptides until one successful candidate is found is of central technical interest. we have investigated the integration of b-cell epitope prediction with the variability of antigen and the conservation of patterns for post-translational modification (ptm) prediction to improve over state of the art in the field. in particular the application of machine-learning methods shows promising results. results: we find that protein regions leading to the production of functionally altering antibodies are often characterized by a distinct increase in the cumulative sum of three presented parameters. furthermore the concept to maximize antigenicity, minimize variability and minimize the likelihood of post-translational modification for the identification of relevant sites leads to biologically interesting observations. primarily, for about % of antigen the approach works well with individual area under the roc curve (aroc) values of at least . . on the other hand a significant portion reveals equivalently low aroc values of < = . indicating an overall non-gaussian distribution. while about a third of antigens are seemingly intangible by our approach our results suggest the existence of at least two distinct classes of bioinformatically detectable epitopes which should be predicted separately. as a side effect of our study we present a hand curated dataset for the validation of protectivity classification. based on this dataset machine-learning methods further improve predictive power to a class separation in an equilibrated dataset of up to %. conclusion: we present a computational method to automatically select and rank peptides for the stimulation of potentially protective or otherwise functionally altering antibodies. it can be shown that integration of variability, post-translational modification pattern conservation and b-cell antigenicity improve rational selection over random guessing. probably more important, we find that for about % of antigen the approach works substantially better than for the overall dataset of proteins. essentially as a side effect our method optimizes for presumably best applicable peptides as they tend to be likely unmodified and as invariable as possible which is answering needs in diagnosis and treatment of pathogen infection. in addition we show the potential for further improvement by the application of machine-learning methods, in particular random forests. the applicability of peptides for the generation of preventive vaccines, therapeutics and diagnostics is an actively investigated field. although historically disfavored the application of peptides in vaccine design is currently experiencing a renaissance [ ] . while the focus is often on tcell responses especially the generation of b-cell responses is of relevance against certain pathogens such as hiv to prevent initial infection [ ] . it is thus not surprising that since the early days of computational biology scientists have attempted to predict the relevance of protein domains and peptides in several areas of application. initial hallmarks of the field are represented, among many others, by work of hopp and woods [ , ] . during the following and more recent years various methods and problems concerning the prediction of continuous b-cell epitopes have been proposed [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . recently the usability of amino acid scales for the prediction of b-cell epitopes has been profoundly questioned [ ] and common standards regarding the validation of epitope predictions have been discussed [ ] . generally, b-cell antigenicity predictions should probably be understood as a measure of the likelihood to develop antibodies against a particular determinant or part of a surface, rather than another. in addition, most proposed classifiers of continuous epitopes are ultimately a composite of accessibility and charge-interaction potential prediction with a strong focus on delivering a few experimentally applicable peptides rather than an overall complete probability distribution for raising antibodies. in addition, continuous epitopes make up an undefined but presumably small part of the complete "epitope space" of an antigen. this even so when assuming distinct epitopes rather than a continuous surface and accepting dominant continuous elements of structural epitopes as continuous epitopes. in this study we extend previous work by investigating a subgroup of continuous b-cell epitopes, namely protective continuous epitopes. this aspect has to the best of our knowledge not been systematically tackled so far. from a biological point of view several principles should govern the availability and evolutionary behavior of protective amino-acid sites on proteins. one of the questions we ask is whether these principles or constraints lead to signals which can be used for predicting or rather detecting candidate epitopes. we assume that an antibody (and hence it's epitope) is protective because the function of the antigen (target protein) is inhibited and the activity of the organism is thus reduced. or alternatively because immunological processes are activated leading to the destruction of the organism. the prior (protectivity class i) might most likely be expected in adhesion molecules or pathogenicity factors like matrix degrading proteases and toxins. the second category (protectivity class ii) would primarily refer to downstream events of antibody induced complement activation such as pore formation and opsonization leading to phagocytosis. it can be considered likely that the two mechanisms would often lead to differently characterized epitopes. consequently as different functional constraints can be expected separate strategies for detecting them may be required. basically any protein of high expression and density on the surface with at least one good epitope can be the target of class ii protectivity. we define a "good" epitope as a surface area of high interaction potential, shape complementarity to the basic layout of an antibody [ ] and dissimilar to self. predicting that class therefore also requires to assess (or estimate) the density of a protein on the cell during pathogenesis, optimally experimentally or by inference from related organisms. a practically applicable continuous epitope could then be any exposed, possibly evolutionarily highly variable loop with an amenable antigenicity and solvent-accessibility profile. class ii protectivity may often be comparably straightforward to predict as soon as a target protein has been identified because selection of high scoring b-cell epitope scores often seems to be relatively straightforward and selection routines primarily falter in the domain of suboptimal scores as has been indicated by sollner and mayer [ ] . however, immunologically subdominant or even cryptic b-cell epitopes can be of special interest regarding protectivity and inter-strain cross-reactivity [ ] and are sometimes consistently immuno-silent during natural infection [ , ] . as a consequence we focus on conserved and therefore presumably functionally constrained epitopes without post-translational modifications which still exhibit amenable antigenicity scores. regarding the previous definition these epitopes may often fall into class i of protective continuous epitopes. such principles are primarily valid for pathogens already adapted to the host. organisms in the process to adapt to new hosts or receptors can undergo significant alterations in their antigenic structure as has been demonstrated for sars virus [ ] [ ] [ ] and hiv [ ] , respectively. we speculate that by means of functional constraints centers of biological activity can exert conserving pressure on closely associated potential epitopes while less relevant regions can be more variable. it may also be viable to suggest that conservation of posttranslational modification patterns may be different when comparing highly variable exposed loops and sites of functional relevance as modifications can play a major role in the masking of protective epitopes [ , ] but may often be undesired near functional centers. as class ii protectivity is to a certain degree already approached by standard b-cell epitope prediction (the maximization of antigenicity) and on the other hand depends on a bioinformatically more elusive factor (expression levels of pathogen protein) we see reason to focus on the prediction of conserved, functionally constrained epitopes (class i protectivity). this work assesses in how far correlation between antigenicity, variability, post-translational modifications and protectivity/functional relevance can be put to use in a predictive model without the availability of d data. to compensate for the lack of d data multiple alignments of selected proteins are harnessed to derive information regarding the conservation of post-translational modification motifs as well as sequence variability per-se. we believe that understanding evolutionary "movement" of pathogen proteins allows insights into the importance of potential epitopes and we interpret such importance as indicator of protectivity. classification into presumably protective or non-protective epitopes is conducted using three independently determined parameters: predicted b-cell antigenicity, sequence variability and conservation of post-translational modification motifs. as described in the methods section used antigenicity, variability and motif-conservation scores are based on multiple-alignments i.e. each sequence contributes to a composite value. all values are determined within -mers which slide over the alignments and overlap by amino-acids. b-cell antigenicity and sequence variability are averaged within these mers. we chose this size to use an intermediate between common assumptions about sizes of continuous epitopes (usually between and amino-acids). the maximum ratio of post-translational modifications over all constituent alignment columns is used within the same area. in other words, for the prior two the -mer values are calculated as the averages over the averages calculated from individual alignment columns. the latter seeks the maximal ratio of possibly modified amino-acids over all alignment-columns in the area because it presumably is most indicative of actual modifications. an overview over the major steps in the workflow has been highlighted in figures and . logistic regression models based on pca of amino acid propensity scales we examined regression models based on amino acid propensity scales. as described in methods each of these scales characterizes amino acid residues regarding specific properties by assigning a value. from this representation, the information of amino acid propensity scales was transformed into principal components applying principal component analysis. based on these principal components, a logistic regression model was derived. it is in the following mentioned as pca . briefly, a dataset of proteins was obtained by clustering all bcipep sequences with at least % identity. this step removed redundancies potentially biasing validation procedures. in a second step non-antigenic amino-acids were randomized (maintaining the original amino-acid composition) to avoid the misclassification of unknown epitopes. each amino-acid of those proteins was then parameterised using all of the described components. validation on the training set by bootstrap analysis in combination with a logistic regression indicated an aroc value of . . to obtain an unbiased impression of the performance of the pca classifier compared to an accepted gold standard such as abcpred [ ] an independent validation-dataset published by blythe and flower was used. to make methods compatible abcpred predictions were run with standard settings except that the threshold was lowered to . . scores reported for peptides by abcpred were assigned to each comprising amino-acid where larger values superseded the prediction of an overlapping peptide. aroc values were calculated. both methods performed close to random (as is not too astonishing concerning the findings by blythe et.al), with aroc values of . and . for pca and abcpred, respectively. these results are relativated later in this work when using only potentially relevant domains of a protein antigen, indicating systematic problems of the way b-cell epitope prediction validation is usually conducted. to assess the effect of domain accessibility filtering (masking) from protectivity prediction aroc values of the described linear parameter combination before and after filtering were compared. whereas the median aroc over all protein was determined as . before masking of presumably inaccessible trans-membrane or cytoplasmic domains it increased to . afterwards. while the aroc before masking is comparable to the one obtained by antigenicity prediction on the blythe and flower validation dataset the improvement to . strongly indicates the benefit of the procedure. domain accessibility filtering can be considered an aspect of fair evaluation in b-cell epitope classification as a whole as it can be assumed that the figure shows the first part of the overall workflow applied in this project figure the figure shows the first part of the overall workflow applied in this project. the figure shows the second part of the overall workflow applied in this project figure the figure shows the second part of the overall workflow applied in this project. continuous epitopes of accessible domains are more likely mapped or otherwise reported than others, besides the protectivity aspect. while it may be argued that inclusion of uniprot data into the process adds an aspect of human intervention we see that many data-sources can and are used for the annotation of putative ectodomains. among those are also experimental data, which is in itself not a problem for bioinformatical validation strategies as long as the validation dataset was not engineered to fit these data particularly well. that is not the case. the domain filter was simply built by manually collecting different datasources according to simple rules as we considered automated harvesting for proteins and unnecessary effort. to evaluate the prediction of protective linear epitopes a new validation dataset was generated as described in methods and data. briefly, the iedb resource was queried for pathogen proteins with linear antibody determinants which lead to a biological effect upon interaction. in viral polyproteins commonly only the dominant surface proteins were used as could be expected for a newly sequenced pathogen without in detail knowledge. to limit predictions to candidate regions (i.e. possibly immunologically accessible regions) domain accessibility filtering was applied. to do so domain data regarding polyproteins, trans-membrane structures or signal-peptides (predicted or experimentally determined) were taken from uniprot [ ] or predicted using the tmhmm v. . [ ] and signalp . servers [ ] . domains which were not considered relevant for protective b-cell responses were intracellular domains of trans-membrane proteins, the first amino-acids of a putative extra-cellular domain after a trans-membrane region and leader-peptides. briefly, proteins were completely scored for antigenicity/protectivity but amino-acid scores in regions outside domains assumed to be surface exposed were set to thus leading to a generic classification as non-protective. masked amino-acid stretches were still considered for roc calculation to reflect the impact of the analysis as a whole. aroc measures were thus based on completely scored proteins partially set to . for each amino-acid of proteins in the protectivity dataset variability and percentage of modifications (ratio of sequences which carry a modification motif indicating this specific amino acid versus all aligned sequences) based on multiple alignments were calculated as described earlier. finally the average predicted antigenicity was calculated for each alignment column. each of the three sub-scores was then rescaled between and for easier comparability. to asses the power of score-combinations antigenicity, -variability and -ptm ratio were summed for all overlapping -mers where the score was assigned to the central amino-acid. see table for aroc values of individual proteins using any of the three sub-scores alone as well as the linear combination (sum score). the last row of the table indicates the overall aroc when analyzing all unmasked aminoacids together. considering the aroc merged value (resulting from the concatenation of all putatively accessible domains after scoring) antigenicity alone outperforms all other scores, including the combined one. yet, although table indicates overall better performance of antigenicity, the distribution of aroc values for individual proteins indicates a different view. table shows that for the combined score fewer proteins fall in the very low aroc area ( versus are < = . ) whereas substantially more ( versus ) fall in the aroc area we considered good (> = . ). eight of the proteins used for protectivity prediction are similar or identical to sequences used for training the pca classifier (as identified using blastp). to evaluate this bias/contamination the aroc value medians of truly independent versus dependent (contaminated) proteins has been listed in table . interestingly and against expectation contaminated proteins underperform when measured by median compared to independent entries leading to the observation that no pre-emptive separation of shared sequences is necessary in this case. for an overview of aroc distributions see the histograms in figure . taken together or ( or %) of investigated proteins reveal aroc values > = . depending on whether contaminated proteins are neglected or not. this population is characterised by mean and median aroc values of . and . , respectively. we interpret this as a roughly % probability that protectivity prediction will significantly enhance selection of peptides for the stimulation of protective immune responses for a particular protein, given that a relevant protein has been identified beforehand. we also want to point out that overall up to % of proteins show aroc values < = . or > = . . as described in methods a "synthesis score" representing the number of peptides required for likely experimental success is a relevant readout concerning minimization of experimental cost. for vaccine design it is crucial to limit the number of synthesized peptides which enter experimental validation to a practically feasible amount. how many peptides can be synthesized depends on budget and resources as well as ethical considerations regarding the number of lab-animals, entities which are tied to the number of proteins (antigens) to be screened as well as available time. to provide such a measure we defined the size of peptides to be synthesized as -mers (a size commonly used by us) and the minimal overlap with protective epitopes to call it a hit as five amino-acids. selected peptides could overlap, but each new epitope had to be centered on a hitherto uncovered amino-acid. these central amino acids were selected by maximizing the described sum score of antigenicity + ( -variability) and ( -maximal modification ratio). following this procedure we determined how many peptides had to be selected per protein to provide a likely working selection for at least % of screened proteins. the presented combination method required six peptides to be selected compared to eight for random picking. this compares to five versus eight peptides when only the proteins with aroc > = . were looked at. note that random picking of central-amino acids was also restricted to the regions not filtered out by domain-exclusion to warrant fairness in the comparison. proteins highlighted in bold are markedly homologous or identical to sequences used for the training of the pca antigenicity classifier. values derived from those proteins are denoted as "contaminated". the last five rows compare overall (global) classification performances of concatenated (merged) proteins as well as mean and median values between individual parameters and the combined score. note that "merged" does not mean averaged and is thus biased by the length of individual proteins. conserved, optimally unmodified, continuous and protective or at least functionally altering epitopes. to assess the relationship of antigenicity, variability and modifications pearson correlation-coefficients were calculated. after domain-filtering all distinct (non-overlapping) protective regions were analyzed separately to obtain an idea whether a common trend could be identi-fied. in summary, overall average and median correlation were not significant between antigenicity, variability and modification ratio (generally < = . and > = - . ). for each combination a subset of epitopes showed high correlation, however. results have been summarized in table . note that no pair of features shows significant positive or negative correlation for more than % of the total the figure shows the distribution of aroc values for protectivity predictions solely based on antigenicity, ptm (post transla-tional modifications), variability as well as the sum score of the three figure the figure shows the distribution of aroc values for protectivity predictions solely based on antigenicity, ptm (post translational modifications), variability as well as the sum score of the three. note that like for the prediction of protectivity the used modification and variability scores (features) have been used as -value, so actual associations have to inverted as well. each field in the table contains a pair of numbers where the first and second indicate the number of epitopes associated with a pearson coefficient of < = - . and > = . , respectively. high numbers therefore indicate a strong degree of feature association. number of considered epitopic regions. however, for all pairs of sub-scores strong positive and negative associations exist. in the case of antigenicity and variability more than % of analyzed epitopes show strong correlation between the two, but at practically equal numbers positive and negative association. this means that roughly % of epitopes are either markedly antigenic and conserved or non-antigenic and variable. furthermore another % are markedly antigenic and variable or nonantigenic and conserved. protective epitopes seem to be tendencially positively correlated considering antigenicity and conservation of motifs for posttranslational modification while variability and the evolutionary constraint index (eci) are often negatively associated, as could be expected. unfortunately no association between protein functional class and type of correlation could be detected (data not shown). preliminary experiments evaluating the potential power of the correlation coefficient as a new parameter for protectivity prediction indicated close to random performance (data not shown). the previously described observation that for about % of proteins either substantially good or bad predictive power can be seen suggests that the described % correlated epitopes are distinct sets depending on the parameter-combination. alternatively it may also be that weaker correlations than - . and + . can still positively influence aroc. an unweighted linear combination score is a simple way to combine parameters without optimizing a model, i.e. as a first approach strategy or if non independent optimization dataset exists. to extend this strategy, albeit without the option to analyse the unbiased effect on the entire protectivity dataset, machine-learning procedures were applied. in particular, to analyse the validity of using all three parameters as well as the relative merit of the sum-score the protectivity dataset was converted into a format amenable for machine learning procedures such as decision trees. as described in the methods section the entire protectivity set of proteins was subjected to domain filtering to restrict to possibly accessible residues which were then compiled into the cml set. the class separation baseline of this set is , %, due to the massive domination of non-protective residues. using weka standard settings a c . decision tree achieved . % separation on the same set. because relative merits of machine learning are difficult to assess on strongly biased datasets such as cml, balanced and stratified training and validation sets (mts and mvs, respectively) were randomly sampled from cml to make independent validation possible. using the training set (mts) and again applying c . different parameter combinations were assessed. results can be seen in table and figure . the decision tree algorithm was applied because for single parameters this should basically be a search for the entropically optimal class-split whereas for parameter combinations also non-linear relationships (particularly the presence of distinct groups) can be captured. the table shows that each parameter contributes significantly and the best model comprises all three attributes as well as the sum-score as a fourth attribute. independent of variability alone shows the best class-separation among individual parameters. interestingly ptm alone yields no improvement over random classification while the combination with the remaining features significantly boots its impact. to assess the performance of a somewhat more sophisticated machine-learning technique validation using the validation set and cross-validation are compared in table for a c . tree and a random forest. obviously there is much potential for improvement over a single decision tree as the random forest achieves a class separation power of up to - % compared to - % using a single tree. analysis of the c . tree generated from the validation set indicates the merit of all four attributes including the sum-score as an additional parameter as all four were incorporated by the algorithm (tree not shown). in addition the resulting tree is astonishingly complicated ( leaves) when considering that only four parameters were used, again indicating the existence of different clusters. preliminarily, these groups may well be interpreted as distinct epitope signatures pointing towards different types of protective epitopes. in other words, different but recognizable epitope profiles should be considered. it also becomes obvious that a single unweighted combination score has its merit but is outperformed by variability alone and both of them by decision tree based multi-parameter models, at least in the stratified data representation. also, the sum-score proves to be an important additional component in the decision models as can be seen by the increase from % class separation to % class separation by inclusion of this extra feature. to exemplarily show how the described methods may be used for the prediction of continuous, protective epitopes a recently published relevant epitope on borrelia burgdorferi ospc has been used [ ] . the protein was selected because it was the first to show up in a new iedb query for functionally altering epitopes and because it shows no significant homology to any protein in the protectivity dataset. figure indicates the experimentally determined epitope (fat red bar) together with a masked signal peptide (fat gray bar) together with results from three predictive methods. the top-most plot shows the sum-score which does not obviously correlate strongly with the epitope. even after removal of isolated, positively predicted amino-acids (which can be considered as noise) both the c . prediction and the random forest predict amino acids inside the -mer epitope. as the random forest performed best during validation it is used for the selection of five -mer peptides each centered on a cluster of a least two amino-acids. one of these peptides covers more than % of the protective epitope. while the proposed methods do not excel on this independently chosen protein five selected peptides may be enough to sufficiently cover the relevant site. as a remark it is also of interest that the consensus prediction of the c . tree and the random forest would obviously reduce the selection to two peptides with the same epitope coverage. this work describes the generation of a novel b-cell classifier based on the logistic regression of amino acid parameters derived from principal component analysis of a commonly used parameter database. we extend the classical application of antigenicity classifiers from the prediction of continuous b-cell epitopes to the prediction of protectivity by introducing measured variability and predicted modification patterns into the concept. we could show that validation on the data-set by blythe and flower revealed close to random performance for both the gold standard abcpred as well as for pca , although our method exhibited marginally better performance in comparison. this result is clearly relativated as we could show far better performance on a different dataset when considering only potentially relevant domains. in detail, to assess the benefit of excluding presumably irrelevant domains from the prediction of protective continuous determinants a manual selection of protein regions was created based on standard bioinformatical tools. for this selection we used a curated set of proteins with known protective or otherwise functionally altering, continuous epitopes. on this compilation protectivity prediction using pca in combination with variability and modification likelihood performed significantly better after domain-accessibility filtering as measured by aroc values, while without filtering performance was comparable (although again slightly better) to antigenicity validation results on the blythe et.al validation-set. even when disregading eight proteins which contaminate the validation process due to similarity with the pca training data the difference persists and is even enhanced. another primary observation is the gross variance in aroc values observed for various proteins. approximately % of sequences exhibited aroc values < = . or > = . , indicating a pattern substantially different from random noise. it may be wise to investigate the per-formance on a per-protein or per-epitope basis and to register how many percent of known, distinct sites were found with high reliability. that may help to avoid an averaging effect leading to the underestimation of the predictive power of classifiers. if a method performs well on every second protein or on certain epitopes that may be sufficient for many practical applications in the life-sciences field. this is especially so where high throughput is involved. interestingly no correlation between the functional category of a protein or pathogen class (bacterial or viral) and the aroc could be established, indicating a more complex situation than just two types of epitope each associated with a distinct functional class. by determining the number of theoretically required synthetic peptides to achieve satisfying protectivity in a vaccine or mab approach we conclude that up to six peptides would be required to achieve success in every second protein. such success naturally requires the existence of protective, largely conserved and possibly unmodified epitopes. we also want to point out that the selected validation set presumably represents a combination of differently well mapped proteins. in addition, each of these can contain one or several protective b-cell epitopes of both class i and ii. as we tried to detect only one of these in the set, neglecting the other, validation is skewed against our method. it may also be good to remember that calculated aroc values are averaged over entire proteins, not distinct epitopes. the application of machine-learning procedures potentially combines the prediction of different epitope classes and allows an estimation of the information content in the data. a random forest model achieved % class separation in an equilibrated model, pointing towards the potential to significantly improve upon the single-score method. although the selection of pathogen proteins relevant for the stimulation of protective immune-responses can be enhanced by bioinformatics that is a topic distinct from the prediction of likely protective epitopes on these antigens [ , ] . by combining in-silico ranking of likely protective targets and likely protective peptides from these targets completely automatized screening for applicable peptides is possible. although methods do exist now for both aspects of in silico protectivity screening caution is still necessary. for example, the method published by doytchinova and flower predicts % of the proteins in our dataset as protective antigens (or at least antigens). on the other hand % of the proteome of staphylococcus aureus col ( of proteins) are also predicted to be relevant antigens which although possible seems to be a very high number suggesting a higher false positive rate than approximated by validation procedures in the publication. on the other hand all proteins are ranked by a score, so an order of predicted relevance is available, in our view essential for practical use. unfortunately proteins would be selected to cover all four proteins for which s. aureus protectivity data is available in the iedb and which have close homologues also in strain col (fibronectin-binding protein a and b, enterotoxin b and enterotoxin type a), yet not all may be required for a protective immune response and others may contain unmapped or discontinuous epitopes [ ] . these findings indicate that several predictive methods and experimental data should be combined when selecting candidates for the generation of protective immune responses. our work solely focuses on the prediction of candidate peptides after such a selection has taken place, independent whether by means of experimental data, literature mining or purely bioinformatical/biological considerations. we can show that prediction of protective or at least functionally relevant continuous b-cell epitopes can be efficiently done for approximately % of analyzed proteins of pathogen origin. by minimizing sequence variability and probability of post-translational modification it can also be assumed that selected peptides are particularly suited for vaccine or monoclonal antibody generation. exclusion of rationally selected domains strongly enhances the prediction of protective sites, indicating the relevance of a filtering step to restrict to immunologically likely accessible regions. furthermore, analysis of correlation between variability, conservation of modification profiles and predicted antigenicity shows different and opposed categories of correlation thus indicating the existence of distinct epitope types. at this point it is difficult to verify or falsify our basic assumption of the twoclass nature of protective epitopes. on the other hand the high percentage of epitopes with either positive or negative feature correlation indicates the existence of at least two types. also, decision tree based models significantly outperform the single score-model pointing towards more complex relationships as well as possibly several distinct epitope signatures. future paths may lie in the detailed unraveling of parameter-associations and the utilization of more sophisticated classification methods such as regression trees for the assessment of biological relevance and protectivity in the selection of peptides for biotechnological application, as indicated by initial random forest classification. the set of unique amino acid propensity scales taken from the aaindex database [ ] forms a × descriptor matrix of rank . calculation of sequence properties from propensity scales can be expressed as a matrix multiplication of the amino acid sequence in matrix representation with the descriptor matrix. hence, the resulting property matrix is of rank as well, corresponding to linearly independent vectors. from this only coefficients plus the intercept can be estimated in regression models. therefore principal component analysis (pca) was employed to transform the propensity scales from the -dimensional space to a -dimenstional subspace spanned by all principal components with non-zero eigenvalues, comparable to how parameter reduction has been done before [ , ] . the × descriptor matrix was centered and scaled to unity variance before the application of pca. the full information contained in the original × descriptor matrix is retained, because no component is omitted. from this pca-transformation, a new × descriptor matrix was built. these pca-derived propensity scales were used in turn to calculate sequence characteristics for the data set. the values were averaged over a sliding window of nine amino acids. logistic regression employing the logit function was used to build models for the classification of single amino acid residues as epitopic or non epitopic. in order to estimate the generalization error, bootstrap validation was employed. error estimates were acquired from out-of-bag validation -i.e. using those residues that are not part of the bootstrap sample as validation data. replicates were calculated for the validation of each investigated model. to numerically represent variability of a protein at a certain amino acid position an information-entropy measure has been applied. for each protein where variability should be determined the sequence was blasted against the non-redundant protein database (nr) and hits were selected manually. the aim was to choose a diverse but not too diverse set of sequences to optimally represent the degree of evolutionary freedom of each amino acid position. those proteins were downloaded and multiply aligned using clustalw. for each alignment column a variability value was calculated as follows: randomly draw samples of size (i.e. times amino acids which is a combination with repetition) from each column, independent of how many sequences have been aligned. that way the evaluation should be less dependent on the number of homologues as for some proteins only five elements can be found whereas for others hundreds are available. determine the most abundantly found amino-acid in the column. then calculate the shannon-entropy weighted by the emboss [ ] eblosum variant of the blosum [ ] substitution scores between each amino acid and the most abundant one in the column. after averaging over all samples to obtain the mean variability the final variability score for each alignment column computes as where f(j) is the score at alignment column j, f x is the frequency of the most abundant letter x in column j, f i is the frequency of letter i in column j and w ix is the substitution weight between letters i and x. for each alignment variability scores are independently rescaled between and . the variability score ultimately used for protectivity scoring is -rescaled f(j). note that each individual gap is regarded as a new character which occurs only once (thus extending the letter alphabet to a potentially high number leading to the perception of strongly gapped positions as highly variable). as an independent strategy an evolutionary constraint index (eci) was calculated for each alignment column. this constraint is essentially the difference between the standard shannon entropy and the entropy after reducing the amino acid alphabet according to the same substitution matrix as above. briefly, amino acid identities were grouped as follows: e < = e, d, q, k, r ; i < = i, l, m, v; y < = y, w, f, h; s < = s, t, a. other amino-acids remained ungrouped. the eci is primarily discussed when parameter correlations are analyzed. to predict posttranslational protein modifications prosite [ ] patterns were used. in particular we considered patterns ps , ps , ps , ps , ps , ps , ps , ps , ps , ps , ps , ps , ps , ps . all sequences in the previously created multiple alignments were searched for the occurrence of these patterns. for each hit the amino acid putatively carrying the modification was marked and for each column in the alignment the ratio m of modified amino-acids was calculated. as for sequence variability the value used for protectivity scoring is -m. the advantage of using motif predictions on aligned sequences is the possibility to derive the degree of conservation of the motif. combined with the assumption that conserved motifs of post-translational modification are more reliable and do otherwise carry a high false positive rate this increases the weight of the prediction. a reference data set was generated from the antibody binding site repository bcipep [ ] . this database holds a collection of experimentally determined b-cell epitopes. the bcipep data set is highly redundant with plenty of entries showing relation to more than one source protein. to realize a non-redundant data set, homologue proteins of this collection were grouped. members of two different groups differed by at least % in sequence identity. after this partitioning, the number of epitopes with length between to amino acids that could be localized on each protein was determined. finally, the proteins bearing the largest number of epitopes were selected as the representative for their group. this procedure leads to a diverse set of protein sequences with experimentally determined immunogenic regions for each protein. this data set holds in total proteins with an average length of amino acids. the prevalence of amino acids being part of an epitope is . %. the mean length of an epitope is residues. out of the proteins originate from bacteria, from viruses, from fungi, from human, from allergens, and from other sources (e.g. eukaryotic parasites). the data set holds in total continuous epitopes, i.e. about epitopes per protein. since no systematic epitope mapping of all the proteins in the data set has been performed, categorizing sequence regions as non-antigenic is problematic, as these could be categorized false negative. epitopes classified as nonepitopes do exist, yet it is problematic to discern whether those are the results of organism or individual (i.e. responses varying from individual to individual) or yet other biases. we chose to declare the non-defined regions of our reference dataset as non-epitopes. regions not part of an epitope were randomized while maintaining the average amino-acid frequency of bcipep. the resulting was used for training b-cell epitope classifiers. each amino-acid functioned as the central amino acid of a -mer (peptide), inheriting its class (antigenic or nonantigenic) to the peptide which was then used for parameterization and training/validation. nine-mers were used due to the desire to use an intermediate between common assumptions about sizes of continuous epitopes (usually between and amino-acids). each sequence in the generated multiple alignments was independently scored for its antigenicity using the pca classifier. pca classification resulted in the assignment of an antigenicity value for each amino acid in the multiple alignment with the exception of the flanking amino acids due to a window effect. for each alignment column overall antigenicity was calculated as the average antigenicity over the corresponding amino acids of individual sequences. proteins with known b-cell determinants were downloaded from the "the immune epitope database and analysis resource" (iedb) [ , ] . the iedb allows filtering by various criteria. we applied the following step-wise exclusion filters to obtain a protectivity-related dataset: . remove entries from non-pathogens (including pathogenic plants). src and src were removed because no uniprot or genbank ids were specified. a neutral protease (gi ) of bacillus anthracis str. ames was manually added from a literature source [ ] . all proteins were then clustered and identified groups multiply aligned using the standard tools blastclust and clustalw, respectively. epitopes of all sequences present in the alignment were manually mapped to the homologous sequence where the fewest remapping steps were necessary, or where all epitopes could be represented as can be the case for large deletions or proteins with precursor variants. the process is thus similar to the one applied at the los alamos national laboratory hiv database where all reported epitopes are remapped to the reference strain hxb [ ] . after removal of all redundancies and impractically short proteins entries ( amino acids), with an average peptide coverage (and thus protectivity prevalence) of . % remained, which are from now on referred to as "protectivity dataset". it has to be cautioned that the functional effect of antibodies directed against these determinants is classified only as "leading to biological activity", not necessarily protectivity. for our purposes we consider this close enough an approximation. this dataset can be found in the supplementary materials [see additional file ]. validation results were analyzed using the rocr package) [ ] where specifically aroc (area under the curve of true-positive rate versus false-positive rate plots) calculation has been most relevant. the weka package [ ] was used where machine-learning functions were needed, in particular a c . and a random forest implementation. from a practical point of view predictions of continuous epitopes should be measured by the number of synthesized peptides required to cover known epitopes. the synthesis score is defined as the number of peptides required to cover at least five epitopic amino-acids in the protectivity validation dataset. five has been selected as a minimum requirement for an epitope. to analyse the relevance of the used parameters simple machine learning techniques were applied as implemented in the weka package. for these analyses a dataset was generated based on the entire protectivity dataset (i.e. not the antigenicity dataset) after exclusion of likely inaccessible regions. essentially all residues which were likely immune-accessible according to the rules mentioned earlier were represented by the sum score (individually rescaled between and for each protein), antigenicity, ptm pattern conservation and variability. this dataset of dimension with antigenic (protective) and non-antigenic residues (baseline prediction . %) will be termed complete machine learning set (cml set). in a second step for each antigenic (protective) residue a non-antigenic residue was randomly sampled to obtain an equilibrated set. this set was then randomly resampled into two stratified sets representing % and % of cml for training and validation, respectively. the training set ( instances) and validation set ( instances) are termed mts and mvs, respectively. more than one reason to rethink the use of peptides in vaccine design cellular immunity elicited by human immunodeficiency virus type / simian immunodeficiency virus dna vaccination does not augment the sterile protection afforded by passive infusion of neutralizing antibodies prediction of protein antigenic determinants from amino acid sequences a computer program for predicting protein antigenic determinants a semi-empirical method for prediction of antigenic determinants on protein antigens predictive estimation of protein linear epitopes by using the program people. vaccine bepitope: predicting the location of continuous epitopes and patterns in proteins selection and combination of machine learning classifiers for prediction of linear b-cell epitopes on proteins machine learning approaches for prediction of linear b-cell epitopes on proteins predicting antigenic determinants in proteins: looking for unidimensional solutions to a three-dimensional problem? mapping epitope structure and activity: from one-dimensional prediction to four-dimensional description of antigenic specificity improved method for predicting linear b-cell epitopes benchmarking b cell epitope prediction: underperformance of existing methods towards a consensus on datasets and evaluation metrics for developing b-cell epitope prediction tools identification of discontinuous antigenic determinants on proteins based on shape complementarities protective immunity to rabbit oral and cutaneous papillomaviruses by immunization with short peptides of l , the minor capsid protein universal influenza b vaccine based on the maturational cleavage site of the hemagglutinin precursor formalin inactivation of the lactate dehydrogenase-elevating virus reveals a major neutralizing epitope not recognized during natural infection receptor and viral determinants of sars-coronavirus adaptation to human ace crosshost evolution of severe acute respiratory syndrome coronavirus in palm civet and human adaptive evolution of the spike gene of sars coronavirus: changes in positively selected sites in different epidemic groups cryptic nature of a conserved, cd -inducible v loop neutralization epitope in the native envelope glycoprotein oligomer of ccr -restricted, but not cxcr -using, primary human immunodeficiency virus type strains evolutionary dynamics of the glycan shield of the human immunodeficiency virus envelope during natural infection and implications for exposure of the g epitope influence of n-linked glycosylation of porcine reproductive and respiratory syndrome virus gp on virus infectivity, antigenicity, and ability to induce neutralizing antibodies prediction of continuous b-cell epitopes in an antigen using recurrent neural network uniprot: the universal protein knowledgebase a hidden markov model for predicting transmembrane helices in protein sequences machine learning approaches for the prediction of signal peptides and other protein sorting signals characterization of a unique borreliacidal epitope on the outer surface protein c of borrelia burgdorferi vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines spaan: a software program for prediction of adhesins and adhesin-like proteins using neural networks a novel staphylococcus aureus vaccine: iron surface determinant b induces rapid antibody responses in rhesus macaques and specific increased survival in a murine s. aureus sepsis model aaindex: amino acid index database peptide quantitative structure-activity relationships, a multivariate approach a new set of amino acid descriptors and its application in peptide qsars emboss: the european molecular biology open software suite amino acid substitution matrices from protein blocks prosite: a dictionary of sites and patterns in proteins bcipep: a database of b-cell epitopes an ontology for immune epitopes: application to the design of a broad scope database of immune reactivities curation of complex, context-dependent immunological data effective antiprotease-antibiotic treatment of experimental anthrax rocr we acknowledge emergentec biodevelopment gmbh for funding our research. the author(s) declare that they have no competing interests. js designed the study, assembled the protectivity dataset, performed all analysis based on this data and drafted the manuscript. rg and rr prepared the bcipep dataset (removed redundancies) and developed the pca antigenicity classifier. rg, rr, pp and al worked on the validation of the pca classifier and investigated the contribution of individual sequence features. bm conceived of the study, and participated in its design and coordination. key: cord- -v plz c authors: ghosh, shyamasree title: nanotechnology and sialic acid biology date: - - journal: sialic acids and sialoglycoconjugates in the biology of life, health and disease doi: . /b - - - - . - sha: doc_id: cord_uid: v plz c nanotechnology is the science of matter at size in a scale of / , , , of a meter. in the last century, considerable progress has been made in the field of nanotechnology and its finds application in major spheres of human life encompassing personal products, medicines, biosensors, disease diagnosis, food, chemicals, energy, agriculture, and industry with application in human health and environment. nanotechnology application to glycobiology is a considerable new development. in the recent times, nanotechnology finds promising applications in the study and targeting of sialic acid encompassing (i) detection of sialic acids in minute quantities and (ii) enabling their targeting in diseases. we discuss in this chapter the recent advances in the application of nanotechnology to sialic acid biology as (i) imaging agents, (ii) spectroscopic tools for their detection, (iii) monitoring of cellular systems, and (iv) application in drug delivery. nanotechnology encompasses the science of matter at dimensions and tolerances at nanoscales of less than nm, with manipulation of individual atoms and molecules. their unique size-dependent properties enable their superior applications to human use. bionanotechnology is the science encompassing the application of biotechnology and nanotechnology and has applications in products used in our everyday lives including personal care products to drugs and medicine. nanoparticles (nps) are now gaining much importance due to their application in biology and medicine. the major biological applications include as fluorescent biological labels, detection of pathogens and proteins, probing of the dna, engineering applications in tissues, targeting tumor and targeted delivery of drugs, genes, and small molecules, identification, estimation separation, purification, and characterization of biological molecules and cells, as magnetic resonance imaging (mri) contrast enhancement agents and in phagokinetic studies [ ] . nps with similar size as proteins enable applications of nps in biotagging or labeling. to interact with biological target, a biological molecule antibodies, including biopolymers like collagen, or monolayers of small molecules acting as bioinorganic interface rendering the property of biocompatibility. to enable optical detection, nps with fluorescent properties of that alter their optical properties are applied [ ] . some of the modifications of nps for biomedical applications are enlisted in fig. . nanotechnology has been applied to glycobiology forming the new science of glyconanotechnology and is a synergy between nanotechnology and glycans playing role in biological and medical applications [ ] . more recently, they have been applied in the sialic acid biology. glycans occur as surface lining macromolecules and form the first line of contact for any other cell or pathogen and protein-carbohydrate interactions and are known to play role in cell signaling, molecular recognition, immunity, and inflammation. carbohydrate comprising of wood, insect shells, or cartilage, with mechanical properties serve as biomaterials of importance. cellulose nanocrystals find importance in processes for degradation of biomass to biofuels and chemicals. glyconanomaterials ( fig. ) with properties of nanomaterials of better solubility, biocompatibility, lower cytotoxicity with the uniqueness of their size, chemical properties, surface engineering, surface charge and electronic, photonic, and magnetic like physical properties and properties of glycans of water solubility, biocompatibility, structural diversity, and specific targets [ ] have major applications in biology encompassing the domains of (i) as sensitive biological probes in cells and tissues enabling building of different scaffolds, (ii) as imaging agents, (iii) as spectroscopic tools for their detection, (iv) monitoring of cellular systems, and (v) application in vaccination and drug delivery. sialic acid has been associated with the disease pathology in several diseases including autoimmune disorders, infection, and cancer. recently, the sialic acid-siglec axis discussed in the earlier chapters is revealing to be an emerging target to prevent or affect several diseases. however, the study of deeper role of sialic acid-siglec axis in immune modulation and therapy suffers from the limitation of suitable sensitive methods. natural sialic acid ligands can be modified by chemical methods leading to the development of sialic acid mimetics (sams) that reveal improved and selective binding affinity toward siglecs. glycobiotechnology involving bioorthogonal synthesis is enabling the presentation of sams on nps, polymers, and living cells which finds application in the study of the sialic acid-siglec axis and its role in immune modulation and therapeutic potential [ ] . although sialic acid and its derivatives reveal promises as stealth carriers with properties including targeting ability, cancer inhibition, viral and inflammation recognition, brain targeting and effective targets in several disorders chapter .) by in vivo and in vitro studies, design of nanocarrier in drug delivery and targeting, based on sialic acid remains as a major challenge and suffers from the limitations of multi-target side effects and calls in for more research [ ] . we discuss in this chapter (i) the glycans and nanotechnology, (ii) role of nanotechnology in detection and quantitation of sialic acid, and (iii) nanotechnology and their applications in sialic acid biology and associated biology of disease, the application, and challenges. glycoproteins or glycolipids that take part in cellular communication, inflammation, and immune responses using carbohydrate-protein or carbohydrate-carbohydrate and are known for their multivalent interactions [ ] with larger variation in affinity/avidity in normal individuals and reported to be disease markers in different diseases as cancer, asthma, and diabetes. nanotechnology enabling estimation, creating, manipulation of matter at nanoscales, is finding application in study and manipulation of glycans. various scaffolds such as glycodendrimers or glycopolymers with high surface/volume ratios, allowing better surface contact with improved multivalency effects have been constructed from diverse nanomaterials including semiconductor, carbon-based nanomaterials and have been studied for carbohydrate-protein interactions and find applications in drug delivery, imaging, diagnostics, or sensitive quantitation tools. polysaccharides nanomaterials including chitosan, dextran, hyaluronic acid, and heparin have been designed drug delivery devices [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and in pharmaceutical application with the advantages of biocompatibility, with reduced toxicity/nontoxicity, prolonged persistence, and drug release. hybrid substructures are also constructed with metal-cored nps coated with polysaccharides. inorganic nanostructures of iron oxide, noble metal, and semiconductors enable formation of synthetic scaffolds to multimerize glycans and enhance the affinity for receptors. magnetism and fluorescence of hybrid materials finds applications in sensing, delivery, or imaging. gold nanoparticles (aunps) [ , ] in conjugation with glycans [ ] [ ] [ ] [ ] [ ] [ ] enable them with high aqueous solubility/dispersibility, biocompatibility and their high surface area/volume ratio of aunps allows enhanced sensitivity. carbohydrate-lectin analyses have been enabled by the application of aunps. mannosylated aunps find application in detection of complement activation and opsonization processes in macrophagemediated endocytosis and to target escherichia coli-containing type pili mannose-specific receptors. magnetic nanoparticles (mnps), including iron oxide and manganese oxide nps, find application as contrast agents for mri [ , ] . glyco-mnps with high surface/volume ratio have enabled detection of early stage disease by mimicking leukocyte recruitment during inflammation [ , ] . tetrasaccharide sialyl-lewis x (sle x )-functionalized mnps have found application in targeting e-/p-selectins and find application in detection of inflammation [ , ] . quantum dots (qds) including binary cadmium or zinc selenides or sulfides are luminescent semiconducting nanomaterials and can emit light with broader excitation spectrum and sharper emission bands [ , ] . glyco-qds functionalized with carboxymethyldextran and polylysine have found application in study of carbohydrate-protein interactions. qds can be stabilized with glycodendrimers [ , ] . buckminsterfullerene c , and carbon nanotubes (cnts) [ ] [ ] [ ] [ ] glycosylated as in α-d-mannosyl fullerenes and fullerenols have been known to inhibit erythrocyte aggregation [ , ] . single-walled cnts (swcnts) and multiwalled cnts (mwcnts) linked to c -lipid tail with α-galnac residues have been applied as probes or radiotracers in developing sensitive in vivo imaging or radiation delivery systems with high radioisotope loading [ , ] . glycan-linked graphene has been reported to enable agglutination and inhibition of bacterial motility. chitosan-based nps [ ] are reported to deliver proteins, oligonucleotides, and plasmid dna. multifunctional glycol-chitosan nps with a near-infrared (nir) fluorophore for fluorescence imaging [ ] have found application in encapsulating anticancer drugs or complex small interfering (sirna) as drug delivery device. chitosan-polyethylene glycol (peg)-coated iron oxide nps have been reported to make better intracellular delivery of a dna repair inhibitor (o -benzylguanine) to glioblastoma multiform cells and enable treatment monitoring by mri. dextran enables improved water solubility and stability of iron oxide mnps while sulfated dextran can electrostatically interact with positively charged polycations. functionalization of dextran-coated iron oxide nps with sle x tetrasaccharide has enabled recording of inflammation in mouse brain. hyaluronic acid and heparin-based nps offer promises in cancer therapy by targeted, magnetic, photodynamic, and gene therapy [ ] . glycodendrimers enabled formation of three-dimensional ( d) supramolecular sugar scaffolds of sugars with a ru (bpy) core [ ] that could bind to e. coli expressing mannose receptor of bacterial pili, displayed on virus-like particles [ ] enabled picomolar inhibition of adhesion of ebola virus. nanoengineered glycan sensors probes of aunps and cnts may help with glycoprotein profiling. glyconanomaterials [ ] [ ] [ ] of gold and silver find application in cancer detection by quantifying cell-surface mannose glycans. mannan-coated aunp incubated with a human gastric cell line in the presence of the mannose-binding lectin cona enabled detection of aberrant glycosylation in cancer [ ] . cona-functionalized cnts finds application in surface glycan detection [ ] . sialic acid is known to be present as components of mucin component, glycoproteins, and other microbial polymers in nature food, further emphasizing the need of sensitive tools to detect them even in traces. the glycome of cells and glycoproteins and their detection and estimation finds importance in understanding glycan functions, development of diagnostics tests, and monitoring of glycoprotein pharmaceuticals. sialic acid-containing carbohydrates, collectively grouped as sialosides, are known to play major roles in the physiology of health and disease-like infections by virus and bacteria, tumor cell metastasis, but limitation of suitable methods to study sialosides forms the major challenge in the study of their structure and function. appropriate quantitation of sialic acid finds importance in health and disease to understand the levels correlating with the homeostasis and pathophysiology of the body in infection and disease. although several biochemical tests find importance in detection and quantitative estimation of sialic acid in the body, the detection of minute quantities of sialic acid and the perturbation in disease states is far from complete. nanotechnology and its diverse application and application in sensitive methods finds importance in the quantitative detection of n-glycans and sialic acid in the body even in very small amounts. synthetic sialoside chemistry, by chemoenzymatic or stereochemical approach, have produced homogeneous size-and structure-defined sialosides with array application, to mimicking cell-surface display and aids in understanding sialoside-mediated interactions. application of nanotechnology in sialoside arrays [ ] is suggested to lead to promising results in study of sialic acid biology. n-glycans are isolated and characterized by conventional methods of enzymatic treatment, followed by their release and derivatization with a fluorochrome and separation by normal-phase high-performance liquid chromatography (hplc). nano quantity analyte detector (nqad) has been designed to quantitate the nonderivatized sialic acid in glycoproteins, separated by hydrophilic interaction chromatography, detected by measuring size differences in dry aerosol and by converting the particle count rate into chromatographic output signal. this sialic acid quantitative sensitive method lacking requirement of active chromophore or fluorophore finds importance over conventional methods and hplc/nqad method offers advantage in reproductive results over the conventional hplc/dmb method. while hplc/dmb method involves derivatization of glycoproteins using , -diamino- , -methylenedioxybenzene (dmb) and dionex-based high-ph anion-exchange chromatography with pulsed amperometric detection (hpaec-pad), the hplc/nqad method is designed with elimination of the derivatization step and efficient polyglycoplex amide column adding to the sensitivity to the detection method [ ] . simultaneous quantification and characterization of the n-glycans including both neutral and sialylated glycans suffers from lack of appropriate methods of identifying them. this is circumvented by applying a weak anion-exchange hplc separation step to fractionate glycans by their sialic acid content followed by a mild acid desialylation step and then resolved by nano-lc-coupled electrospray ionization (esi)-mass spectrometry with an intercalated nanofluorescence detector by which neutral glycans can be separated and characterized [ ] . esi-ms method finds applications in detection and characterization of the heavily polysialylated n-glycans in human serum with improved sensitivity [ ] . isomeric glycan profiling using nanolc-ms with porous graphitized carbon (pgc) as the stationary phase has found importance in detection of sialylated serum proteins by high detection sensitivity and chromatographic resolution [ ] . subambient pressure ionization with nanoelectrospray (spin) using advanced data processing tools has increased the efficiency and sensitivity and has enabled high-resolution ms in detecting sialic acid polymer chains over conventional esi-ms [ ] . a quick, sensitive fluorimetric detection method by using a sensitive lectin-cdte qds nanoprobe made by conjugating sambucus nigra bark lectin (sna) as probe for sialic acid forming sna-cdte qds has been designed to detect sialic acid in egg products. sialic acid and sna-cdte qds, interaction lead to generation of fluorescent signal and is able to detect as sialic acid as low as . ng/ml [ ] . n-glycolylneuraminic acid (neugc), is produced in animals, including cattle and mice, but not in human and is considered to be immunogenic in humans. therefore, neugc contamination in human embryonic stem cells cultured xenogeneic serum due to accumulation indicated its harmfulness and raised concerns over its safety of cell therapy products. to detect femto level the presence of neu gc, a nano-flow liquid chromatography/fourier transformation ion cyclotron resonance mass spectrometry (nanolc/ftms) and nanolc/ms/ms has been designed with promising results [ ] . gangliosides (ggs) are involved in many brain functions at the cell and molecular level and their study detection and characterization suffers limitation of suitable sensitive methods for detection and analysis. sialic acid-coated nps are finding applications in targeting in cancer [ ] . nanotechnologybased detection of glycans and sialic acid conjugates is finding application in detection of gg composition. in human hemangioma, gg composition and structure has been detected by highly sensitive methods of mass spectroscopy (ms) methods based on fully automated chip-nanoelectrospray (nanoesi) high-capacity ion trap (hct) and collision-induced dissociation (cid) all integrated in the chip-nanoesi approach revealed detection of the presence of one modified o-ac-gd and o-ac-gm ggs and the presence of gt a and gt b isomers and unusual gt c and gt d glycoforms in brain hemangioma tumor [ ] . the nanotechnology-based method offers advantage over conventional methods in its sensitivity and detection of unusual forms of ggs hitherto undetected by conventional methods in this disorder. in all, gg components were detected in human caudate nucleus (cn) by chip-nanoelectrospray ms performed on a nanomate robot coupled to a hct instrument in only . min revealing structures of mono-, di-, and trisialylated ggs and finds importance in detection of gg in cnrelated neurodegenerative disorders [ ] . sensitive detection of neu gc-containing ggs from neu accontaining analogs was separated from the lymphoma cell line derived from mouse (yac- ) lymphoma cell monosialoganglioside fraction by nano-high-performance liquid chromatography (nanohplc) in online conjunction with esi quadrupole time-of-flight (esi-qtof) mass spectrometry and served as a promising glycolipidomic tool [ ] . evading the reticuloendothelial system (res) is a major obstacle in drug delivery and targeting in cancer. sialic acid is known for its reduced interaction with the innate immune system by siglec, thus regulating phagocytic evasion. surface engineered aunps conjugated to sialic acid have revealed that sialic acid-mpeg-aunps can escape uptake by res and therefore, efficiently target tumor cells and by targeted delivery can enhance accumulation in tumor [ ] (fig. ) . influenza a infection is initiated by binding of viral envelope hemagglutinin (ha) glycoproteins to cell membrane sialic acid. free toxic sialic acid monomers cannot block ha adhesion in vivo. polyvalent, generation (g ) sialic acid-conjugated polyamidoamine (pamam) dendrimer (g -sialic acid) has been found to inhibit three influenza a subtypes (h n , h n ) indicative of the fact that polysialic acid (psa) inhibitors have potential in antiviral therapeutics [ ] . amongst dendritic polymeric inhibitors, including spheroidal, linear, linear-dendron copolymers, combbranched, and dendrigraft polymers are known for the ability to inhibit virus hemagglutination (ha) and to block infection of mammalian cells in vitro. comb-branched and dendrigraft inhibitors revealed to be the most effective inhibitor, with up to , -fold more antiviral activity [ ] . targeted delivery of sialic acid inhibitors like sialic acid-blocking glycomimetic has been reported to block cancer metastasis [ , , ] . sialic acid-blocking glycomimetic [p- f (ax)-neu ac] coated antibodies allowed targeted delivery into melanoma cells successfully preventing cancer metastasis in murine lung cancer model [ ] (fig. ) . linkage-specific sialylated glycans could be characterized from reaction with condensation reagent -( , -dimethoxy- , , -triazin- -yl)- methylmorpholinium chloride (dmt-mm) in methanol with nanoscale liquid chromatographic separation prior to accurate mass orbitrap ms analysis and improve separation and enrichment of trisialylated n-glycan fraction from haptoglobin and human plasma, as trisialylated fraction has been linked with cancer-associated changes in the serum n-glycome [ ] . the identification of sialylated thomsen-friedenreich antigens in proteins finds importance in cancer research. sialylated antigens in minute quantities ( . μg) and plasminogen ( . μg) could be detected from gels by reductive β-elimination, permethylated and analyzed by nano-lc-matrix assisted laser desorption/ionization (maldi)-tof-ms and using a computational algorithm to filter spectral noise and enhance/isolate the signals of interest [ ] . integrating a fast preparation protocol of mucins with high-throughput nanolc/ms have enabled the study the o-glycosylation of the colon muc mucin from biopsy of sigmoid colon during routine colonoscopy of normal control patients [ ] . negative ion nano-liquid chromatography/mass spectrometry (nano-lc/ms) and tandem mass spectrometry (nano-lc/ms [ ] , using graphitized carbon as separating medium, could analyze neutral and acidic o-and n-linked oligosaccharide alditols. automated glycofragment mass fingerprinting using the glycosidiq software confirmed the oligosaccharide sequence for both neutral desialylated as well as sialylated structures in membrane proteins from ovarian tissue [ ] . attachment of α-n-acetylneuraminic acid (neu acα) to the terminal glycine residues tetraantennary peptides [glycine (n)-nhch [ ] ] [ ] c is reported to give rise to water-soluble assembled glycopeptides that can to bind influenza virus multivalently and inhibit adhesion of the virus to cells more effectively is a promising antiviral strategy in the design of multivalent antivirals [ ] . polyacrylamide hydrogel-based lectin microarray with lectins on colorectal cancer (crc) cell lines sw , sw , and hct revealed high glycan expression of d-galactose, d-glucose, and/or sialic acid residues with uelx europaeus agglutinin-i (ueai) showing specificity to sw cells. uea-i conjugated with silica-coated nagdf : yb + , er + @ nagdf has been reported to be effective designs to target tumor molecule in sw tumor detected by upconversion luminescence imaging, t -weighted mri, and x-ray computed tomography (ct) imaging [ ] . cd finds importance as an important drug target in autoimmune diseases and b cell-derived malignancies. nanoprobe of sialic acid/nacetylneuraminic (nana) acid conjugated to carboxyl groups modified cdse/zns quantum dots (cooh-qds) by the nhs/edc esterification chemistry led to the formation of functionalized qd nanoconjugate which has been applied to target cd and the targeting could be detected by fluorescence imaging [ ] . an immobilized mercaptophenyl boronic acid (mpba) nanochip with nanocone-array substrate on au and ag nps for dynamic electro-optical by the metal-s bond could detect selective sialic acid as low as μm [ ] . nano-tio has been proved to have cytotoxic and phototoxic effects on different crystalline phases for human skin keratinocytes (hacat cells) under ultraviolet (uv) irradiation revealing increased α , -sialylated glycans. although mixture of crystalline p revealed highest cytotoxicity and phototoxicity, followed by pure anatase a , and pure rutile r but a and r did not affect sialic acid expression on hacat cells [ ] . nanomaterials find application in tumor targeting and find application in cancer therapy. pegylated, borate-coordination-polymer-coated polydopamine np (pda@cp-peg) with dox (doxorubicin) reveal synergetic targeting of sialic acid-overexpressed tumor cells. photothermal effect of the polydopamine core and the dox-loading capacity of the polymer layer enable their potential for chemo-photothermal combination therapy with less toxicity, efficient tumor targeting ability, and chemo-photothermal activity for tumor inhibition with promising potential clinical applications [ ] . -mpba, a surface-enhanced raman scattering (sers) nanoprobe (glucose-mpba-agnps) prepared with times stronger sers enhancement ability as compared to mpba-agnps could detect sialic acid expression by amplifying their expression on cancer cells, by the differential accumulation of glucose-mpba-agnps on cancer vs normal cells due to the differences of sialic acid expression on cancer vs normal cells enabling detection of cancer cell with diagnostic and prognostic potential [ , ] . carbon dot (cdot) nps offer promising potential for drug delivery and bioimaging applications. j . macrophages have been shown to take up phenylboronic acid (pba)-modified nps as pb binds to sialic acid residues overexpressed on diseased cell surfaces and finds application in drug targeting to macrophages associated with tumors [ ] . sialic acid as a ligand by dexamethasone (dm)-loaded solid lipid nps has been used for targeting renal ischemia-reperfusion injury (iri)-induced acute kidney injury (aki). dm-loaded sialic acid-conjugated pegylated nps (sialic acid-nps) could reduce apoptotic human umbilical vein endothelial cells (huvecs) via downregulating oxidative stress-induced bax, upregulating bcl-xl, and inhibiting caspase- and caspase- activation being internalized by inflamed vein endothelial cells (vec) mediated by specific binding between sialic acid and e-selectin receptor expressed on the inflamed vec and could effectively ameliorate renal functions in aki mice, causing improved blood biochemical indexes, histopathological changes, oxidative stress levels, and pro-inflammatory cytokines proving to be efficient and targeted delivery of dm for ischemia-reperfusion-induced injury-induced aki, with improved therapeutic outcomes and reduced side effects [ ] . β-amyloid (aβ) plaques in the brain are pathological features of alzheimer's disease (ad). np contrast agents capable of binding with aβ highly selectively enable early detection of ad. but the major obstacle is provided by the blood brain barrier (bbb) that preclude the entrance of nps into the brain for aβ binding. bovine serum albumin (bsa)-coated nps are designed with sialic acid (np-bsa x -sia) has been reported to overcome the challenges in aβ imaging in vivo due to biocompatible and high magnetic relaxivity, indicating their suitability as contrast agents for mri [ ] . cona-conjugated dox-loaded mesoporous silica nanoparticles (msns) find applications as delivery devices in bone cancer treatment as cona and can recognize and bind sialic acid overexpressed in human osteosarcoma (hos) cell line [ ] . neutrophils by forming neutrophil extracellular traps (nets) with dna fibers and histones can combat pathogens and antimicrobial components to kill pathogens, but nets could lead to pathological conditions like sepsis or acute lung failure due to histone-mediated toxicity. poly sialic acid nps with property as an antagonist of the cytotoxic properties of extracellular histones neutralize histone-mediated cytotoxicity and initiate binding of these polysialylated particles to net filaments [ ] . middle east respiratory syndrome coronavirus (mers-cov) targets the epithelial cells of the respiratory tract in human and camel host, binding to the cell-surface receptor dipeptidyl peptidase (dpp ) by s b and sialic acid by s a domain. binding is hampered by modification of sialic acid including -n-glycolylation and ( ) -o-acetylation or depletion of cell surface sialic acid by neuraminidase treatment indicating that virus-sialic acid interactions are vital to viral entry and infection [ ] . inhibition of influenza a virus infection by multivalent sialic acid inhibitors is a promising strategy [ ] . a red blood cell (rbc) cytosensor has been designed employing sialic acid on a quartz crystal microbalance (qcm) by immobilizing rbcs on a cona-modified gold chip employing recognition between cona and mannose. -aminobenzeneboronic acid (apba)-functionalized gold nanoparticles (aunps/apba) were used to label sialic acid and acted as a signal amplification nanoprobe and find importance in detection of sialic acid in diabetic individuals as compared to the normal individuals [ ] . mnps find importance in molecular targeting therapy in cancer but have limitations in targeting. sialic acid-binding lectins, wheat germ lectin (wga) conjugate, or nanomagnetolectin, can target sialic acids overexpressed in prostate cancer and promoted apoptosis under magnetic field (magnetofection) [ ] . early diagnosis of metastatic cancers can prevent mortality in cancer. as aberrant overexpression of sialic acid has been reported in tumors correlating with progressive metastasis, pba-installed pegylated aunps coupled with toluidine blue o (t/ba-gnps) as sers probes has been reported to target surface overexpressed sialic acid revealing strong sers signals from metastatic cancer cell lines (breast cancer; mda-mb and colon cancer; colon- cell lines) [ ] . cd is a member of the siglec family and cd -ligand-targeted nps with therapeutic functions have proved successful in preclinical settings for blood cancers, autoimmune diseases, and tolerance induction [ ] . biosensors for detection of virus were developed by utilizing plasmonic peak shift phenomenon of the aunp and viral infection mechanism of ha on virus and sialic acid on animal cells [ ] . molecularly imprinted polymers (mips) as artificial receptors; can be designed to bind targets like hyaluronan and sialylatic acid and their conjugates and find application in labeling and imaging of cellular targets. fluorescent-labeled mip nps with glucuronic acid (mipglca) and sialic acid could target extracellular hyaluronan and mip-coated inp/zns qds could target hyaluronan and sialylation sites in both their intra and extracellular expression. green and red-emitting qds functionalized with mipglca and mipsialic acid, respectively, have been reported to enable multiplexed cell imaging [ ] . ( -aminomethylphenyl)boronic acid (ampb)-installed hyaluronic acid (ha)-ceramide (hace)-based nps, including manassantin b (mb), forming hace-ampb/mb nps ( nm), when targeted on cancer cells revealed increased cellular accumulation and efficient antitumor activity and were hypothesized to react with sialic acid overexpressed in cd receptor-positive human adenocarcinoma cells [ ] . hydrophobically modified polysialic acid (hpsa) nps, prepared by -ethyl- -( -dimethylaminopropyl)-carbodiimide (edc)/n-hydroxysuccinimide (nhs) coupling between n-deacetylated psa and β-cholanic acid loaded with dox forming (dox-hpsa) are reported for its anticancer drug nanocarrier activity, therapeutic efficacy, and specific targeting of cancer cells in a cells [ ] . fluorescent-conjugated polymer nps with their optical properties and low cytotoxicity find applications in imaging and the fluorescent intensity was reported to further improve when these polymers were modified with a pba group, covalently linked with sialic acid, forming a sialic acid-imprinted nps ( nm) size with selective staining for du cancer cells [ ] (fig. ) . protamine nanocapsules (ncs) linked with psa acted as drug delivery devices and revealed properties of enhanced stability and facilitated transport of macromolecules across the intestinal epithelial cells in cell line including caco- [ ] . magnetic relaxation nanosensors (mrns) made by conjugating entry blocker peptides to iron oxide nps through targeted binding with ha. , and , -sialic acid ligands on cell surface could detect ha variants (h and h ) in fats and detect the different influenza subtypes [ ] . sialic acid coatings on polymeric micelle consisting of poly(sarcosine)block-poly(l-lactic acid) (lactosome) targeting the immunosuppressive receptors of siglec-g and cd could prevent accelerated blood clearance (abc) phenomenon due to the reduction of the anti-poly(sarcosine) igm production [ ] . sialoglyco-conjugated nps synthesized from highly branched αglucuronic acid-linked cyclic dextrins (glca-hbcd) forming sialoglyco-np (neu acα , lacnac-glca-hbcds, sialoglyconp [sagnp]) could recognize and interact with human influenza virus strain a/beijing/ / (h n ) detected by ha inhibition assay and sag-np with sialic acid substitution of , have been reported to inhibit virus-binding activity [ ] . au-nps functionalized with sialic acid diluted with a peg forming the sialic acid functionalized gold nanoparticles could detect soluble form of murine siglec-e (msiglec-e-fc fusion protein) on chinese hamster ovary cells (cho cells) and find application in detection of siglec on mammalian cells [ ] . a benzoic group functionalized gold nanoflower was designed as nanoprobes for recognition of target sialic acid and assembly of poly sialic acid by sensitive sers signal [ ] . fluorescent biocompatible polymeric nps designed with a hydrophobic monomeric core, fluorescent monomer, and a protein-binding monomer that conjugates lectin to target sialic acid is reported to detect and monitor progression of influenza viral infection by detecting the sialic acid expression level changes in human lung epithelial cells [ ] . fluorescent dye rhodamine and two inp/zns qds emitting in the red and green-mip particles with d-glucuronic acid (glca), a substructure of hyaluronan, and sialic acid capable to localize hyaluronan and sialic acid has been designed for bioimaging of human keratinocytes extracellularly viewed by epifluorescence and confocal microscopy and proves to be a promising tool toward monitoring of disease progression [ ] . sna forms strong bonds with aunps as compared to saraca indica (saracin ii), in the ground state as detected by uv-vis absorption, steady state, time-resolved fluorescence coupled with circular dichroism (cd) spectral studies, finding application in drug delivery systems [ ] . aunps with sialic acid-terminated complex bi-antennary n-glycans, synthesized with glycans isolated from egg yolk, found application as sensor in detection of both recombinant ha and whole influenza a virus particles of the h n subtype [ ] . aggregation of -mercaptophenylboronic acid functionalized aunps ( -mpba-aunps) could bind to sialic acid and detected by colorimetric assays and finds importance in detection of sialic acid in blood serum samples [ ] . pba conjugated with polyethylenimine (pei . k) to generate amphiphilic pba-grafted pei . k (pei-pba) nanovector, encapsulated sirna to form pei-pba/sirna nanocomplexes with properties of biocompatibility, serum stability, and rnase resistance enabled specific delivery to sialic acid overexpressed target cancer cells and significantly decreased polo-like kinase- (plk)- expression in tumors, leading to apoptosis and cell cycle arrest [ ] . monosaccharide-imprinted fluorescent nps comprising of doped silica nps with a shell imprinted with sialic acid, fucose, or mannose as the template with probe fluorescein isothiocyanate (fitc) enabled imaging of human hepatoma carcinoma cells (hepg- ) and human primary tumor cell line michigan cancer foundation (mcf- ) derived from mammary gland [ ] . sialic acid incorporation into the gg molecule could increase fourfold anticancer compound paclitaxel loading capacity forming selfassembled nanostructures of di-and tri-sialogangliosides [ ] . an inductively coupled plasma mass spectrometry (icp-ms) is used to detect sialic acid on the cancer cell surface, recognized by biotinylated phenylboronic acid (biotin-apba) aunps in hepg and mcf- cells [ ] . molecularly imprinted nps were prepared as sers for imaging cancer cells based on targeting of sialic acid overexpressed on cancer cells [ ] . sialic acid coreshell nps with nitrobenzoxadiazole (nbd) fluorescent groups allowing environmentally sensitive fluorescence finds application as a biosensor [ ] . sepsis is known to lead to acute respiratory distress syndrome (ards). murine siglec-e and its human orthologs siglec- and siglec- play role in negatively regulating acute inflammatory responses and may act as targets in sepsis and ards treatment. thus, poly(lactic-co-glycolic acid) nps linked with siglec ligand, di(α → ) n-acetylneuraminic acid (α , nana-np), induced enhanced oligomerization of the murine siglec-e receptor on macrophages [ ] . reduced graphene oxide-tetraethylene pentamine- -butyl- methylimidazolium hexafluorophosphate (bmimpf ) hybrids with bimetallic gold platinum alloy nanoparticles (auptnps) with sna could detect α , -sialylated glycans in serum [ ] . sialic acid-modified selenium (se) nps conjugated with an alternative peptide-b peptide forming b -sialic acid-senps, can cross bbb and enter into cerebral endothelial cells and can act as nanomedicine in ad detected by laser-scanning confocal microscopy, flow cytometry analysis, and icp-atomic emission spectroscopy (icp-aes) [ ] . -aminophenylboronic acid functionalized cdsete@zns-sio qds (apba-qds) probes could detect sialic acid on k cells [ ] . psa can be immobilized on nanoporous silica materials silica nanoparticles (npsnps) of mcm- type [ ] with different applications. raman spectroscopy (sers)-based sensing platform was developed for detecting sialic acid on single cell surface by -(dihydroxyborophenyl) acetylene (dba)-linked aunps hela cell [ ] . super-paramagnetic iron oxide nanoparticles (spio nps), αcd abs and mxd sirna molecules entered leukemia cells and knocked down mxd , leading to apoptosis in reh cell line and in primary preb all samples with synergistic effects by anticancer agents vincristine or dox [ ] . avian influenza viruses preferentially bind to sialic acid α- , -galactose receptors on epithelial cells and magnetic nps coated with chitosan and functionalized with maackia amurensis (maa) lectin (np-lectin) could isolate sialic acid α- , -galactose receptors from porcine trachea [ ] . aunps immobilized with graphite oxide (go), prussian blue (pb), and ptc-nh (an ammonolysis product of , , , -perylenetetracarboxylic dianhydride) nanocomposite go-pb-ptc-nh modified glassy carbon electrode (gce) linked to snas could detect α , -sialylated glycans in serum [ ] . lectin-tagged fluorescent polymeric nps ( nm) could detect cellular sialic acid expression [ ] . qds labeled avian influenza h n virus could enable study of establishment of infection in human bronchial epithelial (hbe) cells using a d spt technique [ ] . dm and methotrexate (mtx) entrapped within psa-trimethyl chitosan (tmc) nps enabled site-specific targeting in rheumatoid arthritis [ ] . qds modified with pba (qds-pba) could target sialic acid expressed on vesicular stomatitis virus (vsv) enabling the virus labeling [ ] . covalent immobilization of sna on a mixed self-assembled monolayer (sam) on planar gold surfaces forming a two-dimensional ( d) sensor and immobilized sna on mixed sam layer on aunps forming d sensor could detect sialic acid [ ] . aunps functionalized with a thiolated trivalent α , -thio-linked sialic acid and a thiolated peg has been designed to detect the human influenza virus x (h n ) as the trivalent α , -thio-linked sialic acid bind to virus hemaglutinin [ ] . multifunctional fluorescent silica nanoparticles (fsnps) with pba were designed to label sialic acid on cancer cell surface with high selectivity and sensitivity [ ] . sialic acid conjugated to poly(ethylene oxide)-polycaprolactone polymersomes could interact with influenza viruses by inhibiting viral ha binding to host cell sialic acids, thus preventing viral entry. targeting by design of neuraminidase inhibitor zanamivir into the polymersome core, inhibited viral replication [ ] . aunps attached to polycrystalline gold modified by an aminoalkanethiol linker layer with covalently immobilized sna on a mixed sam formed on aunps could detect sialic acid and finds application in arthritis or cancer [ ] . siglec- ligand, displayed on liposomal nps, allowed targeting of siglec- positive cells in peripheral human blood [ ] . signals between qds and aunps-sialic acid-binding proteins (sbps) and sialic acid moieties, respectively, enable biosensing based on the nanometal surface energy transfer (nset) and could enable detection of glycosylation linkages (α - vs α - ), and -o-acetyl and n-glycolyl group modifications [ ] . gold nanocluster probe was developed to detect cell surface sialic acid [ ] . sialic acid reduced and stabilized aunps synthesized by a simple onepot, green method for colorimetric detection of influenza virus by hasialic acid binding [ ] . the liposomes targeting sialioadhesion or sn or cd could selectively bind to sn-expressing cells and macrophages accumulating intracellularly overtime enabling antigen delivery to macrophages for their presentation to t cells [ ] . a novel electrochemical strategy for in situ detection of cell surface sialic acids by chemoselective labeling technique and a dual-functionalized nanohorn probe [ ] was developed. -aminophenylboronic acid functionalized qds (apba-qds) synthesized by covalently binding apba to mercaptopropionic acid-capped cds qds, and polysialic acid stabilized gold nanoparticles (psa-aunps), were prepared by a one-pot procedure. the apba-qds recognized the sialic acid on bgc- human gastric carcinoma (bgc) cells and then the psa on aunps, therefore, amplifying signal [ ] enabling detection of sialic acid. semiconductor qds with small molecular pba tags enabled labeling of sialic acid and imaging of cells [ ] . sialic acid surface-decorated selenium nanoparticles (sialic acid-se-nps) have been reported to penetrate cervical carcinoma cells and induce apoptosis by proapoptotic enzymes caspase- and poly(adp-ribose) polymerase (parp) cleavage in cancer cells [ ] . sialic acid-terminated glycerol dendron functionalized aunps have been reported to inhibit influenza virus infection [ ] . lectin-au-thionine bioconjugates linked to aunps revealed mannose expression and expression of biomarker sialic acid in cancer detection, diagnosis, and treatment [ ] . plga np modified with bbb penetrating peptide (similopioid peptide) and a sialic acid residue could cross the bbb and interact with brain receptors. [ ] . polymeric (poly(d,l-lactide-coglycolide), plga) nps surface modified with sialic could be devised [ ] . the application of nanotechnology in the study and biomedical applications of glycosylated molecules and sialic acids and their conjugates on the cells and serum, in human health and disease is a recent development and considerable work has been progressed in the last decade. different designs of nanoparticles have enabled (i) sensitive detection of sialic acid in free forms and in conjugated form inferring on their structures and discovery of novel molecules, hitherto unknown in health and disease due to their sensitive and improved specialized nature of detection systems and (ii) targeting of sialic acids as drug delivery targets with tremendous application in targeting of infectious, pathogenic diseases and cancer. the ongoing research is their application in biomedicine, imaging, and sensor applications is thought to have a major impact on the human lives. with the 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of glycoproteins by the lectin biosensor down to attomolar level using gold nanoparticles paulson jc on-chip synthesis and screening of a sialoside library yields a high affinity ligand for siglec- quantum dot nanometal surface energy transfer based biosensing of sialic acid compositions and linkages in biological samples in situ tracing of cell surface sialic acid by chemoselective recognition to unload gold nanocluster probe from density tunable dendrimeric array colorimetric viral detection based on sialic acid stabilized gold nanoparticles antigen delivery to macrophages using liposomal nanoparticlestargeting sialoadhesin/ cd in situ electrochemical assay of cell surface sialic acids featuring highly efficient chemoselective recognition and a dual-functionalized nanohorn probe highly sensitive fluorescent analysis of dynamic glycan expression on living cells using glyconanoparticles and functionalized quantum dots quantum dots with phenylboronic acid tags for specific labeling of sialic acids on living cells sialic acid surface decoration enhances cellular uptake and apoptosis-inducing activity of selenium nanoparticles haag rinhibition of influenza virus infection by multivalent sialic-acid-functionalized gold nanoparticles lectin-based biosensor strategy for electrochemical assay of glycan expression on living cancer cells sialic acid and glycopeptides conjugated plga nanoparticles for central nervous system targeting: in vivo pharmacological evidence and biodistribution vandelli ma plga nanoparticles surface decorated with the sialic acid, n-acetylneuraminic acid key: cord- - lc fcpe authors: rekha, kaliyaperumal; thiruvengadam, muthu title: secondary metabolite production in transgenic hairy root cultures of cucurbits date: - - journal: transgenesis and secondary metabolism doi: . / - - - - _ sha: doc_id: cord_uid: lc fcpe cucurbits are important group of vegetables due to their nutritional significance and are also used for valuable traditional medicine. the infection of plants by agrobacterium rhizogenes results in a hairy root (hr) phenotype characterized by rapid growth in hormone-free medium, an unusual ageotropism and extensive lateral branching. these genetically transformed root cultures (hairy roots) can produce levels of secondary metabolites comparable to that of intact plants. hairy root cultures offer promise for high production and productivity of valuable secondary metabolites in many plants. high stability and productivity features allow the exploitation of hrs as valuable biotechnological tool for the production of plant secondary metabolites. while these chemical compounds are employed by plants for interactions with their environment, humans have long since explored and exploited plant secondary metabolites for medicinal and practical uses. the main constraint for commercial exploitation of hairy root cultivations is the development and scaling up of appropriate reactor vessels (bioreactors) that permit the growth of interconnected tissues normally unevenly distributed throughout the vessel. emphasis has focused on designing appropriate bioreactors suitable to culture the delicate and sensitive plant hairy roots. to this end, hairy root culture presents an excellent platform for producing valuable secondary metabolites. for these reasons, this chapter describes the establishment of hairy roots and production of secondary metabolites from hairy roots of cucurbits and also phytochemicals uses for biological activity. cucurbits are the popular name to the plants of family cucurbitaceae, which include over genera and species [ ] , among the economically most important plant families [ , ] . it is a large group of plants which are medicinally valuable. cucurbitaceae members are primarily established in the tropical regions of the world. global production of cucumbers, including gherkins, was among the top ten vegetables produced globally (http://faostat.fao.org). cucurbits are a prominent source of secondary metabolites, and many genera of this family received a great level of scientific interest because of the extensive range of pharmacological and nutraceutical properties [ ] . it is reported that the bitter flavor of cucurbits is due to tetracyclic triterpenoids [ ] . various phytochemicals such as alkaloids and saponins are extracted from momordica, citrullus, cucurbita, and lagenaria [ ] . the family proved itself as a strong source of food and medicine. major species of importance include: citrullus lanatus (watermelon), cucumis sativus (cucumber), cucumis melo (musk melon), cucumis anguria (bur gherkin), cucurbita pepo (pumpkin), momordica charantia (bitter gourd), momordica dioica (spine gourd), coccinia grandis (ivy gourd), and praecitrullus fistulosus (tinda). in recent years, consumption of cucurbits in the average diet has been highlighted for its contribution towards lowering the risks of several life-threatening diseases such as coronary heart disease, stroke, pulmonary disease, and different types of cancer. plant species are capable of producing different types of secondary products which can be harnessed by humans for their beneficial properties in a large domain of industrial or medicinal applications [ ] . world health organization (who) estimates that up to % of people rely mainly on traditional herbs as remedies for their medicines [ ] . extracted from entire plants, secondary products are used by food and pharmaceutical industries, although most often numerous natural plantderived molecules remain undiscovered or unexplored for their pharmacological properties [ ] . roots play most important roles in plants and they anchor plants to the ground, take up minerals and water from the soil, store nutrients for perennial plants, and produce a diverse array of chemicals for symbiotic interactions or defensive with other plants or microbes in the rhizosphere. these plant-produced chemicals have traditionally been referred to as secondary metabolites and more recently tagged as specialized metabolites. bioactive compounds are extra nutritional constituents that naturally occur in small quantities in plant and food products [ ] . most common bioactive compounds include secondary metabolites such as antibiotics, mycotoxins, alkaloids, food grade pigments, plant growth factors, and phenolic compounds [ , ] . many secondary metabolites not only protect plants from pathogens, insects, and environmental stresses but also are valuable for human health. many plant species, including crop plants, are capable of producing and releasing biologically active compounds (allelochemicals). allelochemicals (e.g., phenolics, terpenoids, alkaloids, coumarins, tannins, steroids, and quinines) are released by the plant into the environment by root exudation, volatile emissions, leaching from the leaves and other aerial parts, and the decomposition of plant material [ , ] . plant roots release a range of compounds that are not directly involved in the growth and development of the plant but are very much important for plants during stress conditions (biotic/abiotic). these compounds include aliphatic acids, aromatic acids, fatty acids, sterols, phenolics, enzymes, and other secondary metabolites, including flavonoids [ , ] . many plant secondary metabolites of interest are accumulated in roots. however, plant cultivation is often time consuming and metabolite extraction from plant roots is destructive to plant growth. agrobacterium rhizogenes is a gram negative soil-borne bacterium of the family rhizobiaceae, which causes the hairy roots disease by infecting wounded higher plants. the transformed roots can be excised to establish axenic root cultures and indefinitely propagated in growth regulator free medium. the root exhibit fast, plagiotropic growth characterized by profuse lateral branching and rapid root tip elongation [ , ] . root loci (rol) genes harbored by the root-inducing (ri) plasmid of this bacterium are incorporated into the host plant genome, causing hairy root. rol genes are thought to affect growth and development of transformed roots and induce secondary metabolite synthesis by turning on the transcription defense genes [ , ] . the rolb and rolc genes are absolutely essential for induction of hairy roots [ ] . fast growing and genetically stable hairy roots can be efficiently cultured in large scale bioreactors [ ] . besides, hairy root cultures are usually capable of producing the same compound(s) of identical chemistry found in wild-type roots of the naturally occurring parent plant without loss of structural integrity and/or quantity or concentration of the product, which is frequently observed in callus or cell suspension cultures [ ] . a. rhizogenes to regulate the genes that were involved in the plant secondary metabolite production [ ] . hairy roots induced from different plant tissues generally grow fast, are genetically stable, and often, but not always, simulate the biochemical profiles of plant roots, which makes hairy roots an attractive system for producing valuable secondary metabolites. plant roots can synthesize, store, and secrete a vast array of compounds, and transformed root cultures have a wide range of biosynthetic capacities [ ] . various advantages of hairy root culture over cell suspension culture include genotypic and biochemical stability, cytodifferentiation, and growth in hormone free medium. these factors play a vital role during secondary metabolite production. fast growth, low doubling time, ease of maintenance of hairy roots, and their ability to synthesize a large range of chemical compounds offer an additional advantage as a continuous source for the production of valuable secondary metabolites [ ] . a number of secondary metabolites have been reported to be produced from hairy root cultures [ ] . progress has been made on commercialization of hairy root products. rootec bioactives ltd., founded in in switzerland, currently produces phytochemicals from hairy roots induced from plant species in their proprietary mist bioreactors. in the future, more investigations could be directed toward determining the efficacy of crude hairy root extracts or hairy root-produced chemicals. there have been few reviews in the literature on a wide variety of hairy root applications in secondary metabolites of medicinal plants. previously, very few studies of secondary metabolite production in hairy root cultures of cucurbits have been reported. first time, we focus this chapter on establishment of hairy roots and production of secondary metabolites in cucurbits. hairy roots were induced from various explants (leaf, cotyledon, hypocotyl, node, and root) after - weeks of culture. control explants failed to induce hairy root formation. high induction of hairy roots was observed in leaves compared to other explants in gynostemma pentaphyllum, cucumis anguria, momordica charantia, and m. dioica [ ] [ ] [ ] [ ] . cotyledon explants produced higher frequency of hairy root induction in cucumis melo [ ] [ ] [ ] and c. sativus [ , ] . transgenic frequency ( %) of the infected stems of luffa cylindrica formed vigorous hairy roots within weeks from the inoculation of the bacteria [ ] . table shows the different strains of agrobacterium rhizogenes (maff - , k , r , c c , a , , mtcc , atcc , r , kctc , and kctc ) that were examined for their ability to induce hairy roots of various cucurbits such as melon, pumpkin, cucumber, sponge gourd, chinese cucumber, southern ginseng, bitter melon, spine gourd, and bur gherkin [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . two strains of a. rhizogenes differed in their ability to induce hairy roots, with strain kctc being more effective than kctc [ ] [ ] [ ] . monocyclic phenolic compound, acetosyringone incorporated into the nutrient medium showed enhanced transformation frequency than the medium without it. acetosyringone was used for co-cultivation in c. sativus [ ] and m. charantia [ ] . acetosyringone is an amino acid derivative which served as a nutrient source for the invading agrobacterium and enhanced the transformation rate. it was reported that acetosyringone would induce the vir gene of agrobacterium cultures. the established hairy roots show typical morphological characteristics with rapid growth on phytohormone-free medium, lack of geotropism, and extensive lateral branching [ ] [ ] [ ] [ ] . the transformed root was confirmed by pcr to determine the presence of a t-dna sequence in their genomes in gynostemma pentaphyllum [ ] . the pcr products from the hairy roots for rolb regions but not from untransformed roots of g. pentaphyllum. this finding indicated that the rolb genes from the ri plasmid of a. rhizogenes were integrated into the genome of g. pentaphyllum hairy roots. the negative results of pcr amplification for the virc gene demonstrated that no bacterial dna was involved in rolb amplification leading to false positives [ ] . the transgenic nature of hairy roots was confirmed by pcr using rolc and aux gene specific primers, and transgenicity was also confirmed by polymerase chain reaction (pcr), reverse-transcriptase pcr (rt-pcr), and sequencing in c. anguria, m. charantia, and m. dioica [ ] [ ] [ ] . the integration of ri t-dna into the genome of plant cells caused the formation of hairy roots, in which rol and aux genes were harbored in m. dioica [ ] . pcr analysis targeted the a. rhizogenes rolc, aux , and vird genes in m. dioica. the rolc and aux genes, located on [ ] independent t-dnas (tl-dna and tr-dna, respectively) of the ri plasmid of a. rhizogenes strain, are diagnostic for t-dna integration into the host genome. the vird gene, located outside the t-dna, is diagnostic for the presence of any remaining agrobacteria in the root tissue [ ] . the rol and aux genes are essential for the induction of hairy roots, and they act as a potential activator of secondary metabolites in cucurbits [ ] [ ] [ ] . the vird gene was used to verify the complete absence of a. rhizogenes in the hairy roots lines of c. anguria and m. dioica. this result indicates that pri t-dna fragments of a. rhizogenes were successfully integrated into the genome of c. anguria and m. dioica without bacterial residues [ , ] . the obtained full length coding sequence of rolc gene of m. charantia and m. dioica [ , ] . the use of pcr combined with dna sequencing instead of southern blotting for the characterization of transgenic plants has the advantage that the newly inserted genes can be detected at an earlier stage with less dna and less plant material [ , ] . the presence of pri t-dna in pumpkin long-term hairy root cultures was determined by southern hybridization [ ] . integration of the t-dna region of ri-plasmids into the plant genome was confirmed by both opine assay on paper electrophoresis and pcr-based detection of rol genes in trichosanthes kirilowii var. japonica [ ] . successful integration of the t-dna into chromosomal dna of the kmh- was first examined by pcr amplifying the rolc gene located on the integrated t-dna in cucumis melo [ ] . an immunoblot analysis of the oriental melon transgenic hairy root extract revealed kda single bands coincident with the molecular weight of the gfp gus fusion proteins. elisa demonstrated that the highest level of gfp-gus fusion protein expression was . % of the total soluble protein in a transgenic hairy root of oriental melon [ ] . the integration of t-dna containing a gus reporter gene in hairy root lines was confirmed at low copy numbers ranging from to copies using quantitative real-time pcr, and histochemical staining of cucumber hairy roots showed overexpression of the gus gene when driven with the camv s promoter in c. sativus [ ] . the presence of gus activity and its localization were observed in all of the tissues of the root, especially in transgenic cucumber hairy root lines with the camv s and camv st/amv promoters. the transgenic cucumber hairy roots lines with the camv s promoter or the camv st promoter showed localized gus activity only in the vascular bundles in c. sativus [ ] . quantification of the copy number of the gus gene using absolute quantification in real-time pcr revealed a low copy number of the gus gene per genome [ ] . the transgenic plants looked normal and were positive for the neomycin phosphotransferase ii. southern blot analysis of the transgenic plants revealed that all plants contained vector dna, but only some of them contained dna from the ri plasmid [ ] . enzyme-linked immunosorbent assay (elisa) revealed the highest levels of the recombinant t-pa accumulation in transgenic hairy roots carrying the t-pa transgene under the control of single and dual rold promoters as compared to triple and quadruple rold promoters [ ] . previously, it was reported that changes in secondary metabolite production in hairy roots and ri plants correlate with changes in the phenotype induced by the insertion of rol genes and with the quantity of the polypeptide encoded by the rolc gene [ , ] . interestingly, both the capacity to grow and produce nicotine in hairy roots and ri plants of nicotiana tabacum cv. xanthi were higher after integration of the three rol genes (a, b, c) together than with rolc alone. in addition, the level of nicotine accumulation was positively correlated with the levels of the polypeptide encoded by the rolc gene, as detected by immunoassays [ ] [ ] [ ] . the rola gene appears to be an activator of growth and secondary metabolism. although the rolb gene has emerged as the most powerful stimulator, its use is presently disputed owing to its growth-suppressing effect. more positively, the self-activation of rolc gene seems to be promising [ ] [ ] [ ] . the time profile of the growth of hairy roots in liquid culture was reported in c. anguria, m. charantia, and m. dioica [ ] [ ] [ ] . the sixth day was the lag period of hairy root growth; then it began to increase gradually during the eleventh day. the exponential growth stage during the days was followed by the stationary phase during the - days. the higher fresh mass (fm) and dry mass (dm) was observed at , , and days of culture of c. anguria, m. charantia, and m. dioica [ ] [ ] [ ] . the culture duration of days of hairy roots increased about -fold compared to inoculum and the gypenoside content in g. pentaphyllum [ ] . hairy root cultures showed a sigmoidal growth curve, and crude extracts showed a progressively increasing translational inhibitory activity that reached the maximum value during the early stationary phase of l. cylindrica [ ] . sucrose is the most significant carbon source for plant tissue cultures and helps as the chief energy source and an important constituent in secondary metabolite biosynthesis in cucurbits [ ] . the amount of sucrose usually affects the accumulation of secondary metabolites in cultures. about % of sucrose produced the higher amount of biomass accumulation and metabolite production in c. anguria, m. charantia, and m. dioica [ ] [ ] [ ] ] . about % of sucrose induced hairy root induction in trichosanthes kirilowii var. japonica [ ] . many previous reports focus on the composition of medium nutrients to achieve maximum accumulation of metabolites in cultured cells [ ] . the different media, full and half strength ms, b , nn, and ls were employed in hairy root culture and the results shown that ms medium was superior for biomass accumulation in g. pentaphyllum, c. anguria, m. charantia, m. dioica, cucumis melo, c. sativus, and t. kirilowii [ - , , , ] . however, other media like b was also used to induce hairy roots in l. cylindrica [ ] . hairy root induction of cucurbits using carbohydrate source is by sucrose and nutrients media is by ms or b . increased secondary metabolite production in hairy roots cultured in vitro, over their wild-type counterparts, may be seen as one of the most exciting spin-offs of biotechnology. due to their great richness in secondary products, such as triterpenoids and phenolic compounds, plants represent an immense source of therapeutic and/or industrial compounds. for example, plant-derived biomolecules, such as saponins (g. pentaphyllum), triterpenoids of bryonolic acid, and chondrillasterol (trichosanthes kirilowii var. japonica), ribosome-inactivating protein (luffa cylindrica), charantin (momordica charantia), hydroxybenzoic acids, hydroxycinnamic acids, and flavonols (c. anguria, m. charantia and m. dioica), are efficient in the treatment of different pathology types relating to cancer, cardiovascular and metabolic disorders, and/or other infectious diseases (table and fig. ). many plant metabolites are commercially available as drugs, flavors, food additives, cosmetics, fragrances, and insecticides. here, several important phytochemicals from hairy roots of cucurbits are discussed (fig. ). phenolic compounds are secondary metabolites, ubiquitous in plants and plant derived foods. they show a large diversity of structures, including rather simple molecules (e.g., vanillin, gallic acid, caffeic acid) and polyphenols such as stilbenes, flavonoids, and polymers derived from these various groups [ ] . phenolic compounds are classified into three major groups based on the number and binding position of exchangeable hydroxyl groups on aromatic compounds: simple phenol and phenolic acid group, hydroxycinnamic acid derivative group, and flavonoid group. a majority of the plant phenolic metabolites are derived from the aromatic amino acids that are synthesized from the shikimate pathway. phenolics are collectively valued for their wide variety of health-promoting activities. flavonoids are phenylpropanoid metabolites, most of which are synthesized from p-coumaroyl-coa and malonyl-coa, and share their precursors with the biosynthetic pathway for lignin biosynthesis [ ] . flavonoids are low-molecular-weight compounds having approximately atoms of carbon, which are organized in a c Àc Àc configuration [ ] . more than flavonoids have thus far been identified in plants [ ] . phenolic acids are considered as simple phenolics, and they are categorized into two groups, i.e., the hydroxybenzoic and hydroxycinnamic acids. fig. (continued) antiallergenic, antimicrobial, cardioprotective, anti-inflammatory, antioxidant, artherogenic, and vasodilatory effects [ ] [ ] [ ] . phenolic compounds are synthesized via the phenylpropanoid pathway that begins with conversion of phenylalanine to cinnamic acid by phenylalanine ammonia lyase (pal). in the last few years, great attention has been paid to the bioactive compounds due to their ability to promote benefits for human health, such as the reduction in the incidence of some degenerative diseases like cancer and diabetes [ , ] , reduction in risk factors of cardiovascular diseases [ , ] , antioxidant, antimutagenic, antiallergenic, antiinflammatory, and antimicrobial effects [ , ] , among others. due to these countless beneficial characteristics for human health, researches have been intensified aiming to find fruits, vegetables, plants, agricultural, and agro-industrial residues as sources of bioactive phenolic compounds. the qualitative and quantitative analysis of phenolic compounds from hairy roots and untransformed (roots from in vitro seedling) root extracts of c. anguria, m. charantia, and m. dioica [ ] [ ] [ ] were studied using ultra-hplc. the phenolic compounds in the c. anguria, m. charantia, and m. dioica extracts were identified by comparisons of the retention time and uv spectra of authentic standards and the quantitative data were calculated from calibration curves [ ] [ ] [ ] . both transgenic and nontransgenic roots contained flavonols, hydroxycinnamic, and hydroxybenzoic acids. hairy roots contained higher amounts of flavonols compared to nontransgenic roots of c. anguria, m. charantia, and m. dioica [ ] [ ] [ ] . myricetin, quercetin, catechin, kaempferol, and rutin levels were higher in hairy roots compared to nontransgenic roots of c. anguria [ ] . the contents of naringenin and biochanin a were lower in concentrations in hairy roots than nontransgenic roots of c. anguria [ ] . myricetin, quercetin, catechin, kaempferol, rutin, biochanin a, and naringenin levels were higher in hairy roots compared to nontransgenic roots of m. charantia [ ] . naringin was presented in nontransgenic roots, but it was absent in hairy roots of m. charantia [ ] . quercetin, kaempferol, catechin, and rutin levels were higher in hairy roots compared to nontransgenic roots of m. dioica [ ] . myricetin, naringenin, and biochanin a contents were lower in concentrations in hairy roots than nontransgenic roots of m. dioica [ ] . kaempferol, myricetin, naringin, quercetin, and rutin have antimicrobial activity against human pathogenic microorganisms with some mechanisms of action such as inhibition of nucleic acid synthesis, cytoplasmic membrane function, and energy metabolisms [ ] . caffeic acid and chlorogenic acid were major hydroxycinnamic acid derivatives in hairy roots and nontransformed roots compared to p-coumaric acid, ferulic acid, ocoumaric acid, and t-cinnamic acid in c. anguria [ ] . caffeic acid, ferulic acid, ocoumaric acid, and t-cinnamic acid levels decreased in hairy roots compared to nontransformed roots of c. anguria [ ] . chlorogenic acid and p-coumaric acid contents were higher in hairy roots than nontransformed roots of c. anguria [ ] . chlorogenic acid containing plant materials have been shown to have antiviral, antifungal, and strong antibacterial activities [ ] . chlorogenic acid, p-coumaric acid, and ferulic acid levels were higher in hairy roots compared to nontransformed roots of m. dioica [ ] . caffeic acid, o-coumaric acid, and t-cinnamic acid contents were lower in hairy roots than nontransformed roots of m. dioica [ ] . caffeic acid, p-coumaric acid, o-coumaric acid, chlorogenic acid, and m-coumaric acid levels were higher and ferulic acid content was lower in hairy roots than nontransgenic roots of m. charantia [ ] . protocatechuic acid, β-resorcylic acid, syringic acid, gentisic acid, and salicylic acid levels were higher and gallic acid, p-hydroxybenzoic acid, and vanillic acid were lower in hairy roots than nontransgenic roots of c. anguria [ ] . gallic acid, p-hydroxybenzoic acid, gentisic acid, and salicylic acid levels were higher and protocatechuic acid, β -resorcylic acid, and vanillic acid were lower in hairy roots compared to nontransformed roots of m. dioica [ ] . gentisic acid has an effective role in the anticarcinogenetic activity [ ] . gallic acid, protocatechuic acid, β-resorcylic acid, vanillic acid, syringic acid, gentisic acid, and salicylic acid levels were higher and p-hydroxybenzoic acid was lower in hairy roots compared to nontransformed roots of m. charantia [ ] . veratric acid was higher and vanillin, hesperidin, and homogentisic acid were lower in hairy roots compared to nontransformed roots of c. anguria [ ] . vanillin was higher and veratric acid, hesperidin, and homogentisic acid levels were lower in m. charantia and m. dioica [ , ] . previous studies have revealed that polyphenolic compounds are commonly found in both edible and nonedible plants and that they have multiple biological effects, including antioxidant activity [ ] . flavonoids and other phenolic substances may play a preventive role in the development of cancer and heart disease [ ] . biological activities related to antibacterial and antioxidant activities may be correlated with total polyphenol and flavonoid contents [ ] . the total phenolic and flavonoid contents were higher in hairy roots compared to untransformed roots of c. anguria, m. charantia, and m. dioica [ ] [ ] [ ] . gypenosides (gyp) are the major components of gynostemma pentaphyllum makino, a chinese medicinal plant. phytochemical studies of g. pentaphyllum have identified approximately dammarane-type saponin glycosides, known as gypenosides, which are responsible for its pharmacological activities [ ] . saponins are a class of chemical compounds found in particular abundance in various plant species. more specifically, they are amphipathic glycosides grouped phenomenologically by the soap-like foaming they produce when shaken in aqueous solutions and structurally by having one or more hydrophilic glycoside moieties combined with a lipophilic triterpene derivative. triterpenoid saponins are triterpenes which belong to the group of saponin compounds. triterpenes are a type of terpene containing carbon atoms. triterpenes are assembled from a five-carbon isoprene unit through the cytosolic mevalonate pathway to make a thirty-carbon compound. cucurbitacins are triterpenoids that confer a bitter taste in cucurbits such as cucumber, melon, watermelon, squash, and pumpkin. these compounds discourage most pests on the plant and have also been shown to have antitumor properties [ ] . gypenoside content was higher compared to roots of control parent plant of gynostemma pentaphyllum hairy root cultures [ ] which was significantly higher than that previously reported for hairy root cultures [ ] . transformed roots can synthesize and store significant quantities of secondary metabolites. although the hairy roots under these conditions produced approximately % to % less gypenosides than commercial sources of g. pentaphyllum, the growing time was much shorter when compared to field-grown plants. with hairy root cultures, product quality and quantity are easy to control because natural variances in seasonal climates and geographical environments are excluded and culture conditions and process variables are easily optimized [ ] . the hairy root cultures have been considered as a potential alternative for production of gypenosides. several strategies for the enhancement of biomass and gypenosides have been adopted like the effects of medium compositions, culture conditions, and elicitations [ ] . ribosome-inactivating proteins (rips) are widely distributed plant enzymes that inhibit protein synthesis by virtue of their n-glycosidic activity, selectively cleaving an adenine residue from a highly conserved and surface-exposed stem loop structure in the s rrna [ ] . this cleavage prevents the binding of the ef- /gtp complex, with the subsequent arrest of protein synthesis leading to autonomous cell death [ ] . rips are either enzymatically active single polypeptides (type i) or heterodimers (type ii). a type ii rip consists of an a chain, functionally equivalent to a type i rip, which is attached to a sugar-binding b chain [ ] . besides rna n-glycosidase activity, some rips have ribonuclease, dnase, dna glycosylase, and apurinic/apyrimidic lyase activities [ , ] . in addition, rips from trichosanthes kirilowii cell cultures have been demonstrated to possess chitinase activity [ ] . certain type i rips display a variety of antimicrobial activities, including antifungal, antibacterial [ ] , and broad-spectrum antiviral effects against different plant and animal viruses [ ] , including human immunodeficiency virus [ ] . rips have been studied as potential tumor cytotoxic agents, both in their native form and after conjugation with monoclonal antibodies. rip activity of l. cylindrica plantlets, grown in vitro on ms medium, was evaluated in crude extracts from different parts and organs and compared to the inhibitory activity shown by extracts from seeds and from transformed roots [ ] . the inhibitory activity, as far as normal, nontransformed tissues are concerned, is in agreement with what was already known from previous reports of l. cylindrica [ , ] . rip-producing hairy roots promise to be much more stable than conventional in vitro grown calluses and cell suspensions [ ] . this study tested the sc-rip extracts from the seeds and hairy root tissue cultures of luffa cylindrica (established by transformation with agrobacterium rhizogenes strain ) for inhibitory effects on the growth of in vitro melanotic and amelanotic human melanoma cell lines [ ] . the results reported that rips can be produced and purified from hairy root cultures, in good agreement with what has been recently reported [ ] for hairy root lines of trichosanthes kirilowii. ribosomeinactivating proteins (rips) from plants catalytically damage eukaryotic ribosomes, making them unable to perform the elongation step of protein synthesis. type rips are single-chain proteins, whereas type rips consist of two polypeptide chains and possess a galactose-specific binding domain to cell surfaces. type rips are more common and have been identified and purified from more than plants. interest in type rips has been growing due to their widespread physiological activities as abortifacient agents and immunotoxins [ ] . the antiviral activity of rips has also focused attention on their potential use as anti-hiv agents [ ] . there have been few reports on the production of rips by plant tissue or cell cultures. a low level of trichosanthin was reported to accumulate in transformed hairy root cultures of trichosanthes kirilowii var. japonica [ ] . trichosanthin was also identified in cell extracts of the transformed callus tissues resulting from infection by agrobacterium rhizogenes but not in the untransformed callus of t. kirilowii [ ] . the major protein in the basic protein fraction was tentatively identified as a class iii chitinase based on the n-terminal amino acid sequence. this is consistent with the report [ ] , who identified two major extracellular basic proteins and one intracellular basic protein produced by t. kirilowii var. japonica hairy roots as class iii chitinases. however, the n-terminal sequence of hr-pb was very similar to but not identical with the sequence of any of these proteins. a molecule of charantin consists of aglycone or a steroidal portion, which is highly soluble in relatively nonpolar solvent such as chloroform and dichloromethane. however, the glucosides attached to its molecules make it slightly soluble in polar organic solvents such as ethanol or methanol. conventionally, isolation of this compound involves extraction with mixtures of these solvents using soxhlet apparatus. chloroform is highly toxic and carcinogenic, and its use has now been replaced with its much less toxic relative, dichloromethane, which still carry some health risks. chronic exposure to dichloromethane has been linked to cancer of lungs, liver, and pancreas in laboratory animals. it is a mutagen and may cause birth defect if women were exposed to it during pregnancy [ ] . this compound could be used to treat diabetes and can potentially replace treatment by injection of insulin which has not been successful in stimulating the pancreas of the diabetic patients to lower blood sugar to the desired level [ ] . in some cases, the injected patient shows signs of side effects. plant derived compounds that show antidiabetic property such as charantin and others are now being widely accepted as an alternative medicine for diabetes mellitus, and they are free from side effects [ ] . charantin, a naturally occurring steroidal glycoside, is widely distributed throughout the plant of momordica charantia. the presence of charantin was confirmed by performing thin layer chromatography (tlc) in hairy roots as well as in fruit and leaf [ ] . the charantin content was lower in hairy roots compared to leaf and fruit of m. charantia [ ] . the typical cucumber flavor results from the enzymatic action of lox on linolenic and linoleic acids, which introduces molecular oxygen at c or c , forming -hydroperoxylinolenic acid ( -hpot) or -hydroperoxylinolenic acid ( -hpot). hpl cleaves -hydroperoxide ( -hpo) and -hpo to produce the c and c aldehydes that are responsible for the cucumber flavor [ ] . these aldehydes can then be reduced to the corresponding c alcohols by alcohol dehydrogenase (adh). studies have reported that only the oxylipin metabolic pathway contributes to aldehyde and alcohol content and hence flavor [ ] . to date, volatile compounds have been identified in cucumber fruits, including aldehydes, alcohols, esters, alkanes, furfurans, and others [ ] , and (e,z)- , nonadienal and (e)- -nonenal are the main aroma compounds [ ] . the hairy roots could newly synthesize some essential oils such as (z)- -hexenol, (e)- hexenal, -nonanol, and (z)- -nonenol, which were reported to be important aroma volatiles in melon [ ] . the stable production of the fruity aroma volatiles by the kmh- was assessed by comparing the yields of the compounds from the hairy roots repeatedly subcultured for more than years. the data revealed that the essential oils for aroma scent were constantly synthesized with no relation to the increased number of times for subculture, and the constant production by this clone was successfully maintained in the hairy roots repeatedly subcultured of cucumis melo [ ] . the volatile compounds were extracted and identified by glc-mass spectrometry. some essential oils such as (z)- -hexenol, (e)- -hexenal, -nonanol, and (z)- -nonenol were stably synthesized by these hairy roots despite the increased number of subcultures. the productivity of these compounds by the best hairy root line was shown to be considerably higher than naturally ripened melon fruits [ ] . phenolic compounds have multiple additional roles in plants, including attracting insects for seed dispersion and pollination. they are also part of the natural defense system against insects, fungi, viruses, and bacteria, and they can act as plant hormone controllers. moreover, in recent years, phenolic compounds have been intensively investigated because of their potential health-promoting effects [ , ] . they have been reported to possess many useful properties for human health, including anti-inflammatory, enzyme inhibition, antimicrobial, antiallergic, vascular, and cytotoxic antitumor activity, but the most important action of phenolics is their antioxidant activity [ , , ] . it has been demonstrated recently that quercetin and kaempferol synergistically suppress cell proliferation in human gut cancer lines [ ] . the translational inhibitory activity found in extracts from our hairy root cultures is the highest that has been found in various tissues of l. cylindrica, including seeds [ ] . the antioxidant potential of hairy roots and nontransformed roots were determined using free radicals scavenging, reducing potential, phosphomolybdenum assays, and chelating effects on ferrous ions. the highest antioxidant activity was exhibited in hairy roots compared to nontransformed roots in c. anguria, m. charantia, and m. dioica [ ] [ ] [ ] . reducing capacity of extracts suggests that hairy roots were more potential when compared to untransformed roots in c. anguria, m. charantia, and m. dioica [ ] [ ] [ ] . the antioxidant capacity shown by phosphomolybdenum method was higher in the hairy root extract than nontransformed root extract of c. anguria, m. charantia, and m. dioica [ ] [ ] [ ] . the percentage of metal scavenging capacity of transgenic hairy roots was higher than nontransgenic roots of m. charantia, c. anguria, and m. dioica [ ] [ ] [ ] . hairy roots exhibited higher antioxidant activity in m. charantia [ ] . the hairy roots and nontransformed roots of c. anguria, m. charantia, and m. dioica revealed varying antibacterial activity, as exposed by the growth inhibition zones [ ] [ ] [ ] . the results from the disc diffusion method indicated that both hairy roots and nontransformed root extracts had comparable antibacterial effects against gram positive and gram-negative bacteria. hairy roots exhibited highest activity with both gram-positive and gram-negative bacteria compared to nontransformed roots of c. anguria, m. charantia, and m. dioica [ ] [ ] [ ] . gram-positive (s. aureus) bacteria exhibited greater inhibition compared to gram-negative (p. aeruginosa and e. coli) bacteria in m. charantia, c. anguria, and m. dioica [ ] [ ] [ ] . by using the disc diffusion method against the fungal strains, it can be seen that extracts of m. charantia, c. anguria, and m. dioica hairy roots and nontransformed roots exhibited good antifungal activity [ ] [ ] [ ] . hairy roots exhibited greater inhibition of fungus (f. oxysporum and a. niger) in hairy roots than nontransgenic roots of c. anguria, m. charantia, and m. dioica [ ] [ ] [ ] . hairy roots exhibited higher antibacterial and antifungal activity compared to nontransformed roots [ , ] . flavonoid derivatives have also been reported to possess antiviral activity against a wide range of viruses such as hsv, hiv, coxsackie b virus, corona virus, cytomegalovirus, poliomyelitis virus, rhinovirus, rotavirus, poliovirus, sindbis virus, and rabies virus [ ] . cytotoxicity activity and quantitative assay of virus yields using plaque assay were carried out for hairy roots and nontransgenic roots of m. dioica [ ] . hairy roots exhibited higher antiviral activity compared to nontransgenic root extracts of m. dioica [ ] . gypenosides (gyp) are compounds found in the crude extracts from g. pentaphyllum and they have been shown to exert various biological effects such as anti-inflammatory and antioxidative [ ] , antihyperlipidemic, anticardiovascular [ ] , and anticancer [ ] [ ] [ ] . our previous studies have shown that gypenosides induced apoptosis in human colon cancer colo cells [ ] and human tongue cancer scc- cells through endoplasmic reticulum stress and mitochondria-dependent pathways [ ] . although gypenosides have been shown to induce cell cycle arrest and apoptosis in several human cancer cell lines, there is no available information to address whether gypenosides induce dna damage or affects dna repair genes in sas human oral cancer cells. diabetes mellitus is an endocrine metabolic disorder in which the body does not produce sufficient insulin or lack of responsiveness to insulin, resulting in hyperglycemia (high blood glucose level). the classical symptoms include polyuria, polydipsia, weight loss, lethargy, polyphagia, visual blurring, frequent or recurring infections, cuts and bruises that are slow to heal, tingling and/or numbness in hands and/or feet, drowsiness, nausea, and decreased endurance during exercise [ ] . a number of potential medicinal components from bitter gourd, such as α and β momorcharin, momordin, and cucurbitacin b, have been isolated. a number of reported clinical studies have shown that bitter gourd extract from fruits, seeds, and leaves contain several bioactive compounds that have hypoglycemic activity in both diabetic animals and humans [ ] . fruits, seeds, and leaves extract of momordica charantia possess hypoglycemic activity in antihyperglycemic activity in alloxan [ ] or streptozotocin [ ] . the major compounds that have been isolated and identified as hypoglycemic agents include charantin, polypeptide-p, and vicine. charantin is a steroidal glycoside shown to possess powerful hypoglycemic properties when administered orally and intravenously in diabetic rabbits [ ] . hairy roots produced higher amount of charantin which used for antidiabetics [ ] . the vast potential of hairy root cultures as a stable source of biologically active chemicals has focused the attention of the scientific community for its exploitation. scaling up of hairy roots in novel bioreactors can provide the best conditions for optimum growth and secondary metabolite production, comparable to or higher than that in native roots. though the need for developing bioreactors suitable for the hairy root cultivation has long been recognized, root cultures present unique challenges [ ] . the complex fibrous structure of the roots makes the growth analysis and development of a large-scale culture system difficult. hairy root growth is not homogeneous, which affects the reactor performance. furthermore, the hairy root morphology is quite plastic as the roots respond to the changes in the local environment. changes in morphology, including changes in the density and length of the root hairs, directly affect the secondary metabolite production from hairy roots [ ] . thus, bioreactor design for root cultures is a balancing act between the biological needs of the tissues, without inducing an additional, undesirable biological response [ ] . reviews on hairy roots briefly discuss the importance of the use of bioreactors for hairy root cultures [ ] . mechanical agitation causes wounding of hairy roots and leads to callus formation. due to branching, the roots form an interlocked matrix that exhibits resistance to nutrient flow. hairy roots are hetrotrophic, respiratory organisms that rely on oxygen for energy generation and other metabolic functions. substantial progress has been made in understanding the mechanisms of oxygen limitation, one of the principle challenges for large-scale growth of hairy root cultures [ ] . because of the solid phase nature of the roots and the development of oxygen gradients within root tissues, relatively small reductions in the dissolved oxygen concentration in the medium can lead to a significant decrease in growth rate and may also affect the synthesis of certain secondary metabolites. in fact, hairy roots can be oxygen limited even in shake flask cultures [ ] . restriction of nutrient oxygen delivery to the central mass of tissue gives rise to a pocket of senescent tissues. mass transfer resistances near the liquid and solid boundary affect the oxygen delivery to the growing hairy roots. thus, exploitation of hairy root culture as a source of bioactive chemicals depends on the development of suitable bioreactor system where several physical and chemical parameters (nutrient availability, nutrient uptake, oxygen, and hydrogen depletion in the medium, mixing, and shear sensitivity) must be taken into account. the design of bioreactors for hairy root cultures should also take into consideration factors such as the requirement for a support matrix and the possibility of flow restriction by the root mass in certain parts of the bioreactor. several bioreactor designs have been reported for hairy root culture taking into consideration the above factors that permit the growth of interconnected tissue unevenly distributed throughout the culture vessel. reactors used to culture hairy roots can roughly be divided into three types: liquid-phase, gas-phase, or hybrid reactors that are a combination of both [ ] . previously, there are no reports on the large scale production of hairy roots using bioreactor system in cucurbits and the production of phenolic compounds. various biotic and abiotic elicitors applied to hairy root cultures and their stimulating effects on the accumulation of secondary metabolites. according to their origin, elicitors can be divided into different types: (a) biotic and (b) abiotic. abiotic elicitors can be considered as substances of nonbiological origin, being predominantly inorganic compounds such as salts or physical factors [ , ] . inorganic chemicals like salts or metal ions have been used to increase the production of bioactive compounds by their modification of plant secondary metabolism. among the many elicitors applied to hairy root cultures, the most common and effective elicitors are fungal cell extracts, polysaccharides from fungal and plant cells, and heavy metal salts. with the crude fungal cell extracts, it is essential to observe the preparation conditions carefully for achieving reproducible effects. in addition to the chemical agents, uv-radiation, hyperosmotic stress, and temperature shift have been shown effective for some plant species/metabolites. elicitor type, dose, and treatment schedule are major factors determining the effects on the secondary metabolite production. in addition to the accumulation of products in roots, elicitor treatments often stimulate the release of intracellular products. although elicitation is mainly effective to increase specific product yield on per unit mass of roots, the incorporation of nutrient feeding strategies can be applied to enhance the volumetric product yield. the integration of in situ product recovery from the roots/liquid medium is another synergistic strategy with the elicitor treatment to improve the process. so far, there are no reports on the elicitation of hairy roots and production of phenolic compounds from hairy root cultures of cucurbits. further, researchers can use the elicitation to improve the contents of secondary metabolites in cucurbits. hairy root technology has been significantly improved in various fields for past few years. overall, the major groups of secondary metabolites have already been produced from hairy roots of cucurbits. compared to plant cell suspension cultures, hairy root cultures appear to be potential systems for continuous production of valuable secondary metabolites because of their fast growth rates, ease of maintenance, genetic and biosynthetic stability, and ability to synthesize a vast array of compounds. environmental factors, such as light, oxygen, and temperature, as well as abiotic and biotic stress factors, such as phytohormones, heavy metals, and fungal elicitors, have all been applied to hairy roots for increased yield of phytochemicals. in addition to these external stimuli, secondary metabolic pathways have also been modified for enhanced metabolite production, such as overexpression of biosynthetic genes and transcription factors, and suppression of catabolic or competing pathway genes. a better understanding of the biosynthetic pathway and regulation architecture of valuable secondary metabolites is crucial for genetic engineering and fully realizing the biosynthetic potential of hairy roots. the discovery of new genes that participate in the metabolic pathways from hairy root studies increases the tremendous potential of such cultures. it is also predicted that this model of pharmaceutical production is relatively safe. driven by the demand for productive, robust, and stable hairy root cultures for the production of active agents for the food, cosmetics, and pharmaceutical industry, the development of a direct available measuring method for the biomass concentration of hairy root cultures in liquid medium still does not exist. transgenic hairy roots grew rapidly than nontransgenic roots in standardized liquid culture conditions and produced greater amount of biomass and phenolic compounds. the higher amount of secondary metabolites possibly contributes to greater biological activity of hairy roots in cucurbits. the genetic and biochemical stability of the hairy roots as well as its high productivity offers an effective platform for further studies on the biosynthetic pathways of phytochemicals. this prediction is strengthened by the observation that emerging private companies have converted this technology to allow production at a commercial scale. plant biologists can work closely with engineers to tackle the challenges with scaling up hairy root cultures, such as optimal biomass growth and adaptation of the extraction methods to industrial-scale metabolite 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(curcurbitaceae) fermentation studies of transformed root cultures characterization of fluid-flow resistance in root cultures with a convective flow tubular bioreactor the growth of single roots of artemisia annua in nutrient mist reactors advances and challenges in bioreactor design for the production of chemicals from plant tissue culture the extent to which external oxygen transfer limits growth in shake flask culture of hairy roots elicitation: an underutilized tool in the development of medicinal plants as a source of therapeutic secondary metabolites plant cell elicitation for production of secondary metabolites: a review acknowledgements this paper was supported by the ku research professor program of konkuk university, seoul, republic of korea. key: cord- - kpxhzbe authors: das, jayanta kumar; pal choudhury, pabitra title: chemical property based sequence characterization of ppca and its homolog proteins ppcb-e: a mathematical approach date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: kpxhzbe periplasmic c type cytochrome a (ppca) protein is determined in geobacter sulfurreducens along with its other four homologs (ppcb-e). from the crystal structure viewpoint the observation emerges that ppca protein can bind with deoxycholate (dxca), while its other homologs do not. but it is yet to be established with certainty the reason behind this from primary protein sequence information. this study is primarily based on primary protein sequence analysis through the chemical basis of embedded amino acids. firstly, we look for the chemical group specific score of amino acids. along with this, we have developed a new methodology for the phylogenetic analysis based on chemical group dissimilarities of amino acids. this new methodology is applied to the cytochrome c family members and pinpoint how a particular sequence is differing with others. secondly, we build a graph theoretic model on using amino acid sequences which is also applied to the cytochrome c family members and some unique characteristics and their domains are highlighted. thirdly, we search for unique patterns as subsequences which are common among the group or specific individual member. in all the cases, we are able to show some distinct features of ppca that emerges ppca as an outstanding protein compared to its other homologs, resulting towards its binding with deoxycholate. similarly, some notable features for the structurally dissimilar protein ppcd compared to the other homologs are also brought out. further, the five members of cytochrome family being homolog proteins, they must have some common significant features which are also enumerated in this study. amino acids play the vital role for determining the protein structure and functions. but it is informative to know how the functionality of the group of proteins is changed while amino acid patterns are getting changed from one protein to another. it becomes quite harder and mostly time consuming to identify the uniqueness of proteins and their functionality from the wet lab experiments while working with complete sequence. in this regard, several techniques have been developed for the analysis of primary protein sequence that is helping the plos biochemist to work with only specific domain instead of the whole sequence which reduces the experiment time. geobacter sulfurreducens is one of the predominant metal and sulphur reducing bacteria [ ] . the organism geobacter sulfurreducens is known to act as an electron donar and participate in redox reaction [ ] . periplasmic c type cytochrome a (ppca) protein along with its four additional homologs (ppcb-e: ppcb, ppcc, ppcd, ppce) are identified in geobacter sulfurreducens genome [ ] [ ] [ ] [ ] . altogether, five proteins are highly conserved around "heme iv" but are not identical, and mostely differ in two hemes, "heme i" and "heme iii" [ ] . these two regions are known to interact with its own redox partner. deoxycholic acid (conjugate base deoxycholate), also known as cholanoic acid, is one of the secondary bile acids, which are metabolic byproducts of intestinal bacteria used in medicinal field and for the isolation of membrane associated proteins [ , ] . among the five members of cytochromes c family, only ppca can interact with deoxycholate (dxca) while its other homologs cannot. while interacting with dxca, it is observed that few residues are utilized [ , , ] . it would be worthy if the reason of such an amazing difference towards recognizing a single compound can be found through the amino acids sequence viewpoints. further, one can also see the reason of the structural dissimilarity of ppcd compared to the other homologs [ ] . in literature, in-silico techniques have been used to tackle the various problems through the analysis of dna, rna and protein sequences in bioinformatics field. specially, the authors are searching the protein blocks which are highly similar and conserved among the sub-group or entire family members [ ] [ ] [ ] [ ] . there are twenty standard naturally occurring amino acids which are diverse, arises complexity in the sequences, and have some group specific susceptibility. various reduced alphabet methods are established which can perform much better in certain conditions [ ] [ ] [ ] [ ] . sequence similarity is the most widely reliable strategy that has been used for characterizing the newly determined sequences [ ] [ ] [ ] [ ] . finding the functional/structural similarity from homolog sequences with low sequence similarity is a big challenging task in bioinformatics. to tackle this problem, several methods have been introduced that can identify homolog proteins which are distantly distributed in their evolutionary relationships [ ] [ ] [ ] [ ] . again, in microrna field the authors have developed a new identification technique of microrna precursors emphasizing on different data distributions of negative samples [ ] . further, phylogenetic analysis are also studied from different viewpoints to find the evolutionary relationship among various species [ ] [ ] [ ] . some authors have used the statistical tools for sequence alignment, alignment-free sequence comparison and phylogenetic tree [ ] [ ] [ ] [ ] . although every amino acid has individual activity, group specific function of amino acid is also obvious. methods have been introduced for the d graphical representation of dna/rna or protein sequences [ ] [ ] [ ] [ ] [ ] [ ] [ ] where methods are based on individual score and position wise graphical representation. so, in this field establishment of a new methodology is always welcome with distinct findings. combining with various features for dna, rna and protein sequence a web server called pse-in-one (http:// bioinformatics.hitsz.edu.cn/pse-in-one/home/) is developed [ ] which is user friendly and can be modified by users themselves. recently, the authors have classified the twenty standard amino acids into the eight chemical groups and have found some group and/or family specific conserved patterns which are involved in some functional role specially in motor protein family members [ ] . in this study, the previously defined method [ ] of reduced alphabets are used as an application into the cytochrome c family protein members. we introduced a new method of phylogenetic analysis based on chemical group dissimilarity of amino acids. in addition, we build the graph from primary protein sequence. in the designing of graph, we have designated the various chemical groups of amino acids as thevertices in the graph. the primary protein sequence is read as consecutive order pairs serially from first amino acid to the end of sequence, and each order pair is nothing but a connected edge between the two nodes where nodes in the graph are involved with different chemical groups of amino acids. the graph is drawn for every individual protein sequence and we look for various unique edges/ cycles among the entire family members. so any unique findings from the graph may be hypothesized as having a significant functional role in the primary protein sequence. because the variation in the graph is directly affected by the amino acid residues in some specific domain where a change of chemical group has taken place. we highlight all the significant points which are differing from one sequence to other. further, working with reduced alphabets and designing the graph require less complexity and easy visualization even if working with the larger sequences. order pair directed graph a directed graph g = (v, e) is a graph which consists of a set of vertices denoted by v = {v , v , . . ., v i }, and a set of connected edges denoted by e = {e , , e , , . . ., e i, j } where an edge e i, j exists if the corresponding two vertices v i and v j are connected and the direction of edge is from the vertex v i to the vertex v j . from the graph, various graph theoretic properties like edge connectivity, cycles, graph isomorphism etc. can be investigated to differentiate the graphs. given an arbitrary amino acids sequence, it is first transformed into the numerical sequence as described previously where amino acids are categorized into eight chemical groups according to the side chain/chemical nature of the amino acids [ ] . the transformation is done using the following rules (eq ) as per the classification. if a particular amino acid is read as a i , then the corresponding transformed group is g k and the numerical value k is defined by the following eq ( ). : ifa i fd; eg here, g , g , . . ., g are the acidic, basic, aliphatic, aromatic, cyclic, sulfur containing, hydroxyl containing and acidic amide groups respectively [ ] . the eight numerical values are considered as the vertices of the graph g i.e. v i { , , . . . }. algorithm is used to generate the directed graph from the primary protein sequence using matlab b software. here, we obtain the graph which is the order pair digraph because an edge is constructed through the pair (source node, target node) which is obtained from the consecutive order pair list of amino acids in the primary protein sequence. so given an arbitrary amino acid sequence, we can find an order pair directed graph having at most eight vertices/nodes. output: an adjacency matrix and the corresponding order pair directed graph. define a null matrix (m) of size by ; define a -d array (t) of size l,; find x as the chamical group number of a i uisng eq ( ); the phylogenetic tree is an acyclic graph showing the evolutionary relationship among the various biological species based on their genetic closeness. although various phylogenetic tree methods have already been studied, based on chemical nature of amino acids are not yet explored in the literature as per our knowledge. our method of phylogenetic tree formation used the dissimilarity matrix which is obtained for every pair of sequence on the basis of chemical group specific score of amino acids. so this method is completely alignment free and requires less computational complexity. firstly, we calculate the percentage of occurrence of amino acids from each chemical group using the following equation eq ( ) . if there are n number of sequences which are denoted as s , s , . . .s n , then the corresponding length of the sequences are denoted as l , l , . . .l n . and a particular sequence s i is read as for the sequence s , the first amino acid is read as s , the second amino acid is read as s and so on. for each g k group and a particular sequence s i , we count the total number of amino acids s i (t k ) and score per hundred s i (g k ) on using the following eqs ( ) and ( ) respectively. for example, if the primary protein sequence length is aa, out of which aa are from acidic group i.e. g , then the score per hundred of the acidic group is  À Á ¼ %. secondly, we measure the dissimilarity measure for every possible pair of sequence. the dissimilarity of two sequences s i and s j is denoted as d s i ; s j . for each group g k , we count the percentage of amino acid differences of the two sequences taking the mod value of the score obtained on using eq ( ). this is done for all the respective eight chemical groups and all the values are added. finally, we get the dissimilarity matrix d of size n by n as shown below. dðn; nÞ ¼ to draw the phylogenetic tree, we use the nearest distance (single linkage) method. the pair wise distances are the entities of the obtained dissimilarity matrix and the whole procedure is written in matlab b software. five homologous triheme cytochromes (ppca-e) are identified in g. sulfurreducens periplasm and gene knockout studies revealed their involvement in fe(iii) and u(vi) extracellular reduction [ , ] . cytochromes have been thoroughly studied for laboratory experiments because of their small size (about amino acids). table shows the gene name, accession number, protein name, length (#amino acids). the primary protein sequences are collected from http://www.uniprot.org/. sequence identity and the phylogenetic tree firstly, our analysis is directed to measure the primary protein sequence for every member. we obtain the percentage identity matrix of every pair of sequences ( sequence characterization of ppca and its homolog proteins ppcb-e: a mathematical approach exported from clustalw. it is observed that sequences are at least % similar. the maximum similarity is % which is found between ppca and ppcb. if we consider the ppca sequence which shows the minimum of % similarity with ppce and the maximum of % similarity with ppcb, we are not able to differentiate the ppca from other homologs on using the similarity percentage. secondly, we count rate of occurrence (frequancy of amino acids) of every individual amino acid of the respective five sequences which are shown in table . then, we look for chemical group specific frequency for every sequence shown in table using eq ( ). now, we obtain the dissimilarity score of all possible two sequences (using eq ( )). say for an example, we compare the seq. no. and seq. no. , we get the difference for acidic group is . ( . - . ), basic group is . ( . - . ) and so on (from table ). total score after summing the eight groups is . which measures the dissimilarity percentage of the said two sequences. similar results we get for all other pairs which are shown in table . this table shows the biological distances between each pair of sequences. from this pair wise distance matrix, the phylogenetic tree is constructed as shown in fig , also discussed in method section. based on the phylogenetic tree of five members, we find that the ppca and ppcd, ppcb and ppce are mostly closed with regards to the frequency of amino acids of respective eight chemical groups. from fig it is not obvious that ppca differs from other homologs, but if we go through the dissimilarity matrix (table ) , we find some variations. here, it is observed that ppca differs by minimum of . % with ppcd, whereas for other homologs minimum dissimilarity sequence characterization of ppca and its homolog proteins ppcb-e: a mathematical approach is found for ppcd with ppcc which is . %. therefore among all the pairs, the high dissimilarity of ppca shows its uniqueness compared to its homologs. if we have a closer look into the list of amino acids, it is observed that the amino acids d, e, h, k, f, i, l, v, a, g, p, m, c, t are present among all the sequences. other amino acids are not common to all the member sequences. therefore, on the basis of chemical groups, all the amino acids from acidic, aliphatic, cyclic and hydroxyl containing groups are present. it is observed that the acidic, basic and hydroxyl containing groups percentage distinctly differ while compared ppca with other homologs. further, it is observed that only one proline(p) from cyclic group is present in ppcd while in other homologs, proline (p) is present at least times. and another important observation is that the amino acid tryptophan (w) from aromatic group is present only in ppcd sequence. for every member of cytochrome c family, we draw a order pair directed graph using algorithm which are shown in fig . there are maximum of eight possible nodes and the various directed edges among the nodes. we try to highlight the connected edges that show the uniqueness, specially in between the ppca and its homolog members and ppcd with other members separately as well as commonality to all members. details of the edge connectivity information for ppca and its homologs are shown in table . we say two nodes (direction is from row to column) are connected or present if the cell symbol is , not present if the cell symbol is , and common to all the members if the cell symbol is à . an edge between two nodes (in order) is basically a pattern https://doi.org/ . /journal.pone. .g table . existance of unique edges comparison between ppca and ppcb-e groups obtained from directed graph (fig ) . ppcb-e node vs. node * * * * * * (two distinct nodes or two distinct amino acids from two different chemical groups) of length . we find two particular edges, one edge ( ) is present only in ppca sequence (approx. residues - , s table) that is not found in other member sequences, and one edge ( ) which is present in ppcb-e sequences (approx. residues - , s table) , but this edge is not present in ppca sequence. while considering all the members, we find many edges which are common to all. further, ppcd is structurally dissimilar among the homologs [ ] . while looking into the order pair directed graph, we find only one variation i.e. there is an edge ( ) node to node among the ppca-c and ppce sequences which is not observed in ppcd (table ) . this node transition where amino acid changes proline(p) to glycine (g) for ppca-c and ppce and for ppcd this transition is from glycine (g) to glycine (g), located in approximately residues - (s table) . again existence of edges between any two nodes either common to all or individual member specific have some significant role in the primary protein sequences. because node to node connectivity is the point of changes from one chemical group to the other in the primary protein sequence positions and this could be the effective characteristic for the structural or functional variation of proteins. although few residues are being responsible while interacting with dxca, the neighbouring residues of amino acids must be having a role for their unique characteristics. so the subdomain identification involving with different unique cycles would be worth mentioning in this regard. here, we have calculated the various cycles of length c l ( c l ) for group specific and individual member specific which are shown in in s table. say for an example, the cycle of length i.e. the directed edges are ! ! ! ! ! ! . for completing this cycle a particular subdomain is responsible. interestingly, we find various unique cycles for ppca, ppcd and ppcb-e. so there are some unique cycles which are distinctly present for ppca and its homolog proteins and vice versa. there are some unique cycles which are present in ppcd, but no unique cycle is present for ppca-c and ppce. highlighiting the sub-domain for some of the unique cycles of length , and are shown in fig (a) for ppca and fig (b) for ppcd. from fig , the cycle ( ) of length whose sub-domain residues are within to , that is the numerical sequence is . . . from fig (a) . one can see the corresponding amino acids residues from s table. for some cycles, there is a possibility of different sub domains because some edges are repeating more than once in the different positions of the sequence that can be counted for the same cycle. similarly, on varying the cycle length, we get different sub-domains or amino acid residues. these sub-domain findings might be of immense help to the bio-chemists for the understanding of physicochemical nature and the unique activity of various proteins. table . existance of unique edges comparison between ppcd and ppca-c, ppce groups obtained from directed graph (fig ) . ppca-c, ppce node vs. node * * * * * * we take all the five sequences of ppca-e members, obtain the alignment sequence from clus-talw . the alignment figure is shown fig . we mark the various blocks as r , r . . .r which are conserved. rectangular with highlighted regions are chemically conserved, and only highlighted regions are conserved based on individual amino acid. we find two highly conserved regions r and r which are having some variations. the first region (r ) is with residues block (hkk/rh or ) among the members ppcb-e where all the amino acids from basic group, but in ppca this block is hkah or i.e. the rd position k/r is replaced by aliphatic amino acid alanine (a). the second region (r ) is gche/k or / where th position amino acid is either from acidic or basic group i.e. both fall under charge group. if we look into the ppca sequence some dissimilarities are found in "heme i" region [ ] [ ] [ ] . the two consecutive amino acids between regions r and r in ppca is kk (from basic group), but for ppcb-e only one amino acid is from basic group. previously it is observed that ppcd is structurally dissimilar [ ] and the authors have shown that there is an addition of amino acid threonine (t) for ppcd sequence after the r region in fig . but, from figure we can see that another one amino acid valnine (v) insertion is viewed in region of r and r . besides, various patterns which are common to ppca, but not in ppcb-e and vice versa shown in table with bold color. for the pattern " " which is located with the combined regions of r and r ("heme iii" region), there is a change of amino acid threonine (t) for ppcd and lysine (k) for others. apart from these, we find an amino acid deletion both for the ppcd and ppce before the "heme iii" region. further, on combining the regions r , r and sequence characterization of ppca and its homolog proteins ppcb-e: a mathematical approach r (pattern " "), the change for ppca sequence is phenylalanine (f) which is from arometic group whereas other sequences are from aliphatic group, and the change for ppcd sequence is histidine (h) which is from basic group whereas other sequences are also from aliphatic group. again the region between r and r ppcd contains the amino acid methionine (m) from the sulfur containing group while the other homologs contain phenylalanine (f) from aromatic group. altogether, group specific changes have significant role towards the binding with the dxca for ppca and the structural dissimilarity of ppcd. in this work, we have presented the sequence based characterization of cytochromes c family members. we specifically emphasize the distinguished features of ppca and ppcd compared to the other homologs. although the study suggests that percent identity among the five members varies between % and %, on the basis of chemical groups these are shown between % and %. we highlight some of the chemical groups and their percentage that can distinguish ppca and ppcd. the dissimilarity features of ppca may play significant role towards its binding with dxca. similar is the case that may happen for ppcd for its structural dissimilarity. our proposed graph theoretic model can easily show the instant change of amino acids from one group to the other in the sequences. further, the unique cycles for ppca and ppcd may expose their outstanding nature. and finally from the alignment graph, chemically conserved regions are highlighted. we observe some special patterns where amino acid(s) from some of the sequences are abruptly changed. all the cases will provide the features for ppca and ppcd that would explain their unique functionality and/or structural dissimilarity. it may be noted that there are some existing methodologies [ , , , , , , ] which would reflect the sequence pattern information or key features of the observed sequence. many characteristics of the dna, rna and protein sequences can be found out from the web servers and standalone existing tools, one of the important web servers in this regard is defined in [ ] . we look at the problem in a different manner, one dealing with embedded chemical properties of amino acids and various mathematical structures. in general, methodology defined in this article is very easy to implement to get the unique features of observed sequences. so, collectively our methodology will add to be combined for the machine learning algorithms to develop refined computational predictors. hence, the use of reduced alphabets (amino acids) technique involving mathematical basis with the embedded chemical properties of amino acids will be very much useful for the protein homology detection. supporting information s table. amino acids and transformed numerical sequence based on eight chemical groups for c five members. (pdf) s table. unique cycles for ppca-e, ppca, ppcb-e, ppcd. these cycles are involved in various sub-domains, some of which are shown in fig . (pdf) electricity production by geobacter sulfurreducens attached to electrodes geobacter sulfurreducens sp. nov., a hydrogen-and acetate-oxidizing dissimilatory metal-reducing microorganism. applied and environmental microbiology thermodynamic characterization of a triheme cytochrome family from geobacter sulfurreducens reveals mechanistic and functional diversity family of cytochrome c -type proteins from geobacter sulfurreducens: structure of one cytochrome c at . Å resolution † structural characterization of a family of cytochromes c involved in fe (iii) respiration by geobacter sulfurreducens structure of a novel dodecaheme cytochrome c from geobacter sulfurreducens reveals an extended nm protein with interacting hemes lipomas treated with subcutaneous deoxycholate injections guide to protein purification dissecting the functional role of key residues in triheme cytochrome ppca: a path to rational design of g. sulfurreducens strains with enhanced electron transfer capabilities conservation within the myosin motor domain: implications for structure and function identification of common molecular subsequences selection of conserved blocks from multiple alignments for their use in phylogenetic analysis amino acid substitution matrices from protein blocks reduction of protein sequence complexity by residue grouping reduced amino acid alphabets exhibit an improved sensitivity and selectivity in fold assignment protein sequence analysis based on hydropathy profile of amino acids mathematical characterization of protein sequences using patterns as chemical group combinations of amino acids an introduction to sequence similarity ("homology") searching. current protocols in bioinformatics similarity/dissimilarity studies of protein sequences based on a new d graphical representation improved tools for biological sequence comparison analysis of similarity/dissimilarity of protein sequences combining evolutionary information extracted from frequency profiles with sequence-based kernels for protein remote homology detection application of learning to rank to protein remote homology detection. bioinformatics protein remote homology detection by combining chou's pseudo amino acid composition and profile-based protein representation a comprehensive review and comparison of different computational methods for protein remote homology detection imirna-ssf: improving the identification of microrna precursors by combining negative sets with different distributions phylogenetic analysis of protein sequence data using the randomized axelerated maximum likelihood (raxml) program. current protocols in molecular biology phylogenetic analysis of protein sequences based on conditional lz complexity analyzing and synthesizing phylogenies using tree alignment graphs a probabilistic measure for alignment-free sequence comparison simplification of protein sequence and alignment-free sequence analysis phylogenies and the comparative method progressive sequence alignment as a prerequisitetto correct phylogenetic trees graph theory with applications to engineering and computer science protein flexibility predictions using graph theory dictionary of protein secondary structure: pattern recognition of hydrogenbonded and geometrical features use of information discrepancy measure to compare protein secondary structures -d graphical representation of protein sequences and its application to coronavirus phylogeny a d graphical representation of protein sequence and its numerical characterization similarity/dissimilarity analysis of protein sequences based on a new spectrum-like graphical representation pse-in-one: a web server for generating various modes of pseudo components of dna, rna, and protein sequences we thank dr. pokkuluri, phani raj (argonne lab, usa) for the initial discussions of the problem. key: cord- -ita mjr authors: zinn, marc-kevin; bockmühl, dirk title: did granny know best? evaluating the antibacterial, antifungal and antiviral efficacy of acetic acid for home care procedures date: - - journal: bmc microbiol doi: . /s - - - sha: doc_id: cord_uid: ita mjr background: acetic acid has been used to clean and disinfect surfaces in the household for many decades. the antimicrobial efficacy of cleaning procedures can be considered particularly important for young, old, pregnant, immunocompromised people, but may also concern other groups, particularly with regards to the covid- pandemics. this study aimed to show that acetic acid exhibit an antibacterial and antifungal activity when used for cleaning purposes and is able to destroy certain viruses. furthermore, a disinfecting effect of laundry in a simulated washing cycle has been investigated. results: at a concentration of % and in presence of . % citric acid, acetic acid showed a reduction of > -log steps according to the specifications of din en and din en for the following microorganisms: p. aeruginosa, e. coli, s. aureus, l. monocytogenes, k. pneumoniae, e. hirae and a. brasiliensis. for mrsa a logarithmic reduction of . was obtained. tests on surfaces according to din en showed a complete reduction (> -log steps) for p. aeruginosa, e. coli, s. aureus, e. hirae, a. brasiliensis and c. albicans at an acetic acid concentration of already %. virucidal efficacy tests according to din en and din en showed a reduction of ≥ -log-steps against the modified vaccinia virus ankara (mva) for acetic acid concentrations of % or higher. the results suggest that acetic acid does not have a disinfecting effect on microorganisms in a dosage that is commonly used for cleaning. however, this can be achieved by increasing the concentration of acetic acid used, especially when combined with citric acid. conclusions: our results show a disinfecting effect of acetic acid in a concentration of % and in presence of . % citric acid against a variety of microorganisms. a virucidal effect against enveloped viruses could also be proven. furthermore, the results showed a considerable antimicrobial effect of acetic acid when used in domestic laundry procedures. people have been using natural products like vinegar to clean and sanitize surfaces in the domestic environment for decades [ ] . however, there is little scientific evidence on the antimicrobial efficacy of these traditional cleaning methods. inter alia, an appropriate, yet effective use of antimicrobial active products must be considered important to prevent the spread of infections. at home, especially young, old, pregnant and immunocompromised persons (yopis) are at higher risk. many potential pathogens such as pseudomonas aeruginosa, members of the enterobacteriaceae family or even methicillin-resistant staphylococcus aureus (mrsa) have already been found to be present on household surfaces [ ] [ ] [ ] [ ] [ ] . in order to achieve an adequate hygiene at home, many people use bleaching agents, as these are readily available, relatively inexpensive and have a very good antimicrobial effect [ ] [ ] [ ] . on the other hand, consumers do not want to use "chemical" cleaning agents and thus like to use "green" alternatives such as vinegar. already in , rutala et al. were able to show that undiluted white distilled vinegar has a strong effect against salmonella spp. and p. aeruginosa at an exposure time of s, but does not work well against s. aureus and escherichia coli [ ] . vinegar is mainly comprised of acetic acid, a weak organic acid, for which an antimicrobial effect is mainly delivered by its undissociated form, by passive diffusion through the cell wall of the bacteria. the resulting change of the internal ph is believed to have an inhibitory effect on the bacteria by releasing protons [ ] . acetic acid has already been used in the food industry to inhibit food pathogens. various studies have shown a protective effect of acetic acid on various types of meat [ ] , tomatoes [ ] , carrots [ ] and some salads [ ] . further studies were also able to proof an inhibitory effect against certain microorganisms such as enterobacteriaceae [ , , [ ] [ ] [ ] . not only bacteria, but also viruses such as the norovirus, which belongs to the caliciviridae family [ , ] the annually occurring influenza virus [ ] and above all the new coronavirus sars-cov- [ ] , must be considered important for domestic hygiene procedures. norovirus is the leading cause of non-bacterial gastroenteritis in both industrialised and developing countries [ ] . here, infection usually occurs via the faecal-oral route, e.g. by ingesting contaminated food or water or via contact to droplets and aerosols of an infected person [ ] [ ] [ ] [ ] . sars-cov- , as a member of the coronaviridae family, is an enveloped virus, which can cause a severe form of pneumonia and has impacted the global community in an unseen manner since its emergence in december [ ] . apart from changing the daily life of billions of people, the covid- -pandemic has also led to a special perception for the proper inactivation of microorganisms in home care procedures and a fallback to traditional cleaning options with hygienic effects for lack of available disinfectants. as mentioned above, vinegar is widely believed to be an effective means for hygienic cleaning [ , ] . however, there is little scientific evidence for the antimicrobial efficacy of acetic acid based products for domestic cleaning and laundering. hence, the present study aimed to provide data on the antimicrobial efficacy of acetic acid, especially when used in domestic cleaning and laundering procedures. for this purpose, antibacterial, antifungal and antiviral efficacy tests based on existing and adapted standard protocols have been conducted to evaluate the hygienic potential of acetic acid [ ] [ ] [ ] [ ] . to assess its possible use for hygienic cleaning, acetic acid in different concentrations and combined with citric acid, was first evaluated in suspension tests according to din en and din en . the logarithmic reduction factors (lr) for an extended spectrum of test strains are summarised in fig. . the results show that acetic acid in all tested concentrations lead to a complete reduction for p. aeruginosa and a. brasiliensis. for e. coli, a complete reduction could be achieved when using % acetic acid concentration, either alone or in combination with % citric acid. the (lr) of s. aureus increased with increasing concentrations of acetic acid and reached a maximum when % acetic acid and % citric acid was used, without, however, being able to exhibit a complete reduction. furthermore, a complete reduction was achieved with a test concentration of % acetic acid with the addition of % citric acid for the microorganisms l. monocytogenes and k. pneumoniae. for mrsa a maximum reduction of . was achieved. at an acetic acid concentration of %, a complete reduction was achieved for p. aeruginosa, e. coli and a. brasiliensis. for s aureus an lr of . could be detected. at acetic acid concentrations of . and % respectively, no sufficient reductions (lr . to . ) could be achieved for the microorganisms e. coli and s. aureus. the microorganisms l. monocytogenes, mrsa, k. pneumoniae and e. hirae were only tested with % acetic acid + . % citric acid, as this was the only concentration at which a lr of > log steps was acheived for the other microorganisms. the antimicrobial efficacy of and % acetic acid as well as a combination of % acetic acid and . % citric acid was evaluated on surfaces, since suspension tests are not reflecting this application very well. the logarithmic reduction factors (lr) for an extended spectrum of test strains are summarised in fig. . the results show that for all tested microorganisms in the three tested concentrations a complete reduction could be demonstrated. in order to test the virucidal activity of acetic acid, the tests were carried out in accordance with the standards en [ ] and en [ ] , where the effect against the modified vaccinia virus ankara (mva) was tested in suspension and on surfaces, respectively (fig. ) . the results of the virucidal tests show that a complete reduction (≥ log) could be achieved for all tested acetic acid concentrations ( , . and %) after min contact time. according to the standards used (din en and din en ), a product is considered virucidal as soon as it has achieved a reduction of ≥ log. to assess a putative effect of acetic acid in a laundry application, the lr of selected microorganisms was determined in a simulated main wash cycle using a labscale washing machine (rotawash). in contrast to the previous tests, a total concentration of . % or . % acetic acid was added to the wash liquor, alongside with a standard laundry detergent. the lr achieved in these tests are shown in fig. . the results show that for s. aureus, m. luteus and p. aeruginosa there was no significant difference in the lr between a wash cycle where only detergent was used or a cycle where . % acetic acid was added to the wash liquor including the detergent. in contrast, a significant increase of the lr could be demonstrated for the e. coli and s. hominis when . % acetic acid was added. furthermore, a significant increase in lr could be observed for all tested microorganisms when . % acetic acid was added to the wash liquor. here, a complete reduction could be observed for all bacterial test strains, except for s. aureus, for which a lr of . was determined. the current study aimed to investigate the antimicrobial, antifungal and antiviral effects of acetic acid for domestic cleaning and laundering based on different standard procedures and comprehensive tests. although there are many studies that have investigated the antibacterial and antifungal effects of acetic acid [ , [ ] [ ] [ ] [ ] [ ] [ ] there is no available data on how acetic acid does perform in standard procedures for the testing of disinfectants in suspension or on surfaces. likewise, the potential of acetic acid for laundry procedures has not been investigated before, although it is known that consumers sometimes use this substance as an additive to increase the hygiene performance of laundering [ ] . finally, it turned out that the covid- -pandemic in lead to an increased demand for pragmatic, yet effective solutions to improve domestic hygiene, particularly with regards to viruses. the results of this study showed that formulas containing an % acetic acid and . % citric acid are able to meet the standard requirements for disinfectants (i.e. a lr of > ), for all tested bacterial and fungal strains except for mrsa, which fits well with the findings of numerous other studies [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in addition to the suspension tests (din en and din en ), din en was used to test the disinfectant effect on a surface. here, the results obtained clearly show that a complete reduction could achieved for all tested microorganisms even at lower concentrations of acetic acid. ayhan and bilici could show that acetic acid disrupts the cell wall structure and thus causes a loss of atp in the cell [ ] . another study suggests that polyphenol compounds may also play a role in the antimicrobial effect of acetic acid. it was proven that polyphenols combine with the peptidoglycan structure of the cell wall and the phospholipid bilayer in the outer membrane of gram negative bacteria and thus impair the integrity of the cell. furthermore, polyphenols were shown to interfere with the activity of the intracellular bacterial enzymes by inhibiting the formation of amino and carboxyl groups of proteins [ ] . this supports the findings that polyphenols present in the acetic acid possess antimicrobial activity against a broad spectrum of microorganisms [ , ] . nastou et al. tested the effects of household washing treatments to control l. monocytogenes from lettuce. it was shown that application of % acetic acid resulted in a reduction of microorganisms by log. according to the results of nastou et al., which were able to disrupt an inhibitory activity of acetic acid, this effect is proportional to the concentration used [ ] . medina et al. also showed that vinegar (acetic acid) led to a complete reduction of l. monocytogenes and killed a high number of e. coli and s. aureus [ ] . these results support the data obtained in this study, as the maximum lr of l. monocytogenes of . was achieved with a % acetic acid concentration and a citric acid concentration of . %. furthermore, lrs of . and . for e. coli and s. aureus were achieved, respectively. gopal et al. ( ) showed that an acetic acid concentration of % led to a complete reduction of b. subtilis, e. coli and p. aeruginosa. these results are consistent with some pre-tests of the work presented here (data not shown). furthermore, the study of gopal et al. indicated that a % acetic acid led to a complete reduction for aspergillus niger (now: a. brasiliensis [ ] ) and a reduction of more than log steps for candida albicans (c. albicans) [ ] . the results obtained in the present study largely agree with these findings. the study at hand also obtained a complete reduction for a. brasiliensis already at an acetic acid concentration of %. the results for c. albicans, however, are different in the current study, since were also able to achieve a complete reduction of c. albicans at a low acetic acid concentrations ( %). these differences might be explained by the vinegar used, since gopal et al. used an apple cider vinegar, whereas in the current study vinegar made from acetic acid diluted with water and purified to a high degree of purity was used. ryssel et al. investigated whether acetic acid might be used as an alternative for common local antiseptics. they mixed . ml bacteria solution (bacterial count approx. - cfu/ml) with . ml acetic acid ( %) and incubated the mixture for , and min at a temperature of °c. they showed that at % acetic acid concentration no colonies of p. aeruginosa, of p. vulgaris, of a. baumannii and of β-haemolytic group b streptococci could be detected after min incubation. furthermore e. coli, e. faecalis and mrsa were eliminated after a exposure time of min [ ] . our experimental design used an incubation time of min and also showed a complete reduction of p. aeruginosa. in contrast to the attempt of ryssel et al. the current study also tested up to a concentration that would be required to pass disinfection tests, which for most observed microorganisms was % acetic acid and . % citric acid. at this concentration, a complete reduction for e. coli and a lr of . for mrsa could already be demonstrated with an incubation time of min. overall, there has been little research in the literature on the virucidal effect of acetic acid against enveloped viruses. in , rabenau et al. investigated the stability and inactivation of the sars coronavirus and could show that an acetic acid concentration of % leads to a reduction of > log levels within s [ ] . in contrast to the present study the authors aimed to investigate the stability of the sars coronavirus. nevertheless, the data of rabenau et al. confirm the current results, which suggest a complete reduction against enveloped viruses (see fig. ) at a concentration of %. in greatorex et al. were able to show in a study that acetic acid in a concentration of % is effective against the influenza virus a/h n [ ] . this result agrees with those of the present study, which showed that acetic acid is effective against the mva at a concentration of %. this could be demonstrated on the basis of the standards din en and din en , which apply to disinfectant tests with regard to virucidal activity. the present study could confirm the virucidal effect of acetic acid on the basis of existing standards. however, no tests were carried out on the virucidal effect in washing machines, because heinzel et al. were able to show in that conventional household washing detergents achieve a complete reduction of enveloped and non-enveloped viruses already at °c [ ] . the acetic acid concentrations tested in the present study were chosen based on the results of antibacterial and antifungal tests. as the results suggest that acetic acid concentrations of % + . % citric acid showed the highest reductions, the tested concentrations ( , . and % acetic acid) were taken for the virucidal activity tests. the results of the simulated washing process using the rotawash showed that a complete reduction of four microorganisms (m. luteus, p. aeruginosa, e. coli and s. hominis) could be achieved by adding an acetic acid concentration of . % to the wash liquor. likewise, a high lr of . could be achieved for s. aureus. thus, a disinfecting effect of acetic acid was proven for all tested microorganisms at an effective concentration of . % acetic acid. the acetic acid concentration of . % was used since it corresponds approximately to the dosage of commercially available laundry sanitizers [ ] . assuming that a common washing machine uses approx. l of water for each wash step, a final concentration of . % would equal a dosage of ml of a commercially available vinegar essence containing % acetic acid. consequently, a final concentration of . % would require the use of ml vinegar essence, which is still in the range that can be considered to be applied by consumers. the results showed that for s. aureus and m. luteus no additional antimicrobial effect was detected for the lower concentration of acetic acid compared to a simulated wash cycle with detergent alone. however, a significant difference ( -way-anova) for an additional dosage of . % acetic acid could be demonstrated for e. coli and s. hominis, which also exhibits a disinfecting effect with an lr of . . these findings suggest that a considerable antibacterial effect may be expected, when acetic acid is used a hygiene additive for laundry. the results of this study show that acetic acid in a concentration of % and an addition of . % citric acid has a disinfecting effect against a variety of microorganisms. in addition to the typical pathogens e. coli, s. aureus and l. monocytogenes, also p. aeruginosa, k. pneumoniae, e. hirae, a. brasiliensis and c. albicans are among the microorganisms that achieve a reduction of > -log steps against acetic acid in the concentration mentioned. furthermore, this study was able to show that acetic acid in a concentration of , . and % is also effective against enveloped viruses. moreover the present study showed that acetic acid above a certain concentration also has disinfecting properties on the laundry in a washing machine. it could be shown that an above-average dosage of the acetic acid s. aureus, m. luteus, p. aeruginosa, e. coli and s. hominis > log-steps are reduced. the suspension tests were performed according to the standards din en : and din en : [ , ] for bacteria and fungi and to the standard din en [ ] for viruses. all tests were performed under clean conditions, i.e. in presence of an organic challenge ( . g/ l albumine) at room temperature. as specified in the standards, the contact time was min for bacteria and min for yeasts. likewise, the microorganisms suspension used ranged between . and × cfu/ml. unlike described in din en and din en ml was used in the experiments instead of ml. all products were tested at room temperature. neutralization was carried out by dilution using an inactivator solution comprised of tween ( g/l), lecithin ( g/l), l-histidine ( g/l), sodium-thiosulfate ( g/ l) and saponin ( g/l). the virucidal tests were carried out strictly according to din en . the calculation of the reduction factors was done as described in chapter 'microbiological and statistical analysis'. all strains were purchased at the german collection of microorganisms and cell cultures (dsmz, brunswick, germany), except from the methicillin resistant staphylococcus aureus (mrsa), which was derived from the culture collection of the university of gothenburg (ccug). the corresponding strain code of the american type culture collection (atcc) is provided in table for information only. the determination of bactericidal an fungicidal activity on surfaces was performed according to din en [ ] and for virucidal activity according din en [ ] . all tests using bacterial strains were executed at rhine-waal-university of applied sciences; for virucidal tests, an external lab (dr. brill und dr. steinmann institute for hygiene and microbiology, hamburg, germany) was commissioned by the funder of this study. all tests were performed under clean conditions, i.e. in presence of an organic challenge ( . g/l albumine) at room temperature. as specified in the standards, the contact time was min for bacteria and min for yeasts. likewise, the microorganisms suspension used ranged between . and × cfu/ml. for the tests according to din en p. aeruginosa, e. coli, s. aureus, e. hirae, a. brasiliensis and c. albicans were used; for the tests according to din en the modified vaccinia ankara virus (mva) was used. the calculations of the reduction factors were performed as described in chapter 'microbiological and statistical analysis'. determination of the antibacterial activity in laundering procedures using a laboratory washing machine to assess the antimicrobial performance of products containing acetic acid for use in laundry detergents, a laboratory washing machine (rotawash m c, sdl atlas, rock hill, sc, usa)) was used as described in schages et al. [ ] . to simulate a normal household washing machine all quantities were downscaled adequately, i.e. a l vessel was filled with . l of water in addition to the ballast load textiles, the soil ballast, the detergent and eight steel beads (to simulate the mechanics of a washing machine) as described below. in this study, cotton (wfk a, g/m , wfk testgewebe, brüggen, germany) was used as the ballast load. in addition to the ballast load, sbl (sbl , wfk testgewebe, brüggen, germany) was used as a source of organic soil. all materials used are calculated based on the volume of water in a vessel of the laboratory washing machine: ballast load ( g/vessel) consisted of . g textile ballast of standard cotton and of . g textile comprised by the sbl swatches equalling approx. . g standard soil. a liquid heavy duty detergent (ariel actilift, procter & gamble, germany) was dosed according to the detergent manufacturers' instructions ( ml/ l) and adjusted to the volume of one vessel of the rotawash ( ml/ . l). the duration of the wash cycle in the rotawash was min, and the temperature was adjusted to °c, at a water inlet temperature of approx. °c - °c. in every test run five artificially contaminated biomonitors (one swatch per microorganism) are added to one vessel. in this series of experiments s. aureus, m. luteus, p. aeruginosa, e. coli and s. hominis were tested. all tests in the rotawash were run in triplicates. the evaluation is performed as described in chapter 'microbiological and statistical analysis'. the microbial count on each contaminated biomonitor was quantified by extraction with ml tsb-tlh-thio (tryptic soy broth with g/l tween , . g/l lecithin, g/l histidine, g/l sodium-thiosulfate) followed by investigating the colony forming units (cfu/ml) on surface culture on tryptic soy agar (tsa) (oxoid, wesel, germany; incubation at °c for h). rotawash-tests were carried out in a . ml reaction tube (sarstedt, nürmbrecht, germany) for min at °c and rpm in an orbital incubating shaker (thermomix comfort, eppendorf, hamburg, germany). the colony forming units (cfu/ml) were investigated in surface culture either on tsa for bacteria (incubation at °c for h) or malt extract agar (mea) for c. albicans and a. brasiliensis (incubation at °c for h). after laundering, the microbial count on the test swatches is determined similarly. the number of colony forming units (cfu/ml) on plates was used to calculate the microbial load in the extraction liquid (c wei ) (eq. ( )): Þþ n à : ð Þ Ãd ð Þ c wei = weighted arithmetic average. ∑c = sum of viable cell count of all agar plates, used for calculation. n = count of agar plates with the lowest evaluable dilution. n = count of agar plates of the next higher dilution stage. d = dilution factor of the lowest evaluable dilution stage. plates with less than cfu or more than cfu were not considered. to calculate the lr, the logarithmic cfu value of the biomonitors was subtracted from the logarithmic mean of the initial microbial count of the respective biomonitors. an investigation of microbial contamination in the home a critical evaluation of methicillin-resistant staphylococcus aureus and other bacteria of medical interest on commonly touched household surfaces in relation to household demographics community-based infections and the potential role of common touch surfaces as vectors for the transmission of infectious agents in home and community settings reduction of faecal coliform, coliform and heterotrophic plate count bacteria in the household kitchen and bathroom by disinfection with hypochlorite cleaners characterization of microbial communities in household washing machines effectiveness of laundering processes used in domestic (home) settings. in: international scientific forum on home hygiene does improving surface cleaning and disinfection reduce health care-associated infections? towards a lab-scale efficacy test method for the evaluation of hygienic laundry rinsestage disinfectants antimicrobial activity of home disinfectants and natural products against potential human pathogens perspectives on the use of organic acids and short chain fatty acids as antimicrobials organic acids as antimicrobials to control salmonella in meat and poultry products antibacterial activity of chaff vinegar and its practical application effectiveness of lemon juice, vinegar and their mixture in the elimination of salmonella typhimurium on carrots (daucus carota l.) effectiveness of household natural sanitizers in the elimination of salmonella typhimurium on rocket (eruca sativa miller) and spring onion (allium cepa l.) microbiological properties of pork cheek meat as affected by acetic acid and temperature death of salmonella, escherichia coli o :h , and listeria monocytogenes in shelf-stable, dairy-based pourable salad dressings effects of sodium lactate and acetic acid derivatives on the quality and sensory characteristics of hot-boned pork sausage patties gastroenteritis caused by norovirus ggii. , the netherlands statistical analysis of attack rate in norovirus foodborne outbreaks recurring influenza b virus infections in seals report on the current situation of pneumonia in wuhan molecular detection of human calicivirus in young children hospitalized with acute gastroenteritis in melbourne, australia, during stat -dependent innate immunity to a norwalk-like virus. science ( -) replication of norovirus in cell culture reveals a tropism for dendritic cells and macrophages laboratory efforts to cultivate noroviruses inactivation of a norovirus by high-pressure processing a novel coronavirus from patients with pneumonia in china the effectiveness of three home products in cleaning and disinfection of staphylococcus aureus and escherichia coli on home environmental surfaces efficacy of home washing methods in controlling surface microbial contamination on fresh produce deutsches institut für normung e. v. din en -chemische desinfektionsmittel und antiseptika -quantitativer suspensionsversuch zur bestimmung der bakteriziden wirkung (basistest) chemischer desinfektionsmittel und antiseptika -prüfverfahren und anforderungen deutsches institut für normung e. v. din en chemische desinfektionsmittel und antiseptika -quantitativer suspensionsversuch zur bestimmung der fungiziden oder levuroziden wirkung (basistest) chemischer desinfektionsmittel und antiseptika -prüfverfahren und anforderungen (phase ) deutsches institut für normung e. v. din en chemische desinfektionsmittel und antiseptika -quantitativer oberflächen-versuch nicht poröser oberflächen zur bestimmung der bakteriziden und/oder fungiziden wirkung chemischer desinfektionsmittel in den bereichen lebensmittel a new method to evaluate the antimicrobial efficacy of domestic laundry detergents deutsches institut für normung e. v. din en -chemische desinfektionsmittel und antiseptika -quantitativer suspensionsversuch zur bestimmung der viruziden wirkung im humanmedizinischen bereich -prüfverfahren und anforderungen deutsches institut für normung e. v. din en chemische desinfektionsmittel und antiseptika -quantitativer versuch auf nicht porösen oberflächen ohne mechanische einwirkung zur bestimmung der viruziden wirkung im humanmedizinischen bereich -prüfverfahren und anforderungen antimicrobial efficacy of household sanitizers against artificially inoculated salmonella on ready-to-eat spinach (spinacia oleracea) evaluation of alternative methods for the disinfection of toothbrushes antimicrobial activity of olive oil, vinegar, and various beverages against foodborne pathogens authenticating apple cider vinegar's home remedy claims: antibacterial, antifungal, antiviral properties and cytotoxicity aspect antibacterial action of vinegar against food-borne pathogenic bacteria including escherichia coli o :h in vitro assessment of antibacterial activity of pomegranate vinegar and rose water compared with persica mouthwash against oral bacteria toplu beslenme sistemlerinde kullanılan gıda dezenfektanları food disinfectants which are used in general food service systems vinegar functions on health: constituents, sources, and formation mechanisms efficacy of household washing treatments for the control of listeria monocytogenes on salad vegetables antimicrobial effects of vinegar against norovirus and escherichia coli in the traditional korean vinegared green laver (enteromorpha intestinalis) salad during refrigerated storage the antimicrobial effect of acetic acid-an alternative to common local antiseptics? technological and microbiological aspects of traditional balsamic vinegar and their influence on quality and sensorial properties antioxidant activities, phenolic profiles, and organic acid contents of fruit vinegars aspergillus brasiliensis sp. nov., a biseriate black aspergillus species with world-wide distribution stability and inactivation of sars coronavirus effectiveness of common household cleaning agents in reducing the viability of human influenza a/h n evaluation of the virucidal performance of domestic laundry procedures sagrotan desinfektion hygienespüler publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors like to thank speyer & grund gmbh & co. kg for funding this study. lr = logarithmic reduction factor. k = common logarithmic of the microbial count per ml of the initial load on the swatches before laundering. k s = common logarithmic of the microbial count per ml of the initial load on the swatches after laundering.unless otherwise stated, the tests were performed in triplicates and statistically evaluated in the case of a non-gaussian distribution using students t-test, kruskal-wallis or in the case of a gaussian distribution using a -way anova. authors' contributions mkz performed antibacterial and antifungal tests, analysed the data and wrote the manuscript. db designed the study, analysed the data and edited the manuscript. all authors have read and approved the manuscript. this work was financially supported by speyer & grund gmbh & co. kg. the funding body had no role in designing the study, sample collection, analysis, and interpretation of data or writing the manuscript. open access funding provided by projekt deal. the datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests.received: may accepted: august key: cord- -pcp i vb authors: troesch, barbara; eggersdorfer, manfred; laviano, alessandro; rolland, yves; smith, a. david; warnke, ines; weimann, arved; calder, philip c. title: expert opinion on benefits of long-chain omega- fatty acids (dha and epa) in aging and clinical nutrition date: - - journal: nutrients doi: . /nu sha: doc_id: cord_uid: pcp i vb life expectancy is increasing and so is the prevalence of age-related non-communicable diseases (ncds). consequently, older people and patients present with multi-morbidities and more complex needs, putting significant pressure on healthcare systems. effective nutrition interventions could be an important tool to address patient needs, improve clinical outcomes and reduce healthcare costs. inflammation plays a central role in ncds, so targeting it is relevant to disease prevention and treatment. the long-chain omega- polyunsaturated fatty acids (omega- lcpufas) docosahexaenoic acid (dha) and eicosapentaenoic acid (epa) are known to reduce inflammation and promote its resolution, suggesting a beneficial role in various therapeutic areas. an expert group reviewed the data on omega- lcpufas in specific patient populations and medical conditions. evidence for benefits in cognitive health, age- and disease-related decline in muscle mass, cancer treatment, surgical patients and critical illness was identified. use of dha and epa in some conditions is already included in some relevant guidelines. however, it is important to note that data on the effects of omega- lcpufas are still inconsistent in many areas (e.g., cognitive decline) due to a range of factors that vary amongst the trials performed to date; these factors include dose, timing and duration; baseline omega- lcpufa status; and intake of other nutrients. well-designed intervention studies are required to optimize the effects of dha and epa in specific patient populations and to develop more personalized strategies for their use. life expectancy is increasing globally [ ] and the prevalence of age-and lifestyle-related non-communicable diseases (ncds), such as cancer, heart disease, respiratory disease, type diabetes, obesity, chronic kidney disease and dementia is rising [ , ] . this has led patients to present with multiple co-morbidities [ , ] creating more complex needs (e.g., need for multiple medications), putting significant pressure on healthcare and social systems. undernutrition and overnutrition can both seriously impact an individual's risk for developing an ncd [ , ] . there is therefore a growing demand for appropriate nutrition interventions and targeted medical nutrition supplements or formulas to address patient needs, improve outcomes and help to reduce the costs of healthcare. inflammation is considered to play a central role in age-and lifestyle-related ncds [ ] , in loss of muscle mass and strength (sarcopenia) in frailty and cancer [ ] [ ] [ ] , and in the response to surgery and in critical illness [ ] . hence, targeting inflammation is thought to be appropriate to disease prevention and treatment. the long-chain omega- polyunsaturated fatty acids (lcpufas) docosahexaenoic acid (dha) and eicosapentaenoic acid (epa) are known to have roles in supporting human health [ ] , with one of their primary actions being to reduce inflammation [ ] [ ] [ ] and promote its resolution [ ] [ ] [ ] . this suggests a broad role for dha and epa in prevention and treatment of disease including, but not restricted to, specific therapeutic areas such as age-related decline in muscle mass, oncology, perioperative care and cognitive health. humans, like all mammals, cannot synthesize the essential omega- fatty acid α-linolenic acid. furthermore, endogenous synthesis of epa and dha from α-linolenic acid is described as being poor in most humans [ ] and is influenced by a range of factors such as age, sex, genetics and disease [ ] . therefore, preformed epa and dha must be obtained from the diet or supplements. it is now generally accepted that an intake of at least mg epa and dha per day is required for optimal nutrition [ ] [ ] [ ] [ ] , although the exact intake required for specific populations or health conditions is not known and in many cases is likely to be in excess of this suggested minimum intake. blood levels of epa and dha are highly related to intakes [ ] . global mapping indicated low or even very low blood levels of omega- lcpufas (i.e., dha and epa) in a large proportion of people for whom data were available [ ] , suggesting low intakes in those populations. reliance on endogenous synthesis of epa and dha is challenged by the low activity of this pathway [ ] which is further impaired in conditions such as insulin resistance [ ] . therefore, the benefits of dha and epa might be particularly pronounced in those population groups with insulin resistance or other features that limit endogenous synthesis. the anti-inflammatory and inflammation resolving effects of dha and epa have been shown to be relevant to improved clinical outcomes in a number of specific therapeutic areas [ ] [ ] [ ] [ ] [ ] [ ] ] . furthermore, evidence suggests that dha and epa support independence in the older population, improving quality of life and significantly lowering healthcare costs [ ] . moreover, they appear to be crucial for a well-functioning immune system [ ] and play an essential role in the maintenance of muscle mass and function [ ] , both important considerations for older people. adequate supply with dha and epa should therefore be seen as a critical component of both the prevention and treatment of many, but particularly age-related, conditions. this review aims to summarize the available evidence for dha and epa to promote healthy aging and to improve prognosis in a selection of medical conditions as discussed at an expert group meeting in september . dha and epa appear to act via overlapping, as well as distinct, mechanisms of action, modifying cellular function to benefit overall health and wellbeing, as well as to reduce the risk and severity of disease; these mechanisms are discussed in detail elsewhere [ , , ] . it is their membrane-mediated mechanisms that are most well established and understood [ ] [ ] [ ] [ ] and it is considered that through alterations at the membrane level in different cell and tissue types, dha and epa play an important role in cell signaling, gene expression and lipid mediator production [ ] . these mechanisms are quite well explored in the context of omega- lcpufa regulation of inflammatory processes, as described in detail elsewhere [ ] [ ] [ ] (figure ). for example, increased intake of epa and dha results in enhanced appearance of those fatty acids in the membrane phospholipids of cells involved in inflammation (see [ ] [ ] [ ] for references). this has multiple effects. firstly, cell membranes become more fluid, affecting the behavior of several membrane proteins, including their aggregation into signaling platforms, so-called lipid rafts (see [ ] [ ] [ ] for references). as a result, transmission of inflammatory signals within cells, for example from lipopolysaccharide or saturated fatty acids, becomes blunted, resulting in reduced activation of pro-inflammatory transcription factors like nuclear factor kappa-light-chain-enhancer of activated b cells (nfκb) (see [ ] [ ] [ ] for references). such transcription factors control expression of genes encoding many cytokines, chemokines, adhesion molecules, inflammatory enzymes (e.g., cyclooxygenase- ) and proteases. thus, though these effects are initiated at the cell membrane level, omega- lcpufas can affect multiple inflammatory mediators and their anti-inflammatory actions could be wide-ranging as a result. the second effect of increased epa and dha in the membranes of inflammatory cells is that they partially replace the omega- pufa arachidonic acid (see [ ] [ ] [ ] for references). arachidonic acid is the usual substrate for cyclooxygenase, lipoxygenase and cytochrome p enzymes producing eicosanoids [ , ] ; these eicosanoids (e.g., prostaglandin e , leukotriene b ) are recognized mediators of inflammation [ ] . therefore, through the epa-and dha-mediated decrease in arachidonic acid availability, production of these inflammatory eicosanoids is decreased (see [ ] [ ] [ ] for references). the third effect of increased epa and dha in the membranes of inflammatory cells is that they can be released upon cellular activation. the "free" epa and dha can then have further actions. for example, they can act as ligands and activators for anti-inflammatory transcription factors such as peroxisome proliferator activated receptors (see [ ] [ ] [ ] for references) and they can act as substrates for synthesis of eicosanoid and docosanoid lipid mediators. eicosanoids formed from epa such as prostaglandin e and leukotriene b often have only weak pro-inflammatory activity (see [ ] [ ] [ ] for references). however, probably more importantly, both dha and epa are substrates for the synthesis of highly active lipid mediators important in the resolution of inflammatory processes, including resolvins, protectins and maresins [ , ] . together, these mediators have been termed specialized pro-resolving mediators, and they have been shown in many cell culture and animal-based models to terminate inflammatory processes by decreasing cellular activation and the production of inflammatory cytokines, chemokines, adhesion molecules, proteases and enzymes (see [ , ] for references). nutrients , , x for peer review of inflammatory processes, as described in detail elsewhere [ ] [ ] [ ] (figure ). for example, increased intake of epa and dha results in enhanced appearance of those fatty acids in the membrane phospholipids of cells involved in inflammation (see [ ] [ ] [ ] for references). this has multiple effects. firstly, cell membranes become more fluid, affecting the behavior of several membrane proteins, including their aggregation into signaling platforms, so-called lipid rafts (see [ ] [ ] [ ] for references). as a result, transmission of inflammatory signals within cells, for example from lipopolysaccharide or saturated fatty acids, becomes blunted, resulting in reduced activation of pro-inflammatory transcription factors like nuclear factor kappa-light-chain-enhancer of activated b cells (nfκb) (see [ ] [ ] [ ] for references). such transcription factors control expression of genes encoding many cytokines, chemokines, adhesion molecules, inflammatory enzymes (e.g., cyclooxygenase- ) and proteases. thus, though these effects are initiated at the cell membrane level, omega- lcpufas can affect multiple inflammatory mediators and their anti-inflammatory actions could be wide-ranging as a result. the second effect of increased epa and dha in the membranes of inflammatory cells is that they partially replace the omega- pufa arachidonic acid (see [ ] [ ] [ ] for references). arachidonic acid is the usual substrate for cyclooxygenase, lipoxygenase and cytochrome p enzymes producing eicosanoids [ , ] ; these eicosanoids (e.g., prostaglandin e , leukotriene b ) are recognized mediators of inflammation [ ] . therefore, through the epa-and dha-mediated decrease in arachidonic acid availability, production of these inflammatory eicosanoids is decreased (see [ ] [ ] [ ] for references). the third effect of increased epa and dha in the membranes of inflammatory cells is that they can be released upon cellular activation. the "free" epa and dha can then have further actions. for example, they can act as ligands and activators for anti-inflammatory transcription factors such as peroxisome proliferator activated receptors (see [ ] [ ] [ ] for references) and they can act as substrates for synthesis of eicosanoid and docosanoid lipid mediators. eicosanoids formed from epa such as prostaglandin e and leukotriene b often have only weak proinflammatory activity (see [ ] [ ] [ ] for references). however, probably more importantly, both dha and epa are substrates for the synthesis of highly active lipid mediators important in the resolution of inflammatory processes, including resolvins, protectins and maresins [ , ] . together, these mediators have been termed specialized pro-resolving mediators, and they have been shown in many cell culture and animal-based models to terminate inflammatory processes by decreasing cellular activation and the production of inflammatory cytokines, chemokines, adhesion molecules, proteases and enzymes (see [ , ] for references). the foregoing discussion has emphasized the importance of the incorporation of dha and epa into cell membranes in order to elicit their anti-inflammatory and inflammation resolving actions. in this regard, it is important to recognize that the incorporation of dha and epa into the membrane phospholipids of cells involved in inflammatory responses, and into other cells and tissues such as skeletal muscle, is dose-dependently related to their intake (see [ ] [ ] [ ] for references). it is possible that the membrane changes induced by low intakes of dha and epa are insufficient to significantly alter cell and tissue function and therefore no biological or clinical impact would be observed. thus, the dose of dha and epa used in human studies is likely to be important in terms of determining the effect seen and too low a dose could result in the absence of an effect. with the increasingly aging population, cognitive decline has become a growing public health concern: the number of persons living with dementia is expected to nearly double every years [ ] . increasing evidence indicates that poor status of essential nutrients such as omega- lcpufas is associated with increased risk of cognitive decline and of developing alzheimer's disease [ ] . dha is a major fatty acid in membrane phospholipids in the grey matter of the brain and makes up approximately % of total fatty acids in the human cerebral cortex and % of all polyunsaturated fatty acids in the central nervous system [ , [ ] [ ] [ ] . brain dha levels decrease with adult age [ ] and seem to be particularly low among alzheimer's patients [ ] . it is conceivable that low brain dha contributes to the decrease in cognitive functions observed with advancing age in general and to a greater degree in dementia [ , ] . the link between low omega- lcpufa status and the risk of cognitive decline is supported by the observation that a higher proportion of total omega- lcpufas in the membranes of erythrocytes, considered to be a marker of both intake and status of these fatty acids, was associated with a reduced risk of developing cognitive decline in a french cohort [ ] . assessment of individuals with alzheimer's disease showed lower omega- lcpufa intakes and plasma phosphatidylcholine levels compared to healthy controls, but the study design did not allow to draw conclusions on causality [ ] . higher dha in plasma phosphatidylcholine was also associated with a % reduction in the risk of developing all-cause dementia (rr = . , % ci . - . ; p = . ) and a % reduction in risk of alzheimer's disease (rr = . , % ci . - . ; p = . ) in a cohort from the framingham heart study [ ] . the study also showed that higher dietary dha intake was associated with a non-significantly lower risk of developing dementia in general and alzheimer's disease in particular (upper quartile versus lower three quartiles: rr = . , % ci . a meta-analysis of observational studies showed a positive association of dha intake or plasma levels with memory in adults in general [ ]. the observational studies described above cannot establish a causal link and therefore intervention trials with omega- lcpufas are important to verify that these fatty acids can beneficially modify cognitive decline. findings from such intervention trials with omega- lcpufas are not consistent [ ] . however, there are relatively few trials and these differ in the dose of dha and epa and type of placebo used, the duration of supplementation, sample size, the severity of cognitive decline at baseline as well as the omega- lcpufa status of the participants (where this was even assessed) and the cognitive outcomes/tests used. supplementation with omega- lcpufas had a small effect on memory [ ] and executive function [ ] in non-demented older people. a meta-analysis of three randomized, placebo-controlled trials with omega- lcpufa supplements found no effect on severity of dementia, quality of life or mental health in patients with mild or moderate alzheimer's disease over , and months [ ] . intake of mg epa and mg dha per day for four months showed no effect on cognition or mood in individuals with alzheimer's disease [ , ] . however, this was a very small study and it has also been suggested that olive oil, which was used as a placebo, may have a protective effect for alzheimer's disease [ ] and might therefore have masked the effect of the supplementation with omega- lcpufas. similarly, an intervention comparing mg epa plus mg dha daily for months compared to olive oil did not find an effect on the california verbal learning test in cognitively healthy older adults (mean age years) [ ] . daily supplementation with mg dha and mg epa for six months did not affect the mini-mental state examination (mmse) score in acetylcholine esterase inhibitor treated patients with alzheimer's disease compared to a placebo [ ] . however, the intervention had a significant effect on cognitive functioning measured with the alzheimer's disease assessment scores as well as the sub-items, and a correlation was found with the increase in plasma omega- lcpufas [ ] . this suggests that the effect of omega- lcpufas depends on the specific aspect of cognitive health assessed. moreover, subgroup analysis showed a benefit of omega- lcpufas in the group with very mild cognitive decline (mmse score > ) at baseline [ ] . this is in line with the results from other trials indicating that interventions with dha and epa are less likely to have a beneficial effect on individuals experiencing dementia that has progressed beyond the mild stage [ , [ ] [ ] [ ] [ ] . a recent systematic review also reached the conclusion that the most beneficial effect of epa and dha supplementation in alzheimer's patients can be expected in the early stage of the disease [ ] . while individuals with mild cognitive decline are a promising target group, it might make sense to start the intervention even earlier, in older individuals with subjective cognitive decline [ ] . it has been shown that supplementation in healthy older people has a beneficial effect on white matter microstructural integrity, grey matter volume in specific brain areas and vascular parameters accompanied by improved executive function [ ] . this indicates that there might be a potential for preventive uses of omega- lcpufas to maintain cognitive health in older people. however, such an effect is difficult to show as the decrease over time in the placebo group will likely be too small to show a significant difference between the groups as seen in a supplementation trial in cognitively healthy older people [ ] . therefore, careful selection of the study population is required to find the window of opportunity during which the disease has not progressed too far but is already accelerating at a sufficient speed to be able to detect a difference in the decline between the intervention and the placebo groups. the multidomain alzheimer preventive trial (mapt) assessed whether a multimodal intervention consisting of nutritional counseling, physical exercise and cognitive stimulation, in combination with dha and epa, is effective in slowing cognitive decline in older at-risk adults [ ] . three years supplementation with mg dha and mg epa showed no significant effect on cognitive decline in older people with memory complaints [ ] . however, in a subgroup analysis only including individuals with low omega- lcpufa status at baseline, the supplementation had a beneficial effect on cognition [ ] . this indicates that people with low intakes or status of dha and epa should be targeted with such interventions as they may be more likely to experience the greatest benefit. not surprisingly, the dose of dha and epa provided in the intervention group also plays an important role and doses below mg have not had a major effect on cognitive health in older people with some degree of cognitive decline [ ] . several trials investigating the effect of omega- lcpufas on cognitive outcomes, including decline, have been relatively short, perhaps too short to significantly affect these outcomes. it has even been suggested that the three years of supplementation evaluated in the mapt might have been too short [ ] . as neurodegeneration develops over a considerable time, longer-term intervention might be required for a benefit to manifest. a systematic review and meta-analysis of available data from animal studies suggest > % of average total lifespan interventions had significant effects on cognitive function, neuronal loss and the amount of amyloid-beta deposits in the brain [ ] , but this period is considerably longer than the interventions in humans performed to date. in addition to omega- lcpufa dose, study duration and the rate of cognitive decline, other factors may also be relevant to whether an effect of these fatty acids is seen. these include the status of other nutrients and an individual's genotype. a re-analysis of the patients assessed in the omegad trial [ , ] found that those with low blood homocysteine, indicating good b vitamin status, benefitted cognitively and clinically from the combined dha and epa treatment, whereas those with high homocysteine did not [ ] . similarly, it had been shown that those older people with mild cognitive impairment who had the highest levels of plasma omega- lcpufas benefited most from supplementation with b vitamins [ , ] . in addition, adequate intake and status of antioxidants might be required for an optimal effect of dha and epa on cognitive health [ ] . it has been well established that apolipoprotein e (apoe) is a very important genetic risk factor for age-dependent chronic diseases, including alzheimer's disease [ ] , but not all trials have controlled for this. due to two major polymorphisms on the encoding exon of this gene, three major protein isoforms, apoe ε , apoe ε and apoe ε , exist [ ] . clinical and preclinical evidence suggests that carriers of apoe ε are at a higher risk of low omega- lcpufa status [ ] . moreover, it has been shown that homozygous carriers of the apoe ε allele have a more than -fold increased risk of developing alzheimer's disease, possibly due to increased cholesterol levels, altered brain development early in life [ ] or increased oxidative brain damage [ ] . a meta-regression by zhang et al. [ ] showed that stratification by apoe ε genotype had a significant effect on the association between dha, but not epa, intake and cognitive impairment. another analysis found a beneficial effect of omega- lcpufa supplementation on the progression of cognitive decline at an early stage in those with the apoe ε genotype [ ]. thus, individuals with certain genotypes may benefit more from omega- lcpufas than those with other genotypes. in summary, there is good evidence from observational studies for an association between dha and slower cognitive decline or reduced risk of alzheimer's disease. intervention trials are less clear, but there is some evidence that dha and epa can prevent or slow cognitive decline, particularly in the early stages. the inconsistent findings from trials likely relate to a number of factors including dose, duration and timing of the intervention, stage and rate of cognitive decline, status of other relevant nutrients (e.g., b vitamins) and genotype. with increasing age, achieving adequate intake of energy and essential nutrients becomes challenging due to alterations to appetite (anorexia of aging) and gastrointestinal physiology [ , ] . in addition, aging can affect dentition, gum and mouth health, and swallowing, so reducing food intake. cognitive decline, systemic disease and use of some medications can also impact food intake. reduced mobility, increased isolation and limited finances can restrict access to food in older people. as a consequence of these factors, malnutrition (i.e., undernutrition), frailty and sarcopenia are common and frequently overlapping conditions in older people [ ] [ ] [ ] . malnutrition is defined by espen as "a state resulting from lack of intake or uptake of nutrition that leads to altered body composition (decreased fat free mass) and body cell mass leading to diminished physical and mental function and impaired clinical outcome from disease" [ ] . inflammation is an important contributor to the outcome of malnutrition. espen recognizes disease-related malnutrition with inflammation as "a catabolic condition characterized by an inflammatory response, including anorexia and tissue breakdown, elicited by an underlying disease" [ ] . frailty is a state of vulnerability with limited reserve capacity in major organ systems; it involves weight loss, fatigue, low physical activity, slowness and weakness [ ] . frailty is associated with a higher risk of adverse outcomes such as falls, fractures, hospitalization and disability [ ] [ ] [ ] . in older inpatients, frailty was found to be a risk factor for increased length of hospital stay and mortality [ , ] as well as postoperative complications [ ] . moreover, frail patients were more likely to be discharged into care homes after hospitalization [ ] . a decrease in muscle mass was found to be a strong predictor of prognosis in hospitalized older people [ ] . sarcopenia is characterized by the progressive and generalized loss of skeletal muscle mass, strength and function with a consequent increased risk of adverse outcomes; the european working group on sarcopenia in older people defines sarcopenia as "a progressive and generalized skeletal muscle disorder that involves the accelerated loss of muscle mass and function" [ ] . sarcopenia is often part of the aging process preceding the onset of frailty. age-related chronic low-grade inflammation may be an important contributor to sarcopenia [ , , ] . sarcopenia seems to increase the likelihood of adverse outcomes such as disability, poor quality of life and death [ ] [ ] [ ] . both muscle mass and strength were predictive for difficulties in performing activities of daily living after discharge from the hospital [ ] . sarcopenia and particularly sarcopenic obesity (i.e., low muscle mass in association with greater fat mass), have been linked to poorer prognosis, including survival, for a range of cancers [ ] [ ] [ ] [ ] [ ] . pro-inflammatory cytokines have been linked to muscle wasting [ ] , and consequently, the anti-inflammatory effects of omega- lcpufas may be beneficial to prevent the loss of muscle mass and strength associated with aging, sarcopenia and frailty. furthermore, omega- lcpufas may themselves modulate muscle protein synthesis, promoting muscle strength and function [ , ] , likely as a result of their incorporation into membrane phospholipids of the sarcolemma and intracellular organelles [ ] . maintenance of, or an increase in, muscle mass and function seem to be key for healthy aging [ , ] , and also in recovery after surgery or during an intensive care unit (icu) stay [ ] . long-term supplementation with dha and epa in older people is therefore of increasing interest as the medical community looks for safe and affordable ways to slow physical disability and improve quality of life in older individuals. results from cross-sectional and longitudinal observational studies demonstrate that low plasma dha and epa levels are associated with poorer physical performance in older adults [ ] . daily supplementation with mg/d dha and mg/d epa for six months in healthy older men and women increased thigh muscle volume ( . %, % ci . % to . %, p < . ), handgrip strength ( . kg, % ci . to . kg, p < . ) and one-repetition muscle strength ( . %, % ci . % to . %, p < . ) and showed a trend towards increased average isokinetic power ( . %, % ci . % to . %, p = . ) compared to a control group [ ] . the intervention had no significant effect on body weight, total-body fat mass or the intermuscular fat content and raised no safety concerns [ ] . in post-menopausal women aged > years, supplementation with mg/d epa and mg/d dha for six months showed a positive effect on walking speed compared to the placebo group ( . ± % vs. − . ± %, p = . ) [ ] . supplementation for weeks with mg/d dha and mg/d epa in women aged to years resulted in a significant increase in lean body mass, increased resting metabolic rate and fat oxidation as well as decreasing time-to-get-up-and-go as a functional capacity measure [ ] . however, weeks supplementation with mg/d dha and mg/d epa had no effect on muscle mass or handgrip strength in community-dwelling older people (mean age . ± . years) [ ] . in another study, mg/d dha and mg/d epa in combination with physical exercise, cognitive training and nutritional counseling had no effect on different measures of muscle strength in older people [ ] . based on the evidence from these trials, doses of mg/d of dha plus epa or more (with preferably more than mg/d epa) may be required for positive effects on physical performance in older adults [ , ] as lower doses have not had an effect [ , ] . furthermore, the optimal ratio between dha and epa is not known and may differ between specific indications as different body compartments require distinct levels of omega- lcpufas (e.g., the brain is rich in dha and poor in epa). the scarcity of data from interventional studies [ ] has prevented the development of strong recommendations on the use of omega- lcpufas in the prevention of sarcopenia so far. more randomized controlled trials, with different duration and doses, are needed to establish their effect on maintaining muscle mass in the elderly and to decrease the risk of sarcopenia and the related adverse effects on health and well-being, including the onset of frailty. cancer is a major public health concern and both the disease and its treatment are associated with decreased quality of life and significant economic burden due to high healthcare cost and loss of productivity. increasing cancer incidence is due to several factors, including population growth and aging, as well as lifestyle and socio-economic factors. various dietary behaviors are thought to be involved in the pathogenesis and progression of some cancers and they play a crucial role in tumor growth and spreading [ ] . two ways by which diet could exert effects in patients with cancer are by enhancing anticancer therapies, mitigating their side effects, and by favoring the resolution of paraneoplastic syndromes, which in turn impact outcome. paraneoplastic syndromes are disorders triggered by an altered immune system response to new or abnormal growth of tissue. cancer cachexia is the most frequent paraneoplastic syndrome in individuals with cancer [ ] . cachexia is a form of disease-related malnutrition with inflammation [ , ] , and involves reduced appetite, altered utilization of nutrients, increased mobilization of amino acids and muscle protein turnover, loss of adipose tissue and infiltration of skeletal muscle with adipose tissue [ ] . left untreated, cachexia can progress in severity and contribute to the negative outcomes experienced by cancer patients, including mortality [ ] . an international consensus of clinical experts defined cancer cachexia as "a multifactorial syndrome defined by an ongoing loss of skeletal muscle mass (with or without loss of fat mass) that cannot be fully reversed by conventional nutritional support and leads to progressive functional impairment" [ ] . the importance of systemic inflammatory responses in cachexia is increasingly recognized, and it has been proposed to include this component in the definition of cancer cachexia [ , ] . further supporting the causative role of inflammation in the pathogenesis and clinical features of cancer cachexia, it has been recently demonstrated that an elevation of the neutrophil-to-lymphocyte ratio, a simple and reliable marker of systemic inflammation, associates with greater weight loss and cachexia in patients with advanced cancer [ ] . it has been proposed that current malnutrition rates in cancer patients are comparable to those > years ago, but they are less apparent as body mass index is often normal or even high, despite prevalence rates of cachexia and sarcopenia of % and % to %, respectively [ ] . it is estimated that cancer cachexia affects around % to % of cancer patients and is responsible for approximately % of deaths in cancer patients [ , ] . low muscle mass has a negative effect on treatment prognosis, resulting in reduced likelihood to complete at least three treatment cycles, more side effects and a lower chance of progression-free survival [ , ] . moreover, it has a negative impact on toxicity of cancer treatment [ ] [ ] [ ] [ ] and tumor progression during chemotherapy [ ] and causes marked distress to patients and their families [ ] . still, it remains underdiagnosed and is often not treated properly as pharmacological therapies mostly fail to improve the condition significantly [ ] . a review of available clinical trials showed that weight loss often starts very early in the disease progression, potentially even before the cancer itself is diagnosed [ ] . the precise mechanisms are poorly understood, but chronic systemic inflammation seems to play a crucial role in most patients [ ] . inflammation is recognized as a hallmark feature of cancer development and progression [ ] and targeting cancer-related inflammation at the local tumor microenvironment as well as in systemic circulation has the potential to favorably affect patient outcomes [ ] . optimal therapy should take into account the progression of the condition from pre-cachexia to cachexia and eventually refractory cachexia [ ] and would ideally involve a multimodal approach including nutritional interventions targeting inflammation and reduced food intake as well as decreased physical function [ , , ] . given their ability to mitigate inflammation, dha and epa interventions in cancer patients have received increasing attention and the mechanisms are reviewed elsewhere [ ] [ ] [ ] [ ] . there is evidence that dha and epa modulate the inflammatory response, measured as cytokines or c-reactive protein, and affect resting energy expenditure in cancer patients [ ] [ ] [ ] [ ] [ ] [ ] . these findings are relevant, as increased levels of inflammation in cancer patients induce changes in pharmacokinetics of some anti-cancer drugs, resulting in slower clearance and increased treatment-related toxicities [ ] . it has further been suggested that omega- lcpufas might play a role in mitigating the negative effect of disease as well as its treatment on gut health and microbiota composition [ ] . in addition, observations of decreasing plasma levels indicate a depletion of epa and dha in cancer patients [ ] . however, the effects of omega- lcpufas on nutritional status or meaningful clinical outcomes, such as quality of life, survival rates and treatment toxicity, are less well documented. based on evidence from different systematic reviews [ , [ ] [ ] [ ] [ ] , the espen guidelines for nutrition in cancer patients state "in patients with advanced cancer undergoing chemotherapy and at risk of weight loss or malnourished, we suggest to use supplementation with long-chain omega- fatty acids or fish oil to stabilize or improve appetite, food intake, lean body mass and body weight" but the recommendation is graded as weak and the level of evidence as low [ ] . a sub-group meta-analysis found a significant effect of high-protein, omega- lcpufa-enriched oral nutritional supplements (ons) when compared with isocaloric controls on body weight (+ . kg, % ci . to . , p = . ) in cancer patients undergoing chemotherapy [ ] . two of the included studies reported an effect on muscle mass: supplementation with an omega- lcpufa-enriched ons ( mg/d dha and mg/d epa) resulted in a decrease in the loss of fat-free mass after three and five weeks in patients with non-small cell lung cancer (p = . ) [ ] , while an intervention with the same ons resulted in a mean gain of . kg muscle mass in the intervention group versus a mean loss of kg in controls (p = . ) [ ] . a similar intervention resulted in an increase in skeletal muscle mass and lean body mass in cancer patients with omega- lcpufa-enriched ons (p = . , p < . , respectively), while no change was seen in these parameters in the group that received the standard ons (p = . , p = . , respectively) [ ] . moreover, there are indications that supplementation with omega- lcpufas in combination with high protein might have a beneficial effect on quality of life in cancer patients [ ] . importantly, omega- lcpufas were shown to be safe and well tolerated by cancer patients [ , ] . in addition to their effect on lean mass in cancer patients, omega- lcpufas have potential use as adjuvants to cancer therapy [ ] . they are thought to affect tumor activity through a range of mechanisms [ ] . a review of the evidence of omega- lcpufas as an adjunct to chemotherapy found beneficial effects on tumor response to treatment, protection from therapy-related toxicity and maintenance of quality of life [ ] . further benefits of omega- lcpufa supplementation might include reduction in cancer-related pain as well as a decrease in major depressive disorders, which are a frequent consequence of the stress and anxiety caused by a cancer diagnosis [ ] . the lack of consensus on the definition of cachexia has led to the inclusion of patients at different stages of the condition into studies, which is expected to affect the outcomes significantly [ ] . inconsistent or negative outcomes in clinical trials, including those with omega- lcpufas, are often due to suboptimal study design regarding the selection of endpoint [ , ] or due to lack of randomization or (placebo) control group [ ] . moreover, the duration and size of the trials may have been too low in many cases to detect a relevant impact [ ] . the timing of the intervention will likely also play a role, as a recent study only showed a benefit if nutritional interventions were initiated before chemotherapy started [ ] . considerable heterogeneity also exists in the pharmacological treatment as shown in a recent review that found different combinations of chemotherapy used in seven studies on the effect of omega- lcpufas in cancer patients [ ] . dose selection and compliance also play an important role as shown by fearon et al. [ ] in a post-hoc analysis where there was a dose-response between reported intake of omega- lcpfa-enriched ons and total (r = . , p < . ) and lean body mass (r = . , p = . ), as well as a correlation between plasma phospholipid epa and change in total and lean body weight (r = . , p < . ; r = . , p = . ). this provides evidence that doses of mg/d dha and mg/d epa or even more are required for a significant effect on muscle mass. others suggest the use of at least to mg/d dha+epa based on data from the available clinical trials on their use as adjuvants for chemotherapy [ , ] . it is increasingly recognized that multimodal interventions are most promising for the therapy of cancer cachexia, yet most of the clinical evidence is derived from trials using only a single therapy [ ] . in a small feasibility trial, a combination of an omega- lcpufa-enriched ons (~ mg/d dha and mg/d epa), nutritional advice, mg/d celecoxib and exercise compared to standard of care resulted in a stabilization of body weight compared to weight loss in the control group [ ] . the subsequent phase iii study on this intervention is still ongoing [ ] . therefore, studies are needed that combine nutrition, including dha and epa, physical exercise as well as pharmacological interventions. studies highlighting cost-effectiveness might also be helpful in increasing acceptance of such interventions given the potential benefit and the low cost of omega- lcpufa supplements. due to the limited and inconclusive data available, many oncologists are yet to be convinced of the benefits that dha and epa have for cancer patients. their interest in the mechanisms and possible therapies of cancer cachexia could be increased by the recent understanding that some mechanisms leading to cachexia are also involved in the process of metastasis [ ] . if confirmed in clinical trials, early intervention with omega- lcpufas to prevent the development of cancer cachexia may also help to limit the spread of the tumor to distant organs. epidemiological evidence indicates a benefit from supplementation with omega- lcpufas throughout the clinical journey of a cancer patient as higher intakes of these fatty acids in patients diagnosed with colorectal cancer were found to be associated with reduced specific mortality [ ] [ ] [ ] . surgery leads to the release of stress hormones and inflammatory mediators proportional to the magnitude of the procedure, resulting in a metabolic imbalance towards increased catabolism [ , ] . while this serves to support tissue healing and the immune response, it favors the breakdown of muscle protein. this can be detrimental to the patient, especially when there is pre-existing malnutrition, sarcopenia, cachexia, obesity and myosteatosis [ ] or in the presence of low-grade inflammation due to underlying conditions such as cancer or diabetes [ ] . malnutrition in surgical patients has been proposed as "a nutritional state in which nutrient intake does not match nutrient needs-due to underlying disease(s), the surgical stress response, chronic or acute inflammation, intestinal malabsorption (e.g., diarrhea) and/or patient-related factors (e.g., socio-economic status)-leading to losses in lean tissue and diminished function" [ ] . nutritional intervention can help reduce the stress of surgery, thereby preventing and treating catabolism and malnutrition [ ] . this is thought to reduce the risk of complications, decrease the length of hospital stay and promote better functional recovery [ ] . considering the poor general health conditions of at-risk (e.g., many cancer) patients, nutritional conditioning (e.g., in the context of prehabilitation) may prepare individuals for an enhanced recovery after surgery (eras) protocol [ ] . optimal timing for the introduction of nutritional therapy depends on the type of surgery and the general health status of the patient and needs further investigation [ ] [ ] [ ] [ ] [ ] [ ] [ ] . given their effect on inflammation mitigation, it is reasonable to expect a benefit of adding dha and epa to perioperative immunonutrition therapy. however, the evidence to support this is limited and most studies compared an ons containing dha and epa combined with other immune modulating nutrients (i.e., arginine and nucleotides with or without glutamine) with regular hospital diet rather than with a standard ons. a recent meta-analysis focusing on patients with gastrointestinal cancer included studies with patients, where the control group received either no supplements or an isonitrogenous standard ons [ ] . the preoperative administration of immunonutrition resulted in significantly decreased postoperative infectious complications in the combined studies (or . , % ci . - . , p < . ) as well as the studies with a standard ons as a control (or . , % ci . - . , p = . ). for length of hospital stay, significance was only reached in the combined studies (− . days, % ci − . to − . , p < . ) but there was only a weak trend when compared to ons (− . days, % ci − . to . , p = . ). no significant effect was seen on non-infectious complications or mortality. given their effect on post-operative morbidity and length of stay, the current espen guideline for surgical patients advises that standard ons are given pre-operatively to all malnourished cancer and other high-risk patients undergoing major abdominal surgery [ ] . the evidence is somewhat stronger for benefits of postoperative than for preoperative immunonutrition [ ] , although the optimal timing for its introduction to patient treatment plans still needs further investigation. the espen recommendation is that "peri-or at least postoperative administration of specific formulae enriched with immunonutrients should be given in malnourished patients undergoing major cancer surgery" [ ] . based on the duration of supplementation in the trials with positive outcomes, immunonutrition containing dha and epa as well as arginine and nucleotides should start five to seven days before surgery [ ] . similarly, the recommendations from the north american surgical nutrition summit include five to seven days of pre-operative immunonutrition including omega- lcpufas, which should be continued well into the postoperative period [ ] . it has even been suggested that the ideal period for pre-operative nutritional support is seven to days-or longer for severely malnourished patients-in addition to postoperative nutritional support [ ] . patients who are severely compromised (e.g., due to cancer) should ideally receive preoperative nutrition support for more than days [ ] . moreover, attenuation of the metabolic response to the stress of surgery through a range of measures including immunonutrition in the perioperative period is increasingly being recommended [ , ] as the combination of different elements, rather than a single one of them, is thought to produce the optimal outcome for patients [ ] . while many of the trials in this area did not follow an eras program, adherence to such a protocol might further increase the benefits of immunonutrition. this is supported by evidence from a multicenter study in well-nourished cancer patients undergoing colorectal resection comparing peri-operative use of an ons with immune-nutrients compared to a standard ons as part of a more comprehensive eras protocol [ ] . immunonutrition including omega- lcpufas for seven days pre-and five days post-surgery was compared to a standard high caloric ons and led to a decrease in the total number of complications, primarily due to a reduction in infectious complications ( . % vs. . %, p = . ) [ ] . it is evident that dha and epa play a role in perioperative immunonutrition in cancer patients, but more well-designed trials comparing standard to specialized (immunonutrition) ons could provide clearer evidence for their use and confirm the optimal timing. a recent survey among gastrointestinal and oncologic surgeons in the u.s. showed the use of post-operative nutrition support was more common than pre-operative and the use of immune-nutrients was reported by approximately % of responders (versus approximately % use of protein-containing supplements) and lack of awareness was given as the major hurdle to a more widespread use [ ] . sepsis is a severe clinical syndrome defined as "a life-threatening organ dysfunction due to a dysregulated host response to infection" [ ] . in septic patients, inflammatory cytokines trigger the release of even more cytokines, culminating in a so-called cytokine storm that will in turn cause damage to cells and organs [ ] . the outcome can be multi-organ failure and death. in addition to these hyperinflammatory processes, immune suppression also seems to play a role in sepsis and the balance between the two is thought to vary depending on host-, pathogen-and therapy-related factors [ , ] . the factors leading to sepsis are still incompletely understood and attempts to dampen the cytokine storm activation or consequences have failed in clinical trials [ ] . a recent meta-analysis found a lower risk for mortality in patients with sepsis who received omega- lcpufas, mainly intravenously, compared to control groups (or . , % ci . to . , p = . ), while the reduction in infectious complications was only reported in one study and was not significant (or . , % ci . to . , p = . ) and none of the studies reported cases of new onset of organ failure [ ] . a complete interpretation of the findings of this meta-analysis is limited by the low number of included studies. acute respiratory distress syndrome (ards) and multiple organ failure are important complications in patients with sepsis, resulting in prolonged icu stays [ ] [ ] [ ] [ ] . specialized enteral formulations containing omega- lcpufas as well as other ingredients such as antioxidants are available for critically ill patients with ards or acute lung injury (ali). however, the evidence for their effect is inconsistent. early research demonstrated positive clinical outcomes such as improved oxygenation, fewer new organ failures, more ventilator-and icu-free days as well as lower mortality when comparing these with high omega- pufa or standard formulas [ ] [ ] [ ] [ ] . however, subsequent research could not replicate these findings [ ] [ ] [ ] [ ] [ ] [ ] . consequently, the sccm/aspen guidelines for critically ill patients do not recommend the use of these specialized formulas for ards/ali [ ] . in contrast, the canadian clinical practice guidelines recommend that clinicians consider these specialized formulas with fish or borage oil and supplemental antioxidants for patients with ards/ali [ ] . the disparity between the two guidelines is likely related to differences in the studies included in the evaluation and the methods used for analyzing and interpreting the data to develop recommendations. while a recent meta-analysis of patients with ards or ali showed no effect of enteral nutrition enriched with fish oil [ ] , after the exclusion of two studies using a bolus rather than continuous dose, there was evidence that omega- lcpufa-containing formulas decreased mortality in critically ill patients including those with ards/ali [ ] . moreover, a recent cochrane review of these trials identified a significant improvement in blood oxygenation and significant reductions in ventilation requirement, new organ failures, length of stay in the icu and mortality at days when omega- lcpufas were used in patients with ards or ali, although all-cause mortality was not significantly affected [ ] . these findings are important in the context of the current coronavirus pandemic since severe covid- results in ards and there are suggestions that omega- lcpufas could be a viable treatment that is worth investigating [ , ] . for critically ill surgical patients who require parenteral nutrition, intravenous lipid emulsions containing omega- lcpufas are considered safe, but parenteral nutrition should only be considered in patients who cannot be adequately enterally fed [ ] . international consensus exists that a dose of . to . g/kg/d of fish oil would be appropriate for patients who require parenteral nutrition [ ] [ ] [ ] [ ] . a recent meta-analysis of prospective randomized trials showed significant benefits for the fish oil containing parenteral nutrition compared to a standard lipid emulsion [ ] . the risk for infection was lowered by % ( studies: rr . . % ci . to . ; p < . ). mean length of stay in the icu was significantly shortened ( studies: . days; % ci − . to − . ; p = . ) as was the length of hospital stay ( studies: . days, % ci − . to − . ; p < . ). the risk for developing sepsis was also significantly diminished by % (nine studies: rr . , %ci . to . , p = . ). mortality was lower with %, but the difference did not reach significance ( studies: rr . , % ci . to . ; p = . ) [ ] . moreover, fish oil was found to be more cost-effective than parenteral nutrition with a standard intravenous lipid emulsion [ ] . the evidence to date indicates that the provision of dha and epa through capsules, oral nutrition supplements, or enteral or parenteral formulas can help to regulate the inflammatory environment in a number of medical conditions and that this is linked in many cases to improved function, clinical course and outcomes. as dysregulated inflammation is a component of many acute and chronic diseases [ ] , the potential application of dha and epa is broad in terms of prevention and treatment. there is good evidence that dha and epa are a safe and cost-effective treatment that could benefit multiple patient outcomes. use of dha and epa in some conditions is supported by their inclusion in relevant guidelines [ , , , , ] , although the level of evidence has sometimes been considered to be low. this is because of inconsistent data on the effect of dha and epa on clinical outcomes, especially in some settings. this inconsistency has limited stronger support through guidelines and has hindered the wider acceptance of the benefits of dha and epa in the medical community. if omega- lcpufas are effective in disease prevention and in patient care, it is important to understand the reasons behind the inconsistent findings of studies and use this information to design and conduct better clinical trials to determine if poor results may be due to a real lack of effect or to other factors. undoubtedly the dose of dha and epa used is an important factor, but this is not the sole explanation for inconsistencies. other considerations include the timing and duration of supply of dha and epa, epa to dha ratio, baseline epa and dha status, intake of other nutrients including omega- fatty acids, b vitamins and antioxidants, clinical state, and medication use. more well-designed intervention studies are required to address the relevance of these different variables in order to properly identify the effects of dha and epa in specific target patient 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sequential analysis pharmacoeconomics of parenteral nutrition with ω- fatty acids in hospitalized adults inflammatory responses and inflammation-associated diseases in organs the ability of fish oil to suppress tumor necrosis factor alpha production by peripheral blood mononuclear cells in healthy men is associated with polymorphisms in genes that influence tumor necrosis factor alpha production key: cord- -mmdk xom authors: chen, jing; tang, yue; liu, yun; dou, yushun title: nucleic acid-based therapeutics for pulmonary diseases date: - - journal: aaps pharmscitech doi: . /s - - - sha: doc_id: cord_uid: mmdk xom nucleic acid-based therapeutics present huge potential in the treatment of pulmonary diseases ranging from lung cancer to asthma and chronic pulmonary diseases, which are often fatal and widely prevalent. the susceptibility of nucleic acids to degradation and the complex structure of lungs retard the effective pulmonary delivery of nucleic acid drug. to overcome these barriers, different strategies have been exploited to increase the delivery efficiency using chemically synthesized nucleic acids, vector encapsulation, proper formulation, and administration route. however, several limitations regarding off-target effects and immune stimulation of nucleic acid drugs hamper their translation into the clinical practice. therefore, their successful clinical application will ultimately rely on well-developed carriers and methods to ensure safety and efficacy. in this review, we provide a comprehensive overview of the nucleic acid application for pulmonary diseases, covering action mechanism of the nucleic acid drugs, the novel delivery systems, and the current formulation for the administration to lungs. the latest advances of nucleic acid drugs under clinical evaluation to treat pulmonary disorders will also be detailed. due to their location and physiological function, the lungs are directly accessible to pollutants and viruses from the outside, rendering them susceptible to diseases ranging from lung cancer to chronic pulmonary diseases. among these pulmonary diseases, chronic obstructive pulmonary disease claimed . million lives in , while lung cancer caused . million deaths ( ) . since current treatments of these diseases have limited efficacy, many studies are being conducted to find novel effective treatments. though most lung diseases are considered to be the product of a variety of endogenous and exogenous influences, and less obviously are associated with gene replacement therapy. abnormal conditions are likely to arise from an imbalance between destructive and protective mechanisms. nucleic acids can be a new class of therapeutics to reconstitute a homeostatic balance by overexpression of protective genes or the suppression of damaging genes, which offers new strategies for the treatment of respiratory diseases ( ) . the mesh-like network of blood vessels in the lungs, coupled with easy access through the pulmonary airways, enables the lungs to be targeted by both intravenous and topical routes. the latter fact makes the lung unique compared with other organs, allowing specific lung sites such as alveolar cells and bronchial epithelium to be exclusively targeted for different therapeutic applications ( ) . in this review, we focus on nucleic acid-based therapies for pulmonary diseases. we discuss the hurdles nucleic acids face for translation into clinics and recent progress in the product into clinical trials. antisense oligonucleotides (asos) are single-strand dnas or rnas that selectively bind to complementary mrnas to modulate their functions. their hybridization could result in downregulation or upregulation of gene expression by diverse mechanisms. rnase h -dependent asos could bind to target rna to form hybrid through watson-crick base pairing and downregulate translation through rnase h-induced degradation of the mrna. splice switching oligonucleotides could control the way exons skipping, modulate pre-mrna splicing, and generate novel proteins. asos can also interfere with other aspects of rna functions, such as blocking association of specific transcription factors with mrna, antagonizing microrna activities, and inhibiting rna-mediated telomerase activity ( ) ( ) ( ) . antisense oligonucleotides are the first kind of nucleic acid drugs widely used in clinical trials. among the fdaapproved nucleic acids, aso-based drugs account for the majority as for now ( table i) . as of august , only one aptamer drug and sirna drug have been approved by the fda. the first clinically approved nucleic acid drug was aso drug, vitravene (fomivirsen), indicated for cytomegalovirus retinitis in . followed by kynamro (mipomersen) targeting mrna encoding apolipoprotein b for the treatment of familial hypercholesterolemia, exondys (eteplirsen) designed to skip exon of the dystrophin protein for the treatment of duchenne muscular dystrophy, spinraza (nusinersen) inducing the inclusion of exon in the smn and smn mrna to treat spinal muscular atrophy and recently luxturna (voretigeneneparvovec-rzyl) for biallelic rpe mutation-associated retinal dystrophy ( , ) . small interference rnas(sirnas) are double-strand rna molecules of to base pairs in length designed to silence target genes in a sequence-specific manner. after introduction into the cytoplasm, sirnas interact with multifunctional protein argonaute- and form the rna-induced silencing complex (risc), where one of the strands is degraded and the other strand (mostly antisense) is left as a guide to recognizing target mrna sequences. subsequently, mrnas which are perfect or nearly perfectly complementary to the sirna antisense strand are cleaved by the activated riscs ( ) . the specific gene silencing effect of sirnas makes them indispensable tools for target identification and validation in drug discovery and development ( ) . in , onpattro (patisiran) infusion became the first fda-approved sirna drug. it is for the treatment of peripheral nerve disease caused by hereditary transthyretinmediated amyloidosis in adult patients. onpattro is designed to interfere with rna production of an abnormal form of the protein transthyretin. by preventing the production of transthyretin, the drug can help reduce the accumulation of amyloid deposits in peripheral nerves, improving symptoms, and helping patients better manage the condition. micro rnas(mirnas) are - nucleotides long, singlestranded, endogenous noncoding rna molecules that act as key regulators for a variety of cellular pathways. they can regulate gene expression by complementary binding to the core sequence in the ′-untranslated region( ′-utr) of target mrnas ( ) . either sirna or mirna could associate into the risc. unlike sirna, mirna can recognize mrna with partially complementary sequences, which means one mirna may have multiple different mrna targets ( ) . hence, delivery of exogenous micrornas or microrna mimics could be particularly useful in diseases having multiple diseaserelevant targets ( ) . mirnas mediate multiple biological processes, and alterations in mirna function have been associated with different diseases like cancer, metabolic disorders, and viral pathogenesis ( ) . mirnas related to cancer are generally classified as tumor suppressor mirnas or tumorpromoting mirnas. tumor suppressor mirnas (e.g., let- , mir- families, and mir- / ) are responsible for suppressing oncogenes and are mostly downregulated in cancer. restoration of their normal function can be achieved by mirna replacement via administration of synthetic mirna mimics functioning similarly to the endogenous counterparts. tumor-promoting mirnas (e.g., mir- , mir- - cluster, and mir- ) are known to downregulate tumor suppressor genes and have been reported to be overexpressed in cancer ( ) . asos and mirna sponges targeting tumor-promoting mirnas can be used to block aberrantly overexpressed mirnas ( ) . aptamers are short oligonucleotides with unique threedimension structures that enable them to specifically recognize and bind to targeted proteins. aptamers of interest could be selected from a pool of randomized molecules by methods known as systematic evolution of ligands through exponential enrichment. therapeutic aptamers could act as inhibitors of protein function, or as targeting moieties for drug delivery ( , ) . the use of rna-aptamers conjugates for targeted delivery of oligonucleotide molecules has been widely explored and well reviewed elsewhere ( , ) . pegaptanib, the only aptamer that has been approved by the fda, is acting through the former way. vascular endothelial growth factor (vegf) induces angiogenesis, and increases vascular permeability and inflammation, playing a central role in the progression of age-related macular degeneration. pegaptanib could selectively bind to vegf isoform, vegf , thereby preventing vegf from activating its receptors and suppressing pathological neovascularization ( ) . the therapeutic and targeting properties of aptamers could be combined to construct multifunctional molecules. using an aptamer that binds to and antagonizes the receptor tyrosine kinase axl, an aptamer-mirna conjugates was developed with synergistic therapeutic effects, owing to oncosuppressive effects of the mirna and inhibitory function of the aptamer ( , ) . barriers to nucleic acid-based therapies for pulmonary diseases the treatments for pulmonary diseases are mainly by parenteral injection and pulmonary administration through intranasal instillation, aerosol, or inhalation. hence, the first barriers that nucleic acid drugs via these two routes encounter are blood and respiratory tract (fig. ). parenteral administration of unmodified nucleic acids has been set back by their very short half-life in the bloodstream, serum nuclease degradation, quick renal clearance, and poor biodistribution. the parenteral route also exposes the whole human body to nucleic acids, which may hamper the delivery efficiency to target tissues or organs ( ) . to avoid enzymatic degradation and renal clearance, local drug administration routes have been proposed to directly deliver the drugs to the site of interest. pulmonary administration reveals a strong potentiality as it could transport therapeutic agents to diseased lung tissue in a non-invasive manner. while the degradation by nucleases is negligible comparing to systemic administration, delivery through the airway could be hampered by physiological barriers. the mucociliary clearance action, the surface liquid that covers the airway and macrophages along different parts of the airways, limits the transport of nucleic acids to the site of action ( ) . the highly viscous mucus layer in the airways traps and prevents nucleic acids reaching the underlying epithelium and propelled them out with the impact of cillated cells ( ) . thus, the development of particles that could efficiently penetrate the mucus barrier, without compromising its protective properties, is a clear challenge for improving pulmonary drug delivery ( ) . even if the nucleic acids successfully penetrate through and escape from all the extracellular barriers mentioned previously, they still face the challenge to cross the cell membrane and reach the site of action in the cytoplasm or nucleus. negative charge and large molecular weight make it hard for naked nucleic acids to enter the cell. the endocytosis of nucleic acids could be improved with the help of cationic biomaterials or targeting moieties which interact with the negative proteins or receptors on the cellular surface ( ) . one of the most challenging intracellular barriers for nucleic acids delivery is their tendency to remain entrapped in endosomes. intracellular nucleic acids are transported in early endosome vesicles where various nucleases exist and the ph further reduce to . in the process to late endosomes and lysosomes, and most nucleic acids degraded in the endosome before reaching the site of action ( ) . the classic approach has been to use small-molecule endosomolytic agents like chloroquine to disrupt endosomes and release entrapped oligonucleotides from endosomes. two similar types of small molecules have been reported recently with these molecules substantially enhanced the pharmacological activities of oligonucleotides both in cell culture and murine model ( , ) . although these endosomolytic agents significantly enhanced the delivery efficiency, they currently display a narrow therapeutic window for clinical use. to overcome these biological barriers, strategies like chemical modification, conjugation, vector encapsulation, and selection of administration route have been utilized to improve the delivery of nucleic acids to lungs. since naked nucleic acid is prone to degradation in the biological fluid, chemical modifications at the sugar, backbone, or the individual bases have been introduced to improve its stability and efficacy in biological systems. phosphorothioate(ps)-modified backbone is the most widely used chemistry modification to increase the nuclease resistance. based on ps backbones, nucleic acids designed with additional ′-sugar modifications such as ′-o-methyl ( ′-ome) or ′-o-methoxyethyl ( ′-moe) can not only further enhance stability and target affinity, but also largely block the activation of toll-like receptors and reduce immune responses ( ) . besides ps modification, peptide nucleic acids and phosphoramide morpholino oligomers are nucleotide analogs with strong nuclease resistance as the phosphodiester linkage is completely substituted by a polyamide backbone or a phosphorodiamidate group ( ) . however, ′-sugar modifications of asos might block the recruitment of rnaseh. therefore, bgapmers^was developed, that is asos containing a sequence of ps-modified backbone residues(bgap^) to facilitate rnase h activity and sugar-modified residues(bflanks^) on either side of the gap to increase resistance to degradation and enhance binding to target mrna ( ) . beside chemical modification, conjugation strategies are often exploited to enhanced stability and delivery efficiency. representative biomolecules conjugated to nucleic acids conclude targeting ligands and membrane-active molecules, such as lipids, aptamers, peptides, carbohydrates, and polymers ( ) . cholesterol attachment to nucleic acids facilitates cellular import and improves intracellular uptake via lipoproteins-mediated pathways ( ) . intravenous and intraperitoneal injection of anti-mdr cholesterol-sirna conjugate in healthy and tumor-bearing severe combined immune deficiency mice demonstrated efficient accumulation deep in the tissue and the cytoplasm of almost all the liver and tumor cells ( ) . sirnas conjugated to n-acetylgalactosamine molecule, a high-affinity ligand for the hepatocyte-specific asialoglycoprotein, are undergoing clinical trials and provided promising results ( ) . antibodies or aptamers could be conjugated directly to nucleic acids to realize targeted delivery to specific tissues or cell types. because of the advantages like good reproducibility and low system toxicity, chemical modification and conjugation of nucleic acids have been paid great attention and all the four fda-approved asos are chemically modified and used without a delivery vehicle. while compared to vectorbased systems, poor delivery efficiency and limited orientation are still great concerns of nucleic acid-conjugates for their clinical translation. besides chemical modification, vectors offer important opportunities for nucleic acids to overcome delivery challenges. ideal nucleic acid delivery vectors are expected to condense and protect nucleic acids, facilitate their transport to target cells, and subcellular compartments. viruses, as naturally evolved transfection agents, could enter the cells via endocytosis and release viral genome that could replicate and transcribe into proteins for producing multiple copies. due to their higher transfection efficiency, three major classes of viral vectors, namely, adenovirus ( ) , adeno-associated virus ( ) , and lentivirus ( ) have been extensively used in nucleic acid therapy. however, the limitation of payload, inherent immunogenicity, and the difficulty of large-scale production limited their clinical application. the advantage of non-viral vectors lies in low immunogenicity and toxicity, ease of production, and the large payload over their viral counterparts. widely investigated non-viral delivery vectors include polymers, lipids, polypeptides, and inorganic nanomaterials (such as calcium phosphate and quantum dots). most of the vectors for nucleic acids possess cationic charges that assist in loading nucleic acids through charge interactions. common non-viral delivery systems used in pulmonary diseases are listed in table ii . based on various non-viral vectors, hybrid systems made up by condensed nucleic acid/polycation complexes as the core and lipid bilayer membrane as the shell have been developed. th e use of en dogenou s pho sp holipids, su ch as dipalmitoylphosphatidylcholine, can be considered a valid approach to increase the compatibility of nanoparticles with the lung environment ( ) . researchers combined a naturalderived pulmonary surfactant shell with a sirna-loaded dextran nanogel to achieve effective sirna delivery to murine alveolar macrophages, which are difficult to transfect, resulting in a substantial gene knockdown with a relatively low dose ( ) ( ) ( ) . diverse surface modifications and conjugation of targeting agents attached to the vectors could render them desirable properties and enhance the therapeutic efficiency of nucleic acid therapy. surface modification with high molecular weight hyaluronic acid which can mediate active cd targeting in tumors and increase circulation time of cationic sirna lipoplexes improved the delivery efficiency and achieved supported reduction of the expression of luciferase mrna in tumor due to the sirna inhibition ( ) . systemic administration of nucleic acids faces serious challenges, including rapid excretion, low bioavailability, and systemic toxicity. while local administration allows lower delivery doses and reduced side effects, making it an attractive route ( ) . most of the fda-approved nucleic acid-based drugs are locally delivered: fomivirsen is delivered to the eyes by intraocular injection, spinrazais by intrathecal injection, and luxturnais by subretinal injection ( , ) . for pulmonary disease, the target organ could be reached through systemic administration or pulmonary administration. the latter route could potentially enhance retention time of nucleic acids in the desired site of action, reduce systemic toxic effects, and provide a therapeutic solution to a range of pulmonary disorders ( ) . inhalation and intranasal route represent the most common way to deliver nucleic acid into the airways due to the ease of administration and non-invasive characteristic, and are the main administration routes in clinical trials. biodistribution studies of aerosol inhalation of polyester-sirna nanoparticles to mice bearing orthotopic lung tumors showed specific accumulation in the lungs ( ) . nucleic acids can be formulated into liquid aerosol generated by an inhaler or nebulizer, or dry powder aerosol for pulmonary delivery. liquid aerosol formulations were almost exclusively adopted in clinical trials involving pulmonary delivery of nucleic acids. among the three major types of inhalation devices consisting of pressurized metered dose inhalers (pmdis), nebulizers, and dry powder inhalers (dpis), pmdis and dpis are the most portable and commonly-used devices ( ) . pmdis, in which the therapeutic agents are suspended in the hydrofluoroalkane (hfa) propellant, have been regarded as golden standard delivery system for asthma and chronic obstructive pulmonary disease therapies ( ) . a pmdi formulation containing mannitol microparticles which encapsulated sirna polyplex nanoparticles showed good aerodynamic properties for deep lung deposition and significant gene knockdown efficiency in lung a cells ( ) . dpis are usually thought as a better option to deliver therapeutic nucleic acids than pmdis because their dry particle form enhances the stability of nucleic acids and decreases the risk of microbial contamination ( ). chow et al. first formulated naked sirna into inhalable dry powders (at % w/w) using spray drying technology with the incorporation of mannitol and l-leucine; the latter acted as powder dispersibility enhancer, and the integrity of sirna was well retained ( ) . although systemic administration does not provide the aforementioned advantages of local delivery, for some indications like lung metastasis and pulmonary hypertension, the desired target sites might locate on the interstitium and lung alveolar and endothelial cells rather than the airway epithelium. lung metastases are expected to have an endothelial origin and therefore may be better accessible through blood vessel than through airways ( ) . although intravenous injection is not direct delivery to the lung, this route is still able to achieve high levels of transgene expression in the lungs. a multifunctional lipid envelopetype nanodevice developed to target the lung endothelium was found to accumulate in the lung within min after injection. this carrier did not quickly remove to other organs and remain in lungs for h. based on this carrier, systemic administration of anti-cd sirna successfully suppressed the metastatic progression ( ) . therefore, the administration route should be carefully chosen according to the therapeutic application. since the discovery of nucleic acids, their association with multiple diseases and hence the therapeutic potential have been extensively demonstrated. in the last decades, many investigations have been successfully proved the therapeutic efficiency of nucleic acids on various lung diseases ranging from cancer to pulmonary inflammatory diseases. some of the nucleic acid products have entered the clinical stage; recent clinical trials involving nucleic acid drugs for pulmonary diseases are summarized in table iii . lung cancer is the leading cause of cancer-related deaths in the usa and worldwide ( ) . according to the difference in histology, % cases of lung cancer are classified as nonsmall cell lung cancer (nsclc) and % cases are small cell lung cancer (sclc). in addition to sclc and nsclc, malignant pleural mesothelioma is a rare form of lethal cancer developing in the tissue lining of the lungs ( ). current treatments for lung cancers include surgical resection, chemotherapy, radiation therapy, and targeted drug therapy, but these existing therapeutics have limited efficacy, and survival rate of nsclc patients has remained low ( ) . therefore, studies on target treatment of lung cancers with selective nucleic acid against oncogenic pathways have drawn intensive interest and some of them have entered clinical practice. custirsen (ogx- ) is a ps-aso inhibitor of clusterin, an anti-apoptotic chaperone protein upregulated in cancer cells in response to chemotherapy and might mediate resistance ( ) . preclinical data showed that custirsen significantly decreases clusterin production, increases the sensitivity of lung cancer cells to chemotherapies, and inhibits tumor growth in lung cancer models. in the phase trial of custirsen in patients who were treated with a combination of a gemcitabine/platinum doublet, serum clusterin levels were notably reduced. a larger randomized phase study is needed to demonstrate the potential survival benefit of custirsen in patients with nsclc ( ) . imetelstat (grn l) is a base phosphoramidate oligonucleotide conjugated to a -palmitoyl lipid group against the rna component of telomerase, an enzyme responsible for maintaining telomere length and crucial for the indefinite growth of tumor cells. blocking telomerase with imetelstat leads to antineoplastic effects. in a phase study, imetelstat failed to improve progress-free survival rates in advanced nsclc patients with diverse telomere. but there was a trend toward survival improvement for patients with shorter telomeres. further investigations on short telomeres as predictive biomarkers are warranted for clinical development of imetelstat ( ) . a lot of sirna-based therapeutics are being assessed in preclinical and clinical trials of pulmonary diseases. aln-rsv , a sirna therapeutic directing against the mrna encoding the n protein of the respiratory syncytial virus, has completed phase ii clinical trials ( ) . sirnas also hold great promise as therapeutic agents for cancer through rnai silencing oncogene expression. sirna for cancer therapies are beginning to be tested in human clinical trials, such as aln-vsp(alnylam pharmaceuticals) for the treatment of liver cancer and calaa- (calando pharmaceuticals) as tumor inhibitor ( ), and they have shown promising pharmacodynamics and tolerability. however, to extend small rna therapy to other major cancer types, including lung cancer, delivery vehicles that target nonliver tissues and specific delivery route are needed. lung cancer is an attractive cancer type for local or systemic small rna delivery treatment. various therapeutic target genes (e.g., survivin, bcl , hdm ) for lung cancer therapy have already been identified and become targets of sirna therapy ( ) . mirnas play a central and complex role in cancer development and are generally classified as tumor suppressor mirnas or tumor-promoting mirnas (oncomirnas). tumor suppressor mirnas in lung cancer include let- family, mir- / family, mir- / , mir- family, and mir- ; oncomirnas in lung cancers include mir- ~ cluster, mir- , and mir- / ( ) . there are two approaches for mirna modulators to act as cancer therapies: exploiting antisense-based inhibitors of oncogenic mirnas or replacing downregulated tumor suppressor mirnas with synthetic mirna mimics ( ) . to date, there are two tumor suppressive mirna mimics of mirna- (mrx ; mirna therapeutics inc.) and mirna- (targomirs; engeneic ltd.) that have entered clinical trials. mrx is a synthetic version of mir- a encapsulated in liposomes. mir- a is a tumor suppressor often expressed at reduced levels in a broad range of cancer types, which functions to downregulate the expression of more than different oncogenes across multiple oncogenic pathways ( ) . but immune-related serious side effects caused termination of the trial of mrx . targomirs are double-strand synthetic mir- -based microrna mimics delivered by engeneic dream vectors which are deprived from nonliving bacterial minicells with a targeting moiety ( , ) . the mir- family has been implicated as tumor suppressor in a range of cancer types, and their primary targets are genes (e.g., bcl , cdk , and jun) involved in cancer progression. in vitro and in vivo studies showed that the restoration of expression of mir in malignant pleural mesothelioma induced the apoptosis of tumor cells and inhibited tumor growth. long-term survival after a short treatment period was observed in the phase study. however, the safety issue and early signs of activity of targomirs still warrant further clinical trials ( ) . asthma is a kind of chronic inflammatory airway disease with high prevalence, which could induce airway hyperresponsiveness, infiltration of inflammatory cells, and airway remodeling. it has been estimated that about million people suffer from this disease on a global scale ( ) . the current therapeutics for asthma (including inhaled β -adrenergic receptor agonists, inhaled corticosteroids, and monoclonal antibody against ige) could effectively control the disease for most patients while there are still about % of the patients still out of control under the current treatments. ( ) . besides, the current drugs fail to stop or reverse the airway remodeling and some of the drugs followed with concerns of long-term adverse effects, which means there are unmet needs for better drugs ( , ) . choi et al. developed a novel therapy combining traditional drugs with novel therapeutics. in the regimen, dexamethasone (dexa) was attached to peis to act as a controller ingredient to control the airway inflammation. while sirna against vitamin d binding protein, which is a responsible molecule of allergic asthma, was delivered by dexa-pei at the same time ( ) . this multi-target treatment effectively reduced the airway inflammation and secretion of inflammatory factors. asthma is a complex disease associated with the interaction between genetic, epigenetic, and environmental parameters, involved with a plethora of cells and cellular factors ( ) . one direction for developing new drugs to treat asthma is to target central pathways to the pathogenesis of the disease, and nucleic acid-mediated therapies silencing the specific effector or the upstream regulator can be a potential approach. ribosomal protein s (rps ) was found to bind to the subunit of nf-κb complex and enhance the downstream inflammatory effect. intratracheal delivery of rps silencing sirna effectively alleviate airway hyperresponsiveness (ahr) and immune cell infiltration, and decreased serum total ige levels were also observed ( ) . sb , a new class of aso therapeutic sequence-specific targeting and cleaving gata mrna, has entered into phase a clinical trials. the overexpression of gata was found in cells involved in allergic inflammation. the results of the trial showed that inhaled sb significantly attenuate both the early-phase and late-phase allergen-induced asthmatic responses ( ) . another aso drug tpi-asm , developed by pharmaxis, contains two types of asos targeting the βc subunit of the il- , il- , gm-csf receptors (top ), and human ccr (top ) respectively. tpi-asm showed the protective effect against ige-mediated early asthmatic response and reduced eosinophilic airway inflammation ( ) . chronic obstructive pulmonary disease (copd) is one of the most common chronic respiratory diseases of the airways with an increasing morbidity and mortality; it has been forecasted that copd will be ranked the fourth burden of disease worldwide by year ( , ) . copd is characterized by progressive airflow obstruction and airway inflammatory response. current therapeutic strategies are through inhaled long-acting β -agonists, long-acting muscarinic antagonists, and corticosteroids to dilate bronchus and suppress inflammatory, which is similar to the treatment of asthmas ( , ) . emerging drugs in copd focus on the cellular and molecular components regulating airway inflammations ( ) . phosphodiesterases (pdes) are a group of different isoenzymes (pde - ) hydrolyzing camp, increased levels of which promote airway smooth muscle relaxation and bronchodilation with anti-inflammatory responses. among the big pde family, pde is present in many types of cells relating to copd and thought to be a promising therapeutic target. tpi , a dual pde inhibitor comprising two modified asos directing against pde b/ d and a, was designed to reduce the recruitment and activation of inflammatory cells in copd and shown to reduce the neutrophil influx in bronchoalveolar lavage (bal) and inflammation of smoke-exposure or lps-challenge murine models ( ) . the phase i clinical trial of tpi was initiated in but was withdrawn due to drug development suspension. the lungs of copd patients show that the reduction of alveolar elastic fibers and self-healing ability is impaired due to chondroitin sulfate proteoglycan versican inhibiting tropoelastin assembling into fibers. wu et al. employed a small interfering rna (sirna) against versicanin primary pulmonary fibroblasts from copd patients and enhanced the deposition of tropoelastin, which offers a new direction to lung repairment in copd therapy ( ) . mirna expression has been proposed as an accessible biomarker of copd disease ( ) . multiple mirnas were found altered in copd patients and murine models and could serve as potential biomarkers for the copd detection and prognosis. for example, downregulation of mir- a, - p, c- p, , and upregulation of mir- and have intimate association with copd development ( ) . micrornas were also found to play an important role in copd muscle dysfunction and mass loss ( ) . elevated mir- - p expression in patients with muscle wasting might contribute to the inhibition of protein synthesis and loss of muscle mass ( ) . it was demonstrated that mir- a, as a suppressor of tgf-β signaling by reducing the expression of smad protein, might attribute to the maintenance of muscle mass ( ) . cystic fibrosis (cf) is a genetic disorder giving rise to the functional failure of the cystic fibrosis transmembrane conductance regulator (cftr) protein, which acts as an epithelial chloride channel. the interaction of cftr and epithelial sodium channel (enac) is responsible for the homeostasis of the airways epithelial surface. the deficiency or flaw of cftr leads to hyperactivity of epithelial sodium channel. reduced chloride secretion and increased sodium absorption subsequently result in mucus dehydration, chronic infection, and airway inflammation ( , ) . using antisense oligonucleotides that correct the basic defect at the mrna level could restore the crucial balance between enac and cftr. a recent study exploited aerosol delivery of asos in cf-like mouse models to inhibit enac activity by triggering rnase h -dependent degradation of scnn a mrna, which encodes the enac ɑ subunit. this strategy effectively reduced goblet cell hyperplasia and reversed cf-like symptoms, demonstrating that an enac antisense therapy may provide a potential therapy for cf ( ) . the drug qr- is a single-stranded antisense rna-based oligonucleotide sequence designed to hybridize the sequences adjacent to the deleted f region in the cftr mrna to restore the full function of cftr protein in patients with the f del mutation. preliminary studies in cell culture and mouse f del model showed improved chloride efflux after qr- treatment ( ) . data showed that topical administration of qr- to the nasal epithelium improved cftr function by measuring the nasal potential difference of f del cf subjects ( ) . a phase b study to evaluate the safety, tolerability, and pharmacokinetics of qr- is ongoing in cf patients with homozygous f del cystic fibrosis. acute respiratory distress syndrome (ards) is a type of acute diffuse lung injury with a high mortality rate, which is clinically characterized by pulmonary infiltrates, hypoxemic respiratory failure, and edema, ( ) . the mild form of ards is termed as acute lung injury (ali). it is suggested that approximately ~ cases of ards per , population per year. ali is more common, with rates up to per , per year reported ( ) . the common risk factors conclude sepsis, trauma, pneumonia, and toxic inhalation ( ) . current ards therapy is to improve impaired gas exchange and lung mechanics by anti-inflammatory drugs, bronchodilators, and mechanical ventilation, which show limitation in controlling the disease progression. as researchers digging into the mechanisms of ards, crucial regulatory agents participating in the initiation and progression of ards, like mirnas and cytokines, have become appealing therapeutic targets. it was found that murine ali models treated with asos against mir- gained the enhanced recovery of ali as evidenced by the reduction of bal protein and pro-inflammatory cytokines, and the number of bal cells ( ) . nf-κb is a family of dna binding proteins involved in the expression of pro-inflammatory factors and thus the development of ards. depletion of nf-κb by specific sirna targeted nf-κb p in lipopolysaccharide (lps)-induced ali rat models effectively reduced levels of the pro-inflammatory cytokines and ameliorated symptoms induced by lps ( ) . in vivo administration of the sis plyase/hmgb a/r v complex reduced the s plyase level and weakened the inflammatory response and apoptosis in an lps-induced ali model, indicating that sis plyase and hmgb a have a synergistic therapeutic effect for ali ( ) . nucleic acid drugs hold great promises as new classes of therapeutic agents for pulmonary diseases, and some candidates have entered into clinical trials (table iii) . the unique structures of lungs enable the delivery of nucleic acid to be implemented by intravenous and pulmonary routes. inhalation and intranasal routes have been found to be ideal for effective delivery. for proper therapeutic use, researchers have modified the chemical structure of nucleic acids to increase their ability against nuclease degradation and reduce immune responses. the transition from bench to bedside of nucleic acid-based therapy also depends heavily on the availability of a safe delivery system that can facilitate trafficking into site of action. the safety issue, especially the immunogenicity of nucleic acids and their vectors, is the biggest stumbling block before nucleic acid drugs for lung diseases become available in the clinic, and further work in this area need to be thoroughly investigated. it is still necessary to identify suitable carriers with the ability to successfully reach the action site in the lung and protect the activity of nucleic acids during the delivery. with the advances and ongoing clinical trials, the future of nucleic acid drugs for pulmonary diseases remains very promising. world health organization. the top causes of death gene therapy for pulmonary diseases targeted delivery of sirna to activated t cells via transferrinpolyethylenimine (tf-pei) as a potential therapy of asthma pharmacology of antisense drugs nucleic acid therapies for cystic fibrosis. nucleic acid ther [internet] rna therapeutics: beyond rna interference and antisense oligonucleotides advances in the delivery of rna therapeutics: from concept to clinical reality fda-approved oligonucleotide therapies in preclinical and clinical development of sirna-based therapeutics sirna versus mirna as therapeutics for gene silencing development of novel therapeutic agents by inhibition of oncogenic micrornas an overview of micrornas delivering the promise of mirna cancer therapeutics micrornas as cancer therapeutics: a step closer to clinical application therapeutic rna aptamers in clinical trials aptamers: molecules of great potential current progress on aptamertargeted oligonucleotide therapeutics aptamers as targeted therapeutics: current potential and challenges food and drug association on / / for macugen®, nda selective delivery of therapeutic single strand antimirs by aptamer-based conjugates multifunctional aptamer-mirna conjugates for targeted cancer therapy pulmonary administration of small interfering rna: the route to go? sirnabased therapies for pulmonary diseases carrier interactions with the biological barriers of the lung: advanced in vitro models and challenges for pulmonary drug delivery mucus-penetrating nanoparticles for drug and gene delivery to mucosal tissues overcoming cellular barriers for rna therapeutics breaking down the barriers: sirna delivery and endosome escape a novel family of small molecules that enhance the intracellular delivery and pharmacological effectiveness of antisense and splice switching oligonucleotides high-throughput screening identifies small molecules that enhance the pharmacological effects of oligonucleotides oligonucleotide therapy for obstructive and restrictive respiratory diseases nucleic-acid therapeutics: basic principles and recent applications adv drug deliv rev delivery strategies and potential targets for sirna in major cancer types. adv drug deliv rev cholesterol-containing nuclease-resistant sirna accumulates in tumors in a carrierfree mode and silences mdr gene a myeloid cell-binding adenovirus efficiently targets gene transfer to the lung and escapes liver tropism overcoming the cystic fibrosis sputum barrier to leading adeno-associated virus gene therapy vectors impact of trem- gene silencing on inflammatory response of endotoxininduced acute lung injury in mice lipid nanoparticle delivery of a microrna- inhibitor improves experimental pulmonary hypertension delivery of therapeutic sirna to the lung endothelium via novel lipoplex formulation dacc development of spray-freeze-dried sirna/pei powder for inhalation with high aerosol performance and strong pulmonary gene silencing activity tpp-dendrimer nanocarriers for sirna delivery to the pulmonary epithelium and their dry powder and metered-dose inhaler formulations anti-inflammatory effect of anti-tnf-α sirna cationic phosphorus dendrimer nanocomplexes administered intranasally in a murine acute lung injury model dendrimer-inspired nanomaterials for the in vivo delivery of sirna to lung vasculature an inhalable β -adrenoceptor ligand-directed guanidinylated chitosan carrier for targeted delivery of sirna to lung recent advances in chitosan-based nanoparticulate pulmonary drug delivery hybrid lipid/polymer nanoparticles for pulmonary delivery of sirna: development and fate upon in vitro deposition on the human epithelial airway barrier hybrid pulmonary surfactant-coated nanogels mediate efficient in vivo delivery of sirna to murine alveolar macrophages bio-inspired pulmonary surfactantmodified nanogels: a promising sirna delivery system surfactant protein b (sp-b) enhances the cellular sirna delivery of proteolipid coated nanogels for inhalation therapy efficient in vitro and in vivo pulmonary delivery of nucleic acid by carbon dot-based nanocarriers local delivery of sirna-loaded calcium phosphate nanoparticles abates pulmonary inflammation aerosol delivery of stabilized polyester-sirna nanoparticles to silence gene expression in orthotopic lung tumors inhaled gene delivery: a formulation and delivery approach gababreceptor ligand-directed trimethyl chitosan/tripolyphosphate nanoparticles and their pmdi formulation for survivin sirna pulmonary delivery dry powder formulation of plasmid dna and sirna for inhalation inhaled powder formulation of naked sirna using spray drying technology with l-leucine as dispersion enhancer delivery systems for pulmonary gene therapy t=js&page=reference&d=emed &news=-n&an= ovidweb.cgi?t=js&page=reference&d=-emed &news=n&an= lipid envelope-type nanoparticle incorporating a multifunctional peptide for systemic sirna delivery to the pulmonary endothelium micrornas and lung cancers: from pathogenesis to clinical implications nanoparticle-based targeted gene therapy for lung cancer ogx- ): a second-generation antisense inhibitor of clusterin in development for the treatment of prostate cancer phase i/ii trial of custirsen (ogx- ), an inhibitor of clusterin, in combination with a gemcitabine and platinum regimen in patients with previously untreated advanced nonsmall cell lung cancer a randomized phase ii study of the telomerase inhibitor imetelstat as maintenance therapy for advanced nonsmall-cell lung cancer a randomized, double-blind, placebocontrolled study of an rnai-based therapy directed against respiratory syncytial virus therapeutic mirna and sirna: moving from bench to clinic as hyaluronic acid-conjugated lipoplexes for targeted delivery of sirna in a murine metastatic lung cancer model delivery systems and local administration routes for therapeutic sirna pulmonary delivery of therapeutic sirna micrornas in non-small cell lung cancer: current status and future therapeutic promises phase i study of mrx , a liposomal mir- a mimic, administered twice weekly in patients with advanced solid tumors safety and activity of microrna-loaded minicells in patients with recurrent malignant pleural mesothelioma: a first-in-man, phase , open-label, dose-escalation study versatile vectors for targeted drug or si/shrna cancer therapy epidemiology and economic burden of asthma new targets for drug development in asthma new therapies for asthma: is there any progress? asthma: pathogenesis and novel drugs for treatment a new combination therapy for asthma using dual-function dexamethasone-conjugated polyethylenimine and vitamin d binding protein sirna ribosomal protein s gene silencing protects against experimental allergic asthma allergen-induced asthmatic responses modified by a gata -specific dnazyme antisense therapy against ccr and the common beta chain attenuates allergen-induced eosinophilic responses chronic obstructive pulmonary disease emerging drugs for chronic obstructive pulmonary disease emerging therapeutic strategies in copd a multi-targeted antisense oligonucleotide-based therapy directed at phosphodiesterases and for copd deposition of insoluble elastin by pulmonary fibroblasts from patients with copd is increased by treatment with versican sirna targeting microrna function in respiratory diseases: mini-review the role of micrornas in copd muscle dysfunction and mass loss: implications on the clinic mir- - p reduces ribosomal rna and protein synthesis in muscle wasting mir- a suppresses smad protein expression and promotes resistance to muscle loss cystic fibrosis inhaled enac antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice strategies in early clinical development for the treatment of basic defects of cystic fibrosis qr- , an investigational rna therapeutic, improves cftr activity in cystic fibrosis subjects homozygous for the f del mutation mechanisms and clinical consequences of acute lung injury acute lung injury antisense oligonucleotide treatment enhances the recovery of acute lung injury through il- -secreting m -like macrophage-induced expansion of cd + regulatory t cells small interfering rna targeting nf-κb attenuates lipopolysaccharideinduced acute lung injury in rats combined delivery of hmgb- box a peptide and s plyase sirna in animal models of acute lung injury key: cord- -wig wdno authors: xu, qi; shan, yuanyuan; wang, ning; liu, yaping; zhang, maojie; ma, meihu title: sialic acid involves in the interaction between ovomucin and hemagglutinin and influences the antiviral activity of ovomucin date: - - journal: int j biol macromol doi: . /j.ijbiomac. . . sha: doc_id: cord_uid: wig wdno ovomucin (ovm) plays an important role in inhibiting infection of various pathogens. however, this bioactivity mechanism is not much known. here, the role of sialic acid in ovm anti-virus activity has been studied by elisa with lectin or ligand. structural changes of ovm after removing sialic acid were analyzed by circular dichroism and fluorescence spectroscopy. ovm could be binding to the hemagglutinin (ha) of avian influenza viruses h( )n( ) and h( )n( ), this binding was specific and required the involvement of sialic acid. when sialic acid was removed, the binding was significantly reduced . % and . %, respectively. therefore, sialic acid was proved as a recognition site which avian influenza virus bound to. meanwhile, the endogenous fluorescence and surface hydrophobicity of ovm removing sialic acid were increased and the secondary structure tended to shift to random coil. this indicated that ovm molecules were in an unfolded state and spatial conformation disorder raising weakly. remarkably, free sialic acid strongly promoted ovm binding to ha and thereby enhanced the interaction. it may contribute to the inhibition of host cell infection, agglutinate viruses. this study can be extended to the deepening of passive immunization field. ovomucin (ovm) has a unique antiviral activity, the mechanism of which is not entirely known. ovm is a highly glycosylated protein containing sialic acid (sa), which belongs to the mucin family [ ] . mucins are a major component of mucus, which are widely distributed in the body's internal surface and mucosal tissues, such as the respiratory tract and intestines. they provide an important innate immune barrier to potential toxins, particles, and pathogens [ ] . it can prevent pathogens from being in contact with susceptible cells. it is thought that the adhesion of mucins to pathogens is an important mechanism with a potentially significant effect. ovm has antiviral properties, that is similar in structure and composition to the influenza virus receptor and early findings suggest that ovm has an inhibitory effect on swine influenza virus-induced hemagglutination [ ] . the interaction of ha on the surface of the virus with ovm leads to the release of glycopeptide complexes. hemagglutination inhibition assays and enzyme-linked immunosorbent assays have revealed the high affinity of ovm for bovine rotavirus, chicken new castle disease virus and human influenza virus [ ] [ ] [ ] . it was found that nacetylneuraminic acid (neuac, a specific subtype of sialic acid in ovm) in the β-subunit could greatly facilitate the interaction between ovm and chicken new castle disease virus. the alteration of the conformation of ovm by the alkylation of disulfide bonds leads to loss of binding to ovm antibody [ ] . however, evaluations of antiviral activity in these studies focused primarily on the inhibition of viral-induced hemagglutination and did not include other more accurate and intuitive antiviral methods. structurally, ovm is a highly glycosylated protein whose monosaccharides are predominantly in the forms of oligosaccharides and glycosides consisting of fewer than monosaccharides [ , ] , including nglycosidic bonds and o-glycosidic bonds [ ] . n-glycans are linked to the aspartic acid (asp) residues of the polypeptide sequence asn-x-ser/thr, where x represents any amino acid except proline, and oglycans are predominantly linked to the serine (ser) and threonine (thr) residues [ , ] . these oligosaccharides mainly include mannose (man), galactose (gal), n-acetylgalactosamine (galnac), nacetylglucosamine (glcnac), sa, fuctose (fuc) [ ] and sulfuric acid esters [ ] . sialic acid of ovm can promote interaction with chicken new castle disease virus. meanwhile, various viruses such as influenza virus, coronavirus and rotavirus utilize glycoproteins containing sialic acid on the surface of host cells as recognition receptors [ ] [ ] [ ] . sialic acid can be recognized by the epitope on the globular head of the influenza virus ha, thereby inducing interaction with the corresponding ha receptor binding sites and interfering with or blocking the adsorption of the virus to the cells [ ] . sialic acid residues are generally located at the terminus of the n-linked oligosaccharide chain and the o-linked oligosaccharide chain with α , -, α , and α , -linkages [ ] . different influenza viruses are capable of specifically recognizing different linked types of sialic acids. human influenza viruses are more likely to bind to the α , -linkage sialic acid receptor, and avian influenza viruses preferentially recognize α , -linkage sialic acids [ , ] . ovm is a glycoprotein containing a large amount of sialic acid. its antiviral activity has not been studied deeply, and its anti-infective mechanism is barely understood. the role that sialic acid plays and whether it is recognized as the same receptor of the influenza virus and binds to ha remain to be revealed. therefore, this study aims to verify the interaction between ovm and ha and demonstrate the function of sialic acid in this interaction to explain the possible mechanism of ovm for satisfactory antiviral activity and increase the knowledge of the role of ovm in passive immunity. ovm was crude extracted according to a previously reported method [ ] with modifications. in brief, ml of fresh whole egg white was stirred at °c for min and subsequently diluted with ml of mm nacl. the ph was adjusted to . with m hcl, and the solution was incubated overnight at °c. the egg white solution was centrifuged at , g for min at °c, and the precipitate was resuspended with mm nacl for h and then centrifuged under the same conditions. after the precipitate, which was crude ovm, was washed several times with distilled water, the above procedure was repeated. the extract was freeze-dried and stored at − °c. ovm crude extract was purified by gel filtration chromatography (sephacryl s- hr, mm × cm) using the akta purification system (ge, usa). the target elution peak was dialyzed by distilled water, and the product was purified ovm. purified ovm ( mg) was mixed with μl of ph . sodium acetate buffer and μl of na enzyme and incubated in a °c water bath for h. desialylated ovm (dsa-ovm) was dialyzed against kd dialysis bags for h with ph . borate buffer. the solution was removed, stirred for h and centrifuged for min at r/min; the supernatant was saved for subsequent experiments. the dsa-ovm supernatant was centrifuged for min in a kd ultrafiltration tube at g and concentrated to . ml. a standard curve was used to quantify the concentration of ovm. the effect of enzymatic hydrolysis and the chemical bond of sialic acid in oligosaccharide chains was evaluated by elisa using lectins sambucus nigra (sna) and maackia amurensis (maa). a : dilution of sna was added to the elisa plate at μl/well, and the plate was incubated overnight at °c. to the control group was added pbs buffer without sna. the plates were washed times for min each with pbst and then again with pbst dissolved in % skim milk at μl/well and incubated at °c for h for blocking. after the plates were washed, dsa-ovm and ovm diluted to μg/ml were added to the experimental and control wells, respectively, at μl/well, and the plates were incubated at °c for h. after the plates were washed, ovm antibody diluted : was added at μl/well, and the plates were incubated at °c for . h. after the plates were washed, hrp-labeled goat anti-mouse igg diluted : was added at μl/well, and the plates were incubated at °c for min, washed and stained. the function of sialic acid was assessed by the change in the binding of ha to ovm with the removal of sialic acid. the ha proteins of the influenza viruses h n (ha ) and h n (ha ) were added to the elisa plate at a dilution of : at μl/well, and the plate was incubated overnight at °c. to the control group was added pbs buffer without ha. other operations were the same as in section . . the common components of oligosaccharide chains (gal, fuc, man and sialic acid) were separately mixed with ovm for competitive binding analysis with ha. subsequently, the effect of free sialic acid was analyzed by different additional sequences. the additional sequences were the addition of sialic acid followed by the addition of ovm, the addition of ovm followed by the addition of sialic acid, and the addition of preincubation mixture. the elisa procedure was the same as previously described. the protein solution was diluted -fold, the final protein concentration was . mg/ml, the optical path of the quartz cell was . cm, the sensitivity was mdeg/m, the wavelength scanning range was - nm, the speed was nm/s, and the resolution was . nm measured at room temperature. the optical path of the quartz cell was . cm. using tyrosine (tyr) as an intrinsic probe, the excitation wavelength was nm, and the emission spectrum was scanned at - nm. using tryptophan (trp), the excitation wavelength was nm, and the emission spectrum was scanned at - nm. the excitation and emission monochromators each had a bandwidth of nm. ovm was incubated at room temperature for h, and its concentration was diluted to μg/ml. blank samples were measured under the same conditions. using l-anilinonaphthalene- -sulphonate (ans) as a fluorescence probe, the surface hydrophobicity of ovm was measured by the fluorescence method. ovm was diluted to . - . mg/ml with ph . borate buffer. fluorescence spectra of ans were scanned by adding μl of diluted sample to μl of mmol/l ans solution. the excitation wavelength was nm, and the scan range was - nm. the fluorescence intensity at nm excitation and nm emission was used to making a standard curve for protein concentration. the control was blank ans solution with addition of the corresponding sample buffer. all values were expressed as mean ± s.e.m. anova with bonferroni's multiple-comparison test when more than two groups were compared. all the assays were run in triplicate and were representative of at least independent experiments. a p value b . was considered statistically significant and the asterisks in all figures are defined, *p b . , **p b . , ***p b . . in the elisa reaction, a variety of factors together affected the final test results, and the antigenic epitope of the sample had a significant impact. to ensure the accuracy of the experiment, we needed to verify whether sialidase enzymolysis would affect the other functions of ovm. in this experiment, elisa reaction of ovm was the key to analyzing physiological immune activity. the purified ovm was digested with sialidase to obtain dsa-ovm. the concentration of natural ovm was . mg/ml, and that of dsa-ovm was . mg/ml. for a concentration of μg/ml, the binding changes in ovm and dsa-ovm to antibody were analyzed (fig. a) . compared with that of natural ovomucin, the binding of dsa-ovm and antibody did not change significantly after enzymolysis, exhibiting a good consistency and no significant effect on the antibody binding activity. lectins sna binds specifically to sialic acid linked by an α - linkage, while maa specifically recognizes an α - -linkage. as shown in fig. b , the terminal sialic acid was effectively removed in dsa-ovm after enzymatic hydrolysis, and its binding activity was obviously lower than that in the natural ovm. the binding activity of natural ovm to lectin sna was higher than that to lectin maa, indicating that the terminal sialic acid glycosidic linkage in the ovm oligosaccharide chain was mainly α - and that less α - was present. after enzymolysis, all sialic acids in dsa-ovm significantly decreased (p b . ). the surface antigen ha of the influenza virus could bind to related glycoproteins via protein-protein or protein-carbohydrate chain interactions. to verify whether sialic acid is an important site of the interaction between ovm and ha, we designed a deglycosylation experiment. as shown in fig. , ha of both h n and h n bound to ovm and did not react with bsa (negative control). after sialic acid was removed, the binding of dsa-ovm to ha was significantly lower than that of natural ovomucin. at the same dose, the binding capacity of dsa-ovm to ha decreased . %, and the binding capacity to ha also decreased by . %. this indicated that ha recognizes sialic acid on ovm. sialic acid in ovm is distributed on the oligosaccharide chain terminus. therefore, it can be considered that this recognition involves the participation of sialic acid and oligosaccharide chains. based on the above results, it has been demonstrated that the carbohydrate chain is one of the recognition regions for the interaction between ovm and ha. sialic acid is the critical target. to determine whether this interaction occurred with the participation of other sugar chain components, a sugar competition experiment was conducted. different kinds of sugar were added to the plate coated with ha, followed by washing off and then adding ovm to measure the binding changes. the results displayed in fig. indicate that no monosaccharide had an inhibitory effect on ovm binding ha. there was no significant difference in binding after pre-incubation of carbohydrates and ha compared to the negative control, and the addition of free sialic acid did not cause a decrease in binding. this binding trend was consistent with both ha and ha . according to the results in fig. , when the reaction sequence in the experiment was the addition of sialic acid firstly and then the addition of ovm, it was unexpectedly found that free sialic acid promoted the interaction of ovm and ha. to analyze the role played by free sialic acid in the binding of ovm to ha, further experiments were carried out with different sequences of additions. adding ovm at first and then adding sa or adding the two components together were analyzed (fig. ) . when ovm was first added, followed by sa, neither ovm nor dsa-ovm showed any significant change compared with the negative control, and the binding intensity tended to be similar in ha and ha . when ovm was mixed with sa and added at the same time, it is interesting found that the binding of ovm to ha was significantly enhanced far more than the negative control and other groups. this change in ha was as significant as in ha . based on the above results, it was found that free sialic acid enhances the binding of ovm to influenza virus ha. the changes in the secondary structure of dsa-ovm and ovm were analyzed by circular dichroism spectroscopy (cd), and the results were shown in fig. a . the cd spectra of ovm showed weak negative peaks at nm and nm and a positive peak approximately nm, characteristic of α-helical and β-sheet hybrids [ ] . the negative peak of the dsa-ovm spectrum near nm was weaker and closer to the short wavelength direction than ovm. data on specific changes are shown in table . after removing sialic acid, ovomucin α-helix decreased, while the random coil increased, indicating that the degree of disorder increased [ ] . fluorescence spectroscopy is a powerful tool for studying the changes in the protein microenvironment in solution. aromatic amino acid (tryptophan (trp), tyrosine (tyr), and phenylalanine (phe)) residues in ovm molecules can fluoresce. therefore, the changes in the ovm molecule before and after removing the terminal sialic acid can be reflected according to the change in endogenous fluorescence [ , ] . the endogenous fluorescence spectra of ovm before and after removing the terminal sialic acid at excitation wavelengths of nm and nm were shown in fig. b and c, respectively. as shown in fig. . effects of sa different additional sequences on binding. (a) dsa-ovm binding with ha was reduced. adding sa after adding dsa-ovm or ovm was not able to improve the binding. however, the addition of ovm premixed with sa greatly enhanced the interaction with ha , and pre-incubation the dsa-ovm with sa had a significant improvement in its combination with ha . (b) adding sa after adding ovm or dsa-ovm did not influence its binding ability with ha . the addition of dsa-ovm premixed with sa strengthened the interaction with ha significantly, and as much as pre-incubation the ovm with sa prior to addition to the ha . ovm/sa represents the addition of natural ovomucin first then the addition of sa; dsa-ovm/sa represents the addition of dsa-ovm first and then the addition of sa; ovm + sa represents a pre-incubated mixture of natural ovomucin and sa being added at the same time; dsa-ovm + sa represents a pre-incubated mixture of dsa-ovm and sa being added at the same time; ovm, natural ovomucin; dsa-ovm, ovomucin with the removal of the sialic acid residue; sa, sialic acid, here n-acetylneuraminic acid was used as specific subtype of sialic acid in ovomucin. pre-incubation free monosaccharide with ha also did not interfere with the binding. even the addition of free sialic acid induced increase slightly. (−), negative control, without adding monosaccharide; gal, galactose; fuc, fucose; man, mannose; sa, sialic acid, here n-acetylneuraminic acid was used as specific subtype of sialic acid in ovomucin. the data are presented as mean ± s.e.m. the tyr fluorescence spectrum of fig. b , removal of terminal sialic acid results in enhancement of the endogenous fluorescence intensity of ovm, but no obvious migration of the fluorescence peak occurs. the fluorescence spectra of trp was in fig. c . the overall trend in fluorescence intensity agrees with the result of tyr being a fluorescent probe. however, the λ max of the trp spectrum was blueshifted after removing the terminal sialic acid, suggesting that after removal of sialic acid, the ovm microenvironment hydrophobicity is enhanced. in order to verify this result, the changes in the ans fluorescence spectra of ovm before and after the removal of terminal sialic acid were investigated by an ans fluorescence probe. according to the result, the fluorescence intensity of ans was enhanced after ovm removal of the terminal sialic acid. the value of ovm surface hydrophobicity was . and the value of dsa-ovm was . . this shows that ovm is located in a microenvironment with a non-polar increase and enhanced surface hydrophobicity. this is related to the unfolding of the ovm molecule and the increase in disorder, resulting in the exposure of the hydrophobic region, strengthening the binding of ans to the ovm hydrophobic region, and enhancing the fluorescence intensity [ ] . the exact mechanism through which ovm exerts its antiviral function is not entirely clear at present. this research studied the interaction between ovm and the influenza virus ha and the role of sialic acid in this interaction. it was found that the ovm carbohydrate chain contains terminal sialic acid with both α- - and α - -linkages (fig. b) . has cannot bind to other proteins such as bsa but can interact with ovm. this interaction is dependent on the presence of sialic acid (fig. ) . monosaccharides have no influence on the binding of ovm with ha. it is noteworthy that the binding of ha with ovm was strongly promoted by free sialic acid (figs. and ) . it is indicating that sialic acid is involved in the binding of ovm to influenza virus, and additional free sialic acid could enhance the ovm antiviral process. mucin, a natural barrier widely found in animals, always plays critical role in antiviral and antibacterial activities [ ] [ ] [ ] [ ] . ovm is a mucin protein in egg. according to our previous research, ovm contains plenty of sialic acid, and its subtype is neuac. in this study, we demonstrated the glycosidic bond type of sialic acid linking the ovm carbohydrate chain terminus, which contains both α - and α - -linkages. the content of sialic acid with the α - -linkage was higher than that with the α - -linkage (fig. b) . in the process through which the influenza virus infects the body, the first step is to recognize and bind to the cell surface receptor through the viral surface protein ha [ ] . the main receptors are cell surface glycoconjugates, and sialic acid is required for this process. human influenza viruses mainly recognize α - -linked sialic acids, whereas avian influenza viruses prefer α - -linked sialic acids [ , ] . therefore, due to the type of sialic acid linkages in the ovm oligosaccharide chain, we believe that ovm has anti-infective activity against both human and avian influenza viruses. furthermore, the anti-avian influenza virus function is closely related to α - linked sialic acid. to confirm this hypothesis, this study was carried out to enzymatically hydrolyze sialic acid. using the ha of influenza viruses h n and h n as the binding protein, the changes in ovm as an acceptor with or without sialic acid binding ha were measured. firstly, the interaction of ovm before and after removing sialic acid with its antibody was analyzed, and it was found that sialic acid was not the binding site. no significant difference was found in the binding activity before and after the removal of sialic acid. this demonstrates that sialic acid does not participate in all the functions of ovm but also that follow-up experiments can use an elisa reaction between ovm and an antibody. this ensures that the results are real and effective. subsequently, the interaction of natural ovm and dsa-ovm with ha was investigated, and no binding reaction between ha and bsa was found, which indicated that the recognition of glycoprotein was specific (fig. ) . however, the obvious interaction between ha and natural ovm indicates that ha can specifically recognize ovm and associate with it. on the other hand, the binding of ovm to ha was significantly reduced after sialic acid removal to only % to % of the original (fig. ) . these results showed the same tendency in h n and h n , which proves that sialic acid is involved in the recognition of ha and ovm. after sialic acid was removed, this binding could be inhibited. thus, we demonstrate that one of the antiviral activity mechanisms of ovm is associated with sialic acid in the ovm oligosaccharide chain competing with the cellular receptor for binding ha. after removing sialic acid, ovm still retains some ability to bind ha, presumably through other recognition mechanisms. in fact, sialic acid was not completely removed after enzymatic hydrolysis (fig. b) . therefore, this binding may be associated with sialic acid residues, and the role of sialic acid in this mechanism may be underestimated. to further demonstrate the influence of the terminal monosaccharide of carbohydrate chain in the binding of ovm with ha, several monosaccharides that are commonly found near the end of the oligosaccharide chain were selected and tested. to determine whether gal, fuc, man and free sialic acid could bind with ha, we conducted competition experiment. the results show that after adding of these free sugars first, the binding of ovm and ha was not significantly different and basically maintained at the same level. it suggests that these carbohydrates are not involved in binding and cannot prevent this interaction. monomeric sialic acid is thought to be unable to compete effectively with the receptor on target cells for binding to influenza virus, while dendritic sialic acid structure is more effective against influenza virus [ ] . the process by which influenza virus recognizes sialic acid is strongly related to its linked pattern [ , , , ] . this is similar to the finding that free sialic acid does not effectively bind with ha in this study (fig. ) . sialic acid is a highly electronegative sugar molecule, and the role in ovm is worth in-depth exploration. for this reason, we designed the influence of free sialic acid on the interaction between ovm and ha under different sequences of addition. when ovm was added firstly and then free sialic acid, there was no significant change in the binding of ovm with ha, and the interaction remained at the same level as the control. however, it was exciting to note that when ovm was preincubated with free sialic acid, its binding to ha was significantly enhanced compared to the control. it had the same effect on h n and h n (fig. ) . sialic acid in ovm was involved in the recognition of ha. free sialic acid does not directly bind to ha but mixed with ovm can effectively improve the interaction. sialic acid is electronegative and carries multiple o atoms and n atoms that can have many unique physicochemical effects [ , ] . taken together, this study proposes a novel mechanism hypothesis that sialic acid plays an active role in the antiviral activity of ovm (fig. ) . first, sialic acid in the ovm carbohydrate chain is recognized by the influenza virus ha as a receptor [ ] . on the other hand, free sialic acid is not directly involved in ha binding, but it can bind ovm to form an eupolymer by electrostatic interaction or hydrogen bond [ ] , promote ovm to bind and co-precipitate with ha. therefore, ovm maintain effective defense against virus, and enhance its antiviral activity. in a future study, this hypothesis will be explored in vitro and in cell experiments. in addition, we also analyzed the influence of ovm structure after removing sialic acid. the results exhibited that the secondary structure does not change too much but can cause ovm tertiary structural changes and that increasing the surface hydrophobicity causes molecular expansion [ , ] . this may increase the hydrogen bond binding sites of sialic acid to ovm. therefore, the structural interpretation of ovm antiviral activity is worth further study. ovomucin contains sialic acids with both α - and α - linkages, and the α - bond is higher than the α - bond, which helps it against a wide variety of influenza viruses. the hemagglutinin of the influenza virus recognizes and binds to the ovomucin carbohydrate chain terminal sialic acid, and the interaction is greatly diminished after the sialic acid is removed. therefore, sialic acid is an important recognition site for ovomucin to play a role in the binding of hemagglutinin competing with host cell surface receptors. at the same time, the removal of ovomucin sialic acid lead to a slight increase in the random coil of secondary structure and enhance surface hydrophobicity. we also found a unique role for free sialic acid. the addition of free sialic acid can promote the binding of ovomucin to hemagglutinin and enhance ovomucin anti-influenza virus activity. this in-depth study and exposition can complement the mechanism of ovomucin involved in innate immunity and provide new ideas and perspectives for the development of antivirus agents. an inventory of mucin genes in the chicken genome shows that the mucin domain of muc is encoded by multiple exons and that ovomucin is part of a locus of related gel-forming mucins understanding the adhesion mechanism of a mucin binding domain from lactobacillus fermentum and its role in enteropathogen exclusion ovomucin, a substrate for the enzyme of influenza virus; i ovomucin as an inhibitor of haemagglutination by heated lee virus bindings of ovomucin to newcastle disease virus and anti-ovomucin antibodies and its heat stability based on binding abilities structural features of newcastle disease virus-and anti-ovomucin antibody-binding glycopeptides from pronase-treated ovomucin differences in hemagglutination inhibition activity against bovine rotavirus and hen newcastle disease virus based on the subunits in hen egg white ovomucin h and c-nmr assignments for sialylated oligosaccharide-alditols related to mucins. study of thirteen components from hen ovomucin and swallow nest mucin complete h-and c-n.m.r. assignments for two sulphated oligosaccharide alditols of hen ovomucin n-glycosylation of ovomucin from hen egg white structural insights into bacterial recognition of intestinal mucins low abundant n-linked glycosylation in hen egg white lysozyme is localized at nonconsensus sites chemical and physical characterization of ovomucin, a sulfated glycoprotein complex from chicken eggs determination of the structure of sulfated tetra-and pentasaccharides obtained by alkaline borohydride degradation of hen ovomucin. a fast atom bombardment-mass spectrometric and h-nmr spectroscopic study natural and synthetic sialic acid-containing inhibitors of influenza virus receptor binding early detection of influenza a(h ) viruses with affinity for the human sialic acid receptor by maldi-tof mass spectrometry based mutation detection the receptor preference of influenza viruses the biology of influenza viruses inflammation-dependent changes in α , -, α , -, and α , -sialic acid glycotopes on serum glycoproteins in mice investigating the interaction between influenza and sialic acid: making and breaking the link, influenza virus sialidase-a drug discovery target influenza virus sialidase-a drug discovery target a new method of separating ovomucin from egg white the use of circular dichroism in the investigation of protein structure and function simultaneous quantification of protein order and disorder binding interaction of atorvastatin with bovine serum albumin: spectroscopic methods and molecular docking mg + binding affects the structure and activity of ovomucin protein unfolding as a switch from selfrecognition to high-affinity client binding sialic acids: fascinating sugars in higher animals and man evaluation of the porcine gastric mucin binding assay for highpressure-inactivation studies using murine norovirus and tulane virus impact of mucin, bile salts and cholesterol on the virulence of vibrio anguillarum towards gnotobiotic sea bass (dicentrarchus labrax) larvae mucins: a biologically relevant glycan barrier in mucosal protection receptor specificity in human, avian, and equine h and h influenza virus isolates alpha - -and alpha - -n-linked sialic acids allow efficient interaction of newcastle disease virus with target cells inhibition of viral adhesion and infection by sialic-acidconjugated dendritic polymers binding kinetics of influenza viruses to sialic acid-containing carbohydrates identification of the sialic acid structures recognized by minute virus of mice and the role of binding affinity in virulence adaptation sialic acids in molecular and cellular interactions glycosylation of human iga directly inhibits influenza a and other sialic-acid-binding viruses sialic acid-triggered macroscopic properties switching on a smart polymer surface oxidation of c-reactive protein by hypochlorous acid leads to the formation of potent platelet activator glyoxal-induced modification enhances stability of hemoglobin and lowers iron-mediated oxidation reactions of the heme protein: an in vitro study this work was financially supported by national natural science foundation of china [grant no. ]. key: cord- - b xffsh authors: maas, ronald h. w.; bakker, robert r.; jansen, mickel l. a.; visser, diana; de jong, ed; eggink, gerrit; weusthuis, ruud a. title: lactic acid production from lime-treated wheat straw by bacillus coagulans: neutralization of acid by fed-batch addition of alkaline substrate date: - - journal: appl microbiol biotechnol doi: . /s - - - sha: doc_id: cord_uid: b xffsh conventional processes for lignocellulose-to-organic acid conversion requires pretreatment, enzymatic hydrolysis, and microbial fermentation. in this study, lime-treated wheat straw was hydrolyzed and fermented simultaneously to lactic acid by an enzyme preparation and bacillus coagulans dsm . decrease in ph because of lactic acid formation was partially adjusted by automatic addition of the alkaline substrate. after h of incubation, the polymeric glucan, xylan, and arabinan present in the lime-treated straw were hydrolyzed for %, %, and %, respectively. lactic acid ( . g/l) indicated a fermentation efficiency of % and a chiral l(+)-lactic acid purity of . %. in total, g lactic acid was produced out of , g lime-treated straw, representing % of the overall theoretical maximum yield. approximately half of the lactic acid produced was neutralized by fed-batch feeding of lime-treated straw, whereas the remaining half was neutralized during the batch phase with a ca(oh)( ) suspension. of the lime added during the pretreatment of straw, % was used for the neutralization of lactic acid. this is the first demonstration of a process having a combined alkaline pretreatment of lignocellulosic biomass and ph control in fermentation resulting in a significant saving of lime consumption and avoiding the necessity to recycle lime. lactic acid is used throughout the world in manufacturing of food, chemicals, and pharmaceutical products. recently, there is a lot of interest in biodegradable poly-lactic acid, which is an alternative to petrochemically derived plastic (drumright et al. ) . chiral pure lactic acid is produced commercially by microbial fermentation of the carbohydrates glucose, sucrose, lactose, and starch/maltose derived from feedstocks such as beet sugar, molasses, whey, and barley malt (narayanan et al. ). the choice of feedstock depends on its price, availability, and on the respective costs of lactic acid recovery and purification (datta et al. ; vaidya et al. ) . as an alternative to these traditional feedstocks, lignocellulosic biomass is an inexpensive and widely available renewable carbon source that has no competing food value. lignocellulose consists primarily of cellulose and hemicellulose; polymers build up of mainly hexose sugars and pentose sugars, which are embedded in a matrix of the phenolic polymer lignin. the main pathway to derive fermentable sugars from lignocellulose is through enzymatic hydrolysis by cellulolytic and hemicellulolytic enzymes. a mechanical and chemical pretreatment of the lignocellulose is required to reduce particle size, to modify and/or to remove the lignin, and with that to enhance the accessibility of the polysaccharides for enzymatic hydrolysis (claassen et al. ) . various chemical pretreatments of biomass have been studied in research and development of lignocellulose-to-ethanol production technology (mosier et al. ) . one is the use of lime (calcium hydroxide) at relatively mild temperature conditions (chang et al. ). lime as a pretreatment agent has promising potential because it is inexpensive, safe, and its use hardly results in sugar degradation products such as furfural and hydroxymethyl furfural. nevertheless, this alkaline pretreatment features a relatively high ph value (> ) of the treated biomass, and at these ph levels, the activity of common cellulolytic and xylanolytic enzymes, necessary for the depolymerization of (hemi)-cellulose, is negligible low. therefore, lowering the ph is essential to achieve an efficient enzymatic hydrolysis of the polysaccharides. one approach to remove calcium hydroxide is by washing the lime-treated biomass before enzymatic hydrolysis (chang et al. ); however, this leads to the use of high amounts of water. another way to lower the ph of the pretreated material is by neutralizing calcium hydroxide with sulfuric acid. yet, this results in the formation of the low value byproduct gypsum. as an alternative improvement to these approaches, we propose to use the calcium hydroxide present in limetreated biomass as neutralizing agent for organic acids produced in microbial fermentation processes. to examine this proposed concept, lime-treated wheat straw (ltws) was added fed-batch-wise during a simultaneous saccharification and fermentation (ssf) process in a -l controlled stirred fermenter containing hydrolytic enzymes and bacillus coagulans dsm , a thermophilic bacterium capable to convert both hexoses and pentoses homofermentative to l(+)-lactic acid (otto ; patel et al. ) . the objective of this research was to evaluate whether high alkaline-treated lignocellulosic biomass (without neutralization) can be used directly in a ssf process by ( ) providing a carbon source for enzymatic hydrolysis and fermentation and ( ) providing a source of alkali to control the ph in the fermentation process. wheat straw was selected as a lignocellulose model feedstock and was purchased from a farm in the northeast of the netherlands. the wheat straw was air dried ( . % [w/w] dry matter [dm] ) and ground through a -mm screen. the lime pretreatment was performed by filling two -l mixers (terlet, the netherlands), both with , g ground wheat straw, kg tap water, and g calcium hydroxide. this wheat straw suspension was heated and kept at °c for h under continuously stirring at rpm. the ltws suspension was subsequently cooled to °c, dehydrated by placing the ltws in a cotton bag, and pressing the suspension using a manual piston press at pressure up to . kg/m . after dehydration, an amount of . kg ltws with an average dm content of . % (w/w) and ph . was obtained and served as substrate for further experiments. the chemical composition of ltws was determined as described by van den oever et al. ( ) . the enzyme preparation gc (genencor-danisco, rochester, usa) containing cellulase, cellobiase, and xylanase activity of , , and u/ml, respectively (kabel et al. ) , was used for this study. the preparation had a specific gravity of . g/ml and contained . mg/ml glucose, . mg/ml mannose, and . mg/ml galactose. the bacterium b. coagulans strain dsm was used as the lactic acid-producing micro-organism. bacterial cells were maintained in a % (w/w) glycerol stock solution and stored at − °c. chemicals, unless indicated otherwise, were purchased from merck (darmstadt, germany). gelrite plates were prepared with a medium containing (per liter): glucose, g; gelrite, g (duchefa, haarlem, the netherlands); yeast extract, g (duchefa); (nh ) hpo , g; (nh ) so , . g; bis-tris, g (usb, ohio, usa); mgcl h o, . g; and cacl . h o, . g. glucose and gelrite were dissolved in stock solution a (four times concentrated). the ph of this stock solution was adjusted to . with m hydrochloric acid and autoclaved for min at °c. the remaining nutrients were dissolved in stock solution b ( . times concentrated), which was also adjusted to ph . with m hydrochloric acid but was filter sterilized (cellulose acetate filter with pore size of . μm, minisart, sartorius). after sterilization, the medium was prepared by combining stock solutions a and b and gelrite plates were poured. the bacteria were cultivated on gelrite plates for h at °c. an isolated colony was used to inoculate a -ml broth with similar composition and preparation as described above but without the addition of gelrite. the culture was incubated statically for h at °c and functioned as the inoculum for a , -ml broth. this culture was incubated also statically for h at °c and served as a % (v/v) preculture for the ssf experiments. the ssf of ltws was carried out in a -l fermenter (applikon, schiedam, the netherlands) with ph and temperature control (biocontroller adi ). at the start of ssf, the fermenter was filled with . kg tap water and , g dehydrated ltws (dm content of . % [w/w]). the following nutrients were then added to the ltws suspension: yeast extract, g (duchefa); (nh ) hpo , g; (nh ) so , . g; mgcl h o, . g; and cacl h o, . g. the ltws suspension was then heated to °c, and the ph was adjusted to . with g m sulfuric acid (~ g h so ). the ssf process of ltws to lactic acid consisted of three phases: ( ) the prehydrolysis phase of preloaded ltws, ( ) the fed-batch phase with automatic feeding of ltws from a screw feeder, and ( ) the batch phase with ph control by a calcium hydroxide suspension and no ltws feeding. a schematic representation of the experimental setup is shown in fig. . the prehydrolysis was initiated by the addition of ml enzyme preparation ( mg enzyme/g dm substrate) to the ltws suspension and was incubated for h at °c under continuously stirring at rpm. the fed-batch phase was initiated by the addition of , -ml preculture of b. coagulans dsm to the fermenter. the lactic acid produced by the bacteria was neutralized by the automatic addition of , g dehydrated ltws (dm of . %) to the fermenter through a feeder (k-tron soder feeders, canada) and was regulated by the ph of the medium, which was set at . . throughout the fed-batch phase, an amount of ml of enzyme preparation (total enzyme loading of mg/g dm substrate) was added proportional to the ltws addition rate into the fermenter. during the batch phase, the ph was controlled at . by the addition of . % (w/v) calcium hydroxide suspension. samples were withdrawn for dm, substrate, and (by)product analysis. for the analysis of monomeric sugars, the fermentation broth samples were centrifuged ( min at , ×g), and the ph of the supernatant was adjusted to . with barium carbonate using a ph indicator (bromophenolblue) followed by filtration of the liquid. the analysis was performed by high-performance anion-exchange chromatography using a carbopack pa column (column temperature of °c) and a pulsed amperometric detector (ed ; dionex, sunnyvale, ca, usa). before injection, the system was equilibrated with . mm naoh for min at a flow rate of . ml/min. for the separation of monomeric sugars, at injection, the mobile phase was shifted to deionized water for min. postcolumn addition of sodium hydroxide was used for detection of the neutral monomeric sugars. the determination of soluble oligomeric sugars was performed by centrifugation for min at , rpm (centaur , beun de ronde, the netherlands) of preweighed samples and freeze drying the supernatant overnight. pellets were weighed and hydrolyzed with sulfuric acid, and neutral monomeric sugars were determined according to the method as described by van den oever et al. ( ) . for the calculations, an average molecular weight of oligomers from glucan and xylan of and g/mol, respectively, were applied, resulting in a hydrolysis factor of . and . , respectively. for the analysis of insoluble polymeric sugars, samples of g were centrifuged for min at , rpm (centaur , beun de ronde); the supernatant was removed, and the pellet was washed by resuspension in ml fresh demineralized water followed by a centrifugation step of min at , rpm (centaur , beun de ronde). the sequence of resuspension and centrifugation was repeated three times. after the last removal of the supernatant, the pellets were freeze dried overnight. the pellets were weighed (values used for dm calculation), polymeric material was hydrolyzed with sulfuric acid, and neutral sugars were analyzed according to the method as described by van den oever et al. ( ) . for the calculations, a molecular weight of glucan and xylan of and g/mol, respectively, were applied, resulting in a hydrolysis factor of polymer to monomer of . and . , respectively. the analysis of organic acids was performed by highpressure liquid chromatography according to the procedure described by maas et al. ( ) . the chiral purity (%) of lactic acid was determined by derivatization of all lactates using methanol, after which both enantiomers of methyl lactate were separated on a chiral gas chromatography column and detected using a flame ionization detector. the chiral purity was expressed as the area of the main enantiomer divided by the sum of areas of both enantiomers. the theoretical maximum lactic acid (la theor. max . [g]) production was calculated according the following equation (eq. ): where dm substrate =the total dry matter of substrate ltws (g), f polysacch. =fraction polysaccharides per substrate (g/g), hf monsacch./polysacch. =hydrolysis factor of polysaccharides, incorporation of water results in . g hexose from . g glucan and . g pentose from xylan and arabinan (g/g), and ff=fermentation factor of . g lactic acid per gram of monomeric sugar. the efficiency of the enzymatic hydrolysis (%, w/w) was based on the amount of hydrolyzed polysaccharides (g; calculated by the difference between initial amounts and analyzed insoluble amounts) divided by the amount of polysaccharides (g) initially present in the substrate. the fermentation efficiency (%, w/w) is expressed as the amount of lactic acid produced (g) divided by the amount of monomeric sugars consumed (g) by the bacteria. the overall efficiency of the ssf (%, w/w) was calculated by the amount of lactic acid produced (g) divided by the theoretical maximum amount of lactic acid (g) determined as described in eq. . the polysaccharide composition of the ltws consisted mainly of glucan, xylan, and arabinan of . %, . %, and . % (w/w), respectively, whereas the remaining mass constituted of lignin, ash, extractives, and uronic acids. some of the soluble components in wheat straw were partially removed by the solid/liquid separation (dehydration) of the ltws. the focus of this study was on the conversion of glucan, xylan, and arabinan, which are the predominant polysaccharides present in ltws and accounted for . % (w/w) of the total polymeric sugars. previous work showed that the cellulase preparation gc , used for the saccharification of polysaccharides, functioned optimally at °c and ph . (maas et al., submitted for publication), whereas growth conditions for b. coagulans dsm were °c and ph . (otto ) . in this study, both the enzymatic hydrolysis and the fermentation occurred simultaneously in the same reactor at compromising conditions, which were set at °c and ph . . the ssf of ltws to lactic acid was studied in a l controlled stirred fermenter. previous results showed that when this process was performed without a prehydrolysis of an initial amount of ltws, the concentration of monomeric sugars was low and resulted, therefore, in relatively low lactic acid productivity. as a consequence, the fed-batch addition rate of the alkaline substrate to neutralize the produced lactic acid was low (results not shown). to start the fermentation with a substantial initial amount of fermentable sugars (> g/l), a prehydrolysis of g ltws and enzyme preparation ( mg per g dm ltws) in approximately l volume at ph . for h was introduced. this resulted in glucose, xylose, and arabinose concentrations of . , . , and . g/l, respectively (fig. a) . the second phase (ii) was initiated by introducing a , -ml preculture of b. coagulans dsm . a minor amount of lactic acid produced in the preculture caused a slight ph decrease and was automatically neutralized by the addition of ltws (fig. a,b) . after a lag phase of h, the dissolved oxygen concentration decreased rapidly within h from % to oxygen-limiting conditions of below % (results not shown), and lactic acid production started. at that moment, concentrations of glucose, xylose, and arabinose of . , . , and . g/l, respectively, were present (fig. a) . these sugars were consumed simultaneously where glucose was utilized faster than xylose and arabinose. simultaneous with the consumption of these monomeric sugars, lactic acid was produced, which was neutralized by the automatic addition of alkaline ltws. by the addition of alkaline substrate throughout the fedbatch phase, the ph was maintained accurately at . ± . (fig. a,b) . at the end of phase ii, a total amount of , g dehydrated ltws (~ , g dm ltws) and ml of enzyme preparation was added to the fermenter. a lactic acid concentration of . g/l supernatant was detected (fig. b ), corresponding to a total of g lactic acid. the chiral l(+) purity of lactic acid was determined at . %, which is similar to that obtained with xylose as the sole carbon source (otto ) . at the end of phase i, a low acetic acid concentration was detected in the medium, which increased to . g/l throughout phase ii but remained constant during phase iii (results not shown). this indicates that acetic acid was most likely not a fermentation product formed by b. coagulans. acetic acid can be released upon solubilization and hydrolysis of hemicellulose during chemical pretreatment (palmqvist et al. ) . by the dehydration procedure of the ltws, part of the acetic acid was easily separated from the substrate by removing the press water. apparently, a remaining amount of acetic acid was fed together with the substrate to the fermenter. furthermore, minor traces of other organic acids such as succinic acid and formic acid (< . g/l) were detected in the fermentation broth. phase iii was initiated by changing the ph control from the addition of alkaline ltws to a % (w/v) calcium hydroxide suspension. to maintain the ph at . , the addition of calcium hydroxide suspension occurred relatively fast but shifted, however, after a few hours to a lower addition rate indicating a decline of the volumetric lactic acid productivity (figs. b, b) . to exclude limitation (e.g., by inactivation) of enzymes, an extra dosage of enzyme preparation ( ml) was added to the fermenter after . h of incubation. this resulted immediately in a slight acceleration of the calcium hydroxide addition rate indicat-ing an increased lactic acid productivity and limitation of enzymatic activity (fig. b) . nevertheless, after . h of incubation, a decline of the calcium hydroxide addition rate was observed again. therefore, a second extra dosage of the enzyme preparation ( ml) was added and resulted this time in a slight accumulation of glucose and xylose of . and . g/l (fig. a) , respectively, indicating that microbial conversion instead of enzymatic hydrolysis was rate limiting. after h of incubation, a lactic acid concentration of . g/l was obtained, with a chiral l(+)lactic acid purity of . %. continuation of the ssf process to a total incubation period of h resulted in a slightly increased lactic acid concentration of . g/l supernatant (~ . g lactic acid/kg fermentation broth) with an overall volumetric lactic acid productivity of . g l − h − . at this stage, a chiral l(+)-lactic acid purity of . % was analyzed. this slight decline in lactic acid purity is possibly a result of infection with other undesired lactic acidproducing microorganisms. because the substrate used fig. control of ph (a) during simultaneous saccharification and fermentation of lime-treated wheat straw by commercial enzyme preparation gc and b. coagulans dsm (b). the areas between the dotted lines represent the prehydrolysis phase (i), the fedbatch phase (ii) with ph control by addition of alkaline ltws and enzymes, and the batch phase (iii) with ph control by addition of ca (oh) suspension. extra enzyme preparation gc was added at the times indicated by the arrows was not sterile and also the chemical pretreatment and fermentation occurred in an open system under nonsterile conditions, microbial contamination throughout the ssf process is possible. the efficiency of the enzymatic hydrolysis of the polymeric material present in ltws is shown in fig. . the insoluble polymeric fraction was determined at various time points throughout the ssf experiment. at the end of the prehydrolysis ( h) of g ltws, % of the insoluble glucan (fig. a) , % of xylan (fig. b) , and % of arabinan (fig. c) were converted to soluble saccharides including monomeric sugars and oligomeric sugars. after the fed-batch phase ( h), , g ltws was added and resulted in a conversion of % of glucan, % of xylan, and % of arabinan to products including soluble saccharides and lactic acid. between and h of incubation, further hydrolysis of the polymeric sugars was observed. however, during the last h of the ssf, minor hydrolysis of the polysaccharides occurred, and this corresponded with the decline in lactic acid productivity during this phase. after h, g of glucan, g of xylan, and g of arabinan was still present as insoluble polymeric material. with these values, the hydrolysis efficiency of the initial glucan, xylan, and arabinan present in ltws were calculated as %, %, and %, respectively. the monomeric sugars, derived from the ltws, were partly converted to lactic acid ( g) by b. coagulans and accounted for % (w/w) of the theoretical maximum, indicating the formation of other products such as microbial biomass and carbon dioxide. an overall conversion yield of % (w/w) of the theoretical maximum was calculated according to eq. . the fate of polysaccharides initially present in ltws after h of incubation is shown in table . a part of the polysaccharides present in ltws remained as insoluble polysaccharides ( % w/w), whereas a minor part was converted into soluble oligomeric ( % w/w) and monomeric ( % w/w) sugars. another part of the initial polysaccharides present in the ltws was not recovered in the form of saccharides or lactic acid and was therefore ascribed as 'unaccounted.' the lactic acid produced ( g) during the fed-batch phase (ii) was neutralized with alkaline-pretreated wheat straw. presented values are averages based on duplicate analytical measurements. a total of glucan, xylan, and arabinan b part of the initial polysaccharides remained present as insoluble polysaccharides. c part of the initial polysaccharides was not recovered and therefore denoted as 'unaccounted.' during this phase, an amount of , g ltws was added to the fermenter. together with this substrate, an amount of g calcium hydroxide was added to the fermenter and accounted for a ratio of . g calcium hydroxide per gram of lactic acid. the lactic acid ( g) produced during the batch phase (iii) was neutralized with g calcium hydroxide resulting in a ratio of . g lactic acid per gram calcium hydroxide. lignocellulosic feedstocks are considered as potential attractive substrates for the production of bulk chemicals. pretreatment of biomass is required to break open the lignocellulosic matrix, and an enzymatic hydrolysis is necessary for the hydrolysis of polymeric carbohydrates. the lime pretreatment has proven to enhance enzymatic digestibility of the polysaccharides present in lignocelluloses (chang et al. ; kaar and holtzapple ) and results, in comparison to other pretreatment routes, in minor inhibitor formation. however, before the enzymatic hydrolysis, it is essential to adjust the ph to a level optimal for enzymatic activity. in this study, the reduction in ph by washing or neutralization was omitted by using the alkaline character of ltws to neutralize lactic acid produced by microbial fermentation during a ssf process. the results showed that the largest part of the polysaccharides in ltws was converted enzymatically and the resulting sugars were fermented simultaneously to mainly lactic acid by b. coagulans dsm . between and h of incubation, the bacteria utilized the monomeric sugars, as soon as they appeared in the medium, resulting in relatively low monomeric sugar concentrations (< g/l). this indicates that throughout this period, the enzymatic hydrolysis was the rate-controlling step. the highest lactic acid productivity was observed during the fed-batch phase and the initial hours of the batch phase and declined rapidly after approximately h of incubation, as shown in fig. b . an extra addition of enzyme preparation showed a slight improvement of the volumetric lactic acid productivity but shifted within a few hours again to a relatively low production rate. a second extra enzyme addition did not affect the lactic acid productivity significantly (fig. b ). this addition of new enzymes resulted in a modest liberation of hemicellulose sugars (xylose, arabinose), but no further hydrolysis of glucan occurred. this shows that the remaining glucan was too recalcitrant or not accessible for further hydrolysis, resulting in decreasing lactic acid productivity. another possible explanation of the decreased lactic acid productivity is the inhibition of enzymes and/or bacteria by the increasing lactic acid concentration. a lactic acid concentration of . g/l supernatant (~ . g lactic acid/kg fermentation broth) with a relatively high chiral purity was determined after h of incubation, corresponding to an overall lactic acid yield of % of the theoretical maximum. moreover, the efficiencies of the enzymatic saccharification and the fermentation were both determined. these calculations showed that, based on residue analysis, at the end of the ssf process ( h), % of the glucan, % of the xylan, and % of the arabinan present in ltws was enzymatically hydrolyzed, which agree well with previously obtained results from experiments aiming to convert ltws to ethanol. to improve the yield, it is necessary to decrease the recalcitrance or improve the accessibility of polymeric sugars in the ltws by optimization of the pretreatment procedure. the concentrations of soluble monosaccharides and oligosaccharides in the medium were relatively low, which can be expected in a ssf process. a fermentation yield of % was determined (related to the amount of monosaccharides released from the ltws) and is slightly better than the results obtained by otto ( ) who reported the production of g/l lactic acid from g/l xylose as the sole carbon source. because no other soluble fermentation products were detected, the remaining % of the ltwsderived monomeric sugars were most presumably converted to bacterial biomass and some carbon dioxide during the aerobic part of the fermentation. several process parameters can be listed for enhancement of the overall lactic acid yield and productivity such as improving the accessibility of polysaccharides by a more severe lime pretreatment, enzyme dosage, type of enzymes, b. coagulans strain, size and growth phase of inoculum, ph gradient in ssf, and in situ product removal of lactic acid. these issues will be subject to further studies. during the fed-batch phase (ii), it was possible to counterbalance the ph decrease caused by lactic acid production by the addition of the alkaline feedstock. this suggests that it is possible to combine lime treatment with the production of other organic acids from lignocellulosic biomass. throughout this phase, the ratio of calcium hydroxide in ltws added per produced lactic acid was determined at . g/g. the theoretical stoichiometric neutralization of . g lactic acid requires . g calcium hydroxide. therefore, only % of the calcium hydroxide initially added to the wheat straw was used for lactic acid neutralization. on the other hand, throughout the batch phase (iii), an alkaline/acid ratio of . g/g was calculated corresponding to % of the added calcium hydroxide suspension used for lactic acid neutralization. the low efficiency of the calcium hydroxide added with the ltws for lactic acid neutralization during phase ii has three possible explanations. first, part of the calcium hydroxide could have been used during the chemical pretreatment of the wheat straw such as the neutralization of acetic acid or other organic acids and/or irreversible binding to the lignin. second, the calcium hydroxide might be released slowly from the insoluble wheat straw fibers and could therefore partly have been used for lactic acid neutralization in the fed-batch phase. finally, besides lactic acid production, other acidification reactions could have contributed to the decrease in ph and therefore the demand of alkaline substrate, for instance, the decrease in ph caused by the consumption of ammonium as the nitrogen source by microorganisms (guebel et al. ) . the results in this paper show that it is possible to use lignocellulosic materials for the production of lactic acid. lignocellulosic biomass is a relatively inexpensive substrate, and this affects feedstock costs for lactic acid production positively. nevertheless, in comparison to the traditional relatively 'clean' feedstocks with a well-defined composition, using heterogenic lignocellulosic substrates will require a more intensified downstream processing (dsp) to recover and purify the lactic acid from the complex fermentation broth. the costs of feedstock materials and operational costs of the dsp contribute considerably to the overall production costs of lactic acid (Åkerberg and zacchi ) . whether the cost decrease in using lignocellulosic feedstocks outweighs the potential increasing costs of dsp was not analyzed at the moment. in summary, ltws was converted into l(+)-lactic acid by b. coagulans throughout a ssf process at a -l bench scale. the pentose and hexose sugars derived from the polymeric material were utilized simultaneously by b. coagulans resulting in a final lactic acid concentration of . g/l supernatant, which accounted for % (w/w) of the theoretical yield. to our knowledge, this is the first paper demonstrating a process having a combined alkaline pretreatment of lignocellulosic biomass and ph control in organic acid fermentation resulting in a significant saving of lime consumption and avoiding the necessity to recycle lime. an economic evaluation of the fermentative production of lactic acid from wheat flour lime pretreatment of crop residues bagasse and wheat straw utilisation of biomass for the supply of energy carriers technological and economic potential of poly(lactic acid) and lactic acid derivatives polylactic acid technology influence of the nitrogen source on growth and ethanol production by pichia stipitis nrrl y- using lime pretreatment to facilitate the enzymic hydrolysis of corn stover standard assays do not predict the efficiency of commercial cellulase preparations towards plant materials lactic acid production from xylose by the fungus rhizopus oryzae features of promising technologies for pretreatment of lignocellulosic biomass l(+) lactic acid fermentation and its product polymerization preparation of lactic acid from a pentose-containing substrate. patent wo hahn-hägerdal b ( ) main and interaction effects of acetic acid, furfural, and p-hydroxybenzoic acid on growth and ethanol productivity of yeast isolation and characterization of acid-tolerant, thermophilic bacteria for effective fermentation of biomass-derived sugars to lactic acid switchgrass (panicum virgatum l.) as a reinforcing fibre in polypropylene composites acknowledgments this project is supported with a grant of the dutch programme eet (economy, ecology, technology), a joint initiative of the ministries of economic affairs, education, culture, and sciences and of housing, spatial planning and the environment. open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. key: cord- -tjdw uta authors: winter, christine; herrler, georg; neumann, ulrich title: infection of the tracheal epithelium by infectious bronchitis virus is sialic acid dependent date: - - journal: microbes infect doi: . /j.micinf. . . sha: doc_id: cord_uid: tjdw uta avian infectious bronchitis virus (ibv) is a coronavirus that infects chickens via the respiratory epithelium as primary target cells. the binding of coronaviruses to the cell surface is mediated by the viral surface protein s. recently we demonstrated that α , -linked sialic acid serves as a receptor determinant for ibv on vero cells and primary chicken embryo kidney cells. here we analyze the importance of the sialic acid binding activity for the infection of tracheal organ cultures (tocs) by different ibv strains. our results show that α , -linked sialic acid also serves as a receptor determinant on chicken tocs. infection of tocs by ibv results in ciliostasis. desialylation induced by neuraminidase treatment of tracheal organ cultures prior to infection by ibv delayed the ciliostatic effect or resulted in partial loss of ciliary activity. this effect was observed with both respiratory and nephropathogenic strains. inhibition of ciliostasis was also observed when tocs were pretreated with an α , -specific neuraminidase. analysis of the tracheal epithelium for reactivity with lectins revealed that the susceptible cells in the epithelium abundantly express α , -linked sialic acid. these results indicate that α , -linked sialic acid plays an important role for infection of the respiratory epithelium by ibv. infectious bronchitis is one of the most significant diseases of chickens in the commercial poultry industry. the etiologic agent is infectious bronchitis virus (ibv), a member of the family coronaviridae [ ] . the pathology of the disease associated with ibv infection can show different characteristics depending on the tissue where virus replication occurs. ibv enters its host via the oro/oculo-nasal route and the first site of replication is the respiratory epithelium. from there the virus infection can spread to several organs, including kidney and reproductive tract (reviewed in [ ] ). secondary pathogens can complicate the disease resulting in increased morbidity and mortality. the tissue tropism of viruses may be determined by several factors including the distribution of the cellular receptor for the virus. in the case of coronaviruses, the surface protein s is responsible for attachment to cells. the s protein has not only receptor-binding activity, it also mediates the fusion of the viral lipid envelope with the cellular membrane. for several coronaviruses specific proteins have been described that serve as cellular receptors for the initiation of infection. angiotensin-converting enzyme has been identified as a receptor for sars coronavirus [ , ] and several group coronaviruses including transmissible gastroenteritis virus require aminopeptidase n to enter their host cells [ , ] . a protein receptor has not been identified so far for ibv. recently we demonstrated that a , -linked sialic acids serve as receptor determinants on vero-cells and primary chicken embryo kidney cells [ ] . here we show that sialic acids also play a role in the infection of the avian respiratory epithelium by ibv. tracheas were prepared from -day-old spf chicken embryos (lohmann, cuxhaven, germany) and, after removing connective tissue, were cut manually into approximately mm thick rings by using a microtome blade. individual rings were transferred into ml tubes (sarstedt, nümbrecht, germany) containing . ml of medium with hanks salts (biochrom, berlin, germany) and incubated at c on a rotator. the next day, tocs were screened for % ciliary activity (see section . ). stock virus of the ibv strains m and b were obtained by inoculating embryonated spf chicken eggs. following incubation at c, the allantoic fluid was collected, clarified by low speed centrifugation and stored at À c. strain beaudette of ibv was propagated in vero cells. supernatants of infected cell cultures were harvested, clarified by low speed centrifugation and stored at À c. all ibv strains were kindly provided by dave cavanagh, institute for animal health, compton, uk. avian metapneumovirus subtype a, employed as a control virus, was kindly provided by silke rautenschlein, university of veterinary medicine, hannover, germany. tocs were washed with pbs prior to addition of neuraminidase from clostridium perfringens (type ) or streptococcus pneumoniae (sigmaealdrich, st. louis, mo, usa) using mes ( [n-morpholino]ethanesulfonic acid) buffer as a diluant. if not otherwise indicated tocs were incubated with mu neuraminidase per ring. after incubation at c for h, the tocs were washed three times with pbs and infected by ibv-beaudette ( pfu/ring) or any of the other viruses for h at c. following three washes with pbs, the tocs were incubated with medium at c on a rotator. for all experiments groups of four tocs were used to estimate the mean ciliary activity. all experiments were performed in triplicate. each ring of toc was suspended in ml medium containing mu neuraminidase of clostridium perfringens and in some cases . mg of the neuraminidase inhibitor dana ( , -didehydro- -deoxyneuraminic acid). after incubation for h at c tocs were washed and infected by the beaudette strain with pfu/ring. tocs were analyzed daily with a microscope to estimate the ciliary activity. rings were virtually divided into parts and each part was monitored for ciliary movement. only rings with a starting ciliary activity of % were used for the experiments. groups of five tocs were infected in triplicate by  pfu of ibv. at h post infection, the supernatants were collected and titrated on primary chick kidney cells as described previously [ ] . plaques were visualized by immunofluorescence using a polyclonal anti-ibv serum raised in rabbits. tracheas were prepared from three-week old spf chickens (lohmann, cuxhaven, germany). they were cut into rings approximately cm thick. the rings were washed with pbs and either infected by ibv-beaudette (  pfu/ring) for h at c. or subjected to neuraminidase treatment and lectin staining. infected tocs were incubated in medium (biochrom, berlin, germany) at c. after h, they were mounted on small filter papers with tissue freezing medium (jung, heidelberg, germany) and frozen in liquid nitrogen. neuraminidase treatment was performed by incubation with ml medium containing mu neuraminidase from clostridium perfringens for h at c, before freezing in liquid nitrogen. the frozen organs were stored at À c until they were cut with a cryostat. cryosections were fixed with ice-cold acetone for min followed by air drying for another min. the antibodies or lectins were diluted in % bovine serum albumin and sections were incubated with antibodies or lectins for h at room temperature in an incubation chamber. after three washing steps with pbs, the sections were incubated with appropriate second antibodies for h at room temperature in the dark. virus antigen was stained with polyclonal anti-ibv beaudette serum raised in rabbits. sialic acids were detected with lectins from maackia amurensis (binding to a , linked sialic acids) or sambucus nigra agglutinin (binding to a , linked sialic acids) labeled with digoxigenin (dig glycan differentiation kit, roche, basel, switzerland). mucus producing goblet cells were stained with anti muc- ac antibody (acris, hiddenhausen, germany). bound antibodies or lectins were visualized by fitc-and cy -labeled anti-rabbit (sigmaealdrich), anti-mouse (acris) or anti-digoxigenin antibodies (roche). cilia were detected by cy labeled anti-b-tubulin antibody (sigmaealdrich). fluorescence microscopy was performed with a leica inverted- confocal microscope. after having shown recently that sialic acid serves as a receptor determinant for ibv on cultured cells, we were interested to find out whether this type of sugar is also important for an infection in vivo. to address this question we chose tocs as a model for the upper respiratory epithelium. the trachea of chicken embryos was cut into pieces about mm thick. tracheal rings chosen for this analysis showed ciliary activity along the whole contact area of the epithelium with the lumen of the trachea, i.e. of the tracheal ring. infection of tocs by ibv results in ciliostasis that can easily be detected microscopically. as shown in fig. , infection by ibv-beaudette reduced the portion of the epithelium with ciliary activity. at days post infection (d.p.i.), complete ciliostasis was observed at this experimental setting. to analyze the importance of sialic acids we treated tocs with neuraminidase from clostridium perfringens, which releases the sialic acids from the cell surface. as a result desialylated tocs were less sensitive to the ciliostatic effect of the ibv infection. at d.p.i., about % of the epithelium still showed ciliary activity. this effect is accounted for by the neuraminidase itself rather than by a contaminant enzyme, because in the presence of a neuraminidase inhibitor the enzyme was unable to protect the tracheal epithelium from the ciliostatic effect of the ibv infection (fig. ) . the infection of cultured cells by ibv was found to be dependent on a , -linked sialic acid. to find out whether there is also a linkage specificity in the infection of the tracheal epithelium, tocs were treated with neuraminidase from streptococcus pneumoniae, which has a high preference for cleaving a , -linked sialic acid. as shown in fig. , incubation with this enzyme protected the epithelial cells from the ciliostatic effect of the ibv infection in the same way as was observed with the neuraminidase from clostridium perfringens (fig. ) . from this result we conclude that a , -linked sialic acid serves as a receptor determinant for the infection of avian tracheal epithelial cells by the beaudette strain of ibv. the beaudette strain of ibv has been adapted to grow in cultured cells of non-avian origin, such as vero cells. to demonstrate that the sialic acid-dependent infection of tracheal epithelial cells is a general feature of ibv, we included two other strains. the m strain causes respiratory disease, whereas the b strain has a nephropathogenic potential [ ] . after application of an infectious dose of pfu per tracheal ring, the ibv strains caused ciliostasis by day (m ), (b ) or (beaudette) after infection (fig. aec) . strain m was more pathogenic than the other two strains resulting in a loss of ciliary activity at d.p.i. on % of the epithelium in the microscopic field (fig. c) . a comparable reduction was observed with the two other strains only days following infection of tocs (fig. a and b) . for all three strains, a protective effect of neuraminidase treatment was found. in the case of m , ciliostasis was delayed for day; in the case of the other two strains, the ciliary activity was retained in about % of the desialylated epithelium even on day following infection. ciliostasis was also found after infection by avian metapneumovirus. however, in contrast to ibv infection, neuraminidase treatment did not prevent the ciliostatic effect of the metapneumovirus infection. this result confirms that the sialic acid dependence of the toc infection is a characteristic feature of ibv. in the experiments described above, the ciliary activity was used to monitor the course of infection. to determine the effect of neuraminidase treatment on virus production, the amount of infectious virus released from the epithelial cells was measured. groups of five toc rings were treated with neuraminidase prior to infection by either of the ibv strains, beaudette and m . in parallel, toc were infected that had been incubated in the absence of neuraminidase. at h p.i. the supernatants were collected and titrated on primary chicken kidney cells. with both virus strains, desialylation resulted in a decrease of the virus titer by about % (fig. ) . there was a clear difference in the amount of virus released from infected tocs. for the beaudette strain, the titer of infectious virus in the supernatant was e fold higher than that determined for strain m . the respiratory epithelium is the primary target for an ibv infection. cryosections of toc were prepared and ciliated cells were visualized by staining with antibodies directed against btubulin. mucus-producing goblet cells were stained with an antibody recognizing muc ac. as shown in fig. (top two rows), both ciliated and mucus-producing cells are infected by ibv. cryosections were also stained for sialic acid expression using the lectin maa which binds to a , -linked sialic acids and sna which recognizes a , -linked sialic acids. the epithelial cell layer lining the surface of the trachea shows bright fluorescence after staining with maa, indicating that these cells abundantly express a , -linked sialic acid. they colocalize with ibv-infected cells (fig , third row) . this result is consistent with a role of a , -linked sialic acid as a receptor determinant for ibv. staining with sna indicates that basal cell layers of the tracheal epithelium express a , -linked sialic acid. these cells are not infected by ibv (fig. , bottom row) . to visualize the effect of neuraminidase treatment, we prepared cryosections of neuraminidase-treated tracheal rings and stained the sections with the maa lectin. as shown in fig. , neuraminidase treatment resulted in a reduced reactivity of maa with the apical membrane of the epithelial cells. the residual fluorescent signal indicates that the neuraminidase did not release all sialic acids from the epithelial cells. the binding to receptors on the cell surface is an important determinant of cell tropism and viral pathogenesis. recently we demonstrated that a , -linked sialic acid serves as a receptor determinant for ibv on cultured cells [ ] . this binding activity of ibv has been shown for strains that only grow in avian cells as well as for the beaudette strain that has been adapted to grow also in cultured cells of mammalian origin. for the latter strain it has been recently reported that it also recognizes glycosaminoglycans of the heparan sulfate type [ ] . the acquisition of this additional binding activity may have allowed the adaptation to new host cells as has been shown for other viruses, e.g. foot and mouth disease virus [ ] and tick-borne encephalitis virus [ ] . by contrast, the sialic acid binding activity has been detected on all ibv strains analyzed including respiratory and nephropathogenic variants. here we have demonstrated that this also holds true for epithelial cells of the trachea, which are among the primary target cells for this virus. the importance of sialic acid for ibv infection was evident from the delayed appearance of ciliostasis on desialylated tocs. for the m strain, the ciliostatic effect was delayed only by day, whereas for the two other strains analyzed, some ciliary activity was retained even days after infection. this difference in the protective effect of neuraminidase treatment is probably accounted for by the difference in the cytopathogenicity of these viruses. this may also explain the larger amount of virus that is released from tocs infected by ibv-beaudette compared to m -infected tracheal rings. despite these differences, the relative effect of desialylation was similar, i.e. the amount of released virus was reduced by about %. the partial protection of tocs from ibv infection may be attributable to the difficulties in removing all sialic acid residues from the cell surface of the tracheal epithelium. staining by maa revealed that some sialic acids are still present on the apical membrane of the epithelial cells after neuraminidase treatment. whether this low level of sialic acid by itself is sufficient for a low level of infection is not known. an alternative explanation is that binding to surfacebound sialic acids is only a first attachment step in the infection cycle that precedes the binding to a second receptor, though such a receptor has not been identified for ibv so far. in favor of the latter explanation is the fact that ibv recognizes sialic acid with lower affinity when compared to influenza virus and sendai virus [ ] . because of the higher affinity for sialic acids, a second receptor may be dispensable for influenza viruses; however, it may require the presence of a neuraminidase (receptor-destroying enzyme) to allow passage through the mucus layer covering the respiratory epithelium [ ] and to enable virus release from infected cells [ ] . on the other hand, a receptor-destroying enzyme may be dispensable for ibv because of the lower affinity for sialic acids. so far, two groups of coronaviruses with sialic acid binding activity have been described. on one hand, there is bovine coronavirus and related viruses that resemble influenza c virus, because they recognize n-acetyl- -o-acetylneuraminic acid and contain an acetylesterase that acts as a receptor-destroying enzyme comparable to the neuraminidase of influenza a and b viruses [ , ] . these viruses depend on sialic acid for infection of cells. on the other hand, there is transmissible gastroenteritis virus, a porcine coronavirus. this virus uses aminopeptidase n as a cellular receptor and it does not require sialic acid for successful infection of cultured cells. however, binding to sialylated surface components may increase the efficiency of infection, because mutants lacking the sialic acid binding activity have lost the enteropathogenicity [ , ] . ibv takes an intermediate position; sialic acid is important for infection of cultured cells but it lacks a receptor-destroying enzyme. as mentioned above, ibv maydin addition to sialylated surface moleculesdbind to a specific receptor similar to the interaction of tgev with aminopeptidase n. though it fig. . effect of neuraminidase treatment on the staining of the tracheal epithelium by the lectin maa. tocs were incubated in the presence (þna) or absence (Àna) of neuraminidase. after incubation, cryosections were prepared and subjected to maa staining. arrows point to the apical membrane of the tracheal epithelium. it should be noted that the enzyme had access only to the apical side of the epithelium. has been suggested that feline aminopeptidase n may be used by ibv to infect cells [ ] , recently it has been shown that aminopeptidase n does not serve as a receptor for ibv [ ] . our analysis of toc revealed that both ciliated and mucusproducing cells are susceptible to infection. from this result we conclude that in vivo both cell types can act as primary target cells for ibv. the expression of a , -linked sialic acids and the absence of a , -linked sialic acids is consistent with the preference of ibv for the former linkage type ( [ ] and this report). in this respect ibv appears to have developed a similar strategy to avian influenza viruses which also use a , -linked sialic on the cell surface to initiate an infection. however, there are also major differences between avian influenza and avian coronaviruses. a major determinant of the pathogenicity of influenza viruses is the proteolytic cleavage of the hemagglutinin. the hemagglutinins of highly pathogenic viruses have a multibasic cleavage site that is susceptible to furin-like enzymes present in many cell types. viruses of low pathogenicity become fusion-active by a protease that is secreted by respiratory cells. the s protein of all ibv strains has a multibasic cleavage site and is cleaved in all cell types into the subunits s and s . therefore, proteolytical activation of s is not a major determinant of the pathogenicity of ibv. why some ibv strains are predominantly respiratory pathogens whereas others affect other organs to cause disease, e.g. the renal system or the reproductive tract, is not known. if there exists a second receptor for this virus as discussed above, such a protein may be responsible for the host specificity as well as the tissue or organ tropism of ibv. future work should clarify whether a second receptor exists. furthermore, the sialoglyconjugates used for primary attachment should be analyzed. the available lectins and neuraminidases only allow a differentiation between a , and a , -linked sialic acids. different oligosaccharides exist that contain a , linked sialic acid, and the sialoglycoconjugates recognized by ibv may be different from those that are preferred by avian influenza viruses. such differences may also contribute to a different course of infection. expression of a , -linked sialic acid on epithelial cells in chickens has been analyzed for the trachea and the intestine [ , ] . as far as other target organs of ibvare concerned, e.g. kidney and the reproductive system, a , -linked sialic acid has been shown to be present on primary kidney cells [ ] . it will be interesting in the future to find out whether infection of the reproductive system is also mediated by sialic acid. coronavirus avian infectious bronchitis virus angiotensin-converting enzyme is a functional receptor for the sars coronavirus expression cloning of functional receptor used by sars coronavirus aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev human aminopeptidase n is a receptor for human coronavirus e sialic acid is a receptor determinant for infection of cells by avian infectious bronchitis virus vandenbroeck, incidence, characterisation and prophylaxis of nephropathogenic avian infectious bronchitis viruses heparan sulfate is a selective attachment factor for the avian coronavirus infectious bronchitis virus beaudette efficient infection of cells in culture by type o foot-and-mouth disease virus requires binding to cell surface heparan sulfate adaptation of tick-borne encephalitis virus to bhk- cells results in the formation of multiple heparan sulfate binding sites in the envelope protein and attenuation in vivo neuraminidase is important for the initiation of influenza virus infection in human airway epithelium inhibition of influenza virus replication in tissue culture by -deoxy- , -dehydro-n-trifluoroacetylneuraminic acid (fana): mechanism of action the e protein of bovine coronavirus is a receptor-destroying enzyme with acetylesterase activity bovine coronavirus uses n-acetyl- -o-acetylneuraminic acid as a receptor determinant to initiate the infection of cultured cells transmissible gastroenteritis coronavirus, but not the related porcine respiratory coronavirus, has a sialic acid (n-glycolylneuraminic acid) binding activity point mutations in the s protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus the role of feline aminopeptidase n as a receptor for infectious bronchitis virus. brief review feline aminopeptidase n is not a functional receptor for avian infectious bronchitis virus differences between influenza virus receptors on target cells of duck and chicken lectin histochemical investigations of the distal gut of chicks with special emphasis on the follicle-associated epithelium we thank martina kaps for technical assistance. this work was supported by a grant from deutsche forschungsgemeinschaft (ne / - and sfb tp a ). key: cord- -xwd i vu authors: goh, choon fu; ming, long chiau; wong, li ching title: dermatologic reactions to disinfectant use during the covid- pandemic()() date: - - journal: clin dermatol doi: . /j.clindermatol. . . sha: doc_id: cord_uid: xwd i vu infection preventive practice of using disinfectants against sars-cov- has become the new normal due to the covid- pandemic. although disinfectants may not be applied directly to the human body, it remains at high risk of exposure including close skin contact on disinfected surfaces or during handling. this dermal contact, on a regular basis, can induce hazardous skin reactions like irritation, inflammation, and burning in severe conditions. disinfectants are germicide chemicals that can penetrate the skin and create skin reactions that are usually regarded as irritant and allergic contact dermatitis. more importantly, disinfectants can react with skin components (proteins and lipids) to facilitate their skin penetration and disrupt the skin barrier function. whereas the antimicrobial actions of disinfectants are well understood, much less is known regarding their dermatologic reactions, including but not limited to irritation and hypersensitivity. we reviewed the skin reactions created by those disinfectants against sars-cov- approved by the european chemical agency and the united states environmental protection agency. antigen-specific t cells (adaptive immunity). the inflammation is not directly caused by the chemicals but rather the response of t cells to the haptenized protein. we have provided a comprehensive summary of dermatologic reactions due to the exposure of disinfectants. the data on percutaneous penetration and interactions with skin components in facilitating skin penetration are also highlighted. we have included only the disinfectants commonly found in consumer products from the lists of disinfectants approved by (i) the european chemical agency and (ii) the united states environmental protection agency for use against sars-cov- , where these disinfectants have demonstrated efficacy against a harder-to-kill virus or another type of human coronavirus similar to sars-cov- . table shows the list of disinfectants commonly found in consumer products based on the various chemical classes and their examples and have included adverse reactions associated with the use of disinfectants and their skin penetration ability in table and . who recommends the use of alcohols, namely ethanol ( %v/v) and isopropanol ( %v/v) in handrubs. percutaneous penetration of both alcohols is generally low even with extensive use. [ ] [ ] [ ] they may create both irritant and allergic contact dermatitis. , this could be related to the solvent effect on skin components (table ) or pre-irritated skin with disrupted skin barrier. because isopropanol is more irritating than ethanol, emollients, such as glycerol or propylene glycol, may be added to hand preparations. [ ] [ ] [ ] allergic reactions, including contact urticarial, have been reported. [ ] [ ] [ ] the triggers may be due to impurities, aldehyde metabolites, or fragrances in the product. , iodophors or polyvinylpyrrolidone (povidone, pvp) iodines (pvp-i) is non-staining, being relatively less toxic and irritating, when compared to iodine. topical absorption seems to be time-dependent. icd (usually % pvp-i) sometimes occurs with chemical burns, pain, blistering lesions, and tissue necrosis. percutaneous absorption of silver and its nanoparticle forms is usually low in both intact and damaged skin (< %), because its ionized form does not penetrate the skin readily. , acd is known to occur due to the other ingredients. local skin discoloration (brown-black) is occasionally observed and be seen more frequently with topical application to wounds. , this is not true argyria (blue-grayish discoloration) which is more long-lasting and common following chronic exposure through inhalation or oral ingestion. aha, including citric acid (ca), glycolic acid (ga), and lactic acid (la), can penetrate the skin but the dermal absorption is ph-, concentration-, and time-dependent. [ ] [ ] [ ] [ ] burning, dermatitis, skin peeling, itching, and moderate sunburns are frequently reported at a concentration ≤ % or a ph ≥ . . this can be related to the expression of proinflammatory cytokines, such as tnf-α and il- α. an increased epidermal and dermal thickness is common at higher concentrations (ca and ga: - the cause may be due to the excellent lipid solubility of pa and its strong oxidative disruption on the skin lipids and keratin protein. phenol and its derivative, especially ortho-phenylphenol (opp) or biphenyl- -ol and ortho-benzylpara-chlorophenol (obpcp), have excellent skin-penetrating power. , skin reactions can develop with short contact. acd related to opp and obpcp may occur even at low concentrations ( . %). - chemical burns and digital tip gangrene have occurred, following persistent exposure to . % halogenated phenol; the actual causative compound was not stated. depigmentation or leukoderma is another clinical concern when applying opp and obpcp ( %), , although the depigmentation is reversible but with the repigmentation process taking upwards to a year or more. j o u r n a l p r e -p r o o f acd has been reported at very low concentrations ( . %), and immediate hypersensitivity is possible. [ ] [ ] [ ] [ ] [ ] urticaria, swelling, erythema, and itchiness can be found at higher concentrations ( - %). , ddac may produce mixed hypersensitivity that induces both ige-and t-cell mediated responses. - ddac can be a skin irritant and sensitizer, probably stronger than bac. while the use of disinfectants is inevitable, it is crucial to consider the following points to minimize or avoid any potential dermatological reactions:  damaged skin is prone to adverse reactions from a direct absorption of disinfectants, and extra care should be given to avoid contact with disinfectants.  while multiple disinfectants may be used together or formulated as a single product to achieve synergistic effects, an enhanced adverse effect is expected.  whenever dermatitis is known, disinfectants that are weak or non-irritants and sensitizers should be prioritized. patch testing may be considered. it is important to avoid using disinfectants from a similar class that is known to be allergic to the users in consideration of a potential cross-reactivity. journal pre-proof  it is necessary to use protective garments during handling to avoid direct contact from spillage. even with regular use of protective attires, unnoticeable punctures in the gloves on multiple use and the handling of disinfected surfaces can expose users to contamination. possible interactions of disinfectants with protective garments may occur. for example, glutaraldehyde at - . % may penetrate latex gloves after min and thus, butyl rubber and nitrile rubber gloves are recommended.  emphasis is given only on the dermatological reactions in this review but the exposure through other manners such as ocular route and inhalation is often significant and most probably toxic. for instance, chlorine compounds are known to emit chlorine gas during preparation and application. the exposure to the eyes is thus high and toxic. the dermatologic events are usually, but not always, related to prolonged exposure and contact with concentrated disinfectants. many dermatologic adverse events remain unreported. some skin reactions, especially sensitization, can develop for compounds currently known to be a non-irritant or sensitizer. who. who virtual press conference on covid- . march , . table skin irritation, sensitization, and significant skin manifestations of disinfectants a pharmacological and toxicological profile of silver as an antimicrobial agent in medical devices a rare case of localized argyria on the face final report on the safety assessment of glycolic acid, ammonium, calcium, potassium, and sodium glycolates, methyl, ethyl, propyl, and butyl glycolates, and lactic acid, ammonium, calcium, potassium, sodium, and tea-lactates, methyl, ethyl, isopropyl, and butyl lactates, and lauryl, myristyl, and cetyl lactates assessment of in vitro percutaneous absorption of glycolic acid through human skin sections using a flow-through diffusion cell system in vitro percutaneous absorption of alpha hydroxy acids in human skin negligible penetration of incidental amounts of alpha-hydroxy acid from rinse-off personal care products in human skin using an in vitro static diffusion cell model guidance for industry: labeling for cosmetics containing alpha hydroxy acids. us food and drug agency stimulation of epidermal calcium gradient loss and increase in tnf-α and il- α expressions by glycolic acid in murine epidermis interaction of stratum corneum components with isothermal titration calorimetric study strong irritants masquerading as skin allergens: the case of benzalkonium chloride benzalkonium chloride: a known irritant and novel allergen dermatokinetics of didecyldimethylammonium chloride and the influence of some commercial biocidal formulations on its dermal absorption in vitro contact dermatitis from didecyldimethylammonium chloride and bis-(aminopropyl)-laurylamine in a detergent-disinfectant used in hospital foot dermatitis caused by didecyldimethylammonium chloride in a shoe refresher spray immediate-type allergy by occupational exposure to didecyl dimethyl ammonium chloride occupational allergic contact dermatitis from n,n-bis( -aminopropyl)dodecylamine and dimethyldidecylammonium chloride in hospital staff topical application of the quaternary ammonium compound didecyldimethylammonium chloride activates type innate lymphoid cells and initiates a mixed-type allergic response glutaraldehyde permeation: choosing the proper glove hand hygiene and skin health percutaneous absorption enhancement of an ionic molecule by ethanol-water systems in human skin examination of the effect of ethanol on human stratum corneum in vivo using infrared spectroscopy ethanol and water sorption into stratum corneum and model systems role of isopropyl myristate, isopropyl alcohol and a combination of both in hydrocortisone permeation across the human stratum corneum enhanced permeation of polar compounds through human epidermis permeability and membrane structural changes in the presence of short chain alcohols hypochlorite-induced oxidation of amino acids, peptides and proteins chlorinated phospholipids and fatty acids: (patho)physiological the influence of alkane chain length on the skin irritation potential of , -alkanediols safety assessment of , -glycols as used in cosmetics influences of alkyl group chain length and polar head group on chemical skin permeation enhancement allergic contact dermatitis from povidone-iodine: a re-evaluation study citrate salts, and alkyl citrate esters as used in cosmetics a theory for the mechanism of action of the alpha-hydroxy acids applied to the skin nonanoic acid -an experimental irritant acne vulgaris: studies in pathogenesis: relative irritancy of free fatty acids from c to c final report of the cosmetic ingredient review expert panel on the safety assessment of pelargonic acid (nonanoic acid) and nonanoate esters epidermal langerhans cell apoptosis is induced in vivo by nonanoic acid but not by sodium lauryl sulphate biochemical basis for depigmentation of skin by phenolic germicides -toxicity effects of oxygen-containing terpenes as skin permeation enhancers on the lipoidal pathways of human epidermal membrane the effect of terpene enhancer lipophilicity on the percutaneous permeation of hydrocortisone formulated in hpmc gel systems permeability of human epidermis to phenolic compounds ftir microscopy and confocal raman microscopy for studying lateral drug diffusion from a semisolid formulation contact urticaria to chlorocresol contact urticaria with anaphylaxis caused by chlorocresol, chloroxylenol, and thiourea benzalkonium chloride--a relevant contact allergen or irritant? results of a multicenter study of the german contact allergy group interaction of antiseptic compounds with intercellular lipids of stratum corneum correlation of water and lidocaine flux enhancement by cationic surfactants in vitro opinion of the scientific committee on cosmetic products and non-food products intended for consumers concerning benzethonium chloride. european commission effects of single and repeated exposure to biocidal active substances on the barrier function of the skin in vitro factors of importance in the use of triethylene glycol vapor for aerial disinfection. american journal of public health and the nation's health comparative metabolism of ortho-phenylphenol in mouse, rat and man comparative in vitro-in vivo percutaneous penetration of the fungicide ortho-phenylphenol a single dose open label study to investigate the absorption and excretion of c/ c-labeled orthophenylphenol formulation after dermal application to healthy volunteers pharma bio-research clinics bv key: cord- - o f ty authors: zhou, wei; liu, shijia; ju, wenzheng; shan, jinjun; meng, minxin; cai, baochang; di, liuqing title: simultaneous determination of phenolic acids by uplc–ms/ms in rat plasma and its application in pharmacokinetic study after oral administration of flos lonicerae preparations date: - - journal: journal of pharmaceutical and biomedical analysis doi: . /j.jpba. . . sha: doc_id: cord_uid: o f ty abstract the current study aims to investigate the pharmacokinetic study of five phenolic acids (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, , -dicaffeoylquinic acid and , -dicaffeoylquinic acid) following oral administration of flos lonicerae preparations in rats. a rapid and sensitive ultra performance liquid chromatography–tandem mass spectrometry (uplc–ms/ms) method was developed to simultaneously determine the five phenolic acids in rat plasma. after mixing with the internal standard (is) tinidazole, plasma samples were pretreated by liquid–liquid extraction with ethyl acetate/n-hexane ( : , v/v). the separation was performed on an acquity uplc beh c column ( mm× . mm, . μm) at a flow rate of . mlmin− , and acetonitrile/methanol ( : , v/v)- . % formic acid was used as mobile phase. the detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (mrm) via electrospray ionization (esi) source with positive ionization mode. all calibration curves had good linearity (r > . ) over the concentration ranges of . – ngml− for neochlorogenic acid, . – ngml− for chlorogenic acid, . – ngml− for cryptochlorogenic acid, . – ngml− for , -dicaffeoylquinic acid, and . – ngml− for , -dicaffeoylquinic acid. the intra-and inter-day precision were within % and the accuracy ranged from . % to . %. flos lonicerae, a flower bud of lonicerae japonica thunb. that possessed antibacterial, anti-inflammatory, antiviral, antiendotoxin, blood fat reducing and antipyretic activities, has been widely used in traditional chinese medicine to treat exopathogenic wind-heat, epidemic febrile diseases, sores, carbuncles, furuncles and some infection diseases [ ] , and it has also been employed extensively to prevent and treat some serious viral diseases of human and veterinary, such as sasr coronavirus, h n (swine) flu virus [ ] . it was reported that, more than prescriptions containing flos lonicerae have been used to treat various diseases in china [ ] , and its preparations, such as jin-yin-huang oral liquid composed of flos lonicerae alone, shuang-huang-lian tablet, yin-qiao-jie-du tablet, fufang jin-huang-lian granule, qing-re-jie-du oral liquid and fufang qin-lan oral liquid, etc. [ ] , in which flos lonicerae was the main and active composition, were extensively used for treating acute upper respiratory tract infection caused by virus or bacterial infection in clinical practice. it was found that those phenolic acids especially isochlorogenic acids in flos lonicerae were the main bioactive components for antioxidant, antiviral and antibacterial effects by "spectrum-activity relationship" and "knock-out/knock-in" methods [ , ] . besides, we also found that the oral bioavailability of chlorogenic acid in flos lonicerae, the indicator compound recorded in chinese pharmacopeia [ ] , was enhanced largely as the chito-oligosaccharide at the dosage of mg/kg, one of the most important absorption enhancers possessing a loosening effect on the tension of the tight junctions through ionic interactions with negatively charged groups of glycocalix [ ] , was added, and its antiviral activity in vitro was improved significantly [ ] . thus, phenolic acids might be one of the most important compositions to characterize the quality of flos lonicerae. in order to elucidate the action mechanism of phenolic acids in vivo, it is essential to develop a quantitative method for determining these phenolic acids in plasma samples and studying their pharmacokinetic profiles. there were several articles concerning the quantification of chlorogenic acid in plasma [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, apart from chlorogenic acid, simultaneous pharmacokinetic studies of other phenolic acids have not been reported after oral administration of flos lonicerae preparations in rats, and very little attention has been devoted to the pharmacokinetic studies of these components. in the previous pharmacokinetic studies of chlorogenic acid, its concentration in plasma was analyzed by high performance liquid chromatography (hplc) with uv, ecd or ms [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, flos lonicerae contains a series of phenolic acids that have isomer properties with different pharmacological activities [ ] shown in fig. . therefore, it was desirable to develop an analytical method to allow the five analytes to be quantified simultaneously in rat plasma. in this study, a rapid and selective ultra performance liquid chromatography-tandem mass spectrometry (uplc-ms/ms) method for the simultaneous determination of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, , -dicaffeoylquinic acid and , -dicaffeoylquinic acid was developed in rat plasma. the method was fully validated and applied to the pharmacokinetic study of phenolic acids in rat plasma following oral administration of flos lonicerae preparations. chlorogenic acid and tinidazole (using as internal standard, is) were purchased from national institute for the control of pharmaceutical and biological products (beijing, china). neochlorogenic acid, cryptochlorogenic acid, , -dicaffeoylquinic acid and , -dicaffeoylquinic acid ( % pure) were purchased from sichuan weikeqi bio-tech co., ltd. (sichuan, china). six flos lonicerae extracts (product a-jin-yin-huang extract, product b-qing-re-jie-du extract, product c-fufang qin-lan extract, product d-shuang-huang-lian extract, product e-yin-qiao-jie-du extract and product f-fufang jin-huang-lian extract) were manufactured by harbin third pharmaceutical factory (harbin, china). chromatographic analysis was performed on a waters acquity uplc system (waters co., milford, ma, usa), consisting of a binary pump solvent management system, an online degasser, and an autosampler. an acquity uplc beh c column ( mm × . mm, . m) was employed and the column temperature was maintained at • c. the mobile phase was composed of a ( . % formic acid) and b (acetonitrile/methanol : , v/v) using a gradient elution of - % b at - . min, - % b at . - . min, - % b at . - . min, - % b at . - min, - % b at - min, - % b at - . min, - % b at . - . min, and hold for . min. the flow rate was set at . ml min − . the auto-sampler was conditioned at • c and the injection volume was l. mass spectrometric detection was performed using xevo triple quadrupole ms (waters co., milford, ma, usa) equipped with an electrospray ionization source (esi). the esi source was set in positive ionization mode. the analyte detection was performed by using multiple reaction monitoring (mrm) mode at m/z transitions of . → . for cryptochlorogenic acid, chlorogenic acid and cryptochlorogenic acid, . → . for , -dicaffeoylquinic acid and , -dicaffeoylquinic acid and . → . for is, respectively (fig. ) . the parameters in the source were set as follows: capillary voltage, . kv; source temperature, • c; desolvation temperature, • c; cone gas flow, l h − ; desolvation gas flow, l h − ; cone voltage, v for cryptochlorogenic acid, chlorogenic acid and cryptochlorogenic acid, v for , -dicaffeoylquinic acid and , -dicaffeoylquinic acid and v for is, respectively; collision energy, v for cryptochlorogenic acid, chlorogenic acid and cryptochlorogenic acid, v for , -dicaffeoylquinic acid and , dicaffeoylquinic acid and v for is. dwell time was set at . s. stock solutions were separately prepared by dissolving the accurately weighed five standard reference compounds with a mixture of methanol/water ( : , v/v) containing . % formic acid. a mixed stock solution was obtained by mixing all the five stock solutions above, and giving a final concentration of . g ml − for neochlorogenic acid, . g ml − for chlorogenic acid, . g ml − for cryptochlorogenic acid, . g ml − for , dicaffeoylquinic acid, and . g ml − for , -dicaffeoylquinic acid, respectively. the mixed stock solution was diluted with a mixture of methanol/water ( : , v/v) containing . % formic acid to provide working standard solutions of desired concentrations. the internal standard solution of tinidazole was prepared to the concentration of . ng ml − in methanol. calibration standards and quality control (qc) samples were prepared by evaporating to dryness l of standard working solution by a gentle stream of nitrogen, and then l of blank rat plasma was added. the samples were prepared prior to use during validation and pharmacokinetic study. the final calibration concentration ranges were . - ng ml − for neochlorogenic acid, . - ng ml − for chlorogenic acid, . - ng ml − for cryptochlorogenic acid, . - ng ml − for , -dicaffeoylquinic acid, and . - ng ml − for , -dicaffeoylquinic acid, respectively. the qc samples were prepared at concentrations of . , . , ng ml − for neochlorogenic acid, . , . , ng ml − for chlorogenic acid, . , . , ng ml − for cryptochlorogenic acid, . , . , for , -dicaffeoylquinic acid, and . , . , ng ml − for , -dicaffeoylquinic acid in drug-free plasma. the standards and quality controls were extracted on each analysis day with the same procedures for plasma samples as described below. a l aliquot of plasma was vortex mixed with l of % formic acid, l of is solution ( ng ml − ) and l of m hydrochloric acid in an eppendorf tube, and l of ethyl acetate/n-hexane ( : , v/v) was added to extract the five phenolic components from the plasma. the sample was vortexed for . min, and centrifuged at × g for min. l of supernatant was transferred into another eppendorf tube and dried under a flow of nitrogen gas. the residue was re-constituted in l % acetonitrile/methanol ( : , v/v) containing . % formic acid, and centrifuged for min. the supernatant was transferred to an autosampler vial and an aliquot of l was injected onto the uplc-ms/ms system for analysis. the method was validated in terms of specificity, selectivity, calibration curve, sensitivity, matrix effect, accuracy, precision and stability, in accordance with the usa food and drug administration (fda) bioanalytical method validation guidance [ ] . the specificity of the method was evaluated by comparing the chromatograms of six different batches of blank rat plasma samples, plasma samples spiked with the analytes and is, and plasma samples after an oral dose. blank rat plasma samples were analyzed for endogenous interference, followed by spiking with is for the interference of is. the linearity of each calibration curve was determined by plotting the peak area ratio (y) of analytes to is versus the nominal concentration (x) of analytes with weighted ( /x ) least square linear regression. the lloq was defined as the lowest concentration on the calibration curve with an acceptable accuracy (relative error, re) within ± % and a precision (relative standard deviation, rsd) below %. the intra-day and inter-day precision and accuracy were measured through quantifying three concentration levels of qc samples (six samples for each concentration level) on the same day and on three consecutive validation days, respectively. the precision was evaluated by relative standard deviation (rsd %) and accuracy by (mean measured concentration/spiked concentration) × %. the extraction recoveries of analytes at three qc levels were determined by comparing the response obtained from six extracted qc samples with those obtained from pure reference standards spiked in post-extracted blank rat plasma at the same concentrations. the matrix effects were evaluated by comparing the peak areas obtained from samples where the extracted matrix was spiked with standard solutions to those obtained from the pure reference standard solutions at the same concentration. the stability of phenolic acids in rat plasma was assessed using qc samples, which were freshly prepared and immediately mixed with l of % formic acid followed by storing at − • c for one month to evaluate the long-term stability. the post-preparation stability was tested by determination of the extracted qc samples stored in the auto-sampler ( • c) for h. the freeze and thaw stability was determined using qc samples after three freeze-thaw cycles (− to • c). product a, b, c, d, e, f extracts were dissolved in saline to give the concentration of % (v/v) immediately prior to drug administration. the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, , -dicaffeoylquinic acid and , dicaffeoylquinic acid in product extracts respectively were this validated method was applied to monitor the plasma concentrations of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, , -dicaffeoylquinic acid and , -dicaffeoylquinic acid simultaneously following single oral administration of flos lonicerae preparations in rats. male sd rats (∼ g) were obtained from experimental animal center of nanjing university of chinese medicine and kept in an environmentally controlled breeding room (temperature: ± • c, relative humidity: ± %) for week. the animals were fasted for h prior to drug administration. the rats were randomly divided into six groups with six rats in each group to receive various administrations at a single oral dose ( ml kg − ) by gastric gavage. after dosing for , , , , , , , , , , and min, blood was collected from the pre-intubated catheter and put into tubes with heparin sodium injection ( l) and ascorbic acid ( g) at predetermined time points. subsequently, plasma was prepared by centrifugation at × g, • c for min, and immediately analyzed or stored at − • c for further analysis followed by immediately mixing with l of % formic acid. the pharmacokinetic parameters of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, , -dicaffeoylquinic acid and , -dicaffeoylquinic acid, including the peak plasma concentration (c max ), the time to c max (t max ), the auc from to infinity (auc -∞ ), the auc form to time (auc -t ), mean residence time (mrt) and terminal elimination half-life (t / z ), were calculated by the non-compartmental analysis of plasma concentration vs. time data using the "das . . " software (mathematical pharmacology professional committee of china, shanghai, china). the comparison of pharmacokinetic parameters was possessed by spss . (statistical package for the social science). the stock solutions of the analytes and is diluted with a mixture of methanol/water ( : , v/v) containing . % formic acid were directly infused along with the mobile phase into the mass spectrometer with electrospray ion source. the response observed in the positive ionization mode was higher than that in negative ionization mode owning to its ion enhancement effect, though we found that the response in negative ion mode without acid addition in the process of the whole analyzing procedure was better than that in positive ion mode. in the precursor ion full-scan spectra, the most abundant ions were protonated molecules ions [m+h] + for all the analytes (fig. ) . parameters such as desolvation temperature, esi source temperature, capillary and cone voltage, flow rate of desolvation gas and cone gas were optimized to obtain the highest intensity of protonated molecules ions of analytes. the ion pairs of precursor → production for mrm detection were generated by the intellistart procedure (fig. ) , which was embedded in the masslynx software. the mrm transitions at . → . , . → . , . → . , . → . , . → . and . → . were selected to analyze cryptochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, , -dicaffeoylquinic acid, , -dicaffeoylquinic acid and is, respectively. fig. shows the representative uplc-ms/ms chromatograms of blank plasma (a), blank plasma spiked with the five analytes and is in lloq (b), and the plasma sample at min after oral administration of flos lonicerae preparations (c). no interfering peak was observed in blank plasma under the assay conditions. the retention times were . min, . min, . min, . min, . min and . min for neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, , -dicaffeoylquinic acid, , -dicaffeoylquinic acid and is, respectively. all analytes were eluted rapidly within . min. caffeoylquinic acids containing neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid as isomers and dicaffeoylquinic acids containing , -dicaffeoylquinic acid and , dicaffeoylquinic acid as isomers had the same precursor and product ions in mass spectrometry. therefore, it was indispensable to separate the isomers in the two groups with uplc. an acquity uplc beh c column ( mm × . mm, . m) elicited a suitable retention and a base-line separation between the analytes. besides, since caffeoylquinic acids and dicaffeoylquinic acids are phenolic compounds, they needed formic acid in the uplc mobile phase at a concentration of . % so as to not only overcome the peak-tailing effect, but also enhance the response in esi-ms/ms for five analytes in the positive ionization mode to improve their detection sensitivity because of ion enhancement effect [ ] . we found that a mixture solvent system containing acetonitrile/methanol ( : , v/v), not acetonitrile or methanol, could be utilized as organic phase to separate the two groups of isomers well. in addition, it was difficult to separate chlorogenic acid and cryptochlorogenic acid owning to their high similar polarity unless an optimized gradient with seven different segments shown above was selected, and the content of mobile phase b was lowered from to % at the time of . - min. the results (fig. b and c) showed that the gradient elution method was suitable for the caffeoylquinic acids and dicaffeoylquinic acids separation, and the method described above can achieve symmetric peak shape, high resolution (rs > . ) among peaks and short run time for the simultaneous analysis of the five compounds in plasma. caffeoylquinic acids and dicaffeoylquinic acids are phenolic compounds, susceptible to oxidation. however, they were found to be stable in acidic conditions and unstable in neutral and basic conditions (data not shown). therefore, the acidic solvents were used throughout the sample preparation, including collection, treatment and reconstitution procedures. to prevent from potential degradation of caffeoylquinic acids and dicaffeoylquinic acids in the blood, the fresh collected blood samples were stored on ice and then immediately centrifuged at • c for separation of plasma, although ascorbic acid ( g) as antioxidant was added in the eppendorf tube at predetermined time points. in order to extract the analytes and is with high, stable recoveries and no endogenous interference at the retention time, four types of reagents using methanol, acetonitrile, ethyl acetate or a mixture of ethyl acetate/n-hexane were tried for precipitation of protein or solvent extraction in rat plasma. it was found that protein precipitation using methanol or acetonitrile gave an effective absolute extraction recovery of analytes whilst they appeared poor repeatability and non-negligible matrix effects from endogenous plasma components on the ionization of the analytes and the is (data not shown). besides, it was shown from liquid-liquid extraction using ethyl acetate that the extraction was consistent, compared with protein precipitation, but showed also serious matrix effects. as a result, a mixture of ethyl acetate/n-hexane ( : , v/v) was utilized to extract the five analytes and the is, resulting in consistent and precise extraction efficiency (table ) . besides, the matrix effects from endogenous plasma components on the ionization of the analytes and the is were negligible. therefore, it was demonstrated that ethyl acetate/n-hexane ( : , v/v) produced the best extraction solvent for all the analytes and is. the representative chromatograms of blank plasma, blank plasma spiked with standard solution and plasma sample obtained following oral administration of flos lonicerae preparations in rats are shown in fig. . under the established chromatographic condition, there was no endogenous interference in the plasma and all the five analytes as well as is could be well separated from each other. the regression equation, correlation coefficients and linearity ranges for the five analytes are shown in table . the results table regression data and lloqs of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, , -dicaffeoylquinic acid and , -dicaffeoylquinic acid. showed that they all exhibited good linearity. the lloqs for neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, , dicaffeoylquinic acid and , -dicaffeoylquinic were . ng ml − , . ng ml − , . ng ml − , . ng ml − , and . ng ml − , respectively. the results of the intra-and inter-day precision and accuracy of all the analytes in lloq and qc samples are summarized in table . the intra-day and inter-day precisions ranged . - . % and . - . %, respectively. the accuracy derived from qc samples was between . % and . % for each qc level of the five analytes. the assay values on both intra-and inter-day were all within the acceptable range. the mean recoveries of all analytes at different concentrations are shown in table . the extraction recoveries of three level qc samples were stable. the extraction recoveries of is was . ± . %. the matrix effect of blank plasma of all the analytes was found to be within the acceptable range, all values were in the range from . % to % (table ). the matrix effect of is was . ± . %. thus, it was demonstrated that the plasma matrix effect was negligible for the assay. stability of the five analytes during the sample storing and processing procedures was fully evaluated by analysis of qc samples. the results (table ) indicated that these analytes in acidified table recoveries, matrix effects and stability of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, , -dicaffeoylquinic acid and , -dicaffeoylquinic acid (n = ). pharmacokinetic parameters of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, , -dicaffeoylquinic acid and , -dicaffeoylquinic acid in jin-yin-huang extract (n = , mean ± sd). jin-yin-huang plasma were all stable for one-month storage at − • c, h in the auto-sampler ( • c) and three freeze-thaw cycles with accuracy in range of . - . %. this validated uplc-ms/ms method reported for the first time by us was successfully applied to the pharmacokinetic study of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, , dicaffeoylquinic acid and , -dicaffeoylquinic acid in rat plasma following oral administration of flos lonicerae preparations. the assay was proved to be sensitive enough for the determination of these analytes in rat plasma. it is found in fig. and tables - consistently that the rank order of auc -t and c max in six flos lonicerae preparations were all chlorogenic acid > neochlorogenic acid ≥ cryptochlorogenic acid ≥ , -dicaffeoylquinic acid ≥ , -dicaffeoylquinic acid (most of them had significant difference), although the administration dosages of , -dicaffeoylquinic acid were higher significantly than that of other ingredients, which illustrated that the effective table pharmacokinetic parameters of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, , -dicaffeoylquinic acid and , -dicaffeoylquinic acid in qing-re-jie-du extract (n = , mean ± sd). qing-re-jie-du extract intestinal permeability (p eff ) of , -dicaffeoylquinic acid might be the lowest among the phenolic acids, but that of chlorogenic acid was opposite. besides, the rank order of mrt -t and t / z of phenolic acids in six flos lonicerae preparations were , dicaffeoylquinic acid > , -dicaffeoylquinic acid ≥ chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid (part of them were different significantly), but t max of them had little significance, which demonstrated that elimination in vivo of dicaffeoylquinic acids might be slower than that of caffeoylquinic acids, influenced by plasma protein binding rate possibly, it was consistent with the report [ ] that dicaffeoylquinic acids with two coffee acyl groups had higher binding abilities with human serum albumin (has) than caffeoylquinic acids with one coffee acyl group. the results above indicated that there were differences of caffeoylquinic acids containing neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid as isomers and dicaffeoylquinic acids containing , -dicaffeoylquinic acid and , -dicaffeoylquinic acid as isomers in the pharmacokinetic parameters, although they had similar physicochemical properties. further, it was necessary to separate the caffeoylquinic acids isomers and dicaffeoylquinic acids isomers when phenolic acids as one of the most important compositions were used to characterize the quality of flos lonicerae preparations. we also found from tables and that the values of auc -t in , -dicaffeoylquinic acid and , -dicaffeoylquinic acid in shuang-huang-lian were higher significantly than in qing-re-jie-du, table pharmacokinetic parameters of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, , -dicaffeoylquinic acid and , -dicaffeoylquinic acid in fufang jin-huang-lian extract (n = , mean ± sd). fufang jin-huang-lian although the administration dosage of , -dicaffeoylquinic acid and , -dicaffeoylquinic acid between them were approximately similar, which indicated that other ingredients in shuang-huang-lian or qing-re-jie-du might influence the dicaffeoylquinic acids absorption and metabolism in vivo. in this study, a rapid and reliable uplc-ms/ms method was developed for simultaneous analysis of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, , -dicaffeoylquinic acid and , -dicaffeoylquinic acid in rat plasma. sample preparation was carried out by liquid-liquid extraction with ethyl acetate/n-hexane ( : , v/v) and the data acquisition of each sample was . min. the method has been successfully applied to the pharmacokinetic study of five bioactive phenolic acids in rats following oral dose of flos lonicerae preparations. the results also elucidated effectively that the pharmacokinetic parameters of phenolic acids even isomers had significant differences. studies on antiviral effect and immunopotentiating activity of lonicera japonica thunb. and flos lonicerae in vitro research and comprehensive utilization of honeysuckle lonicera japonica thunb.: ethnopharmacology, phytochemistry and pharmacology of an important traditional chinese medicine improvement of intestinal absorption of forsythoside a and chlorogenic acid by different carboxymethyl chitosan and chito-oligosaccharide, application to flos lonicerae-fructus forsythiae herb couple preparations study of effective substances screening for flos lonicerae based on the spectrum-effect combination novel patterns of efficient components recognition and quality control for flos lonicerae japonicae based on constituent knock-out/knock-in china pharmacopoeia committee, pharmacopoeia of people's republic of china chitosan as a novel nasal delivery system for peptide drugs comparative pharmacokinetic study of chlorogenic acid after oral administration of lonicerae japonicae flos and shuang-huang-lian in normal and febrile rats development of an lc-ms method for determination of three active constituents of shuanghuang-lian injection in rat plasma and its application to the drug interaction study of shuang-huang-lian freeze-dried powder combined with levofloxacin injection pharmacokinetic study of major bioactive components in rats after oral administration of extract of ilex hainanensis by high-performance liquid chromatography/electrospray ionization mass spectrometry lc-ms determination and pharmacokinetic study of six phenolic components in rat plasma after taking traditional chinese medicinal-preparation: guanxinning lyophilized powder for injection bioavailability and pharmacokinetics of caffeoylquinic acids and flavonoids after oral administration of artichoke leaf extracts in humans liquid chromatography-electrospray ionization-tandem mass spectrometry for simultaneous analysis of chlorogenic acids and their metabolites in human plasma liquid chromatography/tandem mass spectrometry assay for the simultaneous determination of chlorogenic acid and cinnamic acid in plasma and its application to a pharmacokinetic study comparative pharmacokinetics of chlorogenic acid after oral administration in rats an lc-ms/ms method for the simultaneous determination of chlorogenic acid, forsythiaside a and baicalin in rat plasma and its application to pharmacokinetic study of shuanghuang-lian in rats molecular docking of chlorogenic acid, , -di-o-caffeoylquinic acid and , -di-o-caffeoylquinic acid with human serum albumin us food and drug administration ion suppression and enhancement effects of co-eluting analytes in multi-analyte approaches: systematic investigation using ultra-high-performance liquid chromatography/mass spectrometry with atmospheric-pressure chemical ionization or electrospray ionization the present study is supported financially by the national natural science foundation of china ( , ), "qing lan" project from jiangsu provincial technology innovation team support scheme, the priority academic program development of jiangsu higher education institution (no. ysxk- ) and program sponsored for scientific innovation research of college graduate in jiangsu province ( ). key: cord- -wanbz et authors: varki, ajit title: loss of n‐glycolylneuraminic acid in humans: mechanisms, consequences, and implications for hominid evolution date: - - journal: am j phys anthropol doi: . /ajpa. sha: doc_id: cord_uid: wanbz et the surface of all mammalian cells is covered with a dense and complex array of sugar chains, which are frequently terminated by members of a family of molecules called sialic acids. one particular sialic acid called n‐glycolylneuraminic acid (neu gc) is widely expressed on most mammalian tissues, but is not easily detectable on human cells. in fact, it provokes an immune response in adult humans. the human deficiency of neu gc is explained by an inactivating mutation in the gene encoding cmp‐n‐acetylneuraminic acid hydroxylase, the rate‐limiting enzyme in generating neu gc in cells of other mammals. this deficiency also results in an excess of the precursor sialic acid n‐acetylneuraminic acid (neu ac) in humans. this mutation appears universal to modern humans, occurred sometime after our last common ancestor with the great apes, and happens to be one of the first known human‐great ape genetic differences with an obvious biochemical readout. while the original selection mechanisms and major biological consequences of this human‐specific mutation remain uncertain, several interesting clues are currently being pursued. first, there is evidence that the human condition can explain differences in susceptibility or resistance to certain microbial pathogens. second, the functions of some endogenous receptors for sialic acids in the immune system may be altered by this difference. third, despite the lack of any obvious alternate pathway for synthesis, neu gc has been reported in human tumors and possibly in human fetal tissues, and traces have even been detected in normal human tissues. one possible explanation is that this represents accumulation of neu gc from dietary sources of animal origin. finally, a markedly reduced expression of hydroxylase in the brains of other mammals raises the possibility that the human‐specific mutation of this enzyme could have played a role in human brain evolution. yrbk phys anthropol : – , . © wiley‐liss, inc. the evolution of modern humans from a common ancestor with the great apes occurred in a series of steps, influenced by complex interactions among genetic, developmental, ecological, microbial, climatic, behavioral, cultural, social, and other factors. there are many scientifically valid approaches towards gaining an understanding of how we came to be human. prominent among these approaches are: developmental, cognitive, social, and behavioral comparisons of humans with other primates; the paleontology and archeology of human ancestors; and the systematic genetic comparison of humans with other primates. given the remarkable progress of molecular biology techniques, the deciphering of most of the human genome (lander et al., ; venter et al., ) , and the relative ease of obtaining genomic dna noninvasively from humans and other primates, the genetic approach should, in principle, be somewhat easier than the others mentioned. in fact, apart from the general realization that our genomic dna sequences are remarkably similar to those of the great apes (king and wilson, ; sibley and ahlquist, ; caccone and powell, ; goodman et al., ; ruvolo, ; takahata and satta, ; kaessmann et al., ; chen and li, ) , there have been relatively few specific genetic differences between humans and apes uncovered to date (reviewed in gagneux and varki, ) . this has led some to call for a great ape/ primate genome project (mcconkey and goodman, ; , to accelerate progress in this area. while morphologists have long sought to demonstrate the adaptive significance of divergent anatomical structures, this has generally not been the case for molecular anthropologists. the latter have, to a large degree, used comparative dna (or amino acid) sequences for dating evolutionary events rather than for understanding the evolutionary processes that led to the sequence divergence. thus, very little is known of the selective (or random) forces that led to the - % sequence differences between humans and the african great apes, or about the adaptive molecular differences that emerged during this period of primate evolution. this review discusses one of the few known apehuman genetic differences with a clear-cut biochemical consequence, the selective inactivation of the cmp-n-acetylneuraminic acid (cmp-neu ac) hydroxylase gene in the human lineage irie et al., ; chou et al., ) . the resultant loss of a specific cell-surface sugar on human cells has implications for issues as diverse as human susceptibility and resistance to pathogens, the consequences of human ingestion of animals foods, the human innate immune response, and the development of the human brain. as with most unexpected discoveries, this finding has raised more questions than answers. an attempt is made to address some of these questions, and to suggest directions for future research. every high school graduate should now know about dna, rna, and proteins, and most would understand that a universal dna code defines the genes of all living things. these genes can be transcribed into their corresponding rna sequences, which in turn are translated into proteins. this "central paradigm" of molecular biology, i.e., that dna makes rna makes protein, has dominated recent approaches to understanding how living things work. however, there are several reasons why this scientifically powerful reductionist approach cannot fully explain the structure and function of complex multicellular organisms like humans. one reason is that dna itself is of little use unless the genes it encodes are expressed (transcribed to rna, and hence translated into proteins)-and gene expression is profoundly influenced by the physical, ecological, biological, and social milieu in which an organism exists. indeed, the social and cultural activities of humans must have a major impact on gene expression within the species, as well as in the many other species that humans interact with. a second reason is that besides dna, rna, and proteins, there are two other major classes of molecules that are required to create almost all life forms that we know of: lipids and sugars (glycans). glycans fall into two general categories: the more familiar small sugars like glucose that are a major source of energy, and the less wellknown glycan chains that are attached to many proteins and lipids, particularly on the surface of cells (varki, a) . indeed, there are no extant free- living organisms whose cells are not each covered with a dense coating of these glycan chains, comprising a so-called "glycocalyx" that is no less obvious than the icing on a birthday cake. these complex cell-surface sugar chains are the products of a specialized intracellular machinery whose synthesis and organization are themselves dictated by the expression of several hundred genes (varki and marth, ; varki, ) . this machinery is supplemented to an unknown extent by the incorporation of sugar building blocks from dietary sources into endogenous biosynthetic pathways (freeze, ) . given their ubiquitous occurrence in nature and their dominant presence on the surface of cells, it is not surprising that these sugar chains are intimately involved in the interactions between cells, both within an organism, and between organisms. a third limitation of the dna-rna-protein paradigm for explaining humans is that the human genome seems to have less than , genes (lander et al., ; venter et al., ) . thus, there cannot possibly be a "gene for" each specific biological entity or function. rather, the genetic contribution to the structure and function of most aspects of an organism arise from the combinatorial effects of multiple genes, gene regulation mechanisms, and gene products that act upon one another in various ways, under the constant influence of environmental factors. the attachment of glycan chains to proteins and lipids (so-called glycosylation) is a prime example of such a "postgenomic" process, wherein new biological entities or functions result from the action of one set of genes on the products of other genes. despite these considerations, studies of the structure and biology of glycans have lagged far behind those of dna, rna, proteins, and lipids. there are many reasons for this, including the branching and complexity of these sugar chains, the lack of an easily recognizable template-driven functional "code," and technical limitations in studying their structure and function. recent advances have substantially reduced this technical gap, opening up a new field now called glycobiology (rademacher et al., ; varki, a) . thus, as with genomics (determining the total genomic dna of an organism), transcriptomics (elucidation of the complete set of genes expressed in a given cell type in a given situation), and proteomics (the description of all proteins found in a given cell type in a given situation), we are just beginning to enter the era of glycomics (elucidation of the complete array of glycans found on a given cell type in a given situation). a microbial organism approaching a mammalian cell surface would likely first encounter members of a family of sugars called sialic acids, which tend to be the outermost units on the glycan chains attached to the proteins and lipids below (fig. ) . this family of -carbon acidic sugars (gottschalk, ; rosenberg and schengrund, ; schauer, ; ye et al., ; inoue et al., ; varki, varki, , b is found predominantly in the deuterostome lineage of animals (warren, ; traving and schauer, ; . there are more than kinds of sialic acids known in nature, and most are derived via biosynthetic modifications of a parent molecule called n-acetylneuraminic acid (neu ac) (fig. ) . further complexity arises from the fact that sialic acids can be attached to the underlying sugar chain in several different types of linkages (tsuji et al., ) . while many of these kinds and linkages of sialic acids can be found within a single species, there are also marked species-specific differences with regard to their relative amounts and/or distribution. likewise, even within a single species, there can be substantial differences between different cell types in the pattern and composition of their sialic acids. the evolution of this remarkable structural complexity is probably related to the diverse biological roles of these sialic acids in different cell types. . structure of the two most common sialic acids on mammalian cell surfaces. the -carbon backbone common to all sialic acids is numbered. thick arrow points to the single oxygen atom that differentiates neu gc from neu ac. humans are genetically defective in the gene encoding the enzyme responsible for adding the oxygen atom. by virtue of their negative charge and surface location, sialic acids can mediate many biological roles in normal and pathological situations (fig. ). it is well-known that a wide variety of human and animal pathogens use cell-surface sialic acids to either gain an initial foothold on the cells they infect or to target toxic molecules that they secrete (sandvig et al., ; escalante et al., ; sharon, ; varki, varki, , b gagneux and varki, ; karlsson, karlsson, , fig. for examples). if these were the sole functions of sialic acids, the detrimental consequences to deuterostome animals should have caused these sugars to be eliminated and/or replaced by others during the course of evolution. however, the absence of sialic acids is lethal during early mouse embryogenesis (w. reutter, personal communication), and genetic modifications of sialic acid linkages in mice cause significant pathologies (priatel et al., ; hennet et al., ) , indicating that these molecules also have critical endogenous functions. some of these functions are primarily structural or physical, e.g., aiding the filtration function of the kidneys or providing negative charge repulsion between cells in the blood stream. in the brain, this type of negative charge repulsion becomes enhanced and specialized by the formation of long chains of sialic acids (called polysialic acids). these can serve to physically separate neurons and neuronal extensions and hence participate in what can be loosely called "brain plasticity" at the organizational level (rutishauser and landmesser, ) . over the last two decades, it has become clear that sialic acids are also recognized by specific receptors within the same animals that synthesize these sugars (bevilacqua and nelson, ; kelm et al., b; varki, varki, , powell and varki, ; crocker and feizi, ; kansas, ; collins et al., a; kelm and schauer, ; crocker et al., ; brinkman-van der linden et al., ; crocker and varki, a,b; see fig. for examples). emerging evidence indicates that these receptors, particularly a family called the siglecs (crocker and varki, a,b; , are able to recognize the diversity in sialic acids, and their linkages to underlying sugars. an additional set of biological roles for sialic acids is found in certain microbial organisms that decorate their cell surfaces with sialic acids (troy, ; wessels et al., ; bozue et al., ) , thereby allowing them to evade recognition and destruction by certain vertebrate immune mechanisms. these examples occur despite the fact that sialic acids seem to be otherwise restricted to the deuterostome lineage of animals. the best explanation can be found in the fact that all of these microorganisms are vertebrate pathogens (see examples in fig. ). since surface sialic acids limit activation of multiple functions of the immune system, these pathogenic organisms likely benefit from coating themselves with these sugars. this form of molecular mimicry may have occurred mostly via convergent evolution, wherein these microorganisms evolved a variety of ways either to make their own sialic acids or to procure them from their vertebrate hosts. while there are many kinds of sialic acids in nature (gottschalk, ; rosenberg and schengrund, ; schauer, ; varki, ; ye et al., ; inoue et al., ; varki, b; , the surfaces of most cell types in the mammalian species studied to date tend to be dominated by two major kinds: n-acetylneuraminic acid (neu ac) and n-glycolylneuraminic acid (neu gc). neu gc is different from neu ac only by an additional oxygen atom (see arrow in fig. ). in order for sialic acids to get attached to glycoproteins and glycolipids, they must first be "activated" by conversion to their respective sugar nucleotide derivatives. thus, the common sialic acid neu ac is converted to cytidine-monophosphate-neu ac (cmp-neu ac), which is then used as a high-energy donor for attaching neu ac to newly made glycoproteins and glycolipids that are on their way to the cell surface. the synthesis of the neu gc form of sialic acid takes place initially at the level of this sugar nucleotide precursor (shaw and schauer, ; bouhours and bouhours, ; muchmore et al., ; kozutsumi et al., ; shaw et al., shaw et al., , takematsu et al., ; kawano et al., ; schlenzka et al., ) . thus, as shown in figure , an enzyme called cmp-neu ac hydroxylase (hereafter referred to as cmah) catalyzes the transfer of one oxygen atom to cmp-neu ac, generating cmp-neu gc. the latter can now also be used as a donor to add neu gc to molecules destined for the cell surface from their sites of initial synthesis within the cell. the enzymes that actually transfer sialic acids to glycoproteins and glycolipids (called sialyltransferases) can typically use both cmp-neu ac and cmp-neu gc as donors . thus, the ratio of these two major sialic acids found on a given cell surface is likely to be largely determined by the ratio within the sugar nucleotide donor pool available in that cell type. (varki, b; traving and schauer, ) can be divided into the general groupings indicated. m, microorganism or toxin. see text for discussion. some of the early pioneers who discovered the sialic acids noted that, in contrast to the situation in other mammals such as rodents and ungulates, neu gc was very hard to find in human tissues (reviewed in gottschalk, ; rosenberg and schengrund, ; schauer, ) . however, the methods they used could have missed small amounts of neu gc in humans. an independent line of evidence suggesting that neu gc is lacking in humans came from the medical field of hematology. for the clinical management of certain blood disorders, it becomes necessary to infuse serum from horses into human patients. not surprisingly, these patients often generate an immune response against components of the infused animal serum and manifest a condition called "serum sickness reaction," which contraindicates further infusions. some investigators who studied the serum sickness reaction noted that the immune response was being generated at least in part against neu gc, which is present in abundance on horse serum glycoproteins and glycolipids (kasukawa et al., ; merrick et al., ; higashi et al., ) . this finding strengthened the notion that neu gc is a foreign antigen to adult humans. however, several groups then reported (mostly using indirect methods such as antibody detection) that neu gc could be found in human cancers and possibly in human fetal tissues (kawachi and saida, ; ikuta et al., ; higashi et al., ; stacker et al., ; hirabayashi et al., a; kawachi et al., ; saida et al., ; devine et al., ; kawai et al., ; marquina et al., ; malykh and schauer, ) . moreover, neu gc was also reported in some cultured cell lines of human origin (nakarai et al., ; ohashi et al., ) . taken together, these data suggested that humans might have a func-tional cmah gene, but simply suppress its expression sometime before the postnatal period when "immune tolerization" to self-antigens occurs. our recent studies done with more sensitive modern techniques showed that while neu gc is undetectable (Ͻ . % of total sialic acids) on the red blood cells and blood plasma proteins of adult humans, similar samples from all of the great apes have substantial amounts (neu gc representing between ϳ - % of total sialic acids). since the great apes are our closest evolutionary cousins (king and wilson, ; sibley and ahlquist, ; caccone and powell, ; goodman et al., ; ruvolo, ; takahata and satta, ) , the human loss of neu gc expression must have occurred sometime after the ape-human common ancestor. a secondary consequence of this loss is that humans also have much higher levels of neu ac, the precursor molecule to neu gc. assays of cmah enzyme ( fig. ) showed easily detectable activity in great ape cells, but not in human cells . the next logical step was to examine the gene encoding the cmah, to see if its promoter (regulatory) regions were mutated in some manner in humans, thereby altering its expression in adult humans. indeed, two groups independently found that the cmah gene in humans had suffered a mutation (irie et al., ; chou et al., ) . surprisingly the mutation was not in the regulatory regions of the gene that might have modified its expression patterns between fetal and adult states, but rather in the coding region which dictates the amino-acid sequence of the enzyme itself (fig. ). the mutational event had deleted base pairs of a single stretch of the sequences coding for the protein (corresponding to exon in the mouse gene). since amino acids are dictated by triplets of dna base pairs, the loss of base pairs (bp) also resulted in a "frame-shift," thus markedly truncating the length of the final protein encoded by the human gene (chou et al., ) . moreover, this truncated protein is missing certain amino acids that were known to be critical for the activity of the enzyme itself (schlenzka et al., ) . in contrast, examination of the cmah gene from the chimpanzee and relevant portions from the corresponding genes from other apes (chou et al., ) showed that they all encode an intact enzyme that is not very different from those originally cloned from mice and pigs (kawano et al., ; schlenzka et al., ) . thus, it is clear that humans lost neu gc on their cell surfaces because of an inactivating mutation in the cmah gene. the gene is localized to chromosome band p -p in both humans and great apes (irie et al., ; chou et al., ) , which does not correspond to an area of known chromosomal rearrangement during hominoid evolution (yunis and prakash, ) . furthermore, the remaining human intronic region is very similar in size to that in the intact mouse genome (irie et al., ) , indicating that the deletion eliminating the fig. . factors involved in biosynthesis of cmp-n-glycolylneuraminic acid (cmp-neu gc). the enzymatic mechanism of the cmp-neu ac hydroxylase that is primarily responsible for generating neu gc in nonhuman animal cells is shown. the reaction takes place in the cytosolic compartment. the enzyme product cmp-neu gc is the donor subsequently used for adding neu gc to glycoproteins and glycolipids. cmp-neu ac hydroxylase activity is present in great apes but not in humans. see text for discussion. -bp exon in humans must have been quite small. the corresponding genomic regions from the chimpanzee and other nonhuman primates were recently sequenced (hayakawa et al., ) . the region containing a -bp exon of the cmah gene turns out to have an adjacent alusq element in all nonhuman primates studied, and both were replaced by a newly disseminated aluy element that is specific to humans (alus are parasitic repetitive dna elements found in large numbers throughout the primate genome). thus, it is possible to propose a mechanistic model whereby an alu-mediated replacement event coincidentally deleted the -bp exon and inactivated the cmah gene sometime in the human lineage (hayakawa et al., ) . why did humans lose neu gc production? it is difficult to be certain about why a particular genetic change became fixed in a particular species at some time in the evolutionary past. however, based on current knowledge of the functions of sialic acids (see above), one can propose some possible scenarios to explain the human loss of neu gc. the most likely one is selection of a randomly occurring cmah gene mutation by a lethal microbial pathogen that required cell-surface neu gc for effective infection (see below for some examples of such current-day pathogens). once such a mutation had thus become common in a human ancestral population, it could have undergone further selection because it conferred some additional benefits, or have simply drifted to fixation in the absence of any further selection. a more intriguing possibility is that the inactivated allele of the cmah gene was favorably selected because it conferred valuable new endogenous functions upon individuals who became homozygous for it. when did this mutation occur in relation to the various steps in human evolution? the identical mutation was found in genomic dna in Ͼ individuals representing many different geographic regions, including african kung bushmen and khwe pigmies (among whom the greatest genetic diversity is known to be present; takahata and satta, ; kaessmann et al., ) . this, together with the lack of detectable neu gc in blood samples from many additional humans studied by us, indicates that the mutation is universal to modern humans. thus, the inactivation of the cmah gene must have occurred sometime after our common ancestor with the bonobo/chimpanzee clade (ϳ million years ago; king and wilson, ; sibley and ahlquist, ; caccone and powell, ; goodman et al., ; ruvolo, ; takahata and satta, ) , but prior to (or possibly coincident with) the common origin of modern humans (variably estimated by different authors, with the most recent possible date of about ϳ , years ago; takahata and satta, ; krings et al., ) . a more accurate estimate of the timing of the gene inactivation would be of value for generating hypotheses about the potential consequences of neu gc loss in humans. for example, if it took place about million years ago, this might suggest a possible contribution towards the brain size increase that began with the emergence of genus homo (wood and collard, ) . sequenceable autosomal dna has not been successfully recovered from fossilized mammalian bones that are more than about , years old (greenwood et al., ; hofreiter et al., ) . even the few successes tend to occur when using samples from northern latitudes. indeed, the probability of recovering dna is known to be worse for samples exposed to warmer temperatures (smith et al., ) . the occasional successes in obtaining mitochondrial dna from fossil hominids Ͼ , years old (krings et al., ) are explained by the much higher copy number of mitochondrial dna within each cell. overall, it is not possible (at least with current technology), to obtain and directly study cmah gene sequences from hominid fossils that predated the common origin of modern humans. two other approaches are therefore currently being pursued to try timing the inactivation of the cmah gene. the first relies on sequence comparisons of the great ape and other primate cmah genes with those of the remaining portions of the inactivated human gene. the assumption is that once the human gene became nonfunctional (i.e., became a pseudogene), it was no longer under selection pressure, and would thus accumulate both synonymous and nonsynonymous mutations at similar rates. such data are currently being used to estimate an approximate inactivation date (collaboration with n. takahata). the second approach relies on the direct study of residual sialic acids found in fossils (unpublished observations, in collaboration with s. paabo). these preliminary analyses suggest a dating of a little over million years ago. it should be noted that there are reported strain variations in red blood cell expression of neu gc among dogs (hashimoto et al., ; yasue et al., ) and cats (ando and yamakawa, ; furukawa et al., a) , and in the latter instance, the presence or absence of neu gc on red cells can act as a blood group system (andrews et al., ) . however, systematic studies of other tissues of these dog and cat strains have not been reported. thus, we do not know if it is only red blood cells that show differential expression. chickens are the only species besides humans that have been found to generate a generalized immune response to neu gc when infused with animal serum (fujii et al., ) . in fact, polyclonal and monoclonal antibodies generated by chickens immunized with horse serum or horse red cell glycolipids have been very useful as tools in the study of neu gc expression (hiraba-yashi et al., b; higashi et al., ) . again, a systematic study of chicken tissues has not been carried out to see if the presumed lack of neu gc is true for all organs. furthermore, the presence of neu gc in ducks indicates that this is not a general feature of birds. further studies of the cmah gene in a wide selection of birds and carnivores seem warranted. however, a recent study showed that neu gc is present on the serum immunoglobulins of cows, sheep, goats, horses, mice, dogs, guinea pigs, rats, and rabbits, as well as rhesus monkeys (raju et al., ) . thus, even if genetic variations in neu gc expression are found among members of some of these species, the -bp exon deletion responsible for the human loss of cmah activity remains a unique genetic event that occurred after our common ancestor with the great apes, and is now universal among modern humans. as indicated above, many major pathogens and their toxins gain access to their mammalian hosts by binding to cell-surface sialic acids. the microbial binding proteins involved in such interactions can show exquisite specificity for the precise structure and linkage of the sialic-acid target (sharon, ; karlsson, ; varki, varki, , b . thus, the human loss of neu gc would have conferred protection from animal pathogens that prefer to bind to this sialic acid, while enhancing the success of pathogens that prefer to bind to neu ac. as shown in table , this issue has not been thoroughly studied for most human pathogens. it is clear that certain microbes causing serious diarrheal diseases in farm animals like cows and pigs have a strong preference for neu gc (kyogashima et al., ; ouadia et al., ; willemsen and de graaf, ; lanne et al., ; delorme et al., ; schwegmann et al., ) , and humans are thus immune to infection. it is reasonable to speculate that this mechanism of resistance may have facilitated the domestication of some animal species, by limiting transfer of their pathogens to human caretakers. an even more speculative possibility is that this difference aided the worldwide migrations that brought humans into contact with diverse pathogen regimes of novel species of wild animals they encountered. in this regard, more information is needed about the sialic acid binding specificity of pathogens affecting animals that now live in very close contact with humans, such as dogs and cats. perhaps they will turn out to prefer neu gc, explaining the relatively low rate of pathogen transfer from these domesticated pets to humans. another intriguing issue is that of the influenza a virus, the agent of epidemic and pandemic influenza in humans (wilson et al., ; taubenberger et al., ) . it appears that these viruses originate from wild water fowl and make their way to humans via domesticated livestock animals such as pigs . studies indicate that some animal forms of influenza a virus preferentially bind to neu gc, while human forms can have some preference for neu ac weis et al., ; ito et al., suzuki et al., suzuki et al., , . thus, a switch in specificity from neu gc to neu ac might be a required or facilitatory step in animal-to-human transmission of some influenza strains. on the other hand, if there are microbes that selectively prefer neu ac, these should be more pathogenic in humans. while no clear-cut examples of this situation are known, most of the possibilities have yet to be explored. one potential example is plasmodium falciparum, the causative agent of the most serious form of human malaria that afflicts millions worldwide. the merozoite form of this organism that invades red blood cells uses cell-surface neu ac as one of its targets for initial binding (klotz et al., ; deluca et al., ; reed et al., ) . since chimpanzees appear to be resistant to this form of malaria (ollomo et al., ) , it is possible that the p. falciparum merozoite receptor proteins do not recognize neu gc. conversely, the merozoite stage of the phylogenetically related plasmodium reichenowi that infects chimpanzees (qari et al., ; escalante and ayala, ) might recognize neu gc preferentially. functional comparison of the p. falciparum and p. reichenowi merozoite receptors seems to be worthwhile. overall, it is clear from table that much further work is needed to pursue the consequences of neu gc loss for resistance and susceptibility to infectious diseases in humans. the antibodies generated by a normal adult human exposed to infusion of horse serum (called hanganatziu-diecher or hd antibodies) can agglutinate the red blood cells of various animals, such as horses, pigs, and cows, by virtue of the fact that they all carry surface neu gc. using the same kind of red-cell agglutination assay, spontaneously occurring hd antibodies have also been reported in patients who have never had exposure to animal serum infusion. while such serum reactivities are rare in normal human adults, they are found in a significant proportion of patients with cancer, as well as in diseases such as leprosy, rheumatoid arthritis, liver disease, and infectious mononucleosis (morito et al., (morito et al., , nishimaki et al., ; takiguchi et al., ) . at least two explanations can be considered for these phenomena. the first possibility is that as in cancer, some of these diseases involve proliferation of vascularized tissues (e.g., the granulomas of leprosy and rheumatoid arthritis). thus, a low-grade immune response might be occurring against neu gc that is being gradually incorporated into such tissues from dietary sources over time (see discussion below). the second possibility is that these antibodies emerge as a nonspecific consequence of the disregulation of the immune system that occurs in some of these disease states. individuals with prior exposure to neu gc in the diet may have rare preexisting memory b cells capable of producing antibodies directed against neu gc. such b cells could undergo nonspecific expansion under conditions of generalized immune disregulation. the first possibility could be tested by directly assaying the affected tissues for the presence of neu gc. despite the complete inactivation of the cmah gene in humans, there have been reports of traces of neu gc in normal human tissues and in cultured human cell lines (nakarai et al., ; ohashi et al., ) . the latter finding is easily explained by the fact that human cells are typically cultured in fetal bovine serum, which is rich in glycoproteins carrying neu gc. the fluidphase uptake of such glycoproteins into the lysosomes of cultured cells would eventually result in incorporation of the foreign neu gc into human cellular glycoproteins and glycolipids (see pathway a in fig. ). the best evidence for this route of incorporation is that growing the cells in the absence of animal serum results in the eventual disappearance of neu gc (furukawa et al., b; . however, this pathway cannot explain the traces of neu gc found in tissues from normal humans who are not directly exposed to animal serum . on the other hand, the nature of the cmah mutation in humans (an exon deletion eliminating critical amino-acid residues) neu gc not studied in detail streptococcus sanguis (dental caries) neu gc not studied helicobacter pylori (ulcers) neu gc not studied tetanus toxin (tetanus) neu gc not studied coronaviruses (common cold) neu gc not studied mycoplasma pneumoniae (pneumonia) neu gc not studied polyoma virus (tumors) neu gc not studied makes it hard to postulate a genetic mechanism to restore its function in malignant cells. indeed, the complete sequencing of the corresponding intronic regions of the cmah gene from human and chimpanzee genomes (irie et al., ; hayakawa et al., ) reveals no obvious avenues for such a repair mechanism. meanwhile, searches of gene databases show no evidence for any related proteins that might have a similar activity. two other possibilities must therefore be considered to explain the traces of neu gc found in normal human tissues. the first is that there could be an additional completely differ-ent biochemical pathway for the biosynthesis of neu gc in mammalian cells. there is so far no strong evidence for such a pathway being active in humans. the suggestion has been made that the glycolyl group of neu gc (which replaces the acetyl group in neu ac) could also originate from an unusual high-energy donor called glycolyl-coenzyme a (coa). such a potential donor can in fact be formed via two known biochemical pathways: beta-oxidation of -hydroxybutyrate in mitochondria, or from -hydroxypyruvate by the action of pyruvate dehydrogenase (vamecq and poupaert, ; vamecq et fig. . uptake, metabolism, and turnover of neu ac and neu gc in mammalian cells. the general pathways for production and utilization of sialic acids in mammalian cells are shown. thick lines represent cellular membranes, or intracellular membranous compartments, as indicated. the common sialic acid neu ac is synthesized from a neutral precursor n-acetylmannosamine (mannac) in the cytosolic compartment, activated into cmp-neu ac in the nucleus, and eventually pumped into the golgi apparatus by a specific transporter (lepers et al., ) . there it serves as a donor for adding sialic acids to newly synthesized glycolipids and glycoproteins that originate from the endoplasmic reticulum, and are en route to the cell surface or to be secreted from the cell. sialic acids are eventually released in the lysosomal compartment as part of the overall degradation of glycolipids and glycoproteins and then pumped back into the cytosol (verheijen et al., ) , to be reutilized as shown. conversion of cmp-neu ac to cmp-neu gc occurs in nonhuman cells, and the subsequent fate of neu gc is similar to that of neu ac-recycling and varying degrees of reutilization. double line indicates block in conversion of cmp-neu ac to cmp-neu gc in human cells. two possible pathways (a and b) for incorporation of neu gc of exogenous into human cells are indicated with dotted lines. pathway a involves uptake of glycolipids or glycoproteins carrying neu gc into the lysosomal compartment. this pathway could only operate in cultured cell lines, or in humans given intravenous infusions of animal proteins. pathway b involves uptake of neutral precursor n-glycolylmannosamine (manngc) by passive diffusion across the plasma membrane, with subsequent conversion into neu gc. manngc could originate from breakdown of dietary neu gc in the gut. both these pathways would effectively bypass the enzymatic defect in humans. note that neu ac and neu gc can also be degraded back to mannac and manngc. while there are many known interconverting pathways involving mannac, the cellular fate of manngc is unknown, in human and nonhuman cells. there is also no known pathway for converting neu gc back to neu ac. hence neu gc tends to recycle and accumulate to high levels in cells that express the hydroxylase (muchmore et al., ) . see text for further discussion. al., ). the glycolyl-coa would presumably donate its glycolyl group directed to a de-n-acetylated neuraminic acid (zhou et al., ; sjoberg et al., ; mitsuoka et al., ) , or to a potential precursor form of sialic acids called mannosamine. however, there is so far no evidence for the enzymatic activity of such a pathway in human cells. the other interesting possibility is the incorporation of neu gc from dietary sources. studies of rats fed with radiolabeled sialic acids showed that a large fraction can be absorbed, and while most is excreted unchanged in the urine, a small amount of the label does get incorporated into tissues schauer, , ; nohle et al., ) . the latter is thought to occur by intestinal conversion of the ingested sialic acids into the acylmannosamine derivative, which can be taken up into the circulation, absorbed into cells, and converted back into sialic acids (see pathway b in fig. ). we are currently studying the possibility that a similar pathway exists in intact humans. if so, it is possible that the traces of neu gc found in normal human tissues are all derived from the ingestion of foods containing neu gc. in this regard, it is of note that sialic acids are only found in animal foods, and that neu gc seems to be very common in pigs, cows, goats, and sheep (raju et al., ; wang et al., ) , while probably being much lower in poultry and fish. it remains to be seen whether this pathway has any relevance to diseases associated with the consumption of red meats. there also needs to be a detailed survey of the amounts of neu gc present in common foods of animal origin. regardless of the mechanism, the question also arises whether there is a weak immune response to these traces of neu gc in adult humans, and if so, what the consequences of such a response might be. as mentioned above, both direct and indirect studies have indicated that neu gc is present in some human cancerous tissues and possibly in fetuses. these earlier reports used less sensitive methods that failed to detect the traces of neu gc we subsequently noted in normal human tissues. thus, it is likely that the levels of neu gc present in cancers are simply higher. the possible explanations presented above for the traces of neu gc in normal tissues can be extended to fetuses and cancers as well, with the added suggestion that these rapidly growing tissues might be more efficient at scavenging neu gc (or its breakdown product n-glycolylmannosamine) from dietary sources. of course, we still cannot rule out an alternative pathway for the synthesis of neu gc that becomes specifically activated in tumors and fetuses. regardless of the mechanism(s) involved, the question again arises as to whether the presence of a sugar that is typically immunogenic in humans can in some way affect the outcome of a pregnancy or a malignant disease. perhaps it will be possible to take advantage of the presence of neu gc on tumors to design some novel approach to the treatment or containment of cancer. of course, all of these considerations do not apply to cancers in other mammals, since they naturally have large amounts of neu gc to begin with. thus, the animal model will have to be a mouse with a homozygous "knockout" of its cmah gene. such mice are currently being prepared. it is of great interest to ask if the loss of neu gc in humans resulted in some significant changes in the endogenous functions of sialic acids. since neu gc has a glycolyl group rather than an acetyl group, it is more hydrophilic, and this could result in changes in the physical or structural functions of sialic acids. however, this possibility and its potential consequences have yet to be investigated. as indicated above, the intrinsic receptors that bind sialic acids within animals have only recently been described, and there may be more to be discovered. correspondingly, we also know relatively little about the impact of neu gc loss on the functions of these intrinsic sialic-acid receptors in humans. the limited information available concerning neu gc and intrinsic sialic-acid receptors is summarized in table . the best-studied examples to date are certain members of a family of proteins called the siglecs (sialic acid-binding immunoglobu- lin-like lectins; sgroi et al., ; crocker et al., crocker et al., , kelm et al., a; powell and varki, ; crocker and feizi, ; crocker and varki, a,b; . sialoadhesin (siglec- ) is a large molecule found on tissue macrophage cells in various organs such as bone marrow, spleen, and lymph nodes. the sialic acid-recognizing function of sialoadhesin has been extensively studied, and it is clear that it binds well to neu ac, but not to neu gc (brinkman- van der linden et al., ; collins et al., a; kelm et al., b) . thus, human cells (which have an excess of neu ac) have an excess of binding sites for sialoadhesin (brinkman- van der linden et al., ) . presumably as a secondary consequence, there is an obvious difference in the tissue distribution of sialoadhesin-positive spleen macrophages between humans and chimpanzees. also, while only a subset of chimpanzee macrophages are sialoadhesin-positive, most human macrophages express this molecule (brinkman- van der linden et al., ) . interestingly, in all the above respects chimpanzee spleens are more similar to those of rats than to humans. the biological significance of these facts will not be known until the functions of sialoadhesin itself are elucidated. meanwhile, it is interesting to note that sialoadhesin-positive macrophages are found in large numbers in pathological tissue specimens from patients with diseases such as breast cancer (nath et al., ) and rheumatoid arthritis (hartnell et al., ) , both of which appear to be rare conditions in the great apes (varki, ) . another siglec that strongly prefers neu ac over neu gc is myelinassociated glycoprotein (siglec- a), which is expressed on schwann cells and oligodendrocytes, and appears to be involved in organizing and maintaining the myelin sheath that surrounds the axons in the white matter of the brain and the peripheral nerves (kelm et al., b collins et al., b) . curiously, the brain has very low levels of neu gc, even in nonhuman mammals (see below). thus, it is presently difficult to make a hypothesis about how the loss of neu gc expression in humans might have affected the function of myelin-associated glycoprotein. initial evaluation suggests that siglecs , , , and are probably not affected by the human loss of neu gc (brinkman- van der linden et al., ; collins et al., a; kelm et al., b kelm et al., , . further studies are underway to evaluate of some of the newer siglecs that were recently described. of particular interest is a recently discovered human siglec-like molecule (siglec-l ) that lacks a conserved amino acid (an arginine residue) which is known to be essential for optimal sialic-acid recognition by previously known siglecs . we found that the loss of the arginine residue was caused by a single nucleotide substitution in human genomic dna that occurred after the common ancestor of humans with the great apes but prior to the common origin of modern humans (an-gata et al., ) . the chimpanzee ortholog of siglec-l has the arginine residue, remains fully functional in recognizing sialic acids, and turns out to preferentially recognize neu gc over neu ac. reintroducing the ancestral arginine by "repairing" the cloned human molecule regenerated sialic acid binding, along with a similar preference for neu gc . thus, the single base-pair mutation that replaced the arginine residue on human siglec-l seems likely to be evolutionarily related to the loss of neu gc expression in the human lineage. however, we do not know which came first, and how exactly the two events are related. in the course of doing this work, we also examined the whole human genome for other siglec-like genes. it turns out that the human genome contains many siglec-like pseudogenes (currently inactive components of the genome derived from ancestral active genes). interestingly, some of these pseudogenes have independent mutations that would have replaced the same conserved arginine residue that is required for optimal sialic acid recognition. much further work needs to be done to know if any of these pseudogenes are derived from genes that are still active in other primates. regardless, this work indicates that additional changes in the biology of sialic acids may have taken place during human evolution. the consequence of neu gc loss for most of the other known mammalian sialic acid-recognizing lectins is also not very clear (see table ), and further studies are needed. an intriguing case is that of a sialic acid-binding uterine agglutinin that was purified from the endometrium (the epithelial lining of the uterus) of both humans and rats, and that was shown to prefer neu gc over neu ac (chatterji et al., ) . it was also shown to recognize sialic acids on sperm. while the functions of this molecule are unknown, one can speculate that its preference for neu gc might somehow be connected to anecdotal suggestions of differences between humans and great apes in matters such as menstrual blood loss and early fetal wastage (varki, ) . as mentioned above, a normal adult human directly exposed to neu gc in the form of infused horse serum generates an immune response against this foreign sugar. biotherapeutic molecules produced via recombinant dna technology are typically produced in nonhuman cells, because human cells may allow transmission of human retroviruses and other infectious agents. as many of these products are glycoproteins, they can contain varying amounts of neu gc originating from the nonhuman producer cells used. fortunately, most of the animal cell lines commonly used to produce such agents (e.g., chinese hamster ovary cells and baby hamster kidney cells) happen to produce very low levels of neu gc. furthermore, although erythropoietin (produced by amgen) was later recognized to have very small amounts of neu gc (hokke et al., ) , microgram amounts of this therapeutic glycoprotein had already been injected repeatedly into many humans, without apparent evidence of an obvious immune reaction (noguchi et al., ) . it is possible that the immune system of most humans is partially tolerized to neu gc because of prior exposure to dietary neu gc. if so, the reaction might be dose-dependent, and there should still be concern over recently developed products from the milk of sheep and goats which have very high levels of neu gc (gagneux and varki, unpublished findings) . another possible explanation for the lack of immune response to neu gc on erythropoietin is that the primary antigens in horse serum are glycolipids (not glycoproteins) bearing neu gc. thus, there may be a lesser immune response to neu gc on glycoprotein therapeutic agents. however, neu gc injected in the form of glycoproteins could be directly taken up by human cells via pathway a of figure , and eventually presented on the patient's own cell surfaces, attached to endogenously synthesized glycolipids. this could result in an immune response developing over time. this concern remains unresolved at the present time. similar considerations apply to the currently controversial attempts to transplant organs from other species into humans. all early attempts at such xenotransplantation of organs from other primates to humans failed for unknown reasons. these included attempts to transplant chimpanzee kidneys into humans (deodhar, ; reemtsma, ) which would nowadays be considered unethical. rejections of the transplanted organs were typically delayed by several weeks, and the mechanism of rejection remained obscure. if posttransplant serum from such patients had been saved, one could have determined if at least part of the rejection response was directed against neu gc. regardless, it should be noted that the most popular model animal for xenotransplantation of organs into humans is currently the pig, which happens to be a species that expresses high levels of neu gc in many tissues. no attention has yet been paid (in the published literature) to the potential for delayed immune response to neu gc on such transplanted animal organs. it remains to be seen if this will pose a significant barrier to xenotransplantation. as indicated above, neu gc is common in tissues of nonhuman mammals and is the major sialic acid in most organs and cell types of the great apes. in striking contrast to this situation, neu gc is hard to find in the brains of these animals. indeed, early studies suggested that neu gc was completely absent from the mammalian brain (gottschalk, ; rosenberg and schengrund, ; schauer, ) , and it was only subsequent analyses with more sensitive techniques that showed that there were small but clearly detectable amounts present (tettamanti et al., ; nakao et al., ; mikami et al., ; muchmore et al., ) . the mechanism for this differential expression in nonhuman animals appears to be the downregulation of cmah gene expression in the brain. this represents a rather rare example of a gene that is widely expressed in many tissues and yet selectively downregulated only in the brain. the fact that this unusual situation has been conserved throughout mammalian evolution suggests that there is a strong selection pressure in its favor. in other words, there must be some reason why the mammalian brain has restricted its neu gc content for Ͼ million years of evolution. in humans, of course, the last traces of neu gc are eliminated from the brain by virtue of the genomic inactivation of the cmah gene. a tantalizing possibility is that some significant positive consequence of this change for the human brain offset any negative consequences in other human organs. however, there is at present no direct evidence to support this hypothesis. studies are currently underway in our laboratory to investigate the consequences of eliminating or overexpressing neu gc in the brains of genetically modified mice. of course, the difference between a mouse and a human brain is much greater than the difference between a great ape and a human brain. thus, these experiments in mice may or may not provide final answers concerning this issue. on the other hand, genetic manipulation of the germline of humans or apes would be considered highly unethical. if the mouse experiments do not yield clear evidence, we may have to seek indirect clues towards understanding the implications of cmah loss for the evolution of the human brain. reviewed here is a genetic, biochemical, and structural difference between human and great ape cells that has potential implications for a wide variety of issues related to human evolution, physiology, and pathology. the nature of the genetic mutation, their universality in modern humans, and its direct biochemical consequences to human cells and tissues are clear. there is also good evidence that it can affect the susceptibility or resistance of humans to certain pathogens. circumstantial evidence is consistent with the possibility of human incorporation of traces of neu gc from dietary origins. in addition, limited data show the effects of human neu gc loss on cells of the human immune system such as macrophages. finally, the universal and selective suppression of neu gc expression in mammalian brains raises the possibility that the human loss of neu gc had some beneficial effects in human brain evolution. however, we have at present many more questions than answers. for example, when did this mutation first occur, and when did it become fixed as a universal feature of human ancestors? are there other consequences for infection risk or resistance in humans? what are the functional consequences for varki] the intrinsic human sialic-acid receptors whose neu gc preference has yet to be explored? do the traces of neu gc found in normal humans indeed come from dietary sources? if so, are there any pathological consequences to the human ingestion of neu gc in foods of animal origin? is the mechanism of neu gc reexpression in tumors and fetuses simply an exaggeration of what happens in normal tissues? or is there another biochemical pathway that becomes activated in these situations? does it matter that animal organs intended for xenotransplantation and certain biotechnology therapeutics produced in animal cells can be quite rich in neu gc? and last but not least, what are the consequences, if any, for the evolution and function of the human brain? much diligent work by many investigators will be needed to answer these questions. on a more general note, the discovery of a major biochemical and structural difference between humans and great apes and the elucidation of its underlying genetic basis tend to raise hopes that this molecular change played a major role in the evolution of uniquely human characteristics. indeed, this is the issue likely to be of particular interest to the readership of this journal. however, the fact that this happens to be the first such difference recognized should not be taken as any indication of its relative significance in human evolution. it is indeed possible that this genetic change came under strong positive selection pressure because it contributed towards the evolution of organs like the human brain. on the other hand, it may have simply aided in the ability of humans to evade certain pathogens of animal origin. a less likely possibility is that this gene inactivation was a fortuitous event that simply drifted to fixation in the immediate ancestors of modern humans, because of a population bottleneck caused by other unrelated factors. further studies will eventually elucidate which of these possibilities is correct. on the minor gangliosides of erythrocyte membranes of japanese cats n-glycolylneuraminic acid and n-acetylneuraminic acid define feline blood group a and b antigens chemical diversity in the sialic acids and related alpha-keto acids: an evolutionary perspective a second uniquely human mutation affecting sialic acid biology hydroxylation of cmp-neuac controls the expression of n-glycolylneuraminic acid in g m ganglioside of the small intestine of inbred rats haemophilus ducreyi produces a novel sialyltransferase-identification of the sialyltransferase gene and construction of mutants deficient in the production of the sialic acid-containing glycoform of the lipooligosaccharide loss of n-glycolylneuraminic acid in human evolution-implications for sialic acid recognition by siglecs dna divergences among hominids paracrine effects of a uterine agglutinin are mediated via the sialic acids present in the rat uterine endometrium genomic divergences between humans and other hominoids and the effective population size of the common ancestor of humans and chimpanzees a mutation in human cmp-sialic acid hydroxylase occurred after the homo-pan divergence binding specificities of the sialoadhesin family of i-type lectins-sialic acid linkage and substructure requirements for binding of myelin-associated glycoprotein, schwann cell myelin protein, and 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hanganutziu and deicher (h-d) antibodies and avian antisera against h-d antigen-active glycosphingolipids identification of nglycolylneuraminic acid-containing gangliosides of cat and sheep erythrocytes. cf fission fragment ionization mass spectrometry in the analysis of glycosphingolipids analysis of the expression of n-glycolylneuraminic acidcontaining gangliosides in cells and tissues using two human monoclonal antibodies evolutionary considerations in relating oligosaccharide diversity to biological function genetic differences between humans and great apes mitochondrial sequences show diverse evolutionary histories of african hominoids molecular evidence on primate phylogeny from dna sequences the chemistry and biology of sialic acids and related substances nuclear dna sequences from late pleistocene megafauna characterization of human sialoadhesin, a sialic acid binding receptor expressed by resident and inflammatory macrophage populations further studies on the red cell 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ganglioside with hanganutziu-deicher antigen activity on human colon cancers ancient dna structural analysis of the sialylated n-and o-linked carbohydrate chains of recombinant human erythropoietin expressed in chinese hamster ovary cells-sialylation patterns and branch location of dimeric n-acetyllactosamine units hanganutziu-deicher type-heterophile antigen-positive cells in human cancer tissues demonstrated by membrane immunofluorescence identification of -keto- -deoxy-d-glycero-d-galactonononic acid (kdn, deaminoneuraminic acid) residues in mammalian tissues and human lung carcinoma cells the molecular basis for the absence of n-glycolylneuraminic acid in humans host-range barrier of influenza a viruses receptor specificity of influenza a viruses correlates with the agglutination of erythrocytes from different animal species recognition of n-glycolylneuraminic acid linked to galactose by the ␣ , linkage is associated with intestinal replication of influenza a virus in ducks great 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hanganutziu-deicher heterophile antigen in human retinoblastoma cells lack of malaria parasite transmission between apes and humans in gabon detection of the ganglioside n-glycolyl-neuraminyl-lactosyl-ceramide by biotinylated escherichia coli k lectin i-type lectins the st gal-i sialyltransferase controls cd ϩ t lymphocyte homeostasis by modulating o-glycan biosynthesis phylogenetic relationship among the malaria parasites based on small subunit rrna gene sequences: monophyletic nature of the human malaria parasite, plasmodium falciparum species-specific variation in glycosylation of igg: evidence for the species-specific sialylation and branch-specific galactosylation and importance for engineering recombinant glycoprotein therapeutics targeted disruption of an erythrocyte binding antigen in plasmodium falciparum is associated with a switch toward a sialic acid-independent pathway of invasion biological roles of sialic acids polysialic acid in the vertebrate nervous system: a promoter 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acids a chimpanzee genome project is a biomedical imperative oligosaccharides in vertebrate development the sequence of the human genome a new gene, encoding an anion transporter, is mutated in sialic acid storage diseases heterogeneity of hanganutziu-deicher antigen glycoproteins in different species animal sera the distribution of sialic acids in nature structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid definition of a bacterial virulence factor: sialylation of the group b streptococcal capsule multivalent binding of k fimbriae to the n-glycolyl-gm ganglioside receptor structure of the haemagglutinin membrane glycoprotein of influenza virus at a resolution anthropology-the human genus difference in form of sialic acid in red blood cell glycolipids of different breeds of dogs identification of polysialic acids in glycoconjugates the origin of man: a chromosomal pictorial legacy g m directly inhibits tyrosine phosphorylation and de-n-acetyl-g m directly enhances serine phosphorylation of epidermal growth factor receptor, independently of receptor-receptor interaction the author thanks nissi varki, elaine muchmore, pascal gagneux, kurt benirschke, jim moore, margaret schoeninger, chris ruff, and two anonymous reviewers for very helpful comments. key: cord- - xdtbbzb authors: remesar, xavier; alemany, marià title: dietary energy partition: the central role of glucose date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: xdtbbzb humans have developed effective survival mechanisms under conditions of nutrient (and energy) scarcity. nevertheless, today, most humans face a quite different situation: excess of nutrients, especially those high in amino-nitrogen and energy (largely fat). the lack of mechanisms to prevent energy overload and the effective persistence of the mechanisms hoarding key nutrients such as amino acids has resulted in deep disorders of substrate handling. there is too often a massive untreatable accumulation of body fat in the presence of severe metabolic disorders of energy utilization and disposal, which become chronic and go much beyond the most obvious problems: diabetes, circulatory, renal and nervous disorders included loosely within the metabolic syndrome. we lack basic knowledge on diet nutrient dynamics at the tissue-cell metabolism level, and this adds to widely used medical procedures lacking sufficient scientific support, with limited or nil success. in the present longitudinal analysis of the fate of dietary nutrients, we have focused on glucose as an example of a largely unknown entity. even most studies on hyper-energetic diets or their later consequences tend to ignore the critical role of carbohydrate (and nitrogen disposal) as (probably) the two main factors affecting the substrate partition and metabolism. at present, two main lines of study focus on metabolic acquisition, distribution and use of food energy to maintain efficiently human body functions. ( ) the analysis of foods and diets, which largely uses centuries-old methodologies, and its subject of study is the relationship to diet of the body in relation to health and disease. ( ) the analysis and function of cell and molecular effects of diet has developed during the last - decades, using the high pace of methodological advances and accumulated knowledge at the molecular and cellular level, with important inroads into the regulation of the cell, organ, and body homeostasis. unfortunately, both lines run parallel, and there is not enough shared-connecting-knowledge to use the enormous amount of knowledge accumulated to help explain the direct relationships of dietary components with metabolic modulation and regulation, despite the paramount importance of this nexus. the complexity (and variability at all levels) or the problem hampers any approach to the health (and survival) problems related to diet. in this focused review, we intend to present a few critical questions that may help to bridge the gap between these two lines. in most cases, the lack of hard research data is surprising [ ] . the excessive narrowness of these study goals is even more severe [ ] . the forfeiting of a large mass of knowledge accumulated just before the present century [ ] , and limited use of quantitative glucose, however, has considerable advantages as a primary energy-substrate source: it can be easily converted in two c fragments even under anaerobic conditions (via the classic embden-meyerhoff pathway), well preserved along evolution [ ] . this quality is seldom recognized, especially considering the growing awareness on the perils of oxidative free radicals generated from oxygen metabolic interventions [ , ] . splitting one c unit (i.e., glucose) yields a variety of (essentially) c molecules: pyruvate, l-lactate, glycerol, alanine, serine, and phospho-enol-pyruvate (pep), used for energy or synthetic pathways. c fragments are also generated from a number of key pathways in addition to straight glycolysis: the pentose-phosphate pathway, hexosamine metabolism, the leloir (galactose) pathway, the catabolism of fructose and other monosaccharides as substrates, the incorporation of glycerides-glycerol, lactate, and a number of amino acid hydrocarbon skeletons. not all three-carbon metabolites can be included in this c list. the clearest examples are propionate (yielding succinyl-coa, see section ) and d-lactate, a significant component of bacterial fermentation (including our microbiota), found in some foods. its degradation is difficult [ ] but does not proceed directly via pyruvate. in this review, we will reserve the term "lactate" for l-lactate as in most metabolic studies. pyruvate is the key c fragment from which the core of intermediary metabolism establishes the use of diet (or reserves/turnover) substrates (figure ), via direct oxidation for energy, used as building materials for biosynthesis or transport between pools (cell, organ, body) to achieve energy homeostasis and efficiency in metabolism. pyruvate is also a main source of oxaloacetate (oaa) to regulate the tca (tricarboxylic acid) cycle, i.e., the krebs cycle (the second one) [ ] , or regenerate c units via gluconeogenesis. these processes require a critical control of the crossing of the mitochondrial membrane [ ] . pyruvate helps maintain the redox state of cytoplasm via interconversion to lactate (lactate dehydrogenase) [ ] as well as the transport through the cell membrane [ , ] . in any case, the main drain of pyruvate is the formation of acetyl-coa via oxidative decarboxylation by pyruvate dehydrogenase [ ] ; this way, a c fragment becomes a c fragment and co . regulate the tca (tricarboxylic acid) cycle, i.e., the krebs cycle (the second one) [ ] , or regenerate c units via gluconeogenesis. these processes require a critical control of the crossing of the mitochondrial membrane [ ] . pyruvate helps maintain the redox state of cytoplasm via interconversion to lactate (lactate dehydrogenase) [ ] as well as the transport through the cell membrane [ , ] . in any case, the main drain of pyruvate is the formation of acetyl-coa via oxidative decarboxylation by pyruvate dehydrogenase [ ] ; this way, a c fragment becomes a c fragment and co . the regulation of glucose fate and circulating levels has been studied exhaustively, and it has been found that the alteration of these processes may cause serious harm to energy homeostasis. glucose is strictly controlled by a huge number of regulatory agents, including insulin, glucocorticoids, glucagon, intestinal peptides, catecholamines, cytokines, testosterone, estrogens, etc. [ ] . they regulate glucose splanchnic production [ ] and utilization [ ] [ ] [ ] . glucose modulates the production and secretion of insulin [ ] and, indirectly, its hepatic inactivation [ ] . however, insulin resistance affects very directly glucose uptake and metabolism, because of changes in insulin tissue receptors and signaling [ ] . on the other side, the c fragments (mainly pyruvate, lactate, glycerol) are regulated within the cells, but not as extensively as is glucose in their inter-organ relationships. c substrates, such as lactate or glycerol can be easily taken up by a wide diversity of cells, even using less specific (or quite different) transport systems [ , ] . when the cell takes up lactate, it can immediately provide nadh in the cytosol, and then follow a number of diverse paths to provide c or energy (via conversion to c). in comparison, after a strongly regulated maintenance of its circulating levels, glucose is transported into the cell, converted to glucose- p, and then it can be used for the production of c (and eventually c) through wellregulated mechanisms. the main potential inconvenient for plasma-carried c (specifically lactate) is its charge (a proton) and its reduced state, which necessarily has to be corrected via production of cytosolic nadh, but this is the same problem glucose generates at the level of triose-p dehydrogenase. in any case, glycerol or pyruvate do not comport these hindrances. it is well known the regulation of glucose fate and circulating levels has been studied exhaustively, and it has been found that the alteration of these processes may cause serious harm to energy homeostasis. glucose is strictly controlled by a huge number of regulatory agents, including insulin, glucocorticoids, glucagon, intestinal peptides, catecholamines, cytokines, testosterone, estrogens, etc. [ ] . they regulate glucose splanchnic production [ ] and utilization [ ] [ ] [ ] . glucose modulates the production and secretion of insulin [ ] and, indirectly, its hepatic inactivation [ ] . however, insulin resistance affects very directly glucose uptake and metabolism, because of changes in insulin tissue receptors and signaling [ ] . on the other side, the c fragments (mainly pyruvate, lactate, glycerol) are regulated within the cells, but not as extensively as is glucose in their inter-organ relationships. c substrates, such as lactate or glycerol can be easily taken up by a wide diversity of cells, even using less specific (or quite different) transport systems [ , ] . when the cell takes up lactate, it can immediately provide nadh in the cytosol, and then follow a number of diverse paths to provide c or energy (via conversion to c). in comparison, after a strongly regulated maintenance of its circulating levels, glucose is transported into the cell, converted to glucose- p, and then it can be used for the production of c (and eventually c) through well-regulated mechanisms. the main potential inconvenient for plasma-carried c (specifically lactate) is its charge (a proton) and its reduced state, which necessarily has to be corrected via production of cytosolic nadh, but this is the same problem glucose generates at the level of triose-p dehydrogenase. in any case, glycerol or pyruvate do not comport these hindrances. it is well known that a high number of tissues use lactate for energy under exercise [ ] , limited insulin resistance [ ] , or as a cryptic "universal cell fuel" [ ] . the brain is an active user of lactate [ ] and glycerol [ , ] . liver is a net lactate user, largely for gluconeogenesis [ , ] . white adipose tissue (wat) can take up lactate for lipogenesis [ ] or produce it from glucose in large amounts [ , ] . the intestine also provides c substrates, in part as a product of digestion [ ] . the transfer of energy to tissues via c has been found to be much higher than often considered [ , ] , constituting a clear alternative to intact glucose [ , ] . under conditions of excess glucose availability, its conversion to c eases the pressure over the regulation of glycaemia and allows for the direct use of its energy via c [ , ] , in a way comparable to the "pre-preparation" of fatty acids ( c n ) fragments to plasma-soluble ketone bodies ( c fragments). the relatively lax control and ease of direct metabolic incorporation allows for a loosely regulated use of c anywhere. this possibility is extensive to the nervous system, which can use c fragments to a large extent as a source of energy in addition to glucose (or as its substitute) [ ] . figure shows the relationship between dietary nutrients and the c and c substrates pools. in addition to the carbohydrate paths shown in figure , a large part of amino acid hydrocarbon skeletons can be incorporated to the c→ c pathways (largely glycolysis and gluconeogenesis) but also as intermediates of the tca cycle (which eventually will result in oaa and then to a c fragment used for energy or synthesis. lipids, essentially triacylglycerols (tag), also provide c (as glycerol), but their carbon is essentially structured in c units (acetate, acetyl-coa) or-massively-in c n chains, such as fatty acids. that a high number of tissues use lactate for energy under exercise [ ] , limited insulin resistance [ ] , or as a cryptic "universal cell fuel" [ ] . the brain is an active user of lactate [ ] and glycerol [ , ] . liver is a net lactate user, largely for gluconeogenesis [ , ] . white adipose tissue (wat) can take up lactate for lipogenesis [ ] or produce it from glucose in large amounts [ , ] . the intestine also provides c substrates, in part as a product of digestion [ ] . the transfer of energy to tissues via c has been found to be much higher than often considered [ , ] , constituting a clear alternative to intact glucose [ , ] . under conditions of excess glucose availability, its conversion to c eases the pressure over the regulation of glycaemia and allows for the direct use of its energy via c [ , ] , in a way comparable to the "pre-preparation" of fatty acids ( cn) fragments to plasma-soluble ketone bodies ( c fragments). the relatively lax control and ease of direct metabolic incorporation allows for a loosely regulated use of c anywhere. this possibility is extensive to the nervous system, which can use c fragments to a large extent as a source of energy in addition to glucose (or as its substitute) [ ] . figure shows the relationship between dietary nutrients and the c and c substrates pools. in addition to the carbohydrate paths shown in figure , a large part of amino acid hydrocarbon skeletons can be incorporated to the c→ c pathways (largely glycolysis and gluconeogenesis) but also as intermediates of the tca cycle (which eventually will result in oaa and then to a c fragment used for energy or synthesis. lipids, essentially triacylglycerols (tag), also provide c (as glycerol), but their carbon is essentially structured in c units (acetate, acetyl-coa) or-massively-in cn chains, such as fatty acids. glucose (in general terms, carbohydrate) is required as a main dietary macro-component for humans. [ ] , also probably being an essential component of diet, at least by default [ ] . the commonness, diverse forms, and high proportions of carbohydrates in many diets has masked this condition [ ] . nevertheless, the extended use of high-fat and high-protein diets resulting in figure . relationship between the main groups of dietary nutrients driving to the formation of c and c fragments. in red, the irreversible decarboxylative oxidation path of pyruvate ( c) to acetyl-coa ( c). glucose (in general terms, carbohydrate) is required as a main dietary macro-component for humans. [ ] , also probably being an essential component of diet, at least by default [ ] . the commonness, diverse forms, and high proportions of carbohydrates in many diets has masked this condition [ ] . nevertheless, the extended use of high-fat and high-protein diets resulting in inflammatory responses and metabolic alterations [ ] points to the requirement of a sufficient amount of c substrates (preferably as c n polysaccharides) in any normal health-sustaining diet, in proportions of c n not different at present from our early hominid ancestors [ ] . the notion that the brain only uses glucose as a substrate remains alive in many texts and studies, largely for the disproportionate basal consumption of glucose by the brain in comparison with most other organs [ ] . however, the widespread existence of intercellular lactate-glucose micro-cycles between glia and neurons suggests otherwise [ , ] . the main substrate for brain energy changes with development [ ] , but the nervous system continues using a large proportion of blood glucose under standard conditions [ ] , whether this is a consequence of glia cells (i.e., astrocytes) producing lactate from glucose to feed neurons [ ] or the neurons themselves using glucose directly is a question not yet settled [ , ] . the intensive utilization of lactate and glycerol by the brain as a whole has been known for more than half a century [ , ] and may be part of a system of protection against the chemical dangers posed by glucose reactivity. from the earliest stages of development, the nervous system has specific needs for some nutrients, such as glucose, as key source of energy [ ] ; oligosaccharides containing galactose and other sugars [ ] ; c fragments, as indicated above; specific essential fatty acids [ ] ; and enough amino n and essential amino acids [ ] . the needs of peripheral and enteric nervous systems are less known; in the last case, we have to include the direct relationships with intestinal microbiota [ ] . in any case, it is widely acknowledged that the brain has priority over the rest of the body with respect to glucose supply [ ] . the extent to which the requirements of c can be substituted by c is yet an object of discussion [ , ] . the erythrocytes (at least the non-nucleated mammal-including human-ones) are also peculiar with respect to the need for glucose. they require integer glucose, since they are not able to get energy via oxidation of c. thus, these cells are considered purely glycolytic, releasing lactate from glucose [ ] . since the red blood cells need little energy and are a fundamental component of the blood (which carries glucose), their needs can be easily covered by direct uptake from plasma [ ] . because their consumption is non-oxidative ( c to c), the return of two c units per glucose does not alter the global equation of glucose consumption and do not represent a quantitatively critical factor, as can be the case with parts of our nervous systems. the triad of tag, fatty acids, and acetate/acetyl-coa represents decreasing levels of complexity of most energy-related lipids; their mw is related directly to their physical conditioning and metabolic usage. a critical point is their lipophilic nature, which conditions transport by the blood and the crossing of intracellular and plasma membranes. acetate ( c) and easily metabolizable ketone bodies ( c ), as well as some short-chain fatty acids, are hydrophilic enough to be transported in blood or lymph and may cross easily most membranes [ ] . fatty acids ( c n , medium to long-chain with less than five to more than c units) are transported bound to proteins [ ] , and are incorporated into the cell by specific efficient binding-proteins [ ] and transporters [ ] . tag are too large and insoluble to be carried by blood plasma: they are transported within lipoproteins, structured with apolipoproteins and other lipids, such as phospholipids and other classes of lipophilic compounds as minority components. the intestine and the liver initially build up these complex lipid carriers, mainly chylomicra and vldl. they are fairly efficient systems for transporting large amounts of tag (i.e., heavy energy packages, mostly containing c n ) to provide fuel for energy and organ functions [ ] in addition to the transport to and from of critical lipophilic substrates such as cholesterol, a process reserved for the smaller lipoproteins (ldl, hdl) [ ] . within tissues, large amounts of lipid are transferred between cells by using at least two mechanisms. in the first, very large (often old) adipocytes, (or those marked for apoptosis), when broken up via apoptosis or autophagia, release a large amount of micro-drops of lipid. these vesicles are taken up via pinocytosis by macrophages, functional adipocytes [ ] , or other cells. a selective apoptotic (and packed groups of macrophages [ ] ) process has been proposed as a mechanism for tissue turnover, which is applied to dead or no longer functional adipocytes [ ] . this clearing process has the advantage to constrain the diffusion of cell debris and lipid drops that can cause damage to other cells, as well as limiting the clogging of individual macrophages with the remains of apoptosis [ ] . the other mechanism is the use of exosomes or vesicles, which can transfer signals, proteins, nucleic acids, but also tag between cells [ , ] . this (incomplete) list represents a wide panoply of mechanisms for transfer of c-based substrates, primarily between the splanchnic bed to peripheral tissues, for storage or direct utilization. the question of transport between cells and tissues is critical for the supply of energy on time, in the most efficient chemical form for immediate use, and with a sufficient transfer rate of to cover the needs. this is, probably the main reason why there are two main systems for sending energy substrates to the tissues via blood: a) lipophilic ( c-derived, energy-dense but large and difficult to handle) and b) hydrosoluble substrates, represented by glucose and c, but also the smaller c components soluble in plasma. the logistic advantages of having both systems rely essentially in the highly dense energy tag as substrates sent elsewhere for heavy needs, and a variety of other metabolites, usable directly by the cells faster to supply in a continuous way, and easily subjected to immediate regulation. the irreversible path of c to c is carried out by the pyruvate dehydrogenase complex [ ] (figure ), which is highly regulated [ , ] and controls the entry of c into the mitochondrion [ ] . we can use glucose to build fatty acids, but the reverse (regular fatty acids to glucose) is not possible because of this critical step at the confluence of the c and c worlds. this irreversibility is typical of animals, since plants and other phyla possess the "glyoxylate shunt" that we do not [ ] . probably, we lost this shunt along evolution because the main source of c to feed the tca cycle and fatty acid synthesis remained glucose-derived pyruvate. process has the advantage to constrain the diffusion of cell debris and lipid drops that can cause damage to other cells, as well as limiting the clogging of individual macrophages with the remains of apoptosis [ ] . the other mechanism is the use of exosomes or vesicles, which can transfer signals, proteins, nucleic acids, but also tag between cells [ , ] . this (incomplete) list represents a wide panoply of mechanisms for transfer of c-based substrates, primarily between the splanchnic bed to peripheral tissues, for storage or direct utilization. the question of transport between cells and tissues is critical for the supply of energy on time, in the most efficient chemical form for immediate use, and with a sufficient transfer rate of to cover the needs. this is, probably the main reason why there are two main systems for sending energy substrates to the tissues via blood: a) lipophilic ( c-derived, energy-dense but large and difficult to handle) and b) hydrosoluble substrates, represented by glucose and c, but also the smaller c components soluble in plasma. the logistic advantages of having both systems rely essentially in the highly dense energy tag as substrates sent elsewhere for heavy needs, and a variety of other metabolites, usable directly by the cells faster to supply in a continuous way, and easily subjected to immediate regulation. the irreversible path of c to c is carried out by the pyruvate dehydrogenase complex [ ] (figure ), which is highly regulated [ , ] and controls the entry of c into the mitochondrion [ ] . we can use glucose to build fatty acids, but the reverse (regular fatty acids to glucose) is not possible because of this critical step at the confluence of the c and c worlds. this irreversibility is typical of animals, since plants and other phyla possess the "glyoxylate shunt" that we do not [ ] . probably, we lost this shunt along evolution because the main source of c to feed the tca cycle and fatty acid synthesis remained glucose-derived pyruvate. the c→ c path irreversibility has important consequences for glucose and energy partition: while c→ c and c n→ c relationships are reversible, c→ c is unidirectional. in plants, fatty acid reserves can be converted into c [ ] . however, in humans and other animals, glucose (or other convertible c) is needed to provide the c fragments used in many metabolic paths, since despite the c→ c path irreversibility has important consequences for glucose and energy partition: while c→ c and c n → c relationships are reversible, c→ c is unidirectional. in plants, fatty acid reserves can be converted into c [ ] . however, in humans and other animals, glucose (or other convertible c) is needed to provide the c fragments used in many metabolic paths, since despite the possible excess of c, no c derivatives can be obtained from them. this leaves us with an inefficient system to obtain energy from c when converted to c, and later oxidized via tca cycle. an excess of c can be corrected only either storing it as fat or through its complete oxidation, whilst any excess of c or c can be used by a wider range of metabolic pathways, or ultimately derived into c. the reversible c→ c glycolytic conversion, and the easy entry of c in most cells results in a strategic advantage of distribution, limiting substrate buildups whilst maintaining their full homeostatic availability. this process is sustained by the existence of substrate cycles within adjoining groups of cells in a given tissue, as is the case of muscle [ ] or the brain, between glia and neurons [ ] . the substrate cycles between different organs are widely known, e.g., the cori cycle [ ] or the glucose-alanine cycle [ ] , in which exercising muscle converts glucose to c, later taken up by the liver, prompting gluconeogenesis (i.e., c→ c) with the overall result (cori cycle) of transfer of reducing power from peripheral tissues to the liver [ ] . in this case, the c→ c interconversion helps transfer reducing power (or -amino groups in the case of alanine) to the liver for their reutilization ( figure ) or disposal. this inter-organ coordination prompts a more efficient use of energy and oxygen (and -amino n), helping to maintain the levels and availability of substrates. most of the inter-organ substrate cycles were described as mechanisms preventing the dangers of reducing power or -amino n accumulation in muscle or other peripheral tissues under conditions of active use of glucose or amino acids (i.e., during exercise) for energy; they always present a time-delay component for maximal effectiveness. the exportation of c carrying the excess reducing power or n to the liver has the additional advantage of allowing the return of these c to the blood as glucose, completing a cycle. however, these "cycles" usually work, in real-time, as simple vectors for transfer-"open interrelationships" (figure ), making full use of the differential organ oxygen supply to transfer a reduced c (i.e., lactate) from active muscle to other muscles or the liver to regenerate glucose, resulting in a chain-transfer of reducing power [ ] . this situation can be sustained only when glucose is not in excess (in the liver), since it may block gluconeogenesis [ ] , but the excess reducing power can be easily corrected because of the high liver oxygen supply, removing lactate immediately or after a delay and even after a buildup of acidosis [ ] . the maximal "reduction" of open cycles can be found in the lung, which scarcely uses glucose [ ] but uses blood protons (as the liver do) exported by relatively hypoxic tissues, using their energy at the mitochondria and thus helping limit acidosis [ ] . a special situation is that of adipose tissue; it has been found to generate large amounts of c from glucose [ ] . this process has been suggested to help lower glycaemia (and its associated problems) under conditions of excess substrate availability [ ] . the extent of this transformation is considerable, especially in the mesenteric adipose mass, which receives the mixed c- c results of digestion before sending them via portal vein to the liver [ ] . this peculiarity is not limited to adipocytes, since the adipose stromal cells act in the same way: a practical anaerobic glycolytic conversion of c to c even under full oxygen availability [ ] . this may be considered either as another contribution to regulate glycaemia or as an alternative to produce c fragments (essentially lactate and glycerol) in massive amounts to supply ready-to-use energy to most organs (including the brain) to circumvent the regulatory difficulties of glucose utilization under situations of excess substrate and/or insulin resistance [ , , , ] . the ample use of c as main energy substrate agrees with this interpretation. int. j. mol. sci. , , x for peer review of figure . inter-organ substrate cycles ( c- c). the first and second panels present the cori cycle and glucose-alanine cycle [ ] . the lower panel shows an "open" inter-organ relationship ( c- c) such as that found between the adipose tissue and peripheral organs [ ] . inter-organ substrate cycles ( c- c). the first and second panels present the cori cycle and glucose-alanine cycle [ ] . the lower panel shows an "open" inter-organ relationship ( c- c) such as that found between the adipose tissue and peripheral organs [ ] . curiously, and despite their primeval importance, amino acids seldom are considered quantitatively important substrates for humans in analyses of overall energy metabolism. the most used explanations for this ellipsis are: • the often incomplete hydrolysis of dietary proteins [ ] ; • intervention of microbiota through transformation/catabolism or even synthesis of amino acids [ ] ; • further transformation/catabolism by the intestine and the liver before overall distribution [ ] ; • sparsely known real catabolic pathways for many amino acids [ - ] in humans; • the absence of specific amino acid/protein reserves; • the lack of knowledge on the regulation of essential amino acids; • the gross differences between measured calorimetric pump energy content of proteins and the real energy obtained in vivo from amino acids according to the known (or assumed) metabolic pathways [ ] ; • the assumed relatively small amount of actually oxidized amino acids derived from the diet compared with carbohydrates and lipids; • the uncertainty on how the n of amino acids is processed and excreted [ ] , which directly affects the estimations of dietary amino acid use [ ] ; • the "diversity and complexity" of amino acid catabolism; • the common absence of data on diet protein amino acid composition. in fact, the main question for eluding amino acids from most studies is the lack of knowledge. we do not know yet (in humans) the complete catabolic pathway of several essential amino acids or the implication and compartmentation of the catabolism of amino acids yielding c, c, c, and c/ c intermediates of the tca cycle. to complicate further the analysis, we have only an approximate knowledge on how the dietary protein (in fact, the amino acids resulting from their digestion if assimilated) translates into metabolizable energy. the role of a few amino acids in the maintenance of glycaemia via gluconeogenesis [ , ] is well known, but the focus is now on branched-chain amino acids [ , ] . these amino acids are catabolized to c fragments (leucine and isoleucine) and propionate (valine and isoleucine) [ ] (nor a c but an important anaplerotic precursor of c fragments [ ] ). some amino acids (such as alanine or serine), when oxidized, i.e., after their amino moiety has been removed, yield directly c (pyruvate). others (i.e., leucine, lysine) are mainly broken down to c, but most are oxidized to intermediate metabolites of the tca cycle (e.g., glutamate, aspartate, threonine). a simplified scheme of the fate of hydrocarbon moiety of amino acids is shown in figure . the amino acid hydrocarbon skeletons can, thus, provide c or (in a higher overall proportion) c, but also the high-energy containing c and c fragments, which, eventually will be converted into c along the tca cycle, hence their importance for gluconeogenesis under conditions of severe energy stress. however, the use of c for -ketoacid synthesis is almost nil, widening the c- c chasm. in fact, the variation of amino acid catabolic pathways results in a simplified handling of their complexity-losing in some cases energetic efficiency in the process-for gross energy purposes (as shown in figure ) than usually believed. as to the aspects of regulation cited above, we are yet in a serious state of ignorance, with few exceptions. catabolized to c fragments (leucine and isoleucine) and propionate (valine and isoleucine) [ ] (nor a c but an important anaplerotic precursor of c fragments [ ] ). some amino acids (such as alanine or serine), when oxidized, i.e., after their amino moiety has been removed, yield directly c (pyruvate). others (i.e., leucine, lysine) are mainly broken down to c, but most are oxidized to intermediate metabolites of the tca cycle (e.g., glutamate, aspartate, threonine). a simplified scheme of the fate of hydrocarbon moiety of amino acids is shown in figure . fragments, which revert essentially into c and c, during the human catabolism of amino acid hydrocarbon-skeletons. the pathways used to prepare this graph are the most common, including the main alternate pathways [ ] . the green lines show the carbon paths for each amino acid, the brown lines represent the relationship between the core of the tca cycle and pyruvate dehydrogenase (with the loss of one co between each substrate group box (marked in yellow). nonstandard abbreviations: hyp = l- -hydroxyproline; orn = ornithine; " c" = one-carbon fragment donor systems. the amino acid hydrocarbon skeletons can, thus, provide c or (in a higher overall proportion) c, but also the high-energy containing c and c fragments, which, eventually will be converted into c along the tca cycle, hence their importance for gluconeogenesis under conditions of severe energy stress. however, the use of c for -ketoacid synthesis is almost nil, widening the c- c chasm. in fact, the variation of amino acid catabolic pathways results in a simplified handling of their complexity-losing in some cases energetic efficiency in the process-for gross energy purposes (as shown in figure ) than usually believed. as to the aspects of regulation cited above, we are yet in a serious state of ignorance, with few exceptions. figure . summary of the final conversion of dietary protein amino acids in c, c, c, c, and c fragments, which revert essentially into c and c, during the human catabolism of amino acid hydrocarbon-skeletons. the pathways used to prepare this graph are the most common, including the main alternate pathways [ ] . the green lines show the carbon paths for each amino acid, the brown lines represent the relationship between the core of the tca cycle and pyruvate dehydrogenase (with the loss of one co between each substrate group box (marked in yellow). non-standard abbreviations: hyp = l- -hydroxyproline; orn = ornithine; " c" = one-carbon fragment donor systems. from an evolutionary point of view, our design evolved to preserve -amino n, since we cannot significantly use other sources of -amino n than amino acids themselves (obtained from dietary protein). all amino acids are synthesized by plants (and other phyla) and used to build their own proteins. we use (eat and digest) these same proteins to obtain the amino acids we need to form our self-proteins and other n-containing molecules (e.g., purines, pyrimidines, porphyrins, etc.). all our amino acids have been obtained (from the diet) either preformed-e.g., essential amino acids-or rebuilt by us from available hydrocarbon structures and -amino n suppliers, i.e., amino acids from plant or animal (formerly "plant") sources. since we are omnivores, the dietary protein is just another source of energy, used, for this purpose, in proportions that depend on their availability [ , ] . thus, the complex safety measures established to retain amino acid n necessarily should interfere with their utilization for energy. nevertheless, it is obvious that essential amino acids are continuously oxidized for energy irrespective of their source: if there are not changes in body protein (in addition to the "obligatory" protein losses: box ), the amount of these amino acids ingested in the diet should tally their oxidation, since they cannot be stored. source of energy, used, for this purpose, in proportions that depend on their availability [ , ] . thus, the complex safety measures established to retain amino acid n necessarily should interfere with their utilization for energy. nevertheless, it is obvious that essential amino acids are continuously oxidized for energy irrespective of their source: if there are not changes in body protein (in addition to the "obligatory" protein losses: box ), the amount of these amino acids ingested in the diet should tally their oxidation, since they cannot be stored. the main, basic studies on our handling of diet amino acids, protein synthesis, and recycling of amino n were done essentially more than half century ago, and they were invariably focused on the ways to preserve n, the mechanisms to survive under conditions of starvation [ ] , dietary protein deprivation [ ] and, globally, malnutrition [ , ] . amino acids as energy staple have been, since, considered "secondary" (or at most "complementary"), and their assumedly "complex" metabolism was oversimplified around their possible use as gluconeogenic substrate under glucose deficits or generators of ketones (ketogenic) during starvation [ ] . the epidemic nature of obesity, developed thereafter, and the need to find ways to cope with its ravages were centered on glucose and fats, a situation that continues with limited global interest on amino acids as energy substrates under conditions of plenty [ , ] . the key problems that remain and prevent further advances in the main, basic studies on our handling of diet amino acids, protein synthesis, and recycling of amino n were done essentially more than half century ago, and they were invariably focused on the ways to preserve n, the mechanisms to survive under conditions of starvation [ ] , dietary protein deprivation [ ] and, globally, malnutrition [ , ] . amino acids as energy staple have been, since, considered "secondary" (or at most "complementary"), and their assumedly "complex" metabolism was oversimplified around their possible use as gluconeogenic substrate under glucose deficits or generators of ketones (ketogenic) during starvation [ ] . the epidemic nature of obesity, developed thereafter, and the need to find ways to cope with its ravages were centered on glucose and fats, a situation that continues with limited global interest on amino acids as energy substrates under conditions of plenty [ , ] . the key problems that remain and prevent further advances in this direction are the diversity of metabolic pathways used to oxidize the hydrocarbon skeletons of amino acids, the relatively unknown mechanisms to retain essential amino acids, and, especially, the not yet clarified variable fate of the n moiety of amino acids during their catabolism. the canonic mechanism of excretion of the n moiety waste is the urea (or krebs-henseleit) cycle [ ] (the "first" krebs' cycle [ ] ), a very peculiar pathway between cytosol and mitochondria. it may also harbor a shunt to generate nitric oxide (no·) and citrulline from arginine [ ] . the daily losses of n via the no· pathway are a fraction of the overall n excretion, essentially made up from nitrogen oxides released through respiration [ ] (a few µg/day) or further oxidized to nitrite, nitrate, and other nitroxylated compounds [ , ] , which are excreted via saliva, urine, and stool [ , ] . however, there is proof of an additional, quantitatively significant, excretion of n as gas through respiration [ , ] that is supplementary to the standard and well-controlled urea excretion. when analyzing in detail n balances in experimental animals, the amount of n ingested is higher than the sum of the n accrued in the body and the n excreted and accounted for [ , ] (box ). the proportion of this "nitrogen gap" (assumedly n ) is in the range of - % of all nitrogen ingested and not accrued (i.e., it is actually excreted), and its amount is related to diet and energy status [ , ] . it has been found that this n loss is related to arginine metabolism [ ] (figure ). excretion. when analyzing in detail n balances in experimental animals, the amount of n ingested is higher than the sum of the n accrued in the body and the n excreted and accounted for [ , ] (box ). the proportion of this "nitrogen gap" (assumedly n ) is in the range of - % of all nitrogen ingested and not accrued (i.e., it is actually excreted), and its amount is related to diet and energy status [ , ] . it has been found that this n loss is related to arginine metabolism [ ] (figure ). the nitrogen "gap: in rats. a "nitrogen gap" was found when analyzing all the components of the nitrogen balance in young rats: i.e., the n ingested, that excreted by urine and feces as well as the total n accrued, thus proving the existence of a sizeable part of the n excreted not as urea, or through the other possible ways and means shown in box . different complete (measuring both sides of the n balance equation) studies repeated these findings, dependent on diet, and with a magnitude (in rodents at least) in the range of - % of all nitrogen excreted. redrawn with data from esteve et al. [ ] . in fresh-water plants [ ] or fish [ , ] living under high (cytotoxic) ammonia concentrations, the levels of no· are high, and the production of no· is increased several fold on exposure to ammonia, assumedly potentiating its oxidation [ ] . the most suggestive process is that of the anammox (anaerobic ammonia oxidation) bacteria [ ] , which assumedly use nox, probably via no· to oxidize nh + coupled to the reduction of no· [ ] . both ammonia and nitrite or nitric oxide can react spontaneously in an exothermic reaction, yielding n and water [ ] , provided that ammonia is protonated to ammonium and the extra electrons are removed figure . the nitrogen "gap: in rats. a "nitrogen gap" was found when analyzing all the components of the nitrogen balance in young rats: i.e., the n ingested, that excreted by urine and feces as well as the total n accrued, thus proving the existence of a sizeable part of the n excreted not as urea, or through the other possible ways and means shown in box . different complete (measuring both sides of the n balance equation) studies repeated these findings, dependent on diet, and with a magnitude (in rodents at least) in the range of - % of all nitrogen excreted. redrawn with data from esteve et al. [ ] . in fresh-water plants [ ] or fish [ , ] living under high (cytotoxic) ammonia concentrations, the levels of no· are high, and the production of no· is increased several fold on exposure to ammonia, assumedly potentiating its oxidation [ ] . the most suggestive process is that of the anammox (anaerobic ammonia oxidation) bacteria [ ] , which assumedly use nox, probably via no· to oxidize nh + coupled to the reduction of no· [ ] . both ammonia and nitrite or nitric oxide can react spontaneously in an exothermic reaction, yielding n and water [ ] , provided that ammonia is protonated to ammonium and the extra electrons are removed no + nh this is a reaction very similar to the integral oxidation of ammonia with nitrite [ ] , which can be reproduced very easily in the laboratory with minimal means. n is also produced by the reaction of nitrite with amino acids at low ph [ ] . the abundance of nitrite produced in the inactivation of no· could justify a small part of the production of n , in part via its reaction with amino acids [ ] . however, this process has been studied under conditions seldom found in a living human [ ] . nitrite may react more easily with ammonia, but their low circulating levels [ ] , and the small area of diffusion of newly-created no·, in the µm range, may prevent this reaction taking place, except, perhaps, in the gut lumen, where the levels of ammonium and no· (but not the environmental conditions) may be higher [ , ] . however, the fact that in axenic rodents the excretion of n is unchanged [ ] hint at the existence of a less "occasional" and regulated source for the production of n . the mechanism of nitrogen gas synthesis from ammonia used by anammox bacteria suggests a possible pathway in which this oxidation is carried out in two steps [ ] : evidently, the hypothesis we presented in figure with respect to the postulated origin of n from the mitochondrial reaction of ammonia and no· has a serious weak point, since, as far as we know, the enzymes that allow the oxidation of ammonia by no· (under anaerobiosis) are exclusive of anammox bacteria. however, the possibility of hydrazine being an intermediate step in the process could not be fully discarded given its limited toxicity (it has been used for the treatment of tuberculosis and cancer [ ] ) and its widespread effects on amino acid metabolism [ ] . inactivation of no· could justify a small part of the production of n , in part via its reaction with amino acids [ ] . however, this process has been studied under conditions seldom found in a living human [ ] . nitrite may react more easily with ammonia, but their low circulating levels [ ] , and the small area of diffusion of newly-created no·, in the µm range, may prevent this reaction taking place, except, perhaps, in the gut lumen, where the levels of ammonium and no· (but not the environmental conditions) may be higher [ , ] . however, the fact that in axenic rodents the excretion of n is unchanged [ ] hint at the existence of a less "occasional" and regulated source for the production of n . the mechanism of nitrogen gas synthesis from ammonia used by anammox bacteria suggests a possible pathway in which this oxidation is carried out in two steps [ ] : evidently, the hypothesis we presented in figure with respect to the postulated origin of n from the mitochondrial reaction of ammonia and no· has a serious weak point, since, as far as we know, the enzymes that allow the oxidation of ammonia by no· (under anaerobiosis) are exclusive of anammox bacteria. however, the possibility of hydrazine being an intermediate step in the process could not be fully discarded given its limited toxicity (it has been used for the treatment of tuberculosis and cancer [ ] ) and its widespread effects on amino acid metabolism [ ] . figure . hypothesis for the origin of ammonia and nitric-oxide generation of nitrogen gas in mitochondria. the coexistence in the liver mitochondrial matrix of both ammonia/ammonium and nitric oxide, within well-regulated pathways, in significant amounts, and related to the urea cycle, figure . hypothesis for the origin of ammonia and nitric-oxide generation of nitrogen gas in mitochondria. the coexistence in the liver mitochondrial matrix of both ammonia/ammonium and nitric oxide, within well-regulated pathways, in significant amounts, and related to the urea cycle, suggests the possibility of a reaction which may generate nitrogen gas at the expense of both. this is a speculative hypothesis, which has not been proven so far. in any case it is difficult to justify the presence of both reactants in a relatively high concentration without interacting, since the uncontrolled chemical reaction between them is spontaneous and exergonic. in this hypothesis, we include the findings of kartal et al. [ ] in their study of the mechanism of ammonia oxidation in anammox bacteria, which has an intermediate metabolite between them, hydrazine (n h ). the two enzyme activities of the complex hydrazine synthase and hydrazine dehydrogenase are intimately related to membrane of the anammoxosome particle. ammonia and nitric oxide are both produced in significant amounts in the mitochondria: ammonia through-mainly-glutamate dehydrogenase [ ] and glutaminase [ ] , and no· via no· synthase [ ] (figure ). one can speculate that within the mitochondria, the reaction can take place through a regulated mechanism. the main problems for this hypothesis to be confirmed are the so far unidentified catalyzer system, and the question of the low solubility of n in biological fluids but not on fat tissue, several-fold higher than in plasma [ , ] , to carry the gas away to be exhaled in respiration. this type of shunt is, probably, the base for the known excretion of n gas under certain circumstances (high arginine, high protein) resulting also in excess ammonium in the mitochondria [ ] . this uncharted metabolic path is affected by diet, sex, and excess energy and amino-n availability [ , , ] , and constitutes an example of our extremely low level of knowledge on the mechanisms and factors that govern substrate energy partition. this uncertainty affects seriously any quantitative study on this subject. we need (and thus maintain) reserves of all possibly scarce materials we need: essential metals, ions, sulfur, selenium, some vitamins, and especially -amino-n. evidently, we also store energy. the balance between reserves, their type and the problem of carrying them as part of our body weight has been extensively studied, but often, we tend to maintain excessive long-term reserves of lipid ( c n ). we retain a lower amount of c n reserves (glycogen) for hypoglycemic emergences, and we also keep a small supply of phosphagens for immediate support to the first line of response (atp). metabolic syndrome (ms) and similar conditions related to excess of energy supply create havoc with the management of reserves, since the unmanageable excess of c tends to end as lipid, which is massively stored for lack of alternatives. this problem derives largely from our taste for fat [ ] (inherited from our ancestors because of its high energy density), perhaps helped by our ingrained memories of lactation [ ] and the availability of "tasty" lipid-rich foods [ ] ; but it is also derived from an unbalanced supply of energy (especially in excess) of c vs. c as a main energy staple [ ] . our main (from a quantitative energy content point of view) physiological reserves are made of fat because wat contains up to % tag (in the range of mj/kg) [ ] ; thus, a few kg of wat may provide enough energy for our basic sustenance for months (table ). its main advantage is the high energy densities of fat and the adipose tissue holding it, especially when compared with polysaccharides. our reserves of glycogen are small [ ] and were devised to sustain glycaemia for a short time, since glycogen contains about half the energy than fat (on a dry weight comparison basis). in addition, it is highly hydrophilic and retains a large amount of water (added weight, limiting its accumulation in significant amounts) [ ] . a high weight (including water) with respect to energy content limits movement excessively in exchange for short-lived reserves; thus, the main reserves, the more effective in terms of energy/weight are lipids. however, their real effectiveness depends on the body mass of protein [ ] . [ ] . the data in the a part of the table were taken from frayn [ ] , modified with results of [ ] and including unpublished data in the calculations. we used standard body weights for men ( kg) and women ( kg) to homogenize the data. we assumed that from the whole body protein, only about % may be used (allowing recovery) under starvation to cover the body's energy needs. this datum was calculated from a study of long-term food deprivation in rats [ ] . similarly, an arbitrary limit of % minimum of body fat (i.e., not available for energy mobilization) was introduced in the calculations for lack of direct references. no information has been added with respect to more "immediate: energy reserves, such as phosphagens (e.g., creatine-p) and atp because of their low global energy entity in comparison with the three main storage pools described here. the data in the part b of the table were calculated only for plasma (i.e., estimated from blood volume minus blood cells, using normal standard concentrations, and a hematocrit value of . here, instead of referring the substrates to their energy content, we used molar concentrations, since they were the primary data. the main theoretical problems posed by body fat reserves are essentially two: a) its use as storage of energy may derive into being a c dump when energy intake is excessive, driving to obesity, inflammation and ms [ ] ; and b) we need, specifically, glucose/ c for inter-organ supply of energy. thus, sources of c should be taped to preserve our homeostasis under conditions of severe scarcity; even when there is a large excess of c reserves available, we keep needing a steady supply of c, which often requires the sacrifice of protein [ ] . in practical terms, the main c "reserve" of the body are the proteins (irrespective of their functionality). however, since we do not have protein reserves as such, we have had to rely on misadjusting the overall turnover rates to degrade faster and synthesize less protein [ ] , draining the amino acids freed to generate c, especially during starvation [ ] . the data shown in table are only general approximations, but the figures of storage substrates and circulating compounds used with different intensity as nutrients throughout the whole body illustrate clearly the enormous difference between the make-up of c substrates and the c ones that may be used for functions other than the generation of energy through the tca cycle. the c/ c ratio for all added-up energy reserves in normal weight healthy adults is about -fold higher when considering body reserves than when we analyze the nutrients carried by blood plasma. this highlights the differences (even with all logical caveats considered) between what is the homeostatic internal medium composition, akin to our immediate substrate supply needs, and what we have retained. the weight of c is overwhelming. in table , we have intended to obtain a value of the c /c ratio in the human diet. the problem is the absence of data (especially on protein composition of the enormous varieties of diets) we analyzed the c /c ratio for a "theoretical" adult healthy human eating a standard diet, such as those being recommended for a "healthy nutrition" by most specialists. the results were again shocking: the final ratio of the standard diet was in the range of / th of that of our "normal" body composition, and it was more than five times higher than the plasma nutrient ratio. these differences stress an innate deficit of c yielding substrates, necessary to maintain energy homeostasis with respect to the energy stores, strongly decanted toward the c supply, making a large part of these reserves not physiologically usable and, in fact, only "dead weight". this unchecked domination of c sources hint that our standard diets are probably inadequate, chosen more because of factors different from the best metabolic adequacy (taste, availability, social or historical factors, etc.). the recommendation of such types of diet adds to the problem they pose, since recommendations are based on population statistics with no 'controls' to refer to. they are not based on sound biochemical metabolic comparative data, simply because they have not been (sufficiently) investigated. the consequences are crystal-clear: a growing pandemic of diet-borne disorders, essentially undiagnosed, not treatable and affecting a large (and growing) proportion of our society. we used standard consensus-recommended diet compositions [ , ] adapted for a kg healthy adult man, assuming his body weight remains without changes (goals expressed by the who and efsa in the references cited above), that is with no accrual neither losses. the intake was adjusted to . mj/d ( kcal/d or w). obviously, the distribution in final metabolites for protein may be subjected to wider changes than lipids and carbohydrate, depending on the protein sources of the diet. we have not found published data for population-wide analysis of amino acids for diet protein composition related to complete and time-sustained normal human diets. in order to get an approximation to the sought data, we used instead data for rat cafeteria diet of previous studies from our research group [ , ] , on the assumption that the "cafeteria" diet was devised to mimic the usual consumption of food of young humans late in the past century in urban westernized settings [ ] . in the rat cafeteria diet used, the amino acid residues had a mean mw of . it is important to add an additional commentary on the methodology we use at present with diet (food) analyses. despite the considerable difficulties, assumptions and techniques available, the classical study of atwater [ ] remains the "current" basis for our energy intake/expenditure calculations. in decades, science has changed considerably, and precision is one of the critical points for accepting such widely used data. a recent critical analysis of the methodology used by atwater but applying present-day criteria [ ] showed that a significant deviation of the original data exists, and that we urgently need methodology more adapted to today's conditions. the dynamic situation of lipid reserves is clear and understandable. what is not is the reason why some body fat seems impossible to mobilize even using very-low energy diets [ , ] even with metabolic situations close to starvation, which resulted in scant (if any) mobilization of part of the fat reserves [ ] , often in spite of real body weight losses (water, protein, minerals). the extra weight and the inflammation accompanying the excess of adipose tissue fat are severely detrimental for the health and normal energy handling, and are maintained without any apparent reason [ ] . our energy metabolism overall can sustain, in a large proportion, the substitution (as energy substrates) of part of carbohydrate ( c-givers) by lipid ( c-providers). carbohydrate may be compensated in part by protein [ ] . however, the n moiety of protein and some amino acids could not be substituted (but we can lower our needs by modulating turnover rates) [ ] . these compensations include the use of internal reserves (mainly lipid) but also, in part, the lowering of the body protein pool [ ] . these measures do follow well-established priorities in the use of energy substrates (or, in the end, any substrate) ingested, and, when insufficient, compensated with the use of reserves [ ] . the order of precedence in consumption (that of preservation is just the reverse) [ ] is essentially: lipid ( c)→ carbohydrate ( c)→ protein (amino n) diet takes precedence in their use for energy over reserves, but changes in the diet may modify some of these evolutionary rules of precedence, thus protein is needed for lipid oxidation and to prevent its storage [ ] . these mechanisms were developed, and refined with efficient regulatory mechanisms, to enhance survival by maintaining energy homeostasis under conditions of insufficient food availability (including starvation), and incorporating the hypothesis of thrifty genes and their possible incidence in the development of diabetes/obesity [ , ] . however, we have not established a metabolic process (spendthrift) to cope with a simultaneous excess of energy and of all main nutrient classes in the diet. no process has evolved to solve this situation because this is a peculiarity, affecting practically only our species, which appeared in a very short time (in evolution terms) [ ] . the trend is fueled in part by the setting of epigenetic traits favoring the development of obesity, despite its real deleterious effects [ , ] . we have no historical evolutionary memory on how the problem of excess can be handled, since our tools are useful only to counter scarcity, and thus, the mechanisms/processes applied are often not only inadequate, but self-defeating and harmful. we ingest food for energy and growth/maintenance. evidently, a large part of the main substrates (amino acids, fatty acids, sugars and intermediate metabolites, as well as most essential components) are used in connection with turnover strategies of our own living matter, in order to maintain all mechanisms in their prime efficiency. however, from a theoretical (quantitative) point of view, intake should match excretion and energy loss (assuming that there is no real change in our mass) [ , ] . thus, the main types of substrate we ingest for energy (with all provisos stated above) belong to three groups: amino acids (n providers), c and c providers. after a large portion of amino acids is sieved and selected, they revert largely to "excess n" plus c, c and c (tca cycle intermediates) (figures , and ). the final proportion of c and c available during a certain stretch of time depends on digestion, rhythms etc., but we can just center the question in basic one-day periods. under no-growth conditions (i.e., no additional storage of energy: only turnover) almost all c will end as a source of energy via tca cycle. c have a higher variability of uses, but at last, all excess c is converted to c, which is then oxidized for energy (or stored as fat). if there is a large excess of amino acids, the elimination of n stresses the c/ c setup, both because of anaplerotic enhancement of tca cycle (via c) and an intense pressure for growth, which increases protein synthesis and "storage" as body protein. most of these effects/mechanisms remain rather unknown, because almost never come alone, and are largely mediated via gene expression and hormonal modulation affecting select different groups of cells. a large part of nutrients is used with little change (if any) for turnover (e.g., amino acids, fatty acids, glucose, etc. for, respectively, proteins, tag or glycogen), or to build-up reserves (i.e., fatty acids) or (almost all compounds) for growth and reposition of live matter lost (i.e., hair, epithelial cells or secretions). amino acids could not be substituted from the point of view of reposition and turnover, as well as for the synthesis of other n-containing body components. carbohydrates may be substituted, largely, by protein, but the loss of energy in the complex catabolism of some amino acids, plus the need to dispose-of -amino n markedly limits the extent of this substitution. on the other side, lipids (e.g., tag, but not including the essential fatty acids) can be entirely substituted because their sole specific metabolic contribution is to provide large amounts of c, which we are prepared to compensate with protein and, essentially carbohydrate. an excess of c, on the other side, limits the utilization of dietary fatty acids, which are then incorporated to the stored fat reserves, without previous oxidation to c [ ] . this is an efficient way to store dietary energy, quite different from the energetically expensive lipogenesis from acetyl-coa [ ] . this c molecule is also in excess (limited by the availability of coenzyme a because of insufficient capacity of the tca cycle to oxidize it, since reducing power/atp are produced on demand. this is compounded by limited lipogenesis, restrained because of the excess of dietary fatty acids [ ] . the large acetyl-coa availability, due to its limited need, inhibits the oxidative decarboxylation of pyruvate, the critical c→ c unidirectional process [ ] , thus secondarily provoking an unwanted accumulation of c. in parallel, glycolysis is also limited because of insufficient c removal [ ] , which results in lower glycolytic processing of c, which adds to the excess carbohydrate provided by the diet together with the unneeded excesses of dietary amino-n and fatty acids (figure ). acids [ ] . the large acetyl-coa availability, due to its limited need, inhibits the oxidative decarboxylation of pyruvate, the critical c→ c unidirectional process [ ] , thus secondarily provoking an unwanted accumulation of c. in parallel, glycolysis is also limited because of insufficient c removal [ ] , which results in lower glycolytic processing of c, which adds to the excess carbohydrate provided by the diet together with the unneeded excesses of dietary amino-n and fatty acids (figure ). figure . partition-related substitution of nutrients in the diet to fuel the metabolic processes. this graph intends to explain the absence of a total/complete substitution capability between the three main groups of nutrients: protein, carbohydrate and fat, which constitute most of our diet, at least from an energy point of view. solid lines show the direct relationship between the groups of figure . partition-related substitution of nutrients in the diet to fuel the metabolic processes. this graph intends to explain the absence of a total/complete substitution capability between the three main groups of nutrients: protein, carbohydrate and fat, which constitute most of our diet, at least from an energy point of view. solid lines show the direct relationship between the groups of substrates. the large red line emanating from c represents the oxidation of acetyl-coa in the tca cycle to obtain most of the energy we use. the thinner red dash-lines indicate other main sources of cell atp, adding up to the sum of energy available to cover our needs. part of our food is made up of other components (minerals, organic micro-components, fiber, etc.). their relationships with other compounds are marked with purple lines. sobc stands for: "synthesis of other body components" from the building blocks provided by the four groups of nutrients analyzed. their mixed paths have been marked in black. the lines in blue represent the metabolism of carbohydrates, including the part shared with amino acids and lipid. protein-amino acid paths are marked in green. the lines in dark yellow represent the paths exclusive of lipids. the serious difficulty lies on the iterative nature of the process along time. a few days of excesses can be modulated in part by decreasing appetite and voluntary intake [ ] , increasing protein turnover [ ] , thermogenesis [ ] and topping the reserves of glycogen and fat [ ] . nevertheless, these 'solutions' work only to a certain degree, not in a permanent way, and create a regulatory havoc that cannot be sustained indefinitely. the emergency measures only patch, do not solve the problem, it is just metabolic procrastination, and in short time deep problems begin to develop [ ] ; and shortly afterward, they also become chronic [ , ] . the excesses of substrates could not be disposed of, and the regular mechanisms of partition cannot function in a fluid way. obviously, oxidation of c is enhanced, but what to do with the unused atp? in a global setting, thermogenesis helps, but not at the level of each cell (largely those in the splanchnic bed). then, the priorities list ingrained in our metabolic control systems retake their place of reference. save as many amino n as possible and take special actions to prevent the toxicity of nh , perhaps via the alternative "nh + no·" production of n . • limit the processing of fatty acids to c fragments and thus dump them as tag (with a token c glycerol) [ ] . this process induces obesity [ ] , steatosis [ ] , and drives muscle to be essentially a fat-infiltrated c energy user, with glucose intolerance [ , ] . the toxicity of glucose (the same occurs with the excess of ammonia) represents an immediate danger that cannot be "stored" (as are the fatty acids in tag). the insulin basic controlling mechanisms of glycaemia no longer function properly [ ] , in fact they interfere, limiting the peripheral utilization of glucose as substrate [ , ] . hyperglycemia is a problem [ ] which is in part corrected by the conjunctive tissue (including adipose tissue), which actively takes up glucose and returns c to the blood [ ] . most tissues may work (including the brain) perfectly using c (largely lactate and glycerol) [ ] [ ] [ ] ] . thus, the blockage of liver metabolism, in its ↑ c-↑ c no-win situation, is in part corrected by an adipose tissue (also inflamed, engrossed by the excess lipid storage [ ] ) which generates large amounts of c at the expense of glucose [ ] . it also produces urea cycle intermediates, probably to compensate in part for the decreased capacity of the liver [ , ] . return to normalcy from the excess-driven picture of energy metabolism is difficult. the search for drugs is not a viable solution, because the problems observed have a known origin, and the severity of the damages is largely a consequence of the mechanisms established to ensure survival under scarcity. we cannot fight these mechanisms because a) we do not know them enough, b) we need them to sustain our "normal" homeostasis, and c) they have to be operative to achieve that normalcy. right now, the main systems in use to fight obesity are-essentially, and despite their limited effect-diet and exercise; but, what type of diet? hypocaloric? under starvation, the stored fat is progressively shed to last as much as feasible; our bodies adapt to lower energy, lower carbohydrate and limited protein intake diets, including the absence of fats, following the blueprints for starvation [ ] . the elimination of dietary carbohydrates [ , ] and lowering of energy intake share some characteristics, because of our adaptability to starvation, but there is a considerable discussion on the proposed benefits of ketogenic diets, which, in any case, could not be generalized from epilepsy to obesity [ ] [ ] [ ] . nevertheless, even after prolonged starvation or removal of dietary carbohydrates, a significant portion of body fat remains, even after prolonged exposure. unfortunately, in the practice and for most obese people, dietary treatments remain not effective [ ] . exercise is an alternative to increase energy expenditure with (or even without) restricted intake [ , ] . this approach is not always feasible, since obesity is syndromic with cardiovascular disease and problems of mobility [ ] and, despite numerous claims on the contrary, exercise it not sufficient to revert the damages of an established ms [ ] , and may not improve the health status [ ] . however, well dosed exercise may provide benefits to mild cases and young patients [ ] . in addition, the effectiveness of exercise is also limited from the point of view of energy: limited duration, limited proportion of increase in overall energy consumed, consequences in the availability and transport of oxygen and products of substrate oxidation. in fact, exercise is better suited to maintain functionality [ ] (when applicable) than to fight obesity. the chronicity of the "excess situation" described above, usually continue even when the dietary excesses cease. too often, very low-energy dieting and exercise combined (even using additional anti-obesity approaches) are unable to eliminate the excess fat and reverse the damages already induced by (or accompanying) this excess deposition. the size of fat depots is a critical point for the severity of the disorders and a barrier to a progressive and effective removal, often leaving only the alternative of surgical modification of nutrient intake [ ] . however, none of these procedures can revert the metabolic homeostasis to the situation prior to depot engrossment. nevertheless, drastic treatments may extend the patient life span, improve the lifestyle and ameliorate a few components of the ms [ , ] , but not cure it or its associated pathologies. the corollary to this exposition is our extreme resilience to change in the energy (and nutrient) equilibrium, which allows us to cope with the situation for a time and then alter, in a probably irreversible (and unknown) way, the common mechanisms for nutrient partition in a situation far from normal, which could not be sustained indefinitely. however, these changes allow a number of affected individuals to keep living despite the severe damages to metabolic processes and their control. a deeper layer of thought (and thorough research) are needed to understand how people can survive decades under ms conditions. probably, excess body fat is directly related to the severity of the disorder it causes: it is commonly accepted that the body mass index vs. mortality curves are u shaped [ ] . however, this index is not a real indexed measurement of the body fat content, and parallel studies analyzing body fat vs. mortality showed a lineal direct relationship [ ] . nevertheless, a certain degree of obesity helps protect the patient from some cardiovascular diseases [ ] . the discussion about the causes of the obesity paradox or even its existence continue, but part of the problem may lie on the inadequate use of body mass index to "measure" obesity [ ] , and the question of whether obesity is by itself a disease (or simply a part of a more severe disorder such as ms). the repeated references to "healthy obesity" (i.e., large body fat stores without the associated metabolic disorders of "proper" obesity) [ ] , have been relatively circumscribed by introducing factors such as age and gender [ ] . our group has postulated that a certain degree of obesity may help limit the ravages of type diabetes by removing excess glucose and releasing c, being more a (palliative) consequence than a cause for this disease [ , ] . in any case, the question is not settled, and perhaps there is also a margin for optimal (and not rigidly low, invariable and universal) mass of body fat reserves other than the one used at present. under conditions of excess energy intake, the metabolic handling of substrates is parallel to modifications in the hormonal mechanisms that regulate energy metabolism [ ] , since the main system of regulation of glycaemia (insulin) has been severely damaged [ ] . the other mechanisms complementary to insulin have a wider array of functions: the glucocorticoids, favor liver glucose output under conditions of stress or metabolic distress [ ] . glucocorticoids also affect the fate of the main gluconeogenic precursors, amino acids, and altering the excretion of n [ , ] . testosterone pairs with insulin as a main anabolic hormone [ ] , but glucocorticoids tend to limit testosterone production and availability [ ] , also affecting the availability of estrogen [ ] , which plays a critical function favoring the oxidation of c (i.e., saving c) in females [ ] via direct intervention in mitochondrial function [ , ] , and also preventing liver steatosis [ ] . an estrogen-derivative has been found to down-modulate the adjustment of the ponderostat, i.e., the oxidation/mobilization of lipids from adipose tissue [ , ] , by decreasing food intake and maintaining thermogenesis [ ] . unfortunately, there is insufficient mechanistic information on the effect of estrogen derivatives because only recently these hormones are considered important metabolic regulators [ , ] and act not only in the sex-related way indicated by its etymology. substitutive testosterone treatment normalizes the glycaemia of mature and old men, a situation that is slowly being accepted by endocrinologists (diabetologists, in fact) [ , ] after being a mainstay of the treatment of ms aging-related disorders by gerontologists [ ] . its soothing effect on the ms ravages [ ] is complemented by the already known effects of estrogen on energy partition [ , ] , limiting the storage of lipids. most of present-day studies on obesity (and of ms, as an afterthought) are focused on cytokine and regulatory circulating rna types [ ] and the paths they modulate, which add considerable knowledge on the possible regulation of their mechanisms of action, but not on the causes/mechanisms of the disorders or the ways to prevent and treat them. figure shows a simplified scheme of the main hormone-controlled mechanisms regulating glycaemia. partition [ , ] , limiting the storage of lipids. most of present-day studies on obesity (and of ms, as an afterthought) are focused on cytokine and regulatory circulating rna types [ ] and the paths they modulate, which add considerable knowledge on the possible regulation of their mechanisms of action, but not on the causes/mechanisms of the disorders or the ways to prevent and treat them. figure shows a simplified scheme of the main hormone-controlled mechanisms regulating glycaemia. in the liver, glucocorticoids increase glucose output [ ] and favor lipogenesis [ ] and tag deposition in most tissues [ ] ; testosterone induces the accrual of protein [ , ] and stabilizes the maintenance of glycaemia [ ] . estrogens favor c oxidation [ ] , increase oxidative metabolism in mitochondria [ ] , and limit lipogenesis and tag deposition [ ] . the role of these steroid hormones on the direct modulation of glucose is less clear, despite the large number of agents and effects uncovered. we already know a part of the puzzle, but there are not yet enough dots to draw a sufficiently clear line to understand their real function and help us fight the ravages of our own (effective) systems of protection of energy and protein. in the liver, glucocorticoids increase glucose output [ ] and favor lipogenesis [ ] and tag deposition in most tissues [ ] ; testosterone induces the accrual of protein [ , ] and stabilizes the maintenance of glycaemia [ ] . estrogens favor c oxidation [ ] , increase oxidative metabolism in mitochondria [ ] , and limit lipogenesis and tag deposition [ ] . the role of these steroid hormones on the direct modulation of glucose is less clear, despite the large number of agents and effects uncovered. we already know a part of the puzzle, but there are not yet enough dots to draw a sufficiently clear line to understand their real function and help us fight the ravages of our own (effective) systems of protection of energy and protein. man's main energy staple has been, in the last millions of years, of plant origin: seeds, starchy roots or other plant energy reserves, fruits, leafy foods, complemented with some products of animal origin. humans have evolved in parallel to their nutrition [ ] . foods from animals have been increasing their share with time [ ] , and our diets are clearly adapted to our pre-human (but pervading) basic food taste types. less exercise, better medical attention, longer lives and control of the sources of food have modified our present-day diets. culture, unproven ideas about food have been combined with religious or social taboos, increased availability of different presentations of food and the culture-driven extreme cult to taste. we are leaving starches to consume fats [ ] , and animal muscle protein and collagen instead of aleurone-like plant reserves [ ] . however, our digestive system and metabolic energy handling systems were adapted to a quite different diet [ ] , and have not been able to maintain the pace of change in eating habits occurring in the last centuries [ ] .thus, a gap between digestive system/nutrient utilization and diet composition exists and keeps growing. glucose continues being the main energy substrate, in spite of everything else, but its food form, ingestion, digestion, handling, and oxidation necessarily change with the composition of the diet. however, that of neanderthals showed little differences with our present day optimal diet types [ ] . the periodicity, the ratio of energy consumed (or needed) for a straightforward metabolic equilibrium versus that actually ingested is a powerful destabilizing factor for an effective maintenance of metabolic equilibrium. activity, feeding, light and internal rhythms control our hormones and metabolism, helping us to adapt to the variable conditions of the environment [ ] . probably this imbalance is a main cause of the ms together to the fattening of the diet [ ] . considerable observation and experimentation have shown that amino-n should be a part of our diet (in the range of - % of ingested energy) [ ] ; and, also, that protein should be ever present in our meals [ ] . this implies that we also need to ingest an adequate supply of a few special amino acids we cannot synthesize [ ] . in parallel, we also know that, today, most humans are taking in too much fat in their diets [ ] , often over % of all energy ingested [ , ] . an effect compounded by the ingestion of energy above our physiological needs [ ] . there is little quantitative analysis of necessities, amounts and proportions, as explained in detail above. these approximate proportions are quite different from those of all present-day apes, even more than most of the other primates [ ] . this comparison should include our earlier human ancestors, usually short in protein, and even shorter in lipids. hints on the postulated hunter-gatherer ancestors feeding standards and behavior, and the study of human dietary variability at present suggest that we are a remarkably adaptable species [ ] , but in all cases, c takes priority over c, often with the help of the highest protein intake of all primates [ , ] . evidently, within the enormous variety of human groups and their adaptation to all types of diet (the food available) and environmental conditions, it is expectable that a wide variety will be found -also-in the differential use of the main nutrients; provoked, in general, through epigenetic-driven adaptation to the foods available [ ] . but most humans not living under extreme conditions maintain diets which, despite the ample variability in foods and their proportions, provide enough amino-n, c and c for healthy survival [ ] (figure ). the problems arise when the carefully established compensatory mechanisms for diets markedly deviate from those of our ancestors, which result inoperative or deficient by excess of energy and, especially, of some nutrients [ , ] . it seems that the paleolithic (including ancient historical times) physiological blueprint has evolved little in regulation, despite our admirable adaptation, to occupy almost any ecological niche conceivable on earth, and thus, diet-provoked "inflammatory" diseases appear and remain unchecked [ ] . variable food supply, including in some cases its excesses and early death (for almost any cause imaginable including diseases) shortened the lifespan of our predecessors already in historical times [ ] . notwithstanding, in the present, our main health dangers seem to be related to our "excessive" success in providing food and health care to a large proportion of humans, coupled with an insufficiently fast evolutionary pace to let our bodies to adapt to this, so far, unique success in the extension of a species mean lifespan and its consequences [ ] . enter the problems of aging: until recently, a problem affecting a very small part of developed societies, that became a prime cause of health, lifestyle and social problems because of the success in expanding our longevity [ , ] . despite all that, we have not established (in times of plenty) an estimation of the magnitude of c-precursor nutrients we need to consume. essentially the question is: do we need a minimum intake of carbohydrate? [ ] . glucose is the main energy staple of our diet, and we base on c substrates most of our metabolic function and regulation [ , , ] . instead, we centered our attention on lipid ( c sources) [ ] [ ] [ ] , sugars as such [ ] , some minerals (e.g., salt), and, to a limited extent, on protein [ ] . it is unclear why our flag substrate, glucose, has not been given due importance except as a marker of disease. however, the pervading idea of almost any food-derived nutrient contributing effectively to our energy sustenance, with (almost) full substitution possibilities (as energy fuels) between carbohydrates, fats, protein (and even alcohol) is deeply rooted. this belief would render superfluous to analyze if there were a dietary minimum supply of c- c substrates, in the way we know that this minimum exists for amino-n and "loose" maximums are generally accepted for lipids and alcohol (a toxic substance and "pure" c). from the data available, the quest for lowering glycaemia and reducing body weight [ ] favor the use of low carbohydrate ketogenic diets [ ] . however, high-carbohydrate diets improve glucose metabolism [ ] in healthy individuals, and restriction of diet carbohydrates increases the risk of cardiovascular disorders, depending on the diet energy and composition, especially in patients with disorders of glucose metabolism [ ] (but not in healthy individuals [ ] ). not even glycaemia is better regulated in diabetics with excessive dietary carbohydrate restriction [ ] . the disarrangement provoked by the relative insufficiency of carbohydrate in some "low-carbohydrate" diets depends, essentially on four factors: (a) the metabolic "basal state" of the patients, since the results depend on gender, age and the incidence of ms-related disorders. (b) the overall energy intake with respect to the needs (or slimming goals) of the patient. (c) a diet low in carbohydrates must get the energy from either protein or lipid, the proportion and structure of these nutrients in the diet affects the incidence of lowering carbohydrate intake. (d) the type of carbohydrate present in the diet and its food associations, essentially its relationship to fiber and the mean molecular weight of the polysaccharides (including their digestibility and effects on the microbiota). the enormous variation of results, consequences, and effectiveness of the proposals coupled to the soundness of the results from the data published, results in a near-impossibility to draw conclusions. in addition, almost all diets studied have been applied for short periods of time to small numbers of individuals with scant information even for the point a) and were done in 'comparison' with other different types of diets. even the reviews and meta-analyses could not draw clear consequences from the huge amount of literature accrued on this question. there are too many factors to allow us to draw safe conclusions from partial, incomplete and not superimposable data. to face this type of analysis, a much-needed simplification is required, and reserve on the conclusions (and on their application to people) must be observed. diet studies largely analyze the consumption of food items (a huge, varied and socially peculiar group of products), which in addition have been cooked and mixed in varied proportions. this is "reduced" to the nutrients they contain, but this may forsake aspects such as synergisms, food structure (i.e., fiber), and the nature of the nutrient itself (fructose is as carbohydrate as amylose or a resistant-starch, for instance, and collagen-rich squid is as protein as gluten). we know what is eaten but not what is assimilated (the variable microbiota plays a critical role on this process). we need more information on-at least-what is ingested along longer periods of time. in any case, a multilateral approximation to the data known and the line of thought presented here allows us to assume that a minimum (both in proportion and in absolute terms) of daily dietary carbohydrate does exist [ ] , despite the possibility to substitute a large portion of the c intake by other sources of c. there is abundant literature but a dearth of information on diets. unfortunately, when dealing with the concept of diet industry, policy, fashion, absurdity and taste interact to produce dispersion, unproven assertions, expanded private interests and outright lies, with-unfortunately-scant material usable to advance scientific knowledge and improve the preservation of health. in most of the studies available, an enormous variability in all the factors implied can be observed, and the formal and bona fides results rarely go further than "hinting to," with excessive room for interpretation and discordance (or even actual overall relevance). the most recent and popular case in point is the mediterranean diet, which, in fact, has not been yet defined in nutritional terms [ ] but has generated, nevertheless, a considerable number of studies on its health benefits [ , ] . why do relatively small modifications in diet may induce so deep changes or no changes at all in functional parameters? we do not yet know enough to obtain a defendable and plausible answer. on one side, the nutritionists and dieticians usually analyze diets as a whole, use tables and measure our anthropometric (and metabolic) data and detailed food intake to calculate the needs of nutrients (translated to foods) to cover the energy expenditure, despite the fact that both factors are closely linked. on the other side, a growing number of molecular biologists analyze the specific mechanisms of control of endocrine and paracrine factors (largely those easily measurable), usually using isolated cells or cultures, since this approach is not viable in most in vivo models. both worlds of study are eons apart, and keep publishing large amounts of deep, sometimes excellent studies on "new" signaling factors or mechanisms, and the beneficial properties of some foods. our scientific careers have passed through different consecutive fashion periods: enzymes, hormone receptors, cyclic nucleotides, gene analysis, cytokines, gene expression, micrornas, gene modification, exosomes, stem cells, the transcendence of microbiome, "virome/pandemic" etc. however, surprisingly nobody has devoted sufficient time and funds to study such elementary "unknowns" as how "essential" are some amino acids (and which are the paths of their complete catabolism in humans), how the excess n is eliminated (signals, pathways, mechanisms, sites), or how much c (largely glycosyl monomers) we need to ingest daily? what is our dependence of c as energy substrates? how is regulated the utilization of c/ c for energy (not only in a type of cells, but overall)? where does this take place? why there are not enough quantitative studies on how much lactate or glycerol uses the brain, muscle or heart? is wat a real storage depot or a secondary supplier of c from excess glucose during hyperglycemia? is insulin the main hormone controlling use of glucose by tissues? what is the role of steroid hormones in the control of glycaemia? and so on. perhaps the plausible results of studies trying to answer these questions do not sound "flashy" enough for promotion, or perhaps they are not sufficiently "safe" to merit publication in "prestige" journals, but we need to carry on "risky" studies to get the answers we need in order to understand real problems such as how the substrates provided by the diet are distributed and used. we also need them to understand and fight the negative effects of disorders that right now affect large portions of humankind. right now, it is critical to obtain much more basic (and critical) knowledge to understand the nature (and causes) of "inflammation" (obviously, not that defined by celsus!) that is often used to justify everything that is wrong in ms and related disorders. funding: this study received no funding, and was in part financed by the researchers themselves, colleagues and the sparse funds available from 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features of metabolically healthy obesity phenotype prevalence early endocrine and molecular changes in metabolic syndrome models longitudinal study of the effects of a high-fat diet on glucose regulation, hippocampal function, and cerebral insulin sensitivity in c bl/ mice regulation of glucose homeostasis by glucocorticoids glucocorticoids and acidosis stimulate protein and amino acid catabolism in vivo molecular regulation of urea cycle function by the liver glucocorticoid receptor testosterone supplementation improves insulin responsiveness in hfd fed male t dm mice and potentiates insulin signaling in the skeletal muscle and c c myocyte cell line do the interactions between glucocorticoids and sex hormones regulate the development of the metabolic syndrome? front glucocorticoids antagonize estrogens by glucocorticoid receptor-mediated activation of estrogen sulfotransferase estradiol determine liver lipid deposition in rats fed standard diets unbalanced with excess lipid or protein regitz-zagrosek, v. reduction of apoptosis and preservation of mitochondrial integrity under ischemia/reperfusion injury is mediated by estrogen receptor beta estrogenic control of mitochondrial function estrogen treatment after ovariectomy protects against fatty liver and may improve pathway-selective insulin resistance oleoyl-estrone is a precursor of an estrone-derived ponderostat signal rats receiving the slimming agent oleoyl-estrone in liposomes (merlin- ) decrease food intake but maintain thermogenesis impaired estrogen receptor action in the pathogenesis of the metabolic syndrome estrogen and voluntary exercise attenuate cardiometabolic syndrome and hepatic steatosis in ovariectomized rats fed a high-fat high-fructose diet the impact of testosterone replacement therapy on glycemic control, vascular function, and components of the metabolic syndrome in obese hypogonadal men with type diabetes effect of testosterone replacement therapy on sexual function and glycemic 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epigenetics in the relationships between nutrition and health the metabolic syndrome: prevalence in worldwide populations origins of major human infectious diseases evolution of the human lifespan and diseases of aging: roles of infection, inflammation, and nutrition an upper limit to human longevity? is dietary carbohydrate essential for human nutrition? effect of acute and short-term dietary fat ingestion on postprandial skeletal muscle protein synthesis rates in middle-aged, overweight, and obese men preference for dietary fat: from detection to disease how dieting might make some fatter: modeling weight cycling toward obesity from a perspective of body composition autoregulation high-sugar diet leads to obesity and metabolic diseases in ad libitum-fed rats irrespective of caloric intake adéquation de l'apport en protéines et acides aminés dans les régimes végétariens a randomized trial of a low-carbohydrate diet for obesity a review of low-carbohydrate ketogenic diets a mediterranean and a high-carbohydrate diet improve glucose metabolism in healthy young persons low-carbohydrate ketogenic diets, glucose homeostasis, and nonalcoholic fatty liver disease effects of a carbohydrate-restricted diet on emerging plasma markers for cardiovascular disease systematic review and meta-analysis of dietary carbohydrate restriction in patients with type diabetes concepto de dieta mediterránea: ¿un grupo de alimentos saludables, una dieta o una panacea publicitaria? [the mediterranean diet: a group of healthy foods, a type of diet mediterranean diet and risk of falling in community-dwelling older adults mediterranean diet and health: a systematic review of epidemiological studies and intervention trials key: cord- -ugd kkor authors: giles, ian g. title: a compendium of reviews in biochemistry and molecular biology published in the first half of date: - - journal: international journal of biochemistry doi: . / - x( ) - sha: doc_id: cord_uid: ugd kkor abstract . . a compendium of reviews and mini-reviews in biochemistry and molecular biology published in the first half of is presented. in all titles are listed from different publications. . . this compendium presents the references by journal name. keywords have been included with each reference to increase the value of the collection. keyword and author cross-reference indexes are not included but are available in the electronic database from which this version was constructed. should anyone wish to have this information in electronic form it can be distributed on ms-dos formatted flopppy disks in either reference manager or medline format. the author should be contacted for details of the number of preformatted floppy disks required. krasikov n., thompson k. and sekhon g.s. ( ) brief clinical report-monosomy q . + . -a recognizable aneuploidy syndrom~report of a patient and review of the literature. am. j. med. geti. . - . verloes a., mulliez n.. gonzales m., laloux f.. hemutnnsle t., pierard g.e. and koulischer l. ( ) restrictive dermopathy, a lethal form of arthrogryposis multiplex with skin and bone dysplasiac new cases and review of the literature. am. j. med. genet. , . aplasia cutis ccugenita; pyloric atresia, newborn; sibs. leonard c., huret j.l., imbert mc., lebouc y., selva j. and boulley a.m. ( ) trisomy- p in a liveborn offspring due to maternal translocation t( - )(ql l-p ) and review of the literature. am. j. med. gene; . , [ ] [ ] [ ] [ ] [ ] spontaneous abortions; handing teclm' tques; duplication ~; infant; segregation. xie l.q.. markides k.e. and lee m.l. ( ) biomedical applications of analytical supercritical fluid separation techniques. anal. biuchem. u)o, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] chtumatography-mass spectrometry; amino-acid derivatives; gas-chromatograph stationary phase; chargeexchange; silica-gel; extraction; glycosphingolipids; resolution; interface. hozier j.c. and davis l.m. ( ) review-cytogenetic approaches to genome mapping. anal. biochem. , . chaiken i., rose s. and karlsson r. ( ) quantitative analysis of protein interaction with ligands. ( ) analysis of macromolecular interactions using immobilized ligands. anal. b&km. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . neurophysin self-association; affinity-chmmatography; subunit-exchange chromatography; biosynthetic precursor; equilibtium-carstants; peptide recognition; sense pcptides; binding; purification; elution. ichikawa y.. look g.c. and wong c.h. ( ) review-enzyme-catalyzed oligosaccharide synthesis. anal. b&hem. , - . gal-l - + ( )glcnac a- + sialyltransferase; acetylneuraminic acid synthetase; immobilized l -galactosidase; gdp-l-fucose; sialic acids; esckrichia coli; rat-liver, glycoprotein oligosaccharides; carbohydrate antigens; determines expression. gabriel . and gersten d.m. ( ) staining for enzymatic activity after gel electrophoresis-review. anal. bbckm. , sodium dodecyl-sulfate.; ted blood-cells; phosphoenolpyruvate carboxylase activity; nucleotide-linked dehydrogenases; alkaline-phosphatase isoenzymes; pathogenesis-related proteins; cr-l-fucosidase; polyactylamide-gel; produce phosphate; general-method. wood p.j. ( ) the measurement of parathyroid hormone. ann. cfin. b&km. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] cyclic amp; immtmoradiomettic assay; primary hypatparathytoidism; humoral hypercalcemia; calcium huneostasis; intact parathytin; clinical utility; circadian-rhythm; chromogranin a; lung-cancer. newman d.j.. henneberry h. and price c.p. ( ) particle enhanced light scattering immunoassay. ann. clin. b&hem. , . c-reactive protein; human chorionic-gonadotropin; latex agglutination-test; cell-labeled antibodies; shell corn patticles; counting immunoassay; turbidimetric immunoassay; spectroscopic immunoassay; passive hemagglutinatiom luteinizing-hormone. thompson d.. milfordward a. and whither j.t. ( ) the value of acute phase protein measurements in clinical practice. ann. clin. c-reactive protein; erythrocyte sedimentation-rate; inflammatory bowel-disease; tumornecrosis factor, amyloid a protein; tlteumatoid atthritis; plasma-ptoteins; polymyalgia rheumatica; acute-leukemia; tissue-injury; acute phase. soldi s.j. ( ) drug receptor assays-quo vudis. ann. . allen j.f. ( ) protein phosphorylation in regulation of photosynthesis. biochln. bi@ys. ac& la . light hamsting complex; dtlomphyll a; b protein; cyanobncterium syn&ococc~~ ; excitatiarcnergy distribution; absorptial cross-section; ii reaction center. thylakoid membrane pelypaptides; state-l state- tmnsiticns; amino-acid-sequence; randomixed chlomplast iamellae. anthony c. ( ) the c-type cytochromes of methylotrophic bacteria. b&him. biqhys. acta , l- . methylobacteriwn extorquetu am; electron-transport chain; blue copper proteins; oxidation mutant classes; ammo-acid sequence; sp strain aml; paracoccus dcni~rificons; melhylophilvs mdylotrophur; obligate metbylotmpb; m&and dehydrogenase. hoch f.l. ( ) cardi~lipins and biomembrane function. biochim. biophys. acta , - . rat-liver-mitochondtia; fatty-acid composition; brown-adipose-tissue cytochmme-c-oxidaset munbmne lipid-ccmpositiau adenine-nucleotide translocase; age-related-changes; skeletal-muscle mitochond~, chronic ethanol-consum@n; lateral proton conduction. bandekar j. ( ) amide modes and protein conformation. biochim. biophys. actu . - . transforfn-infrared-spsccpy; laser raman-spectroscopy; secondary-structure-analysis; liver ahohd-dehydmgenase; hydrogadeuteriutn exchange; a transmembrane channel; valyl-glycyl-glycine; iii spectral region; vibratiaral analysis, gramicidin-a. b&him. biophys. acta , - . acetyl-coa carboxylase; coenzyme-a reductase; hormone-sensitive l&se; dependent multipmtein kinase; rat-lives; -hydmxy- -methylglutaryl coenxyme; enxymic activity; phosphorylationdephosphorylatiar; hydmxymethylgfutaryl coenxyme; reversible phosphotylation. w&t k. ( ) origins and fates of fatty acyl-coa esters. biochim. biophys. acfu , - . coenzyme a syntbetase; performance liquid-chromatography; rat-liver micmsomcs; acyltransfemse-cataly~ cleavage; dependent transacylation system; rabbit alveolar macropbages; pemxisomal &oxidation; amcbidonic-acid; brain micmsomes; sbott-chain. low-density~l&protein; high-performance liquid; chrcmatogmphy mass spectmmetry; chicken vitellogam gene; thin-layer chmmatography; apolipoprotein-vldl-ii; fatty-acid composition; laying turkey hens; egg-yolk; plasma-lipoproteins. erlansonalbertsson c. ( ) pancreatic colipase-structural and physiological aspects. biuchim. biophys. acta . - . gastric-inhibitory polypeptide, messenger-rna levels; diabetic rats; pro-colipase; co-lipase; tymsine residues; porcine colipase; sequence; taurodeoxycholrte; triglyceride. coleman r. and rahman k. ( ) wehle e. ( ) reiter r.j. ( ) the ageing pineal gland and its physiological conxequences. bioessays , - . malatonin receptors; admoet@c-receptors; circadian variations; n-acetylscrotonin, plasma melatcoin, serum melatouin; hamsters; gerbil; brain; reduction; downward j. ( ) regulatory mechanisms for ras proteins. bioessap , - . gtaase-activating protein; neurofibranatosis type-l gene; nucleotide exchange-reaction; gap-associated proteins; growth-factor; tymsine phosphorylation; ras-p gtpase; stimulation; p ; recepors. rusciano d. and burger m.m. ( ) adamo m., roberta ct. and lemith d. ( ) how distinct are the insulii and ~sul~-l~e growth factor? -signalling systems. biofwtors . - . human-skin fibroblests; igf binding-protein; messenger ribonucleic-acid; cooh-teminal truncation; monoclonal-antibody; endothelial-cells; factor receptoc amniotic-fluid; dna-synthesis; rat-heart. hehnreich e.j.m. ( ) how pyridoxal s-phosphate could function in glycogen phosphorylaae catalysis. biofbctors , - . aron d.c. ( ) insulin-like growth factor-i and erythropoiesis. biofators , - . factor-binding-protein; erythroid colony formation; cultured human-tibroblasts; factor messenger-rna, n-terminal sequence; fetal bovine serum; igf-i; somatomedin c, clinical-applicstians; stimulating factors. silver b.j. ( ) platelet-derived growth factor in human malignancy. biofmtors , - . terminal coding region; c-sis; b-chain. bmis w.d. and durst r.w. ( ) bajpai p. and bajpai p.k. ( ) arachidonic acid production by microorganisms. biofechnol. appl. biockm. . l- . bellomo m.j., parlier v., muhlematter d., grob j.p. and beris p. ( ) three new cases of chromosome- rearrangement in bandq and band-q with abnormal thrombopoiesis bring further evidence to the existence of a q q -syndrome. cancer genet. cytogenet. . - . acute nonlymphocytic leukemia; chronic myelogcnous leukemia; chronic myeloid-leukemia; acute megakaryoblastic leukemia; chronic myelocytic-leukemia; acute myeloblastic-leukemia; british cooperative group; myelodysplastic syndromes; blast crisis; tmnslocation t(l - ). pedersen b. ( ) survival of patients with t(l- )(pl l-p l&report of cases and review of the literature. cancer genet. cytogenet. , - . acute nonlymphocytic leukemir; trsnslccation i- ; myelodysplastic syndromes; chromosome analysis; mycloid disorders; secondary. nossal g.j.v. ( ) the molecular and cellular basis of affinity maturation in the antibody response. cell . - . mutation. thomas g. ( ) map kinase by any other name smells just as sweet. cell , - . protein-kinsse; insulin; identification; muscle. teach r.e. ( ) type- astrocyte developme ciliaty netttotmphic factoc fibmblast growth-factor. retinoic acid xceptor; chick limb bud; tymsine kinaae m eatiy xatqus embryos; pmto-oncogene int- ; activin-a; w-locus. greenwald i. and rubin gm ( ) mellman i. and simons k. ( ) the golgi complex--in vitro verirus. cell . - . asparsgine-linked oligosaccharides; cell-free system; rough endoplssmic-reticulum; vesicular stomatitis-tims; plasma-manbrane proteins; bmfeldin a; cis-golgi; successive compartments; intracellular-transport; n-acetylglucosamine. chao m.v. ( ) hall a. ( ) signal transduction through small gtpases-a tale of gaps. cell , - . proteins. wetr d.z., peles e.. cupples r., suggs s.v., bacus s.s., luo y., trail g., hu s.. silbiger s.m.. benlevy r.. koski r.a., lu h.s. and yarden y. ( ) neu differentiation factor-a transmembrane glycoprotein containing an egf domain and an immunoglobulin homology unit. cell , - . epidennal growth-factor. human mterleukin- receptor. human-breast-carcinoma; human mammary-tumors; factor-a; nudeotide sequence; molecular cloning; prom-oncogene; tyrosine phosphorylation; signal transduction. travers a.a. ( ) the reprogramming of transcriptional competence. cell . - . position effect variegation; drarophila; protein. helenius a. ( ) unpacking the incoming influenza virus. cell , - . amantscline: protein, virions. jan l.y., jan y.n. and hughes h. ( ) tracing the roots of ion channels. cell . - . protein. gauss p. and walther c. ( ) pax in development. cell , - . genes; conservation; drosophila; nowchord, proteins; domain; box. varshavsky a. ( ) the n-end rule. cell , - . shon-hved protein; dependent pmteolytic system; ubiquitin-conjugating enzyme; repair gene ra&, escherichia coli; transfer rna; sacclurroqces cercvisiue; endoplasmic-reticulum; cell-cycle; amino-acid. cell signailing iwashita s. and kobayashi m. ( ) signal transduction system for growth factor receptors asso&ed with tyrosine kinase activity-epidennal growth factor receptor signalling and its regulation. cell signal. . - . vogt w. and nagel d. ( ) eriksson l.c. and andersson g.n. ( ) nucleotide-sequence; femo-cofacscr. iritani n. ( ) nutritional and hormonal regulation of lipogenic-enzyme gene expression in rat liver. eiu. j. fatty-acid aynthase; acetyl-coa cuboxylase; post-transcriptiaral regulation; chickembryo hepatocytes; messenger rna levels; malic enzyme; thymid hormone; posttmnscriptional regulation; molecular-clcming. gavel y. and vonheijne g. ( ) the distribution of charged amino acids in mitochondrial inner-membrane proteins suggests different modes of membrane integration for nuclearly and mitochondriaily encoded proteins. eur. j. biochem. , - . cytochmme-c-oxidrse; adp-awcartier. beef-heart mitochondria; nicotinamide nucleotide tmnshydrogenase; brown fat mitochondtia; diffemnt genes cdna, imn-sulfur proteirx saccharanyces cerevisiae; unce@ieg potdn; escherichia coli. frrmcklyn c., musierforsyth k. and schimmel p. ( ) youn y.k., lalonde c. and demling r. ( ) haas a. and goebel w. ( ) defromentel c.c. and soussi t. ( ) mackay t.f.c., lyman r.f. and jackson ms. ( ) hpitan n.l.v. ( ) sobell j.l., heston l.l. and sommer s.s. ( ) delineation of genetic predisposition to multifactorial disease-a general approach on the threshold of feasibility. genomics , l- . polymense chain-reaction; fragment length polymorphisms; dependent diabetes-mellitus; sickle-cell anemia; factor-m gene; enzymatic amplificatiat; genomic dna; mutations; sequence; diagnosis; predisposition; genetics. troy f.a. ( ) polysialylation-from bacteria to brains. glycobiology. , - . cell-adhesion molecule; escherichia coli kl; rainbow-trout eggs; deaminated neuraminic acid; endo-n-acetylne.uramiidase; group b meningococci; long oligosaccharide segment: netno-blastoma cells; polysialic acid; sialic-acid. varki a. ( ) diversity in the sialic acids. glycobiology. , - . n-acetylneuramhtic acid, infhtenxa c virus; de-ortho-acetylation; performance liquid-chromatography; hungonuhu d&her antigen; liver golgi vesicles; melanoma-associated ganglioside; bombardment mass-spectrometry; human gastmintestinal-trac deaminated neuraminic acid. stanley p. ( ) glycosylation engineering. glycobiology. , - . hamster ovary cells; mcombinant human erythropoietin; tissue plasminogen-activator. n-linked oligosaccharide, protein glycosylaticm; biological-activity; lysosomal-enzymes; insect cells; animal-cells; sugar chains. harvey d.j. ( ) the role of mass spectrometry in glycobiology. glycoconjugate j. , - . fast-atom-bombardment; -o-methylghtcose pelysaccharide; collisional activation; gas-chromatography; laser desorptien; molecular mass; oligosacdtarides; ionization; proteins. aquit d.a. and b~~~ii~ii c.f. ( ) heat-shock proteins and immunopathology-an overview. heat-shock t-cell receptor; messenger-rna degradation; gamma-delta; stress proteins; antigen-receptor, lymphocytes-t; ~ycobactcrium fuberc&arir. mammalian-cells; cyto-toxicity; dna-binding. fenick d.a. and gemmellhori l. ( ) potential developmental role for self-reactive t cells bearing gamma-delta t cell receptors specific for heat-shock proteins. dendritic epidermal-cells; toxic lymphocytes-$ antigen receptor; intraepithclial lymphocytes; rheumatoid arthritis; athymic mice; mycobacrcritert lubercularis; immune-response; thymic ontogeny; a-chain; cell receptor. christmas s.e. ( ) cytokine production by lymphocytes-t bearing the gamma-delta t cell antigen receptor. antigen; antigen receptor. mceptor. tumor-necrosis-factor; bone-marrow transplantation; a-p+ lymphocytes; interferon-gamma; monoclonal-antibodies; peripheral-blood; cytotoxic activity; fetal thymocytes; different forms; human intestine. wintield j., jarjour w. and minota s. ( ) stress protein autoantibodies and the expression of stress proteins on the surface of human gamma-delta cells and other cells of the immune system. heat-shock proteins and gamma-delta t cells , - . stress; autoantibodies; immune-system; heat-shock protein; rous-sarcoma virus; juvenile rheumatoid-arthritis; t-cells; synovial-fluid; lymphocytes-t, mycobucterium tuberculosis; borrelia bwgdorferi; lupus erythematosus; transforming protein; stmss protein. modlin r.l.. lewis j., uyemura k. and tigelaar r.e. ( ) lymphocytes-t bearing gamma-delta antigen receptors in skin. dendritic epidermaltells; heat-shock protein; mycobacterium-tuberculosis; intraepithelial lymphocytes; limited diversity; murine epidermis; concanavalin a; thy-l antigen; nude-mice; expression. hohlfeld r. and engel a.g. ( ) the role of gamma-delta lymphocytes-t in inflammatory muscle disease. monochmal-antibody analysis; natural-killer cells; mononuclear-cells; cyto-toxicity; receptor; myopathies; recognition; expmssiost; ptoteins; canplex. aquino d.a. and selmaj k. ( ) heat-shock proteins and gamma-delta t cell responses in the central nervous system. heat-shock proteins and gamma-delta t cells , - . experimental autoimmune encephalomyelitis; experimental allergic encephalomyelitis; fibrillary acidic protein; myelin basic-protein multiple sclerosis: stress prcteim spinal-cord, insitu hybridixation; alexander's disease; praxosin treatment. mario t., nagasawa m. and yata j. ( ) gamma-delta t cells in patients with primary immunodeficiency syndrome-their function and a possible role in the pathogenesis. heat-shock proteins und gamma-delta t cells , - . wiskott-aldrich syndrome; heat-shock proteins; receptor-delta; ataxia telangiectasia; transgenic mice; lymphocytes t; bearing cells; recognition; expression; incmase. reardon cl., bom w. and obrien r.l. ( ) murine gammadelta lymphocyte-t recognition of hsp a possible source for bacterial immunity or autoimmunity. he&& j.e. and whitelaw p.f. ( ) the role of cellular oncogenes in myogenesis and muscle cell hyperuqhy. int. j. bkxhem. , . muscle; all; rous sarcoma vins; fibroblast growth-factor; c-fos expression; embtyonal caminoma-cel~, chicken skeletal-muscle; myc messatger-rna; proto-oncogene; geneexpnssion; dna-binding; src gmc. colacicchi s.. ferrari m. and sotgiu a. ( ) in vivo electron paramagnetic resonance spectroscopy imaging -first experiences, problems, and perspectives. bat. j. b&hem. . - . &oxide spin labels; loop-gap resonator. free-radicals; soluble nitroxides; metabolism; phannacokinetics; oxygen; cells; specttusn~, sensitivity. seyer r.. richoux j.p. and aumelas a. ( ) probing angiotensin receptors. iru. j. biochem. , - . message-address concept; if onnplementaty rna, paraventricular nucleus; biological-activities; subfomical organ; binding-sites; amino-acid; rat-brain; antagonists; analogs. tuck m.t. ( ) the formation of internal -methylademne residues in eucaryotic messenger rna. fnt. j. biochem. , - . rekharsky m.v., nemykina e.v. and erokhin a.s. ( ) thermochemistry of n-c bonds hydrolysis in amides, peptides, n-acetyl amino acids and high-energy n-c bonds hydrolysis in n-acetyl imidazole and urea. lnl. konopinska d., rosinski g. and sobotka w. ( ) insect peptide hormones, an overview of the present literature. amino-acid-sequence; bombardment mass-spectranetry; pigment-concentrating hormone; locust adipokinetic hormone; periphneta americana l; akh-rpch family; corpora cardiaca; lencophaea mademe; cotpus caniiaann; matuiuca swrta. dinarello c.a. ( ) the biology of interleukin- . interleukinomolecular biology and immunology . l- . tumor necrosis factor, colony-stimulating factor; smooth-muscle cells; human-immunodeficiency-vims; vascular endothelial-cells; blood mononuclear-cells; recombinant human interleukin- ; human monocyte interleukin- ; hepatic protein-synthesis; autocrine growth-factor. dower sk.. sims j.e., cerretti d.p. and bird t.a. ( ) the interleukin- system--receptors, ligands and signals. interleukin.+molecular biology and hmunology , - . tumor necrosis factor, pmtein kinase c, growth-factor-receptor. nf-kappa-b; factor increase phosphorylation; prostaglandin e production; thaunatoid synovial-cells; high-affinity receptors; gtp-binding pmt+ human t-cells. ihle j.n. ( ) interleukin- and hematopoiesis. interleukim- olecular biology ad i mmunology , - . colony-stimulating factor. human granulocyte-macmphage; protein kinase-c; recombinant human interleukin- ; cell growth-factor, murine bone-marrow; express functional receptors; acute lymphocytic-leukemia; gtpase-activating pm&n; factor-independent growth. ascorbic-acid deficiency; ischemic-heart-disease; low-density-lipoprotein; eastern finnish men; diabetes melli~rcs. guinea-pigs; blood-pressure; oral-contraceptives; experimental atherosclerosis; plasma-cholesteml. mannella ca., forte m. and colombini m. ( ) toward the molecular structure of the mitochondrial channel, vdac. j. bioenerg. biometnbmne , - . outer-membrane channel; neurarpora crassa mitochondria; hexokinase-binding protein; voltage-dependent channel, rat-liver mitochondrip; synthetic polyanion; electron-microscopy; pore protein; sequence; arrays; molecular-structure. depittt~ v. and pahnieri f. ( ) benz r. and brdiczka d. ( ) the cation-selective substate of the mitochondrial outer membrane pore-ainglechannel conductance and influence on intermembrane and peripheral kinases. j. bioenerg. biomembrane x - . rat-liver mitochondria; hexokinase-binding protein, contact sites; synthetic polyanicn; creatine-kinase; inhibition; transport; stae; brain. arora k.k., parry d.m. and pedersen p.l. ( ) khorana h.g. ( ) rhodopsin. photoreceptor of the rod cell-an emerging pattern for structure and function. j. bid chetn , - . schiff-base counterion; bovine rhodopsin; cysteine residue-l ; molecular mechanism; visual excitation; outer segmenl; protein; light; transducin; binding; mds; rhodopsin; photoreceptors. pugh b.f. and tjian r. ( ) diverse transcriptional functions of the multisubunit eukaryotic tfbd complex. j. a-a-crystallin; tissue-specific expmssion; chicken -l crystallin gene; vettebrate lens ctystallins; non-lenticular tissues; x ray-analysis; y-crystaltin; transgenic mice; b-ctystallin; eye lens; gene-regulation. fibroblast growth-faaer; endothelial-cell mitcgen; vascular-permeability factor, tumor necrosis factor. bovine brain; extracellular-matrix; factor-a; neovascularixaticn in viva; dna-synthesis; acidic fgf. sardesai v.m. 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( ) chemistry and biochemistry of taurolipids. prog. lipid res , - . phosphoenolpymvate carboxykinase gtp; diet-induced hypercholesterolemia promoter-regulatory region; tissue-specific expression; enhancer-binding-pn&tu pymvate-kinase gene; rat-liver, messenger-rna, phosphofmcto- kinasefructose , -bispbosphatase; transcriptional regulation. barber j. and andersson b. ( ) lermrd j. ( ) moms d. ( ) smtctura and fum&mal relationships between ~~yl-~f~ rna syt&tases. biodem. sci , - . dimatsiand structure; atp, mechanisms; resohstion; homology; ~ueteim tymsyl; site; ammoacyl-transfer-rna. key: cord- -jif gof authors: jiménez-liso, maria rut; lópez-banet, luisa; dillon, justin title: changing how we teach acid-base chemistry: a proposal grounded in studies of the history and nature of science education date: - - journal: sci educ (dordr) doi: . /s - - - sha: doc_id: cord_uid: jif gof we propose explicit and implicit approaches for the teaching of acid-base chemistry based on research into the history and nature of science (nos). to support these instructional proposals, we identify four rationales for students to understand acid-base processes: daily life, socio-scientific, curriculum, and history of science. the extensive bibliography on misconceptions at all educational levels justifies the need for a change from the usual pedagogical approaches to teaching the acid-base domain (traditionally involving conceptual-focused teaching) to a deeper and more meaningful approach that provides (implicitly or explicitly) a chance to reflect on how scientific knowledge is constructed. controversial moments in science from , when three researchers (bronsted, lowry, and lewis) independently enunciated two theories from two different paradigms (dissociation and valence electron), underpin our first sequence with an explicit nos approach for both lower secondary school and upper secondary or university levels. our inquiry teaching cycle promotes the transformation of a hands-on activity (using cabbage as an indicator) into an inquiry, and subsequently, we use an historical model to propose a sequence of activities based on the modeling cycle of couso and garrido-espeja for lower secondary school. finally, we identify some implications for a model-focused teaching approach for upper secondary and university levels using more sophisticated models. researchers in the area of the nature of science (nos) often provide recommendations for teachers. it is usually suggested, directly or indirectly, that teachers should improve their knowledge about what science is and how it is constructed, so that they can transfer this knowledge to the classroom, transforming it into sequences of activities for their students. for example, nouri et al. ( ) recommend a well-designed history of science (hos) intervention to convey essential lessons about the nos consensus described by mccomas ( ) such as science depends on empirical evidence; cultural, political, and social factors influence science; or science has a tentative or fallible nature. many authors identify multiple potential benefits for learning nos through such approaches: teaching scientific methods, challenging myths related to how science works, and differentiating between idealized scientific laws and observations (niaz ). however, they also highlight that research involving rationales and strategies for teaching hos is scarce and nouri et al. ( ) recommend expanding science teacher educators' rationales for teaching hos to inspire a broader array of orientations and teaching strategies. they also suggest paying special attention to instructors' orientation towards teaching hos which may have an impact on their effectiveness. such recommendations usually arise from studies focusing on the benefits of nos for students and research on what teachers think, their beliefs about nos, or in a less declarative way and close to their educational reality, the connection (or otherwise) between this knowledge and what the teacher really does in their classes (leden et al. ) . one of those recommendations involves the design of nos classroom activities: explicitly and implicitly (duschl and grandy ) and using reflective approaches to nos teaching and learning. these approaches open up the range of possibilities for teachers. for example, proposing explicit general activities, linked (or not) to a specific issue of science content, to develop students' understanding of an aspect of the nos consensus view (lederman ; mccomas ) . such an activity might involve the use of a mystery box to help students to learn about observation, interpretation, and argumentation (cavallo ; rau ). another example involves scientific practices, such as the national research council's ( ) inquiry into the problem of the tsunami on the us west coast or "mrs graham's" class which tackled the problem of leafless trees with explicit reflection such as metamodeling learning progression (schwarz et al. ) on how science is built. in this paper, we use garrido-espeja and definition of a model which is a "small number of big or core ideas (harlen ) that have the potential to explain a lot of different phenomena (izquierdo-aymerich and aduriz-bravo ), such as the particle model of matter" or, indeed, the chemical change model. implicit and explicit nos teaching approaches (duschl and grandy ) have a place in the high school science curriculum ( - years old) because an initial study of what science is is often included. at the same time, in chemistry courses, some topics such as atomic structure, the periodic table, or acid-base are often introduced through their historical developments. this history of chemistry topics present in curricula allows the design of authentic scientific practices (implicit approach burgin and sadler ) . the content overload in the spanish science curriculum forces some teachers to dispense with developing the initial lesson about what science is and how it is built or with spending more time in deepening these nos aspects when working on some historical development present in the curriculum, i.e. atomic structure, periodic table, etc. thus, before deciding the approach on explicit or implicit teaching, the first teacher decision is to develop or omit this initial nos lesson and the second is to decide whether to deepen or not the historical developments present in the curricula. we turn now to address the issue faced by teachers: how to translate these nos teaching approaches to sequences of activities on a specific topic? in this theoretical article examining teaching practice, we want to focus on the historical development of acid-base theories (arrhenius, bronsted-lowry and lewis) to analyse the steps to follow to design sequences of activities for different nos approaches. the main objective of this paper is to translate the explicit and implicit nos approaches using the historical development of acid-base domain into activity sequences that can be used as a reference by teachers. in the next section we will outline the importance of teaching the topic of acids and bases because we understand that the first decision for a teacher is whether to spend time on the historical development of the acid-base domain present in chemistry curricula at secondary level, high schools and university level (in analytical and inorganic chemistry subjects). finally, we discuss how to design sequences of activities. we examine conventional teaching approaches to the topic and its consequences in terms of students' alternative conceptions and their difficulties to transfer and apply knowledge and to recognize acid-base models' limits of applicability. in this section, we will use research results (our own and others) from assessments of high school students, university students, both undergraduates and postgraduates, and pre-service teachers to show the common acid-base teaching (concept-focused teaching) approach and its consequences. these discussions and an acid-base historical development (timeline in section ) will help us to analyse the current situation in order to scaffold the design of sequences of activities utilizing different nos approaches: . we will propose nos sequences with an explicit approach through controversial moments of the acid-base historical development for both lower secondary school and upper or university levels; . we will transform a hands-on activity (using cabbage as an indicator) into an inquiry for lower secondary level (section . ) and from it; . we will propose a modeling sequence based on the modeling cycle of couso ( ) and couso and garrido-espeja ( ) using an historical model (erduran ) for the same level (lower secondary school); . we will identify some implications about a model-focused teaching for upper secondary and university levels using more sophisticated models. finally, we will discuss the change in teachers' awareness of a model-focused teaching approach that extends and gives meaning to the usual concept-focused teaching. in short, in this paper, we are constructing a science teaching learning progression in a theoretical manner (schneider and plasman ) to build models using the history of acids and bases as a theme which could be used as a reference by teachers in their professional practice. universities. acid-base processes also appear in other subjects such as ionic equilibria and chemistry lab work. in these subjects, they are often referred to as "acid-base reactions", "acidbase titrations", "ionic solutions", and "acid-base theories". we have identified four categories of rationales why students need to understand acid-base processes: daily life; socio-scientific; curriculum; and hos argument. we now discuss each in turn, briefly. acids and bases are commonly recognized by students and the general public. people know about acidic sweets, stomach acidity, antacids, etc. (cros et al. (cros et al. , . words such as "acid" and "neutral" are used in some tv advertisements ("fairy is neutral and protects your hands"; "johnson's ph . has natural ph"). nevertheless, understanding of acid-base concepts is still limited. furthermore, the use of scientific concepts is increasing and highlighted in advertisements to show products both as beneficial or a matter of trust despite misunderstanding of scientific concepts . thus, some acid-base contents are necessary to understand the phenomena encountered in daily life. pseudoscientists take advantage of the lack of awareness of the population about scientific expressions using it to promote "scientific credibility" to their unfounded proposals of health and home remedies. poor science is common in advertisements about cosmetics and cleaning "with ph" products, all sorts of diets and foods that reduce acidity in your body to prevent or treat cancer, etc. those adverts can serve us as a context to raise socio-scientific controversies (evagorou and osborne ; sadler and zeidler ) about medicalization (domènech calvet et al. ) or alternative treatments (uskola ibarluzea ). although acids and bases are encountered in students' daily life (and social networks), they are rarely taught well at primary school level. for example, in the spanish primary school curriculum, chemical reactions are only exemplified through oxidations, combustions, and fermentations, with no mention of acid-base reactions. however, if combustion and oxidation are the most used examples of chemical changes, they lead to the establishment of some alternative conceptions such as "all chemical changes are irreversible" (stavridou and solomonidou ) or "mass is not conserved in chemical reactions" (stavy ). not surprisingly, some alternative ideas held by students closely match ideas held by people studying science many centuries ago (wandersee ) . the curriculum rationale could be reinforced by the history of science rationale: the knowledge of historical models (justi and gilbert a) and the context in which they were formulated could improve understanding of acid-base models, their limitations, and, as a consequence, the conditions required to select each model (erduran ) . taken together, these arguments justify why teachers need to develop acid-base content in their chemistry curriculum. in the next section, we justify why current acid-base teaching might be changed in order to improve understanding of scientific content. historical aspects of acid-base domain could constitute an educational resource of great relevance to prevent students seeing science as a finished product and to appreciate how some theories and explanations are provisional. for instance, the acid-base historical development would allow teachers to discuss with their students the limitations of each of the acid-base theories and why they were used in the past or still are used (alvarado et al. ) . in this paper, we use three famous acid-base theories (arrhenius, bronsted-lowry and lewis) , although the historical development of the acid-base domain is as long as the history of chemistry itself. figure represents a timeline of the acid-base domain showing links with the chemical change models presented by justi and gilbert ( b) and also with some acid-base models reviews (de vos and pilot ) or history of science books (taton (taton , taton and goupil ) . the historical development of a domain is usually introduced to students in a very condensed manner in order to focus on the last, the most useful or the longest surviving acid-base theories. in this first part of the paper, we only use acid-base "theories" as it is commonly called, but from section , we will be called acid-base "model" to focus on the explanatory and predictive power of the models. this timeline could be a good illustration of the historical development of ideas which is broader than that usually presented to the students. on the left side of the timeline, we use the term pre-model (justi and gilbert a) to indicate that an acid-base classification does not have to be explanatory. in order to understand scientific models, we need to appreciate that they have been constructed to explain and predict phenomena, so the models are more than a descriptive account of the material world. in this sense, acid-base historical models are a good opportunity to understand how change has taken place in the scientific models over time. many of the situations where people encounter science involve the use of scientific knowledge, alongside other forms of knowledge, to reach decisions about action. this is often the case for lay people, who typically find science through media portrayals of socio-scientific issues, or through consultations with experts such as medical practitioners. lay views of science tend to portray such issues as being easily resolved through simple empirical processes (e.g. driver et al. ) . this position, however, is often not sustainable, as illustrated by the following examples. an example of science in the media is the case of enhanced global warming as a result of increased levels of carbon dioxide in the atmosphere due to the combustion of fossil fuels. it is not difficult to find widely different predictions in the media about the likely environmental impact of the burning of fossil fuels. these differences in predictions are based upon the application of models of the atmosphere. resolving those differences involves a complex interplay between models, empirical evidence and methodological expertise. understanding fig. acid-base timeline how such differences arise, and why they cannot be resolved quickly and simply, involves understanding something about the use of models. it is not only lay people who encounter science in situations that are characterized by uncertainty. many experts will be faced with situations where scientific knowledge has to be drawn upon, alongside other considerations, to inform decisions about action in novel situations. a sad and recent example is the current scientific/political/cultural environment with covid- . the academic literature now includes several accounts of how experts have to create new knowledge in order to answer questions that emerge in specific situations. another, much older example, is brian wynne's account of how experts had to develop new knowledge about the impact of pollution following the chernobyl accident impacting upon the milk produced by sheep which grazed on the cumbrian fells (wynne ) . in order to appreciate why the available scientific knowledge may be inadequate to inform action in specific, local conditions, such experts need to understand something about the nature of models in science. so, to summarize, it is necessary to teach models at university level to make the following points: & in order to have a sophisticated understanding of the conceptual content presented to them in chemistry degree programmes, students need to have some understanding of how the models are built. & in addition, if the students develop this understanding of the nature of models, it may enable them better to understand situations involving uncertainty, whether as educated citizens, or if they go on to become professional scientists, in their professional practice. these general arguments about teaching models can be exemplified in the case of acid/base models. conventional chemistry teaching might begin with questions such as "what is an acid?" or "what is a base?"; "what happens when an acid is added to a base, and vice versa?"; "what does ph mean?"; "is it always possible to reach ph= when an acid is added to a base?" conventional teaching shows some concepts from arrhenius and bronsted-lowry's theories that we summarized in fig. . it is possible to recognize some differences between both theories in relation to considering acids and bases as substances (arrhenius) or as the conjugated acid-base pairs (bronsted-lowry) . as is normally mentioned, arrhenius', bronsted-lowry's, and lewis' theories are usually presented together (tarhan and acar sesen ). acid-base theories are introduced in the style of a short story without any connection with the phenomena they want to explain or with the historical problems that inspired them. by teaching these acid-base theories together we expose the combination of the acid-base concepts (acid, base, neutralization, etc.) of the three theories without appreciating a significant advance between them, which for many students could mean only a terminological change. teachers (and textbooks) usually say that "bronsted-lowry extend the definition of acids and bases" (nyachwaya , p. ) given by arrhenius. in this sense, arrhenius', bronsted-lowry's and lewis' models are often presented several times in chemistry programmes, introducing some inconsistencies in their presentation that lead to "hybrid models" (justi and gilbert a) and some concepts and their definitions are mixed in two or more models (gericke and hagberg ) . the science education literature is replete with examples of the consequences for students' learning of this typical way of teaching acid-base content focused on the definition of its concepts and with two or three theories introduced simultaneously. in the next sections we will use a review of research results (our own and others) on the understandings of high school students, university students (both undergraduates and postgraduates), and pre-service teachers in order to design some proposals focusing on two approaches, one nos explicit and the other nos implicit (sections and respectively). there have been a number of studies into students' misunderstandings of acid-base phenomena (for example mcclary and bretz ; nyachwaya ) . many students have difficulties in learning acid-base concepts and the presence of alternative conceptions (hoe and subramaniam ) can interfere with their understanding. for instance, students think that acids alone are associated with corrosiveness (demircioğlu et al. ; hoe and subramaniam ; Özmen et al. ) and are more dangerous and reactive than bases (hoe and subramaniam ; nakhleh and krajcik ; sheppard ) . they also think that rain water in an unpolluted area is neutral or the solution formed after adding an acid and a base is always neutral (banerjee ; hoe and subramaniam ; scerri ; schmidt ) . as quílez ( ) points out, many of these misunderstandings come from students' terminological difficulties. therefore, students do not understand why the degree of acidity or basicity of two acidic or basic solutions is different, although they have the same concentration (alvarado et al. ) . moreover, a superficial correlation of chemical structures with acidity or basicity may explain why students believed that compounds containing h will produce h + and besides, the belief that a stronger acid is either the one that produces a higher hydrogen ion concentration, or that has more h in the formula, or an acid with a higher initial concentration (demircioğlu et al. ; hoe and subramaniam ; Özmen et al. ; ross and munby ) , reveals that students do not apply correctly the definitions of strong and weak to acids and bases (garnett et al. ; mcclary and bretz ) and both equilibrium and incomplete dissociation of acid and bases are not considered. similar problems are found with students' understanding of bases (hoe and subramaniam ) . another issue is that students do not totally differentiate between the terms acidity and ph (alvarado et al. ) ; they do not consider ph is providing information about both the h + and oh − concentrations (garnett et al. ) , and show either a lack of consideration about the influence of variables such as the temperature or the solvent, using strength and concentration as if they were synonymous or having problems when they must differentiate between an acidbase reaction and a neutralization reaction (alvarado et al. ) . many of those alternative conceptions are consistent with students using the theory in other contexts to which arrhenius proposed it. in the next subsection, we are going to look deeper into the difficulties linked to transferring knowledge to new situations or to recognizing the limits of each of the acid-base theories. based on our own results in the spanish context (author ), the consequences of acid-base conceptual teaching for both undergraduate and postgraduate chemistry degree students become evident due to (a) students' difficulties to transfer knowledge and (b) the problems recognizing the limits of applicability of acid-base theories: (a) transference of knowledge to new situations. despite having been taught about acid-base concepts many times, undergraduate university students (n = ) from three spanish universities showed weaknesses in their abilities to recognize an acid-base process and the proportion giving the correct answer decreased when the complexity of the theory applied increased: -most students ( %) recognized a proton transfer process as bronsted's model. - % recognized the autoprotolysis of solvents (so ) as an acid-base process. -only % considered the electron transference as in lewis' process. -less than % of the students applied all three theories. some spanish university students explained that an electron transfer process (lewis' model) or an autoprotolysis of solvents (so ) is not an acid-base process, for example, "it is not an acid-base process", "it is a redox process", or "it is not an acid-base process because there isn't h + or ohor h o + ". thus, as in many occasions mentioned by other authors (drechsler ; drechsler and van driel ; zoller ) , the bronsted-lowry acid-base process is more recognized by students than other acid-base models. for us, this result is evidence that the university students did not transfer their knowledge about acid-base acquired to new situations, for example, the lewis' acid-base electron transferences. (b) applicability of acid-base models. as a consequence of the previous result, it was expected that most of the graduates in their secondary education teacher civil service examination would cite arrhenius', bronsted-lowry's, and lewis' models (data from research on exams). nevertheless, in approximately % of the cases, the description was wrong. only % of the candidate teachers identified the boundaries of arrhenius' model, three-quarters made no comment on the limitations of bronsted-lowry's theory, and none recognized that there might be limitations in lewis' theory and what they explicitly consider is the currently accepted one (jiménez-liso ) , similar to the results founded by yalcin ( ) with turkish candidate teachers. previous omissions of the limits of applicability of the different acid-base theories are worrying as regards that, if they passed this exam, they would be qualified as secondary education teachers in physics and chemistry. they do not usually follow any continuous professional training as teachers, a fact that also occurs in other countries such as england, france, finland, and cyprus, and could affect the quality of teaching and the improvement of the education system (evagorou et al. ) . teachers must have knowledge about teaching scopes and limitations of different acid-base models; nevertheless, most of them had not developed teaching strategies for this issue and only a few teachers said that they usually discussed the use of models of acids and bases in their teaching (drechsler and van driel ) . although they recognized some difficulties of the students such as confusion between models, only a few emphasized the different models of acids and bases (alvarado et al. ; drechsler and van driel ) . moreover, despite some teachers believing that most students do not understand the use of models, they tried to teach it anyway in order to help the best students in their learning, hoping other students understand that simple models are not the whole truth (drechsler and van driel ) . by presenting two or three theories together, the lewis and bronsted-lowry definitions are just that-definitions-and the validity of one of them does not automatically negate the other (although it may expand the set of substances which are classed as acids). the conceptualfocused teaching mentioned above is insufficient because it comes from a purely theoretical perspective without any kind of application, reduced to the definitions instead of containing a clear explanation of their development (cid manzano and dasilva alonso ) and of the problems or phenomena that gave rise to new ideas. the three favourite acid-base theories are presented as a collection of "agreed upon facts", so students memorize them without questioning their relationship with other scientific knowledge (justi and gilbert a) , and focusing on the products rather than on the processes of science. there is clear evidence that many problems learners have arisen from acid-base theories confusion. when several theories of acid-base are presented together to students the scope to produce confusion is expanded, above all if most learners have a very limited notion of the role of models in science (driver et al. ; grosslight et al. ; taber ) . the role of a model in science is related to developing a scientific understanding of some phenomena, explaining them and predicting other related phenomena, then applying the new knowledge to novel situations or contexts (izquierdo-aymerich and aduriz-bravo ; oh and oh ) . so, the reason for explaining three (or more) acid-base theories together is not related to scientific understanding of the phenomenon. the reasons for introducing the three most used acid-base models together appears to be twofold: firstly, conceptual survival (a concept from past chemical curricula that is retained in modern chemical curricula) and, secondly, to show the history of a chemistry concept in a narrative manner. thus, the emphasis is focused on the differences between each concept, a fact that does not promote a proper understanding of science, instead of comprehending the conditions when the models were built and, consequently, the limitations they have. no advantage is taken of the opportunities to get the students to reflect on the nature of science through the history of acid-base theories. on the contrary, they usually develop a distorted image of science itself and of how it is carried out. before we discuss teaching acid-base models in the chemistry curriculum, it is necessary to clarify terminology. acid-base concepts, definitions, theories, and models are often used as synonymous. students' mistakes often arise due to the ambiguous use of the terminology (jiménez-liso and de manuel torres ). to avoid this difficulty, we have adopted acidbase models as the correct terminology to refer to the models that explain and predict phenomena proposed by arrhenius, bronsted, and lewis, because we understand that the theories in which they are included are the ionic dissociation theory, solvents theory, and valence electron theory respectively. considering that curriculum materials shape teachers' practice and characteristics (as their knowledge or beliefs) and students' opportunities to learn in science (davis et al. ; pareja roblin et al. ) , in the next section, we try to identify activity sequences, firstly with an explicit nos approach using the timeline of the historical development acidbase models. as burgin and sadler ( ) mentioned, the prevalent model for teaching nos in school has been referred to as the explicit/reflective approach (lederman ) . in this approach, the priority object of study is to teach the great consensus about the nature of science (tentativeness, creativity, ...) to avoid the main distorted views of science. typical activities using this approach are discussions about a "paper towel investigation", about the "card exchange" (cobern and loving ) , or about historical cases (readings or movies) (aduriz-bravo and izquierdo-aymerich ; moreno et al. ) and scientific errors (kipnis ) as a particular historical case or some historical controversies (niaz ). all of these strategies for teaching are linked to some rationale, to educational purposes (nouri et al. ). the acid-base timeline (fig. ) could link with the next curriculum purposes for specific educational levels (table ) . more interesting than improving acid-base content understanding are the opportunities to advance the understanding of nature of science content using certain moments of the acid-base timeline, for example, the story of what happened with acid-base models in . in , bronsted and lowry proposed their explanations about acid and base behaviour. both knew arrhenius's model ( ) and a less famous one today: the solvent-solute model proposed by franklin ( ) . some years later, two researchers independently proposed a particular case of solvent model (proton model) where the water acts as solvent and its autoprotolysis as definition of acids (proton donor) or bases (accept protons). (taton ) to link with chemical change models expressed by students and with school science models such as "parts model" described by acher et al. ( ) and their pivotal ideas like transformations and conservation of "parts" to distinguish dilution (colour fading) and neutralization (erduran the main purpose of this model is not to identify acid-base processes with aqueous processes university level proton model (based on water autoprotolysis) given by bronsted ( ) and lowry ( ) we can use the original papers from bronsted, lowry, and lewis to help them to answer the next questions that we can ask to our high school students (or university students): -why does emerge a limited model (proton model, bronsted-lowry model) after a broader model (franklin model) ? we want to scaffold "the epistemic value of simplicity, referred to as ockham's razor, meaning that the simplest applicable model is the most elegant and the best" (rollnick , p. xiv) . the solvent model proposed by franklin ( ) was known by bronsted and lowry but they only used the water as solvent, so for solving their problems, they did not need a broader model and they specify it on a model more simple but more useful. in fact, it is the most widely used and known today because most acid-base reactions are aqueous. -a danish researcher (varde, denmark) (bronsted ) and the same year that (lowry ) from bradford (uk) propose the same proton model, how do you think it was possible for two researchers in different countries (without knowing each other) to propose an identical model? perhaps in our digital era this scenario is unthinkable: two researchers producing identical researches without any previous contact, but in , they heard from each other when they read the papers already published in two different journals. the scientific community recognized the merit of both of them and, thereafter, their model was named bronsted-lowry. what circumstances led both to propose the same theory? bronsted ( ) started from the dissociation electrolytic theory of arrhenius, which initially does not call into question his idea of acid (a →b + h + ) and for which he tries to find a better definition of base: "it is the purpose of the present small contribution to show the advantages that come from a modified definition of a base" (bronsted , p. ) , specifically the difficulties in explaining the basicity of ammonia: if we accept scheme (nh + + oh -<===> nh oh), as a suitable expression for characterizing bases, we will be forced to give a special definition of a base for each special solvent. however, in principle, acid and basic properties are independent of the nature of the solvent, and the concepts of acids and bases are in fact of such a general character that we must consider it a necessary requirement of these concepts in general to formulate a pattern independent of the nature of an arbitrary solvent (bronsted , p. ) and ends by concluding: the equilibrium formulated in scheme ( ) between hydrogen ion and the corresponding acid and base can be called a simple acid-base equilibrium. by mixing two simple systems, a double acid-base system and an acid-base equilibrium result that can always be formulated as follows: this equilibrium includes a number of important reactions such as neutralization, hydrolysis, indicator reactions, etc. (bronsted , p. ) on a different path, lowry ( ) knew the electron valence theory of lewis ( ) and relied on it to distinguish two types of chemical affinity (polar and non-polar) and their links in organic and inorganic substances, which led to the need of proposing h o + as what is exchanged in acid-base reactions overcoming arrhenius h + proposal, the difficulty of the basic character of nh and the relative character of strong or weak acids depending on which substance they react with (fig. ) . -with the previous knowledge, students are willing to answer one last question: how do you think two models emerged in from three different people working independently and in different paradigms (proton or electron paradigms)? was a good year for the acid-base historical development. lewis ( ) also raised his electron model (fig. ) under a totally different paradigm (based on his electron valence theory) and to solve a problem not contemplated by his contemporaries: acid-base behaviour in reactions without solvent, for example, in gas reactions. discussing with upper secondary students (or university students) these acid-base moments of a broad timeline, we could challenge the accumulative-linear and erroneous image of science. for university chemistry or geology degree students, similar questions could be posed using the and lux-flood models for reactions in high pressure such as on geological process (without any dissolution). and also for university chemistry students (pre-and post-graduated), another interesting controversy could be the qualitative and quantitative chemical approaches (chamizo ) between pearson ( ) who proposed empirically his hard and soft acid-base model and drago ( ). implicit approach to nos teaching . inquiry-based teaching proposal barrow ( ) described inquiry firstly as an epistemic practice (kelly ) , secondly, as scientific skills that students should develop, and finally as a teaching approach. we remain with this last meaning of inquiry to propose an instructional sequence of activities. as there are a multitude of research proposals (pedaste et al. ) , we have specified our teaching approach in a cycle (fig. in orange) to connect it with the modeling cycle (fig. in green) proposed by couso ( ) and couso and garrido-espeja ( ) . in the acid-base domain, reactions can be followed with indicators from daily life such as red wine. when we use the red cabbage as an acid-base indicator, we generally emulate boyle's descriptive pre-model in order to recognize the acid-base nature of some daily life products. in this way, we create (as boyle did) a classification of acid, neutral, or basic substances. we transform these hands-on activities about the acid-base classification to an inquirybased teaching where the steps will be easily recognizable by our students so that they become aware of how they have learned (learning and emotions self-regulation) and, therefore, can make an explicit debate about the phases of the inquiry, how they help to learn, and what emotions they felt during this sequence (step in cycle orange, fig. ) . to do this, we begin with a familiar problem (chewing gum tv advertisement stops the acid attack, strengthens the tooth enamel and helps to keep your teeth strong and healthy while in the image we can show that raise the ph of the mouth to prevent the formation of cavities (jiménez-liso et al. ) that engages students to explain their personal ideas: chewing gum is the opposite of acids generated by food in the mouth, the tv ad does not tell the truth and the gum does nothing, warms and destroys the acids, more saliva is generated or the gum traps the remains of food. the key moment in this sequence is the students' proposals for designs of experiments that allow them to find evidence to confirm or reject their hypotheses. the experimental designs raised by our students facilitate the discussion about the usefulness of the designs (what did they measure? with what did they check?). for instance, some students proposed to put some food in a glass with water (to simulate the mouth) along with the gum and measure with ph paper, to which another group responded that they did not check the "before" and the "after" adding chewing gum, that is, the effect of chewing gum. others suggested sucking ph paper after eating and again after chewing gum (lópez-banet et al. ) . taking measurements with a ph meter can be a conflict for the students with their expectations, both because the chewing gum does not raise the ph of the acid-dissolution (mouth simulation) and neither does adding water (dilute). this opens the option of deepening the mathematical conflict that involves a linear scale (ph values of - ) versus a logarithmic scale (which means ph) asking how much water would be necessary to raise the value by one point (lópez-banet et al. ). however, as osborne ( ) mentioned, hands-on activities, such as the acid-base classification using red cabbage indicator, are not normally accompanied by an interpretation or explanation of the phenomena. in our inquiry-based sequence, students built essential descriptive knowledge (acid-base reactions vs dilution with water or saliva) so that they now recognize the need to seek an explanatory model perfect to start the modeling cycle (fig. ) . the use of red cabbage as an acid-base indicator, often carried out by students aged up to years old, is not accompanied by its possible explanation using models, keeping the explanation for higher levels ( - years old and university level). in this sense, the first (lewis , p. ) introduction of an explanatory model is presented for - -year-old students and, generally, arrhenius or bronsted-lowry's model is used to present acid-base processes disconnected from those activities carried out (or not) during previous years (jiménez-liso et al. ) . therefore, if we focus the contents exclusively on the phenomenon and the identification of substances, we are only increasing the students' experiential field, but not their ability to explain phenomena they observe or to foresee what is going to happen in new situations. some hands-on activities about properties of acids and bases emphasize the teaching and learning of chemical knowledge through models and modeling by the formulation, evaluation, and revision of chemical models. when we ask students to express what they think happens "inside" by adding a base to an acid and observing changes in the colour of the indicator or changes in ph values, their initial models are unsatisfactory for some explanatory reasons (steps and in modelling cycle; fig. in green; couso ; couso and garrido-espeja ) . students may have difficulties when it comes to expressing these initial models through drawings, most often represent non-explanatory circles, and only some of them point differently to acids or bases indicating that it is acidic when acids "predominate" over the bases and vice versa (lópez-banet et al. ) . despite these difficulties of the students in explaining "what happens inside", we cannot consider these initial models as students' alternative conceptions described in section . for two reasons: firstly, students' alternative conceptions were the product of punctual and "academicist" knowledge and, secondly, the difficulty for the initial models to be explanatory is the initial step to become aware of the need to build a model, that is an idea that helps explain a phenomenon (change of colour of acid-base reactions with indicator) and to predict new ones (for example the bubbles when we add bicarbonate to the vinegar). students are expected to relate properties of a substance to its shapes, in a similar way to nicolas lemery's model (erduran ; erduran and kaya ) . this seventeenth-century scientist explained that acids consist of keen particles that prick the tongue when they are tasted, differing both in length and in mass from one another. on the other hand, alkalis have pores where the acid points entering into do strike and divide them when oppose the motion of acids. so the difference of the points in acid substances is the cause why some acids can penetrate and dissolve well certain sort of mixts (lémery ) . our version of this model is a pacman model (jiménez-liso et al. ) suggested sometimes by some of our students. they are able to reason as ancient scientist used to and to build their own explanations in a similar way about what happens in a microscopic level by means of descriptions of the reality (macroscopic level) and their intuitive thoughts. this anthropomorphic model is already useful for students because it explains acid-base phenomena but it needs to be refined (steps and of the modeling cycle) because it does not serve them to explain a well-known experiment: why the balloon is inflated by adding bicarbonate to the vinegar. when students must construct a model to explain this precise knowledge of reality (what happens with the balloon), they introduce partial modifications to their useful models (lemery or pacman model with triangles as acid) such as "bow ties" that fly when the pacman eats the triangles, and they argue on its validity (or not) according to the descriptive knowledge they already have. this process leads them to identify the insufficiency of their initial models, the useful of pacman model and its limits, and the need for refinement to explain the production of gases in acid-base processes. figure shows other alternative model based on the fighting idea to form a structure together that explain gases formation in an acid-base reaction, done by other students. as it is necessary to help students to comprehend the nature of models, a possible strategy could be introducing an explanation similar to that one mentioned as past scientists did. the activities previously mentioned encourage pupils to express their own ideas, giving opportunities to evaluate and restructure them, in order to pass from their initial to more scientifically valid conceptual schemes ones. for instance, pupils draw representations trying to explain the way they perceive some common substances and they described their models in class to share their ideas, as drawing "bubbles" in acids and less bubbles or no bubbles in the base substances (erduran ) . lemery, pacman or fighting models are very anthropomorphic. however, these models allow quick connection with the chemical formulation and the arrhenius model (fig. from jiménez-liso et al. ). as couso ( ) mentioned, a model-focused teaching would be to put students in the situation of building themselves "adequate enough" explanations, in other words, to construct school-based scientific models to describe the behaviour of the world and to comprehend how it works (aduriz-bravo and izquierdo-aymerich ; izquierdo-aymerich ). instead of learning the models as the result of the scientific activity, it would be enough to focus on some specific big ideas (harlen ) or key ideas (national research council ) that have the potential to explain a lot of different phenomena (izquierdo-aymerich and aduriz-bravo . thus, a model-based teaching approach offers instructional strategies for improving conceptual learning in science education (shen and confrey ) and permits students go beyond the idea of models as reproduction, allowing them to reach the vision that the relationship between model, experiment, and reality is dynamic and evolutionary (tasquier et al. ) . in order to build a school science using more sophisticated models for upper secondary or university levels, as in our case, we talk about the model associated with phenomena using the concrete term "key connected aspects", which should emphasize: -the purposes of each model: for example, arrhenius' model is an explanation based on the classification of substances in acids or bases and their reactions, bronsted-lowry's model is based on equilibrium, and lewis' model focus on a different paradigm, the electron theory. we want to emphasize on this idea because it changes the acid-base view, from the conceptual-focused teaching (fig. ) because we defined acid that contains h + (arrhenius) or that donates h o + (bronsted-lowry) to a new view with an explanatory power of both models, which in the case of arrhenius explains reactions between substances and bronsted-lowry explains equilibrium, balances, and, therefore, their reversibility ( fig. ) , as we will see below. -acid-base characteristic from each scientific model: our perceptions about acid-base definitions given in fig. change from acid-base as substances to the absolute acid and base properties based on their chemical composition in arrhenius' model ( fig. , left) , from the acid-base pairs conjugated to the relative properties of substances in bronsted-lowry's model (fig. , right) , or from acid-base as accept-donor pair of electrons (the electron paradigm) to its possibilities to explain solvent absence in lewis' model. these decisive acid-base characteristics are connected to the models' educational purposes fig. connected key aspects of arrhenius' and bronsted-lowry's models through a simplification of the historical scientific consensus models and it explains why some historical models can still be used to explain some phenomena (table ) . the comparison and contrast of these key features, the nature, and purposes of models can be addressed in the teaching. -scope, boundaries, and explanatory power: for example, bronsted-lowry's model can explain not only the reason why a reaction between a strong acid and a weak base produces a ph < solution without using hydrolysis concept but also that the reaction between a base and water is possible and that two acids (one stronger than other) can react (however a weak acid, according to arrhenius' classification, reacts like a base). these explanations are not possible using arrhenius' model. it would not be prudent to discard a model that is easy to understand and is well applicable in many cases (ockhams razor (rollnick ) . for instance, many chemical reactions occur in aqueous solutions because many compounds have hydrogen or hydroxide ions. thus, teaching the arrhenius concept is important for the purpose of promoting the recognition of the meaning of the acid-base characteristic from this scientific model in science learning. however, for this purpose, the introduction of new concepts needs to be followed in order to overcome the limitations of the arrhenius concept (paik ) . on the other hand, the key ideas from bronsted-lowry model emphasize five concepts: equilibrium, reversibility, simultaneous, and the relative strength of acids and bases, both in aqueous and non-aqueous solvent (fig. ) . when acid-base reactions occur without solvent, for example, gases reactions, neither arrhenius' model nor that bronsted-lowry's model serves to explain them, so we need other models such as lewis' electron valance model or lux-flood model for geological hard pressure acid-base reactions. the arguments put forward in this paper might convince teachers to deepen their teaching of acid-base processes, at all possible educational levels, by taking advantage of the presence of historical development in upper secondary and to cover the need to advance it to primary or lower secondary levels by the arguments involving the presence of acids and bases in our daily lives and on solving socio-scientific issues about health or the environment. the extensive bibliography on alternative conceptions at all educational levels (including teachers and candidates to be) justify the need for a change in the usual way of presenting it that focuses on the presentation of definitions on acid, base, theirs reactions, ph, etc., in two or three "theories" presented together. this fragment of the history of science that survives in the current curriculum (in upper secondary and chemistry degree, university level) offers a very good opportunity for nos teaching without overloading the already extensive and concentrated chemistry curriculum. thus, the main aim of traditional acid-base teaching is to learn the main concepts by means of conceptual-focused teaching and it is very far from making sense to the students, because it makes them look at the bricks and not in their usefulness as part of a larger and more beautiful castle (meaningful) and useful. inquiry-based teaching (section . ) and the model-conceptual teachings that were exemplified in this paper (sections . and . ) provide implicitly a chance to reflect on how science is constructed. whereas the conceptual-focused teaching only explains definitions and emphasizes on descriptions about behaviours (not always coordinated), model-focused teaching emphasizes on explanations, interpretations, and predictions (stefani and tsaparlis ) . in this way, students should learn to use each model within its application domain to address different phenomena. applicability of acid-base models should be better understood, and knowledge of acid-base models would be transferred to new situations, for example, to recognize a new process as an acid-base reaction. in this paper (section . ), we proposed that considering models include key ideas connected in a particular way they provide coherence to concepts. both approaches are in conflict with each other, so this dual treatment is discussed: teaching isolated ideas in a conceptual-focused teaching or the relationship between connected key aspects through a model-focused teaching. we have attempted to show the differences between nos teaching approaches through several sequences of activities. first, one sequence with an explicitly nos approach, as lederman ( ) points as desirable, and then, three implicit approaches, similar to duschl and grandy's ( ) recommendations. these four sequences can help teachers to perceive the potential results of choosing one of those treatments in acid-base lessons, according to their own teaching goals. also, this concretion in sequences of activities, which is the fundamental tools for teachers to teach, can encourage them to teach acid-base models in a way closer to the recommendations of nos researchers. as we pointed out in the introduction, by specifying the implicit-explicit debate in several sequences of activities, we are also offering, for science teacher training, a theoretical learning progression. pre-service or in-service teachers in training could live inquiry and modeling sequences since lemery to the more sophisticated models and it allows to place the implicit sequence one after this lineal progression to make explicit the awareness of how the science is built. finally, as an agenda for future work, we could follow the steps outlined in this paper in order to develop an evaluation study about the efficiency of consensus nos understandings of each implementation of our implicit, explicit-ibse, explicit-modeling sequences, using frameworks such as burgin and sadler ( ) . modeling as a teaching learning process for understanding materials: a case study in primary education a research-informed instructional unit to teach the nature of science to pre-service science teachers canonical pedagogical content knowledge by cores for teaching acid-base chemistry at high school development of the theory of electrolytic dissociation misconceptions of students and teachers in chemical equilibrium a brief history of inquiry: from dewey to standards some remarks on the concept of acids and bases learning nature of science concepts through a research apprenticeship program: a comparative study of three approaches draw-a-scientist / mystery box química general. una aproximación histórica estudiando cómo los modelos atómicos son introducidos en los libros de texto de secundaria the card exchange: introducing the philosophy of science aprender ciencia escolar implica construir modelos cada vez más sofisticados de los fenómenos del mundo [learning school science involves building increasingly sophisticated models of world phenomena models and modelling in pre-service teacher education: why we need both conceptions of first-year university students of the constituents of matter and the notions of acids and bases conceptions of second year university students of some fundamental notions in chemistry teachers and science curriculum materials: where we are and where we need to go joseph priestley across theology, education, and chemistry: an interdisciplinary case study in epistemology with a focus on the science education context international handbook of research in history, philosophy and science teaching acids and bases in layers: the stratal structure of an ancient topic conceptual change achieved through a new teaching program on acids and bases la medicalización de la sociedad, un contexto para promover el desarrollo y uso de conocimientos científicos sobre el cuerpo humano [the medicalization of society as a context for promoting the development and use of scientific knowledge revista de investigación y experiencias didácticas changing how we teach acid-base chemistry learning from the history and philosophy of science: deficiencies in teaching the macroscopic concepts of substance and chemical change pearson's quantitative statement of hsab models in chemistry education. a study of teaching and learning acids and bases in swedish upper secondary schools experienced teachers' pedagogical content knowledge of teaching acidbase chemistry teachers' perceptions of the teaching of acids and bases in swedish upper secondary schools young people's images of science two views about explicitly teaching nature of science philosophy of chemistry: an emerging field with implications for chemistry education examining the mismatch between pupil and teacher knowledge in acid-base chemistry bonding epistemological aspects of models with curriculum design in acid-base chemistry transforming teacher education through the epistemic core of chemistry exploring young students' collaborative argumentation within a socioscientific issue pre-service science teacher preparation in europe: comparing pre-service teacher preparation programs reactions in liquid ammonia surveying students' conceptual and procedural knowledge of acid-base behavior of substances students' alternative conceptions in chemistry: a review of research and implications for teaching and learning models and modelling as a training context: what are pre-service teachers' perceptions? conceptual incoherence as a result of the use of multiple historical models in school textbooks understanding models and their use in science: conceptions of middle and high school students and experts principles and big ideas of science education on the prevalence of alternative conceptions on acid-base chemistry among secondary students: insights from cognitive and confidence measures fundamentos epistemológicos [ epistemological foundations didáctica de las ciencias experimentales: teoría y práctica de la enseñanza de las ciencias epistemological foundations of school science contenidos relacionados con los procesos ácido-base: diagnóstico y propuestas didácticas al nivel universitario la utilización del concepto de ph en la publicidad y surelación con las ideas que manejan los alumnos: aplicaciones en el aula [the use of the concept of ph in advertising and its relationship with the ideas that students handle: applications in the classroom la neutralización ácido-base a debate enseñanza de las ciencias química y cocina : del contexto a la construcción de modelos chewing gum and ph level of the mouth : a model-based inquiry sequence to promote scientific practices el enfoque de enseñanza por indagación ayuda a diseñar secuencias : ¿una rama es un ser vivo? propuestas de educación científica basadas en la indagación y modelización en contexto aprender ciencia escolar implica aprender a buscar pruebas para construir conocimiento (indagación) penguin random house grupo editorial a cause of ahistorical science teaching: use of hybrid models the history and philosophy of science through models: the case of chemical kinetics inquiry, activity and epistemic practice errors in science and their treatment in teaching science teachers' ways of talking about nature of science and its teaching nature of science: past, present, and future cours de chymie valence and the structure of atoms and molecules (issue ). chemical catalog company steam views from a need: the case of a sensopill gum and ph the uniqueness of hydrogen development and assessment of a diagnostic tool to identify organic chemistry students' alternative conceptions related to acid strength the nature of science in science education: rationales and strategies efecto (¿o no?) de la inclusión de naturaleza de la ciencia en una secuencia para el aprendizaje y la aceptación de la teoría de la evolución influence of levels of information as presented by different technologies on students' understanding of acid, base, and ph concepts progressive transitions in chemistry teachers' understanding of nature of science based on historical controversies changing how we teach acid-base chemistry instructors' rationales and strategies for teaching history of science in preservice settings: illustrations from multiple cases with implications for science teacher education general chemistry students' conceptual understanding and language fluency: acidbase neutralization and conductometry what teachers of science need to know about models: an overview teaching scientific practices: meeting the challenge of change lux-flood basicity of binary silicate melts a comparative study of the effects of a concept mapping enhanced laboratory experience on turkish high school students' understanding of acid-base chemistry understanding the relationship among arrhenius, bronsted-lowry, and lewis theories what are critical features of science curriculum materials that impact student and teacher outcomes? hard and soft acids and bases phases of inquiry-based learning: definitions and the inquiry cycle a categorisation of the terminological sources of student difficulties when learning chemistry an activity to help students learn about observation, interpretation, and argumentation. the science teacher transforming teacher education through the epistemic core of chemistry (pp. xiii-xv) concept mapping and misconceptions: a study of high-school students' understandings of acids and bases scientific literacy, pisa, and socioscientific discourse: assessment for progressive aims of science education five ideas in chemical education that must die a label as a hidden persuader: chemists' neutralisazion concept science teacher learning progressions: a review of science teachers' pedagogical content knowledge development developing a learning progression for scientific modeling: making scientific modeling accessible and meaningful for learners from conceptual change to transformative modeling: a case study of an elementary teacher in learning astronomy high school students' understanding of titrations and related acid-base phenomena conceptual reorganization and the construction of the chemical reaction concept during secondary education pupils' problems in understanding conservation of matter students' levels of explanations, models, and misconceptions in basic quantum chemistry: a phenomenographic study building the structural concepts of chemistry: some considerations from educational research jigsaw cooperative learning: acid-base theories exploring students' epistemological knowledge of models and modelling in science: results from a teaching/learning experience on climate change la science antique et médiévale histoire générale des sciences, t. ii: la science moderne. presses universitaires de france histoire des sciences theory of acids and bases ¿los productos homeopáticos pueden ser considerados medicamentos? creencias de maestras/os en formación can the history of science help science educators anticipate students' misconceptions? sheepfarming after chernobyl a case study in communicating scientific information. environment: science and policy for sustainable development investigation of the change of science teacher candidates' misconceptions of acids-bases with respect to grade level students' misunderstandings and misconceptions in college freshman chemistry (general and organic) acknowledgements this work would not have been possible without the inspired discussions with esteban de manuel (rut's phd supervisor) and john t. leach and the participation of ies murgi students, their teachers lucía, isabel and carmen.funding information this work has been partially financed by the projects edu - -p and pgc - -a-i of the ministry of science and innovation (mci) of spain, the state research agency (aei) and the european regional development fund (feder), and with a visiting scholar in the university of exeter prx / of the ministry of education of the government of spain. conflict of interest no potential conflict of interest was reported by the authors. key: cord- -t b j j authors: laufer, s.d; restle, t title: peptide-mediated cellular delivery of oligonucleotide-based therapeutics in vitro: quantitative evaluation of overall efficacy employing easy to handle reporter systems date: - - journal: curr pharm des doi: . / sha: doc_id: cord_uid: t b j j cellular uptake of therapeutic oligonucleotides and subsequent intracellular trafficking to their target sites represents the major technical hurdle for the biological effectiveness of these potential drugs. accordingly, laboratories worldwide focus on the development of suitable delivery systems. among the different available non-viral systems like cationic polymers, cationic liposomes and polymeric nanoparticles, cell-penetrating peptides (cpps) represent an attractive concept to bypass the problem of poor membrane permeability of these charged macromolecules. while uptake per se in most cases does not represent the main obstacle of nucleic acid delivery in vitro, it becomes increasingly apparent that intracellular trafficking is the bottleneck. as a consequence, in order to optimize a given delivery system, a side-by-side analysis of nucleic acid cargo internalized and the corresponding biological effect is required to determine the overall efficacy. in this review, we will concentrate on peptide-mediated delivery of sirnas and steric block oligonucleotides and discuss different methods for quantitative assessment of the amount of cargo taken up and how to correlate those numbers with biological effects by applying easy to handle reporter systems. to illustrate current limitations of non-viral nucleic acid delivery systems, we present own data as an example and discuss options of how to enhance trafficking of molecules entrapped in cellular compartments. oligonucleotide-based strategies which can be used to modulate a vast variety of cellular functions represent a promising alternative to conventional therapies (for a review see: [ , ] ). among the different oligonucleotides with therapeutic potential are aptamers, transcription factor-binding decoy oligonucleotides, ribozymes, triplex-forming oligonucleotides (tfo), immunostimulatory cpg motifs, antisense oligonucleotides, small interfering rnas (sirnas) and antagomirs. nowadays, these potential macromolecular drugs are generally either relatively easily derived by rational design (e.g. antisense or sirna) or straightforward selection processes (e.g. aptamers). one of their main advantages over protein-or peptide-based approaches comprises the high specificity for their target while being non-immunogenic. however, despite these advances, a major impediment to the development of nucleic acid-based strategies for treatment and prevention of diseases is the relatively inefficient means to effectively deliver these macromolecules into the desired target cells. although viral vectors have been widely used to transfer genetic material into cells [ , ] , they bear an inherent risk for the patient to encounter severe immunological responses or even develop cancer [ ] [ ] [ ] [ ] . as a result of these problems much attention has been paid in recent years to the development of non-viral delivery systems. this conception *address correspondence to this author at the institut für molekulare medizin, universität zu lübeck, ratzeburger allee , lübeck, germany; tel: + - - - ; fax: + - - - ; e-mail: restle@imm.uni-luebeck.de includes an assortment of fairly unrelated approaches yielding various degrees of enhanced cellular uptake of nucleic acids. currently, liposomes and cationic polymers are used as a standard tool to transfect cells in vitro. however, these procedures are characterized by a significant lack of efficiency accompanied by a high level of toxicity rendering them mostly inadequate for in vivo applications. in this context cell-penetrating peptides (see below) represent an interesting alternative as they generally are less toxic than liposomes or cationic polymers. moreover, they are commonly better suited to transfer cargo into different cell types like non-adherent cells and primary cells, which are hard to transfect using commercially available standard protocols. the most advanced approaches in the field, which are not subject of the present article, are complex carrier systems combining vantages of assorted strategies to generate nanoparticles with better defined properties aimed towards enhanced uptake as well as intracellular trafficking in combination with cell-specific functionalities. for example, there are attempts to combine peptides with cationic liposomes [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] or polyethyleneimine (pei) [ ] . other strategies are aimed towards the synthesis of high or low molecular weight branched polymers and/or peptides [ ] [ ] [ ] [ ] [ ] or dendrimers [ , ] . even more complex systems are particularly promising with respect to in vivo delivery [ ] [ ] [ ] [ ] [ ] . in this review we will report about particular aspects of non-viral oligonucleotide delivery in vitro, pinpointing the current limitations, and provide quantitative means for determining where the bottlenecks of such strategies at present are. the focus of this article is recent progress in the field of peptide-mediated cellular delivery of sirna and steric block oligonucleotides in cell tissue culture as a starting point for further developments illustrated by own experimental data. our intention is not to provide the reader with easy solutions on how to solve the existing problems encountered with such approaches but give some hints where to start optimizing a particular approach. the idea of using peptides as carriers goes back some twenty years when it was discovered that the hiv- transactivating protein tat is taken up by mammalian cells [ , ] . a few years later, the antennapedia homeodomain of drosophila melanogaster was shown to act similarly [ ] . later on, it could be shown that peptides derived from tat and antennapedia as well as other proteins are capable of transporting macromolecular cargo molecules into cells [ ] [ ] [ ] . based on such promising results, a rapidly expanding field focusing on the so-called cell-penetrating peptides (cpps), also referred to as protein transduction domains (ptd), began to develop. since the first reports about tat, a large number of naturally occurring as well as engineered cpps have been discovered [ ] [ ] [ ] [ ] [ ] [ ] [ ] . table gives an overview of selected "classical" cpps. generally, cpps are short polycationic sequences of less than amino acids that are able to translocate different cargoes (e.g. nucleic acids, peptides and even entire proteins) into cells. the only common characteristic of these peptides appears to be that they are amphipathic and net positively charged at physiological ph. frequently the cargo is covalently attached to the cpp which can be achieved by expression as a fusion construct or by chemical coupling (for a review see: [ ] ). in particular cases, cargo and carrier bind each other non-covalently through mainly ionic interactions [ , , ] . despite the widespread interest in peptide carriers, the mechanisms underlying the cellular translocation of cpps are poorly understood. early work relied upon fluorescence imaging or flow cytometry analysis of chemically fixed cells to examine intracellular localization of fluorescently labeled peptides in the absence or presence of cargo. according to these experiments peptides appeared to be internalized very rapidly within minutes even at °c. from such observations it was concluded that cpps penetrate cell membranes by an energy-independent mechanism [ ] [ ] [ ] [ ] [ ] . although it had been reported quite early on that certain fixation procedures may cause artefacts leading to an overestimation of the cellular uptake rates [ ] [ ] [ ] the dimension of this problem was not commonly recognized until a side by side comparison of fixed and living cells was published [ ] . based on these findings, many groups re-examined their data. however, despite considerable technical improvements, there are still puzzling controversial results concerning the exact mechanism of cpp uptake. though in most cases endocytosis has been suggested to be the main route of internalization (fig. ( a) ), substantial difficulties are encountered identifying the exact pathway ( [ , ] and references therein). prior to endocytosis cpps interact electrostatically with the extracellular matrix of the cell surface mostly through binding to negatively charged glycosaminoglycans, i.e. heparan sulfate proteoglycans [ ] [ ] [ ] [ ] . recent studies indicate that the uptake mechanism of cpps can be influenced by the attachment of cargos. for example, richard et al. [ , ] reported a colocalization of tat - with markers of clathrin-mediated endocytosis, whereas fittipaldi et al. [ ] found a caveolae/lipid raft-dependent process for a tat-gfp fusion protein and wadia et al. [ ] described a macropinocytotic uptake pathway for a fusion construct of tat peptide with cre recombinase. in summary, the precise mechanism of internalization remains elusive and strongly depends on the properties of both cpp and cargo as well as on the transfection conditions and the cell lines used [ ] [ ] [ ] [ ] [ ] [ ] . as opposed to the majority of cpp applications reported, which rely on covalent linkage of carrier and cargo, limiting their general use considerably as a new construct has to be generated as well as tested for any given nucleic acid cargo, we will focus in this article on a peptide termed mpg which forms highly stable non-covalent complexes with nucleic acids (fig. ( ) ). the peptide is a derivative of the original mpg peptide described by morris and coworkers [ ] and differs by five amino acids in the hydrophobic part. these changes result in an alteration of the overall structure of the peptide towards a higher tendency of adopting a helical conformation [ ] . accordingly, the two peptides behave penetratin (antp - ) rqikiwfqnrrmkwkk [ ] transportan gwtlnsagyllgkinlkalaalakkil [ ] tp agyllgkinlkalaalakkil [ ] oligoarginine (r ) rrrrrrrr [ ] map klalklalkalkaalkla [ ] mpg galflgflgaagstmgawsqpkkkrkv [ ] mpg galflaflaaalslmglwsqpkkkrkv [ ] differently with respect to their interaction with artificial lipids as well as xenopus oocytes [ , ] and most probably, their exact mechanism of uptake is not the same. besides ionic interactions responsible for the initial peptide/nucleic acid complex formation, hydrophobic peptide/peptide interactions drive the maturation of large nanoparticles in a sandwich-like assembly reaction (fig. ( b) and fig. ( ) ). in recent years, rna interference (rnai) has gained a lot of interest as a tool for functional genomics studies and probably equally important as a promising therapeutic approach for the treatment of various diseases [ , ] . rnai is a highly evolutionally conserved and specific process of post-transcriptional gene silencing (ptgs) by which double stranded rna (dsrna), when introduced into a cell, causes sequence-specific degradation of homologous mrna sequences [ , ] . mechanistically the process can be divided into two steps. an initiator step where dsrna is cleaved by dicer, a member of the rnase iii family, into - nt long small interfering rna (sirna) fragments [ ] . in a consecutive step, these fragments are transferred to risc (rnainduced silencing complex) where one of the strands, the so called guide strand, serves as a molecular template to recognize homologous mrna that is cleaved by argonaute [ , ] , a protein component of risc. once the guide strand is bound to risc this complex can undergo many rounds of mrna binding and cleavage ( [ ] , fig. ( a) ). to circumvent application of long double stranded rnas, which inevitably trigger an interferon response, it is sufficient to extracellularly supply nt long dsrnas [ , ] . alternatively, sirnas can be expressed endogenously using dna vectors which code for short hairpin (sh) rnas [ ] [ ] [ ] . these shrnas are than cleaved by dicer to sirnas. short hairpin rna constructs have advantages over sirna because the effects of these constructs can lead to a more stable and long-term result. on the other hand, besides the fact that shrnas might interfere with the microrna pathway [ , ] , this strategy requires a gene therapy approach in the long run [ ] . for this reason we will not cover shrnas. as described above, sirnas represent a valuable tool to inhibit the expression of a target gene in a sequence-specific manner. in the following section, selected examples of cppmediated sirna delivery will be presented which are summarized in table . only a few studies describe the covalent attachment of nucleic acid cargo and peptide carrier (confer table ). in one approach, simple mixing of sirna targeted against gfp or cdk and tat peptide did not generate any measurable rnai effect whereas cross-linked sirna-tat - led to a significant down-regulation of the target proteins. however, high concentrations of sirna (about nm) had to be used [ ] . both lf-and tat - -mediated transfections resulted in a perinuclear localization of sirna. in contrast, fluorescently labeled tat - without cargo was mainly found in the nucleolus, suggesting that interactions with risc influence subcellular localization. in another approach, significant uptake of sirnas targeted against luciferase or gfp could be observed after disulfide coupling the '-end of the sense strand to penetratin or transportan [ ] . compared to lf , slightly higher levels of transfection were achieved. interestingly, after lf -mediated transfection, basal luciferase activity returned to normal levels one day earlier than after cpp-mediated transfection although the same concentration of sirna was applied. a remarkably strong rnai effect in hard to transfect primary neuronal cells was reported by davidson et al. [ ] . here, sirnas directed against several endogenous proteins were coupled to penetratin via a disulfide bond. the observed down regulation of the target proteins after peptide-mediated sirna delivery was found to be far more effective compared to lf . this was in part attributed to the toxicity of the lipids. as one of the first groups to report on tat - -or penetratin-mediated sirna delivery in vivo, moschos et al. showed, that intratracheal administration of the conjugates did not lead to any intensification of the knockdown of the target gene p mitogen-activated protein kinase in mouse lungs in comparison to unmodified non-formulated sirna [ ] . strikingly, it was found that the peptides alone triggered a detectable decrease in target gene expression and that the penetratin-conjugate induced elevated levels of the immune markers ifn-, tnf-, and il- p in lung tissue. besides technical difficulties arising from the syntheses of conjugates consisting of short cationic or hydrophobic peptides and highly negatively charged sirnas, dowdy and his group [ ] present a rather critical point of view referring to previous studies with cpp-sirna-conjugates. they claim that the successful delivery described therein is solely the result of excess free peptide, which leads to additional complexation, and thereby cellular import of the sirna. this is in accordance with turner et al. [ ] , who were the first to observe that careful purification of cpp-antisense-conjugates abrogates their biological effect. among other things, this might be the reason why most of the studies reporting on successful peptide-mediated delivery of sirnas use a noncovalent complexation approach (confer table ). in , simeoni et al. [ ] were the first who noncovalently complexed sirna with the peptide mpg. at a : ratio of negative nucleic acid to positive peptide charges a decrease in luciferase activity of about % was detectable in hela or cos- cells. this effect was further enhanced to about % down-regulation by a mutation in the nls sequence of the carrier peptide (mpg nls ), presumably due to an increased delivery to the cytoplasm, where risc is localized. recently, veldhoen et al. [ ] used a derivative of the mpg peptide for the delivery of sirna, which will be described in the chapter "mpg -mediated delivery of sirna and steric block oligonucleotides". leng et al. [ ] presented promising results with a prospect for cell-specific sirna delivery. different versions of a branched histidine/ lysine-polymer (h k b) yielded up to % knockdown of the target gene in several cell types. structure-function studies revealed an important role of the composition of the histidine-rich domain as well as its position within the peptide and the branches for sirna delivery, whereas size and surface charge did not have any effect. furthermore, the toxicity was much lower than for the commercial cationic lipids oligofectamine and lf . finally, the attachment of the tripeptide rgd, an integrin-ligand, slightly enhanced sirna delivery and turned this carrier into a cell-specific system. a fig. ( ) . simplistic scheme of peptide-based nucleic acid delivery systems (a). interaction of cpp and cargo is either achieved by covalent attachment or by non-covalent complexation through mainly ionic interactions. in case of non-covalent complex formation, a further assembly of cargo/carrier complexes occurs, leading to the formation of large nanoparticles (confer fig. ( ) ). in case of covalently joined molecules a similar scenario is less likely, yet cannot be excluded. prior to the translocation process the particles attach to the cell surface by ionic interactions of positively charged cpp residues with negatively charged membrane components. subsequently, complexes are taken up via an endocytotic pathway. although less likely, direct penetration cannot be excluded and may occur simultaneously. once inside the cell, the cargo has to escape from vesicular compartments, otherwise it eventually gets degraded in the lysosome. red: negative charges, blue: positive charges, green: hydrophobic domains. three-dimensional model of mpg /sirna interactions (b). the model was generated by iterative rigid body docking cycles of sirna (pdb r f) and peptide using the program hex . [ ] . the pdb file of mpg was generated with the program icm (molsoft llc) taking into consideration different secondary structure predictions and energy minimization protocols. out of many docking solutions particular ones were picked for illustration purposes using the program chimera [ ] . the phosphate backbone of the sirna is shown in red, the nucleobases in light gray. aliphatic, aromatic and hydrophobic residues of the peptide are shown in green, positive charged residues in blue and the remaining amino acids in gray. it is assumed that formation of larger particles is driven by hydrophobic peptide/peptide interactions generating free positive charges where other sirna molecules can interact. this eventually drives complex formation in a sandwich or mesh like assembly reaction. in principle such a scenario holds true for any given nucleic acid cargo. similar concept has very recently been used by kumar et al. [ ] for a specific delivery approach into the brain. a peptide derived from rabies virus glycoprotein (rvg) interacts specifically with the nicotinic acetylcholine receptor (achr) on neuronal cells to enable viral entry. the authors could show that the biotinylated form of the -amino-acid peptide (ytiwmpenprpgtpcdiftnsrgkrasng) was taken up by neuronal cells. in order to transport nucleic acids with this vehicle, r was conjugated to rvg peptide. systemic treatment of mice with sirna in a non-covalent complex with this modified peptide promoted a highly specific cellular import of sirna only into cells expressing achr. even more important, an antiviral sirna treatment resulted in successful protection of mice against encephalitis caused by japanese encephalitis virus (jev). this is the first study to report on a non-toxic method to deliver sirna across the blood brain barrier which could help to circumvent dangerous and ineffective injections into the brain. to date it presents one of the most promising tissue-specific delivery approaches which might be expandable to other in vivo applications. along these lines, most studies today are performed with the aim of cpp-mediated sirna delivery in vivo. although many of them are already showing promising results, e.g. concerning tumor-targeting and ocular delivery [ ] [ ] [ ] , this is beyond the scope of this review and will be discussed elsewhere in this issue [ , ] . with the aim to increase the endosomal escape of sir-nas after peptide-mediated delivery, lundberg et al. [ ] rationally modified penetratin to form a cpp (termed eb ) with improved endosomolytic properties. they achieved a ph-dependent conformational change of the peptide to a higher degree of helicity by the replacement of two basic amino acids with histidines and the n-terminal addition of six amino acids. in this study, several cpps were compared in a non-covalent approach by measuring the overall cellular stearyl-r n-c egfp, map b primary rat hippocampal neurons [ ] r -mend (sirna/stearyl-r core) n-c luciferase hela [ ] uptake via fluorescence and biological effect of sirna targeted to luciferase mrna. penetratin-as well as tp mediated transfection did not lead to any silencing of luciferase gene expression, despite high amounts of intracellular sirna [ ] and in contrast to previous reports using sirna-penetratin-conjugates [ ] or tp /dna-complexes [ ] . eb -mediated delivery of nm sirna led to approximately % reduction of luciferase activity. this silencing effect was slightly better than for bprpp and in the same range as for mpg nls , but still not as pronounced as for lf -mediated transfection of nm sirna. as it was described earlier, that addition of a ph-sensitive peptide derived from hemagglutinin (ha ) can promote endosomal escape [ ] , the authors linked ha to penetratin [ ] . it turned out that although ha -penetratin improved the silencing effect when coincubated with penetratin, eb was more potent than this combination of peptides. together with confocal microscopy studies the authors concluded that the lack of biological effect after penetratin-mediated sirna delivery is due to a lack of endosomal escape and that eb has a superior endosomolytic activity in comparison to ha penetratin. endoh et al. [ , ] very recently presented an innovative strategy, called clip-rnai (i.e. cpp-linked rbpmediated rna internalization and photo-induced rnai) combining delivery of a specific rna sequence with enhanced photoinduced release of rna from endosomes. this goal was accomplished by fusing the u a rna-binding domain (rbd) to the tat peptide and extending the sirna with a short stretch of nucleotides specifically recognized by this rbd. these complexes were efficiently internalized but exhibited a punctuate cytoplasmic localization pattern, indicative of endosomal entrapment. however, photostimulation of a fluorophore attached to the peptide led to a redistribution of complex into the cytosol followed by efficient rnai-mediated gene silencing. human pre-mrnas contain on average eight expressed sequences (exons) with an average length of nt and up to intervening sequences (introns) which can vary in length between and , nt, therefore comprising up to % of each transcriptional unit. in the nucleus, ribonucleoprotein complexes called spliceosomes recognize exon-intron boundaries and catalyze the precise removal of introns and subsequent joining of exons in a process called rna splicing [ ] [ ] [ ] . additionally, each primary transcript can yield different mature rnas through alternative splicing, thereby expanding the information content and versatility of the transcriptome, e.g. through the production of protein isoforms. a recent study of , human genes revealed, that at least % of all multi-exon genes are alternatively spliced [ ] . there are several different types of alternative splicing, amongst others affecting transcription start sites, splice sites, polyadenylation sites or even whole introns and exons. disruptions of these intricate splicing patterns are tightly coupled with human pathophysiology, either as a determinant or a direct cause of disease or as a modifier of disease susceptibility and severity [ ] . among these diseases are -thalassemia, cystic fibrosis, muscular dystrophies, frasier syndrome, certain kinds of dementia and cancer. a more de-tailed description of the underlying mechanisms is beyond the focus of this article and can be found in a number of reviews [ ] [ ] [ ] [ ] . lópez-bigas et al. [ ] proposed that % of mutations that cause disease lead to splicing defects rather than changes in the amino acid sequence. two common forms of mutations are depicted in fig. ( b) . on the one hand, a mutation in the splice donor can favor recognition of a cryptic splice donor and result in a mutant mrna containing additional intronic sequences (part i). on the other hand, a mutation in the splice acceptor can lead to skipping of a whole exon and result in a shortened mrna (part ii). both scenarios have been used in the context of antisense oligonucleotide-mediated approaches targeting alternative splicing. the use of antisense oligonucleotides interacting with mrna to affect protein production goes back some years [ , ] . since then, three principle mechanisms have been exploited for this purpose (for a review see: [ , ] ): (i) the oligonucleotide/rna duplex forms a substrate for endogenous rnase h, leading to mrna cleavage; (ii) the oligonucleotide/rna duplex prevents the productive assembly of the ribosomal complex or arrests a ribosomal complex already engaged in translation, in both cases affecting protein biosynthesis; (iii) the oligonucleotide/rna duplex alters pre-mrna splicing in the nucleus. the following section will focus on the last approach with the aim to treat splicing disorders and give examples of possible applications for cpps in this context. different forms of human -thalassemia are caused by mutations within in the -globin intron , which activate cryptic splice sites and thus lead to the formation of nonfunctional transcripts (fig. ( b part i) ). those aberrantly used sites can be blocked by antisense steric block oligonucleotides, which leads to the synthesis of functional protein [ , ] . kole and his group adopted this principle for the development of a splice correction assay [ ] . in this model system, a firefly luciferase construct leads to the synthesis of inactive enzyme because the reporter gene pre-mrna is interrupted by the human -globin intron containing an aberrant splice site. upon binding of a steric block oligonucleotide, correct splicing is restored which in turn yields a functional luciferase protein. to achieve this, the oligonucleotide has to be delivered to the nucleus. furthermore, only oligonucleotides that don't activate rnase h are applicable [ ] , e.g. phosphorodiamidate morpholino oligomers (pmo, [ ] ), locked nucleic acids (lna, [ ] ), peptide nucleic acids (pna, [ ] ) or '-o-methyl-modified oligonucleotides (ome). in addition to their inability to activate rnase h, most of these modifications confer higher affinity to the target rna and increased resistance against enzymatic degradation than unmodified versions. compared to rnaibased model systems, this assay is less susceptible to side effects like cytotoxicity or off-target effects because the reporter gene activity is turned up rather than turned down. the splice correction assay has been successfully applied for the analysis of several carrier systems [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in the following section, selected examples will be presented, which are summarized in table . in contrast to many noncovalent cpp-mediated sirna delivery approaches, efficient splice correction was only achieved with conjugates of peptide and steric block oligonucleotide. astriab-fisher et al. [ ] described delivery of ome rna phosphorothioate oligonucleotides linked via a disulfide bridge to tat peptide and penetratin. a few hours after transfection, the cpp-oligonucleotide conjugates were detected both in cytoplasmic vesicles and in the nucleus and caused a dose-dependent increase in luciferase activity. these findings are in contrast to results of turner et al. [ ] , who could not find a biological effect for several cpp-oligonucleotide conjugates in a hela cell assay for tat-mediated transactivation of the hiv- long terminal repeat. the authors observed vesicular uptake but no nuclear import for their highly pure conjugates. interestingly, the rate of uptake could be enhanced by addition of free cpp to the conjugates, though still no biological activity was detected. based on these findings, turner et al. [ ] concluded that these free cpps form complexes with cpp-cargo conjugates, which play a significant role in the uptake process. this is in accordance with observations by meade et al. [ ] for the uptake of cpp-sirna conjugates described above. moulton et al. [ ] achieved correction of missplicing at low micromolar concentrations of a r f -pmo conjugate but not with complexes of peptide and pmo. the steric block activity of the r f -pmo conjugates could be further increased with longer spacers whereas variations in the conjugation chemistry did not result in any differences. furthermore, transfection rates were higher than for conjugates with tat peptide, penetratin or a tat peptide analogue. using the hiv- transactivation assay mentioned above, turner et al. [ ] could show that most cpp-oligonucleotide conjugates attained biologic activity only through co-administration of the endosomolytic substance chloroquine. fluorescence microscopy analyses revealed that this treatment released fluorescently labeled conjugates from endosomal compartments into the nucleus. besides the addition of chloroquine, different endosome disrupting strategies have been evaluated using the splice correction assay, for example co-treatment with endosome-disruptive peptides [ ] or photochemical internalization [ ] (see chapter "strategies to enhance endosomal escape"). however, the most promising results have been achieved with two newly developed derivatives of classical cpps (reviewed in [ ] ). the modification of oligoarginines with non-natural, uncharged amino acids [ ] led, amongst others, to the peptide (r-ahx-r) , in which ahx represents a six-atom aminohexanoic acid spacer. abes et al. demonstrated that in contrast to tat or oligoargine, pmo-conjugates of this peptide led to dose-dependent splice correction at low micromolar concentrations in the absence of endosomolytic agents. the underlying mechanism for this superior activity is not clear yet, as the uptake of (r-ahx-r) constructs was less efficient than the uptake of tat or oligoarginine constructs and also involved endocytotic routes [ ] . the second peptide is a derivative of penetratin, to which six arginine residues were added at the n-terminus (r pen). r pen-pna conjugates were shown to promote efficient splice correction at low concentrations and in the absence of endosomolytic agents [ ] . again, uptake of r pen-conjugates seemed to involve endocytosis and there was hardly any difference in splice correcting activity regardless of the nature of the linker used for conjugation, e.g. a stable thioether versus a reducible disulfide linker [ ] . part ii of fig. ( b) illustrates a phenomenon that represents a strategy for the treatment of duchenne muscular dystrophy (dmd). dmd is a severe progressive neuromuscular disorder caused by several different mutations in the dystrophin gene that abolish the production of functional protein [ ] . depending on the location of the mutation, the corresponding exon is skipped by covering the responsible splice sites with steric block oligonucleotides. this allows the transcription of internally deleted, but largely functional, dystro-phin proteins and converts a severe dmd into a milder becker muscular dystrophy phenotype. a more detailed description of this approach and its application in a number of animal models can be found in several excellent recent reviews [ , ] . successful systemic delivery of splice switching oligonucleotides with or without chemical modifications (pmo, lna, ome) has been accomplished via injection of naked nucleic acids [ , ] , with the help of viral vectors [ ] , through re-implantation of ex vivo manipulated stem cells [ ] or in combination with cpps [ ] [ ] [ ] [ ] [ ] . for the latter purpose, several studies were carried out with novel derivatives of arginine-rich peptides containing different numbers of non-amino acids, e.g. aminohexanoic acid and/or -alanine. these cpp-pmo conjugates showed higher serum stability, less endosomal trapping and led to efficient exon skipping in myoblasts and mice at lower dosages than the splice switching pmo alone [ , ] . yin et al. [ ] used a pna-modified splice-switching oligonucleotide conjugated to tat, a muscle specific peptide (msp) or different functional domains of the adenovirus capsid protein vp (aav , aav ) and examined exon skipping efficiency in vitro and in vivo. surprisingly, both after transfection and intramuscular injection, the activity of these pna-peptide conjugates was not significantly better than that achieved by naked neutral pna, presumably due to endosomal trapping. intracellular trafficking represents one of the major limitations of current non-viral nucleic acid delivery approaches [ ] . in other words a large percentage of intracellular cargo molecules are entrapped in vesicular compartments and thus will not trigger the desired effect. moreover, degradation or retrograde transport might further reduce the number of active molecules. so in order to determine the overall efficacy of a given delivery approach it is essential to know the numbers of intact cargo molecules inside the cell along with the minimal numbers of molecules required to cause a particular effect. based on such information one can easily calculate the percentage of bioactive molecules. in the following chapter we will briefly describe selected examples of variable suitability for a quantitative determination of nucleic acids in a cellular context. in principle, either the peptide or the cargo can be labeled by a reporter group, e.g. a radioisotope [ ] or a fluorophore [ ] . fluorescent peptides or cargos have been quantitatively evaluated by facs [ ] or fluorescence correlation microscopy (fcs) [ ] or fret [ ] . in all cases, it is crucial to distinguish between internalized and membraneassociated signals. for this purpose, a simple wash step with just buffer is not sufficient to completely remove membranebound peptide/cargo-complexes [ , ] . extracellularly bound complexes can be efficiently removed for example by enzymatic digestion with trypsin [ ] , acid wash [ ] or heparin treatment [ , ] . alternatively, discrimination between intra-and extracellular material is possible through chemical modification of extracellular components [ ] or fluorescence quenching [ ] . having established that only intracellular signals are taken into account, it is still challenging to distinguish between intact and degraded forms of peptide or cargo. in two studies, a fluorescence-based quantification method was combined with either hplc analysis [ ] or "cell activity by capillary electrophoresis" [ ] to verify the integrity of cargo and carrier. recently, a technique to measure cellular uptake of cpps by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof ms) was reported by the group of burlina [ ] [ ] [ ] . this quantification is based on the addition of an internal standard, i.e. a peptide with a stable isotope label. the method has been used to determine the amount and stability of intact internalized peptides, e.g. penetratin, r and several novel cpps, and can also be used for the quantification of peptidic cargoes, e.g. an inhibitor of protein kinase c [ ] . varga et al. [ ] [ ] [ ] developed an "integrative systems" approach, combining quantitative experiments and computational modeling studies of vector uptake and trafficking kinetics, with the aim to take multiple potentially rate-limiting cellular and molecular processes into account. by applying their mathematical model to plasmid delivery with either lipofectamine, several pei-based vector formulations or an adenoviral vector, they could successfully predict experimentally observed effects and identify endosomal escape as the most important rate-limiting intracellular barrier for non-viral vectors. recently, zhou et al. [ ] applied a similar strategy for the characterization of a novel lipopolymer (wlsp). this carrier shows an increased rate of endosomal escape compared to conventional pei-based carriers. with the aim to quantify rhodamine-labeled plasmid dna in cellular compartments while avoiding problems arising from subcellular fractionation, like recovery and leakage, akita et al. [ ] developed a novel quantitative strategy, the confocal image-assisted three-dimensionally integrated quantification (cidiq) method. to distinguish endosomes/lysosomes and the nucleus from the cytosol, they were stained with lysosensor dnd- and hoechst , respectively, and sequential z-series images were captured by clsm. by applying this quantification method, the authors could show that due to a rapid endosomal escape, lipofectamine plus delivered more plasmid into the nucleus than r or stearylated r . hama et al. [ ] used the same method in combination with taqman pcr to evaluate the uptake and intracellular distribution of plasmid dna after delivery with viral as well as non-viral vectors. due to superior cell surface binding, the efficiency of cellular uptake was significantly higher for lipofectamine plus than for adenovirus whereas intracellular trafficking, i.e. endosomal escape and nuclear transfer, were essentially the same. however, to achieve comparable transgene expression, -fold higher intranuclear plasmid numbers were required in case of lipofectamine plus. this finding suggests a difference in nuclear transcription efficiency after non-viral delivery. in another approach, jiang et al. [ ] extended the ' end of the sense strand of a sirna with a nuclease-resistant dna hairpin to obtain a so-called "crook" sirna. this modification had no effect on rnai-mediated reporter gene inhibition and served as a primer for a filling-in reaction followed by pcr. parameters were chosen so that the initial rate of template amplification correlates with the initial con-centration of the "crook" sirna. under these conditions, quantification of attomolar sirna levels per cell was possible after liposomal transfections with oligofectamine. a highly sensitive method was developed by overhoff et al. [ ] for the detection of sirna after phosphorothioatestimulated uptake [ ] and adapted for the quantification of sirna or steric block oligonucleotides after non-covalent peptide-mediated delivery ( [ ] and laufer et al., manuscript in preparation, see chapter "mpg -mediated delivery of sirna and steric block oligonucleotides"). this so-called liquid hybridization assay is based on the extraction of total cellular rna and the subsequent hybridization in solution of a radioactively labeled probe which is complementary to the oligonucleotide to be detected. finally, following page analysis, absolute amounts of internalized oligonucleotide can be quantified with high accuracy down to ~ molecules per cell using internal standards [ ] . in addition to this outstanding sensitivity, no amplification step is needed and only intact oligonucleotides are taken into account. considering the multitude of available cpps, nucleic acid cargos and cellular as well as animal model systems, a comparison of different delivery strategies seems nearly impossible. in this context, we have for the first time undertaken a detailed side by side comparison of two different model systems using the peptide mpg as delivery agent. in the following paragraph we present own experimental data to exemplarily illustrate particular aspects regarding current limitations of peptide-based delivery systems. in contrast to many other cpp procedures, which rely on covalent linkage of carrier and cargo, the peptide mpg forms highly stable non-covalent complexes with nucleic acids, displaying binding constants in the low nanomolar range ( [ ] , a. trampe, unpublished data). the high flexibility of this non-covalent approach can be exploited to easily transport a wide variety of nucleic acid cargos without having to synthesize a new construct for each oligonucleotide. we have used mpg for the delivery of sirnas in the context of an rnai-based reporter system and for the delivery of steric block oligonucleotides in the context of the splice correction assay described above [ ] . after mpg -mediated transfection of a luciferase-targeted sirna, we observed strong inhibition of reporter gene readout with an ic in the subnanomolar range [ ] . after mpg -mediated transfection of a luciferasetargeted steric block oligonucleotide (further on also referred to as on- ), we observed a moderate up-regulation of reporter gene readout, representative of low splice correcting activity (laufer et al., manuscript in preparation). one possible explanation for the different degree of reporter gene regulation could be the different intracellular target sites, i.e. the cytoplasm for sirnas and the nucleus for splice correction oligonucleotides. to attain more information about the subcellular localization of mpg -oligonucleotide complexes, we performed confocal laser scanning as well as conventional fluorescence microscopy studies with fluorescently labeled sirnas or steric block oligonucleotides. in both cases, a punctuate non-homogenous distribution of the nucleic acids inside the cells was observed. this pattern is indicative of an accumulation of nucleic acids in endocytotic vesicles, which was verified by coincubation with lysosensor (fig. ( a) ). a quantitative computational analysis of fluorescence microscopy data yielded an average of approximately % colocalization between endosomes and sirna (fig. ( b) ). in contrast to earlier assumptions that cpps directly traverse the lipid bilayer, it has commonly become accepted that for most peptide-cargo combinations endocytosis plays a major role in cellular uptake. as described above and in the chapter "strategies to enhance endosomal escape" below, administration of endosome disruptive substances like chloroquine can greatly increase endosomal release of trapped nucleic acids. chloroquine is a weak base, non-charged at neutral ph but charged at ph . [ ] . it is able to pass easily through membranes in its uncharged form, but becomes protonated and accumulates within acidic vesicles in its positively charged, membraneimpermeable form. although its exact mode of action has not yet been resolved, it is generally accepted that chloroquine works via prevention of endosome acidification which in turn increases the residence time of cargo within the endosomes eventually resulting in a higher probability of transfer to the cytoplasm. fluorescence microscopy analyses in the presence of m chloroquine yielded two quite contrary outcomes. while the localization of sirna did not change after addition of chloroquine (fig. ( c) ), for the steric block oligonucleotide the picture changed completely (fig. ( d) ). in the latter case, a diffuse fluorescence all over the cytoplasm with an accumulation of on- in the nucleus could be observed. nonetheless, considerable amounts of nucleic acid molecules were still visible as a punctual pattern, which indicates that the chloroquine treatment liberates only a certain fraction. on the whole, these qualitative observations are in full agreement with the observed biologic effects in the absence or presence of chloroquine (fig. ( a) ). for mpg -mediated transfection of sirna, even under conditions where the amount of bio-available sirna was severely limited, only a minor increase in rnai (ca. %) was measurable. for mpg -mediated transfection of steric block oligonucleotide, on the other hand, a dramatic increase of reporter gene up-regulation by a factor of - was observed. however, in both cases, the overall uptake did not change upon incubation with chloroquine ( fig. ( b) ), which proves that the endosomolytic substance does not interfere with uptake but leads to a re-distribution of internalized nucleic acids. the underlying mechanism for the different effects triggered by chloroquine in case of peptide/sirna and peptide/steric block oligonucleotide complexes remains unclear. though, this is a good example that the cargo can substantially affect the properties and thereby intracellular trafficking of a particular carrier system. ultimately, to assess the overall efficacy of this carrier system, we were interested to elucidate which percentage of molecules taken up after mpg -mediated delivery is biologically active. to derive such information, the exact intracellular amount of intact oligonucleotide, the corresponding reporter signal and the minimal number of molecules necessary to trigger a specific degree of reporter gene modulation have to be known. for the quantification of internalized cargo, we adapted a highly sensitive method first described by overhoff et al. [ ] , enabling us to detect intracellular oligonucleotide amounts down to copies per cell [ ] . the method is based on the liquid hybridization of a radioactively labeled probe with the corresponding oligonucleotide in cellular lysates. in this context it should be noted that a stringent heparin wash following the transfection procedure is crucial to avoid an overestimation of intracellular nucleic acid molecules due to complexes attached to the outside of the cell membrane [ , ] . in order to correlate the numbers derived from the quantification experiments with the minimal number of molecules essential to trigger the observed effect, an independent assay had to be established. the gold standard in this case is microinjection as this technique enables one to deliver definite amounts of nucleic acids with a high degree of bioavailability into the cytoplasm or the nucleus of a mammalian cell along with a low toxicity profile and great accuracy. considering that after cytoplasmic microinjection only sirna molecules are sufficient for halfmaximal inhibition of reporter gene expression, one can estimate that of the , molecules measured after mpgmediated transfection, only ca. . % are biologically active ( table ) . for the splice correction assay, the numbers are different in terms of absolute numbers but the outcome remains the same. compared to the , molecules sufficient for maximal splice correction after nuclear microinjection, of the , , molecules required following peptide-mediated delivery only ca. . % are biologically active ( table ). in both cases >> % of internalized oligonucleotides are most likely retained in endosomes and subsequently degraded in lysosomes after peptide-mediated delivery. frankly, this is a sobering result and puts in numbers how much room for improvement there actually is. though it certainly is not legitimate to generalize these findings, there are countless reports in the literature suggesting similar limitations for the majority of non-viral strategies. according to actual conceptions, sirna enters a multiple-turnover pathway with one sirna molecule capable of risc-mediated cleavage of or more mrna molecules [ ] . even though the fate of steric block oligonucleotides is not really clear, it can be assumed that per splicing event one molecule is used up and translated into a functional mrna molecule (e.g. single-turnover pathway). as a result the number of molecules needed to trigger an apparent effect is much higher in the splice correction assay compared to the rnai-based reporter system (confer table and table ). on the other hand, being a single-turnover pathway, the splice correction assay should be much more sensitive to even minor changes in intracellular steric block oligonucleotide concentrations whereas the catalytic nature of the multiple-turnover rnai mechanism might mask such small variations. this is in accordance with the data described above. taken together, based on the results presented as well as unpublished data (laufer et al., manuscript in preparation) , the splice correction assay appears to be the superior tool for a quantitative assessment of nucleic acid delivery strategies. as outlined above, endosomal release is one of the major rate-limiting steps for cellular delivery of macromolecules via cationic lipids, polyplexes and especially cpps. in the following chapter we will present some examples of how to increase endosomal release. transfections were performed with . m mpg and nm sirna or g/ml lf and . nm sirna, i.e. in the range of the ic value [ ] . quantification was performed after h according to the liquid hybridization protocol [ ] . molecules per cell were calculated based on the cell number seeded for transfection. for microinjection experiments, molecules per cell were calculated on the basis of the injection volume. endosome-disrupting substances, like chloroquine, calcium or sucrose, were used to significantly enhance the activity of antisense pna oligonucleotides conjugated to tat, oligoarginines or oligolysines [ , , ] . this effect did not result from increased uptake, but rather improved bioavailability in the cytoplasm or nucleus after endosomal escape. takeuchi et al. [ ] showed that by incubation of the target cells with pyrenebutyrate, delivery of arginine-rich peptides could be shifted from endocytic uptake to direct membrane translocation, yielding a rapid distribution of the peptide throughout the cytoplasm, even at °c. pyrenebutyrate acts as a counteranion and, by interacting with the positively charged peptide, increases the overall hydrophobicity, thereby facilitating a direct translocation through the lipid bilayer, as earlier shown with artificial membranes [ ] . this method, which works only in the absence of a medium or serum, was successfully applied for administration of a fluorescent protein and an apoptosis-inducing peptide into dividing as well as non-dividing cells [ ] . however, in general the strategies described above are not feasible for in vivo applications, due to high cytotoxicity or other undesirable secondary effects. the imidazole group of histidine (his) can absorb protons in the acidic environment of the endosome, leading to osmotic swelling, membrane disruption and eventually nucleic acid escape. accordingly, lo et al. [ ] modified tat, which can bind and condense dna through ionic interactions but has no acidic residues that can promote endosomal release, with different numbers of his residues. highest reporter gene expression could be achieved after plasmid delivery with a tat peptide covalently fused to his residues (tat- h). insertion of two additional cysteine residues into tat- h further enhanced stability of peptide/dna complexes and transgene expression through formation of interpeptide disulfide bonds. youngblood et al. [ ] evaluated the influence of the stability of arginine-rich peptide pmo conjugates on cellular uptake and antisense activity. they could show that the stability is affected by the amino acid composition and the type of linkage to the cargo. moreover, they found that degraded fragments could not escape anymore from endosomal or lysosomal compartments. another concept makes use of photosensitive substances, which induce the release of macromolecules from vesicles by light exposure. this so-called photochemical internalization (pci) has, in the past, been used for intracellular delivery of a large variety of macromolecules (reviewed in [ ] ) and, more recently, for the endosomal release of nucleic acids after delivery mediated by liposomes [ , ] , polyplexes [ ] or cpps [ , ] . shiraishi et al. [ ] investigated the biological activity of pnas conjugated either to tat, r or kla-peptide in combination with a pci treatment. depending on the peptide, nuclear as well as cytosolic antisense effects could be enhanced by up to two orders of magnitude. similar results were presented by folini et al. [ ] for a pna targeting human telomerase reverse transcriptase conjugated to tat. in both studies, lower nucleic acid doses were sufficient, thereby reducing the probability of off-target effects. in light of encouraging data from ongoing anticancer clinical trials employing photodynamic therapy [ , ] , an in vivo application of pci seems feasible and will be discussed in more detail by oliveira et al. [ ] in this issue. furthermore, target specificity could be increased by local illumination of cells or tissue. viral fusion proteins drive the fusion process between the viral membrane and the endosomal host cell membrane in a ph-dependent manner, which is required to translocate the viral genome into the cytoplasm after receptor-mediated endocytosis. fusogenic peptides, usually hydrophobic, rich in glycine residues and found at the amino terminus of these proteins, were shown to have membrane perturbing and lipid mixing activities [ ] . many well studied representatives of this group are derived from the fusion sequence of influenza virus ha or hiv- gp [ ] and have been used to improve the transfection efficiency of non-viral delivery systems [ ] . addition of the influenza-derived dimeric peptide diinf- to lf /sirna complexes had no effect on the particle size of ca. nm, but significantly improved gene silencing activity of sirnas targeting the epidermal growth factor receptor or the k-ras oncogene [ ] . similar results were obtained for plasmid delivery through addition of a fusogenic peptide derived from herpes simplex virus glycoprotein h to lipofectamine/dna complexes [ ] . to the same end, futaki et al. [ ] used the peptide gala, which was specially designed to mimic the function of viral fusion sequences, together with various commercially available cationic liposomes. although they could not detect significant differences in cellular localization, plasmid transfection efficiency was increased and liposomal dosage could be reduced. pei covalently modified with the hiv- gp -derived peptide hgp led to a -fold increase of gene expression after plasmid dna delivery and also enhanced sirnamediated knockdown of gapdh by approximately -fold [ ] . % of cells incubated with pei-hgp polyplexes showed not only the punctuate plasmid dna staining observed with pei polyplexes alone, but also a diffuse fluorescence throughout the cell, indicative of endosomal release of vectors. this would explain the observed increase in transfection efficiency, since the overall uptake was unaffected. wadia et al. [ ] were the first to use the influenza hemagglutinin-derived fusogenic peptide ha in combination with a cpp. delivery of a tat-ha conjugate with increasing concentrations of a tat-cre conjugate enhanced reporter protein activity, presumably through an increased release from macropinosomes. the same fusogenic peptide, linked to a polyarginine-p conjugate, promoted release of p from macropinosomes and subsequent translocation to the nucleus, accompanied by an enhanced anti-cancer effect [ ] . the development of delivery systems for therapeutic oligonucleotides is a fast growing field. owing to the enormous potential of short nucleic acids as alternative drugs such a growth is not unexpected. besides viral vectors there is a highly diverse and constantly increasing number of non-viral systems evolving. however, despite considerable progress achieved in recent years, even the most advanced systems either lack the efficiencies required for downstream drug development or do show a substantial degree of toxicity or both. of the many factors which limit their use, cellular uptake of the cargo/carrier complexes and subsequent intracellular trafficking to reach the target site are the most important. in addition to such essential considerations there are various additional parameters to be taken into account like serum stability, pharmacokinetic features and tissue barriers as well as target cell specificity. now the question arises where to start optimizing a given delivery system. currently, there is a clear trend towards in vivo testing. in principle such a development is a step in the right direction since many of the experimental data derived from artificial cell tissue culture systems with established cell lines are not applicable to the in vivo situation. on the other hand, it is questionable to what extent such animal experiments will eventually pay off as long as important fundamental problems remain largely unsolved. as outlined above, our quantitative studies along with microscopic analyses of sirnas and steric block oligonucleotides using either a peptide or a commercially availably cationic lipid as carrier clearly show that less than . % - % of molecules taken up are involved in a biological response, i.e. rnaimediated down regulation or splice correction-mediated up regulation of reporter gene activity. evidently, the vast majority of internalized cargo never reaches the target. this implies that uptake per se is not the limiting factor here. although there are no such detailed quantitative numbers available in the literature for other systems, there are countless reports showing that to various degrees this applies to almost any non-viral delivery approach currently available. taken together, if one would succeed to optimize intracellular trafficking this holds the potential to boost overall efficacy by up to orders of magnitude. moreover, it is reasonable to assume that such fundamental cellular restrictions can be adequately investigated in tissue culture without the use of animal models. so it might be worthwhile to reconsider the concept of maybe premature in vivo testing by moving backwards one step and first optimizing the systems with regard to intracellular limitations before dealing with the next level of complexity. accordingly, in vitro model systems like the ones described above for sirnas or steric block oligonucleotides are valuable tools to study particular aspects of nucleic acid delivery. however, currently available data are based on studies using a variety of different cell lines and techniques, which renders a direct comparison of different delivery approaches impossible. in this context it would be highly desirable to introduce standardized protocols for in vitro testing (e.g. the splice correction system developed by kole and coworkers [ ] ) together with methods for detailed quantitative analyses (e.g. the liquid hybridization protocol [ , ] ). this would facilitate a direct quantitative comparison of exceedingly diverse approaches on at least the cellular level. one reason for the problem with intracellular trafficking of oligonucleotides arises from the mode carrier/cargo complexes are taken up by cells. today it is well established that the majority of these complexes are taken up via endosomal pathways and therefore end up in vesicular compartments from which they have to escape in order to reach their target. although there are many attempts reported to trigger endosomal escape by various strategies, they either proved to be toxic or did not achieve sustained success. additionally, there might be a further reason for the encountered difficulties to overcome these intracellular barriers. it is not unreasonable to speculate that during evolution cells might have evolved mechanisms to avoid large amounts of foreign nucleic acids freely floating in the cytoplasm and thus safely contain them in vesicular compartments where they are eventually degraded. alternatively they might be exported by retrograde transport. co-evoluting viruses evidently have developed strategies to circumvent such defense mechanisms. so it might be somewhat naive to expect that a rather simple man-made carrier system is capable to efficiently overcome such an intrinsic barrier. current developments towards more complex and elaborate carrier systems take into account such considerations. in any case it appears there is no simple solution for this problem. in conclusion, despite significant progress in the field of nucleic acid delivery in vitro as well as in vivo, there still is a long way to go before this will become a standard procedure in the clinic. certain problems like endosomal escape are known for more than twenty years and still far from being resolved. in order to develop new strategies, more information about intracellular processes involved in nucleic acid trafficking is needed. moreover, it would be desirable if the field would move from a qualitative description towards a quantitative evaluation preferentially using standardized model systems. this would allow for comparison of different approaches with one another. while animal studies are inevitable in the long run, there still is a lot of room for improvement on the cellular level. so it might be worthwhile to fathom how far we can push the different systems on this level. nucleic-acid therapeutics: basic principles and recent applications the versatility of oligonucleotides as potential therapeutics gene therapy with viral vectors gene therapy: twenty-first century medicine a pilot study of in vivo liver-directed gene transfer with an adenoviral vector in partial ornithine transcarbamylase deficiency lmo -associated clonal t cell proliferation in two patients after gene therapy for scid-x fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer gene therapy put on hold as third child develops cancer tat peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors comparison between the interactions of adenovirus-derived peptides with plasmid dna and their role in gene delivery mediated by liposomepeptide-dna virus-like nanoparticles vectors based on reducible polycations facilitate intracellular release of nucleic acids cell transfection in vitro and in vivo with nontoxic tat peptide-liposome-dna complexes hiv- tat protein transduction domain peptide facilitates gene transfer in combination with cationic liposomes unique features of a ph-sensitive fusogenic peptide that improves the transfection efficiency of cationic liposomes a versatile reducible polycation-based system for efficient delivery of a broad range of nucleic acids tat peptide-mediated intracellular delivery of pharmaceutical nanocarriers evaluation of transportan in pei mediated plasmid delivery assay efficient gene transfer by histidylated polylysine/pdna complexes branched co-polymers of histidine and lysine are efficient carriers of plasmids a novel transfecting peptide comprising a tetrameric nuclear localization sequence highly branched hk peptides are effective carriers of sirna macro-branched cell-penetrating peptide design for gene delivery protein transduction by lipidic peptide dendrimers tat-conjugated pamam dendrimers as delivery agents for antisense and sirna oligonucleotides design, synthesis, and characterization of ph-sensitive peg-pe conjugates for stimuli-sensitive pharmaceutical nanocarriers: the effect of substitutes at the hydrazone linkage on the ph stability of peg-pe conjugates smart" drug carriers: pegylated tatpmodified ph-sensitive liposomes octaarginine-modified multifunctional envelope-type nanoparticles for gene delivery multiblock reducible copolypeptides containing histidine-rich and nuclear localization sequences for gene delivery intracellular sirna and precursor 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oligonucleotides and analogues with cell penetrating peptides as gene silencing agents delivery of proteins and nucleic acids using a non-covalent peptide-based strategy peptide-based nanoparticle for ex vivo and in vivo drug delivery cell internalization of the third helix of the antennapedia homeodomain is receptor-independent a new peptide vector for efficient delivery of oligonucleotides into mammalian cells a truncated hiv- tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus in vivo protein transduction: intracellular delivery of biologically active proteins, compounds and dna arginine-rich peptides. an abundant source of membranepermeable peptides having potential as carriers for intracellular protein delivery intracellular localization of oligonucleotides: influence of fixative protocols is vp nuclear homing an artifact? positively charged dna-binding proteins cause apparent cell membrane translocation cell-penetrating peptides. a reevaluation of the mechanism of cellular uptake cellular delivery of small interfering rna by a non-covalently attached cellpenetrating peptide: quantitative analysis of uptake and biological effect ballmer-hofer k. antennapedia and hiv transactivator of transcription (tat) "protein transduction domains" promote endocytosis of high molecular weight cargo upon binding to cell surface glycosaminoglycans internalization of hiv- tat requires cell surface heparan sulfate proteoglycans multiple interactions of hiv-i tat protein with sizedefined heparin oligosaccharides interaction of hiv- tat protein with heparin. role of the backbone structure, sulfation, and size cellular uptake of unconjugated tat peptide involves clathrin-dependent endocytosis and heparan sulfate receptors cell membrane lipid rafts mediate caveolar endocytosis of hiv- tat fusion proteins transducible tat-ha fusogenic peptide enhances escape of tat-fusion proteins after lipid raft macropinocytosis role of membrane potential and hydrogen bonding in the mechanism of translocation of guanidinium-rich peptides into cells effects of cargo molecules on the cellular uptake of arginine-rich cell-penetrating peptides on the mechanisms of the internalization of s ( )-pv cellpenetrating peptide novel human-derived cell-penetrating peptides for specific subcellular delivery of therapeutic biomolecules a novel cellpenetrating peptide, m , for efficient delivery of proteins and peptide nucleic acids protein transduction revisited: novel insights into the mechanism underlying intracellular delivery of proteins primary amphipathic cell-penetrating peptides: structural requirements and interactions with model membranes on the mechanism of non-endosomial peptide-mediated cellular delivery of nucleic acids interactions of amphipathic cpps with model membranes rnai therapeutics: a potential new class of pharmaceutical drugs interfering with disease: a progress report on sirna-based therapeutics potent and specific genetic interference by double-stranded rna in caenorhabditis elegans illuminating the silence: understanding the structure and function of small rnas role for a bidentate ribonuclease in the initiation step of rna interference argonaute , a link between genetic and biochemical analyses of rnai argonaute proteins: key players in rna silencing kinetic analysis of the rnai enzyme complex duplexes of -nucleotide rnas mediate rna interference in cultured mammalian cells rna interference is mediated by -and -nucleotide rnas short hairpin rnas (shrnas) induce sequence-specific silencing in mammalian cells rna interference by expression of short-interfering rnas and hairpin rnas in mammalian cells rna interference: from gene silencing to gene-specific therapeutics fatality in mice due to oversaturation of cellular mi-crorna/short hairpin rna pathways toxicity in mice expressing short hairpin rnas gives new insight into rnai expressing short hairpin rnas in vivo visualizing a correlation between sirna localization, cellular uptake, and rnai in living cells conjugate for efficient delivery of short interfering rna (sirna) into mammalian cells highly efficient small interfering rna delivery to primary mammalian neurons induces microrna-like effects before mrna degradation lung delivery studies using sirna conjugated to tat( - ) and penetratin reveal peptide induced reduction in gene expression and induction of innate immunity enhancing the cellular uptake of sirna duplexes following noncovalent packaging with protein transduction domain peptides synthesis, cellular uptake and hiv- tat-dependent trans-activation inhibition activity of oligonucleotide analogues disulphide-conjugated to cell-penetrating peptides insight into the mechanism of the peptide-based gene delivery system mpg: implications for delivery of sirna into mammalian cells transvascular delivery of small interfering rna to the central nervous system cell-penetrating peptide for enhanced delivery of nucleic acids and drugs to ocular tissues including retina and cornea cholesteryl oligoarginine delivering vascular endothelial growth factor sirna effectively inhibits tumor growth in colon adenocarcinoma cell-penetrating-peptidemediated sirna lung delivery cellular delivery in vivo of sirna-based therapeutics targeting the lung using sirna and antisense based oligonucleotides delivery of short interfering rna using endosomolytic cellpenetrating peptides tp , a delivery vector for decoy oligonucleotides targeting the myc protein photo inducible rna interference using cell permeable protein carrier cellular sirna delivery mediated by a cell-permeant rna-binding protein and photoinduced rna interference split genes and rna splicing the spliceosome: the most complex macromolecular machine in the cell? understanding alternative splicing: towards a cellular code genome-wide survey of human alternative pre-mrna splicing with exon junction microarrays splicing in disease: disruption of the splicing code and the decoding machinery alternative splicing: multiple control mechanisms and involvement in human disease pre-mrna splicing and human disease alternative splicing in disease and therapy alternative splicing in disease are splicing mutations the most frequent cause of hereditary disease? inhibition of rous sarcoma viral rna translation by a specific oligodeoxyribonucleotide inhibition of rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide progress in antisense technology antisense oligonucleotides: from design to therapeutic application restoration of hemoglobin a synthesis in erythroid cells from peripheral blood of thalassemic patients therapeutic potential of antisense oligonucleotides as modulators of alternative splicing up-regulation of luciferase gene expression with antisense oligonucleotides: implications and applications in functional assay development morpholino antisense oligomers: design, preparation, and properties lna (locked nucleic acid): high-affinity targeting of complementary rna and dna endosome trapping limits the efficiency of splicing correction by pna-oligolysine conjugates vectorization of morpholino oligomers by the (r-ahx-r) peptide allows efficient splicing correction in the absence of endosomolytic agents efficient splicing correction by pna conjugation to an r -penetratin delivery peptide conjugates of antisense oligonucleotides with the tat and antennapedia cell-penetrating peptides: effects on cellular uptake, binding to target sequences, and biologic actions induction of splice correction by cell-penetrating peptide nucleic acids cell penetrating peptide conjugates of steric block oligonucleotides evaluation of transfection protocols for unmodified and modified peptide nucleic acid (pna) oligomers cellular delivery of polyheteroaromate-peptide nucleic acid conjugates mediated by cationic lipids comparison of basic peptides-and lipid-based strategies for the delivery of splice correcting oligonucleotides antisense-mediated redirection of mrna splicing structural requirements for cellular uptake and antisense activity of peptide nucleic acids conjugated with various peptides cellular uptake of antisense morpholino oligomers conjugated to arginine-rich peptides cell-penetrating peptide conjugates of peptide nucleic acids (pna) as inhibitors of hiv- tat-dependent transactivation in cells photochemically enhanced cellular delivery of cell penetrating peptide-pna conjugates arginine-rich molecular transporters for drug delivery: role of backbone spacing in cellular uptake dystrophin: the protein product of the duchenne muscular dystrophy locus modification of pre-mrna processing: application to dystrophin expression antisense-mediated exon skipping: a versatile tool with therapeutic and research applications clinical approaches in the treatment of duchenne muscular dystrophy (dmd) using oligonucleotides the therapeutic potential of antisense-mediated exon skipping potential of oligonucleotide-mediated exon-skipping therapy for duchenne muscular dystrophy systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology efficient and persistent splice switching by systemically delivered lna oligonucleotides in mice rescue of dystrophic muscle through u snrnamediated exon skipping restoration of human dystrophin following transplantation of exon-skipping-engineered dmd patient stem cells into dystrophic mice morpholino oligomer-mediated exon skipping averts the onset of dystrophic pathology in the mdx mouse antisense oligonucleotide-induced exon skipping restores dystrophin expression in vitro in a canine model of dmd cell-penetrating peptide-morpholino conjugates alter pre-mrna splicing of dmd (duchenne muscular dystrophy) and inhibit murine coronavirus replication in vivo sustained dystrophin expression induced by peptide-conjugated morpholino oligomers in the muscles of mdx mice effective exon skipping and restoration of dystrophin expression by peptide nucleic acid antisense oligonucleotides in mdx mice cellular uptake and intracellular release are major obstacles to the therapeutic application of sirna: novel options by phosphorothioate-stimulated delivery quantitative analysis of permeation peptide complexes labeled with technetium- m: chiral and sequence-specific effects on net cell uptake quantitative assessment of the cell penetrating properties of ri-tat- : evidence for a cell type-specific barrier at the plasma membrane of epithelial cells a quantitative validation of fluorophore-labelled cell-permeable peptide conjugates: fluorophore and cargo dependence of import cargo delivery kinetics of cell-penetrating peptides translocation properties of novel cell penetrating transportan and penetratin analogues acid wash in determining cellular uptake of fab/cellpermeating peptide conjugates cationic tat peptide transduction domain enters cells by macropinocytosis cellular uptake of an alpha-helical amphipathic model peptide with the potential to deliver polar compounds into the cell interior non-endocytically physico-chemical requirements for cellular uptake of pantp peptide. role of lipid-binding affinity characterization of tat-mediated transport of detachable kinase substrates quantification of the efficiency of cargo delivery by peptidic and pseudopeptidic trojan carriers using maldi-tof mass spectrometry quantification of the cellular uptake of cell-penetrating peptides by maldi-tof mass spectrometry a direct approach to quantification of the cellular uptake of cell-penetrating peptides using maldi-tof mass spectrometry quantitative analysis of synthetic gene delivery vector design properties quantitative comparison of polyethylenimine formulations and adenoviral vectors in terms of intracellular gene delivery processes quantitative comparison of polyethylenimine formulations and adenoviral vectors in terms of intracellular gene delivery processes intracellular kinetics of non-viral gene delivery using polyethylenimine carriers quantitative three-dimensional analysis of the intracellular trafficking of plasmid dna transfected by a nonviral gene delivery system using confocal laser scanning microscopy quantitative comparison of intracellular trafficking and nuclear transcription between adenoviral and lipoplex systems a bi-functional sirna construct induces rna interference and also primes pcr amplification for its own quantification quantitative detection of sirna and single-stranded oligonucleotides: relationship between uptake and biological activity of sirna phosphorothioate-stimulated cellular uptake of sirna: a cell culture model for mechanistic studies weak bases and ionophores rapidly and reversibly raise the ph of endocytic vesicles in cultured mouse fibroblasts calcium ions effectively enhance the effect of antisense peptide nucleic acids conjugated to cationic tat and oligoarginine peptides enhanced delivery of cell-penetrating peptide-peptide nucleic acid conjugates by endosomal disruption direct and rapid cytosolic delivery using cell-penetrating peptides mediated by pyrenebutyrate direct observation of anion-mediated translocation of fluorescent oligoarginine carriers into and across bulk liquid and anionic bilayer membranes an endosomolytic tat peptide produced by incorporation of histidine and cysteine residues as a nonviral vector for dna transfection stability of cell-penetrating peptide-morpholino oligomer conjugates in human serum and in cells photochemical internalization: a new tool for drug delivery photochemically induced gene silencing using small interfering rna molecules in combination with lipid carriers photochemical internalization enhances silencing of epidermal growth factor receptor through improved endosomal escape of sirna photochemical enhancement of dna delivery by egf receptor targeted polyplexes photochemically enhanced delivery of a cell-penetrating peptide nucleic acid conjugate targeting human telomerase reverse transcriptase: effects on telomere status and proliferative potential of human prostate cancer cells photodynamic therapies: principles and present medical applications the present and future role of photodynamic therapy in cancer treatment delivery of small interfering rna to the target cell cytoplasm: photochemical internalization facilitates endosomal escape and imprves silencing efficiency, in vitro and in vivo fusion peptides and the mechanism of viral fusion properties and structures of the influenza and hiv fusion peptides on lipid membranes: implications for a role in fusion application of membrane-active peptides for drug and gene delivery across cellular membranes fusogenic peptides enhance endosomal escape improving sirnainduced silencing of oncogenes a fusogenic segment of glycoprotein h from herpes simplex virus enhances transfection efficiency of cationic liposomes unique features of a ph-sensitive fusogenic peptide that improves the transfection efficiency of cationic liposomes application of an hiv gp -derived peptide for enhanced intracellular trafficking of synthetic gene and sirna delivery vehicles the nh terminus of influenza virus hemagglutinin- subunit peptides enhances the antitumor potency of polyargininemediated p protein transduction docking essential dynamics eigenstructures ucsf chimera--a visualization system for exploratory research and analysis the third helix of the antennapedia homeodomain translocates through biological membranes cell penetration by transportan deletion analogues of transportan stearylated octaarginine and artificial virus-like particles for transfection of sirna into primary rat neurons octaargininemodified multifunctional envelope-type nano device for sirna we apologize to those authors whose work was not cited directly owing to space limitations. we thank alexander key: cord- -qhsymg r authors: sanchez, sergio; demain, arnold l. title: bioactive products from fungi date: - - journal: food bioactives doi: . / - - - - _ sha: doc_id: cord_uid: qhsymg r fungi are amazing producers of natural products. they are crucial to the health and the well-being of people throughout the world. they are excellent producers of hydrolytic enzymes, biofuels, organic acids, polysaccharides, and secondary metabolites such as antibiotics, anticancer drugs, hypocholesterolemic agents, immunosuppressants, and others. this chapter centers on these fungal products, especially valuable secondary metabolites, the discovery of which goes back eighty-seven years when penicillin was discovered by alexander fleming. the microbial drug era began back in when alexander fleming discovered in a petri dish seeded with staphylococcus aureus that a compound produced by a contaminating mold killed the bacterium. the active compound, produced by penicillium notatum, was named penicillin. by using the same strategy, other antibiotics such as streptomycin and chloramphenicol were later isolated from different bacterial and fungal fermentations. antibiotics can be produced by fermentation, an old technique that was utilized for beer and wine production almost years ago, during the ancient egypt and mesopotamia era. similarly, cheese production by penicillium roqueforti can be traced back for almost years. additional examples of traditional fermentations are soy sauce in asia and bread production (hölker et al. ; seviour et al. ) . bread production was common in egypt in bc. beer production using the non-filamentous fungus saccharomyces cerevisiae began in bc by the sumerians and chinese. wine was made in iran in bc and in egypt in bc. natural products (nps) with high commercial value can be produced by microbial primary or secondary metabolism. thanks to the technical improvements in screening programs and techniques for separation and isolation, the number of natural compounds discovered surpasses one million (berdy ) . among them, - % are produced by plants (alkaloids, flavonoids, terpenoids, steroids, carbohydrates, etc.) , and % of these plant products have a microbial origin. about - % of the reported natural products show biological activity and of these, approximately % have been obtained from microbes. microorganisms produce many compounds with biological activity. from , bioactive compounds so far obtained from microorganisms, about are produced by fungi (berdy ; brakhage and schroekh ) . therefore, the role of fungi in the production of antibiotics and other drugs for treatment of non-infective diseases has been crucial (demain et al. ) . with less than % of the fungal world having been cultured, there have been significant advances in microbial techniques for growth of uncultured organisms as a potential source of new chemicals (kaeberlein et al. ) . as more genomes are sequenced, it is found that filamentous fungi grasp the genetic capacity to produce an arsenal of secondary metabolites. in fungi, biosynthetic genes are present in clusters coding for large, multidomain, and multimodular enzymes such as polyketide synthases, prenyltransferases, non-ribosomal peptide synthases, and terpene cyclases. genes adjacent to the biosynthetic gene clusters encode regulatory proteins, oxidases, hydroxylases, and transporters. aspergilli usually contain - secondary metabolite gene clusters. most of these clusters coding for secondary metabolites are still cryptic or silent under standard culture conditions (hertweck ). therefore, mining for these cryptic secondary metabolites can be an excellent source of new drugs by awakening cryptic clusters for secondary metabolism. in addition, recent knowledge on cluster regulation has unlocked many hidden fungal bioactive compounds. regulation of fungal secondary metabolism has been reviewed by brakhage ( ) . emphasized are the regulatory elements that control gene transcription, including the targeted activation of silent gene clusters (brakhage and schroekh ) . a method to predict secondary metabolite gene clusters in filamentous fungi has been devised (anderson et al. ). in addition, metagenomics, i.e., the extraction of dna from soil, plants, and marine habitats and its incorporation into known organisms, allow access to a vast untapped reservoir of genetic and metabolic diversity (colwell ; gaudilliere et al. ) . thus, the potential for discovery of new fungal secondary metabolites with beneficial use for humans is great. of the , antibiotics known in , filamentous fungi produced % (verdine ; strohl ) . the beta-lactams are the most important class of antibiotics in terms of use. they constitute a major part of the antibiotic market. included are the penicillins, cephalosporins, clavulanic acid, and the carbapenems. of these, fungi are responsible for the production of penicillins and cephalosporins (fig. ) . the natural penicillin g and the biosynthetic penicillin v had a market of $ . billion by the late s. major markets also included semi-synthetic penicillins and cephalosporins amounting to $ billion. in , the market for cephalosporins was $ . billion and that for penicillins was $ . billion. production of all beta-lactams in had reached over , tons. the titer of penicillin is over g l − and that for cephalosporin c is more than g l − (masurekar ; yang et al. ) . recovery yields are more than %. there have been over , molecules based on penicillin that have been made by semi-synthesis or by total synthesis. important in penicillin biosynthesis are the regulatory factors. penicillium chrysogenum, the producer of penicillin g, contains global regulatory factor pcrfx , which positively regulates three beta-lactam biosynthetic genes, i.e., pcbab, pcbc, and pende (dominguez-santos ) . this regulatory factor not only controls secondary metabolism but also controls primary metabolism. related factor cpcr is a global regulator found in the cephalosporin c producer acremonium chrysogenum, binding to at least two sequences of the pcbab-pcbc intergenic region and regulating cephalosporin c biosynthesis. , -diaminopropane ( , -dap) is secreted by p. chrysogenum and a. chrysogenum. this and spermidine (which contains , -dap) increase the transcription levels of the penicillin biosynthetic genes pcbab, pcbc, and pende (martín et al. ) . they thus stimulate the production of penicillin g. the mechanism appears to involve stimulation of the expression of laea, a global regulator that acts epigenetically on the expression of secondary metabolism genes via heterochromatin reorganization. , -dap also stimulates the production of cephamycin in amycolatopsis lactamdurans. spermidine's activity appears to be due to , -dap. by the mid- s, antibiotics and their derivatives were already on the market (strohl ; brown ) . the market in was $ billion dollars. despite these impressive figures, more antibiotics are needed to combat evolving pathogens, naturally resistant microbes, and bacteria and fungi that have developed resistance to current antibiotics. a new and approved cephalosporin is ceftobiprole, which is active against methicillin-resistant s. aureus (mrsa) and is not hydrolyzed by a number of beta-lactamases from gram-positive bacteria (shang et al. ) . another antibiotic of note is cerulenin, an antifungal agent produced by acremonium caerelens. it was the first inhibitor of fatty acid biosynthesis discovered (vance et al. ) . it alkylates and inactivates the active-site nucleophilic cysteine of the ketosynthase enzyme of fatty acid synthetase by epoxide ring opening. other properties that are desired in new antibiotics are improved pharmacological properties, ability to combat viruses and parasites, and improved potency and safety. parafungin from fusarium lavarum is a recently discovered antifungal agent inhibiting poly(a) polymerase in candida albicans as well as in a broad range of pathogenic fungi (harvey et al. ) . over the years, non-infectious diseases were mainly treated with synthetic compounds. despite testing thousands of synthetic chemicals, only a handful of promising structures was obtained. as new synthetic lead compounds became extremely difficult to find, microbial products came into play. since microorganisms are such a prolific source of structurally diverse bioactive metabolites, over the years, the pharmaceutical industry extended their antibiotic screening programs to look for additional applications of antibiotics in medicine and agriculture (cardenas et al. ; kremer et al. ) . as a result of this move, some of the most important products of the pharmaceutical industry were obtained. for example, the immune suppressants have revolutionized medicine by facilitating organ transplantation (verdine ) . other products include anti-tumor drugs, hypocholesterolemic agents, enzyme inhibitors, gastrointestinal motor stimulators, ruminant growth stimulants, insecticides, herbicides, antiparasitics versus coccidia and helminths, and other pharmacological activities. stimulated by the use of simple enzyme assays for screening, prior to testing in intact animals or in the field, further applications are emerging in various areas of pharmacology and agriculture. in , there were more than secondary metabolites derived from marine fungi in clinical trials (bhatnagar and kim ) . many of the new natural products from marine sources are polyketides. s. cerevisiae and pichia pastoris are used for the production of biopharmaceuticals (berlec and strukelj ) . biopharmaceuticals have the fastest growth rate of products on the market. s. cerevisiae produces % of these. of biopharmaceuticals approved by , were produced by yeasts, by s. cerevisiae, and one by p. pastoris. the production of biopharmaceuticals by s. cerevisiae has been reviewed by nielsen (nielsen ) . the yeast is used to make insulin and insulin analogs. the insulin market was $ billion in . other products are human serum albumin, hepatitis vaccines, and virus-like particles used for vaccination against human papilloma virus. the advantages of s. cerevisiae include proper folding of human proteins and their secretion into the extracellular medium, facilitating purification and proper post-translational modification of the protein. this includes proteolytic processing of signal peptides, disulfide bond formation, subunit assembly, acylation, and glycosylation. human serum albumin is produced at g l − . more than million new cases of cancer were diagnosed in the world in ; . million cases were in men and . million in women, resulting in . million cancer-related deaths. the tumor types with the highest incidence were lung ( . %), breast ( . %), and colorectal ( . %). some of the anticancer drugs in clinical use are secondary metabolites derived from plants and fungi. among the approved products are taxol and camptothecin. taxol (paclitaxel) was first isolated from the pacific yew tree, taxus brevifolia (wall and wani ) , and later found to be a fungal secondary metabolite (stierle et al. ) . it is a steroidal alkaloid diterpenoid that has a characteristic n -benzoylphenyl isoserine side chain and a tetracycline ring (fig. ) . it inhibits rapidly dividing mammalian cancer cells by promoting tubulin polymerization and interfering with normal microtubule breakdown during cell division. the benzoyl group of the molecule is particularly crucial for maintaining the strong bioactivity of taxol. the drug also inhibits several fungi (species of pythium, phytophthora, and aphanomyces) by the same mechanism. in , taxol was approved for refractory ovarian cancer and today is used against breast cancer and advanced forms of kaposi's sarcoma (newman and cragg ) . a formulation in which paclitaxel is bound to albumin is sold under the trademark abraxane ® . taxol sales amounted to $ . billion in for bristol-myers squibb, representing % of the company's pharmaceutical sales and its third largest selling product. it has reached $ . billion annual sales in international markets. although synthetic methods for taxol production have been tried, the chemical molecular structure is so complex that commercial synthetic production is unfeasible. currently, italy, the uk, the netherlands, and other western countries are engaged in the production of taxol by plant cell fermentation technology. taxol production by a plant cell culture of taxus sp. was reported to be at mg l − (sabater-jara et al. ). however, the addition of methyl jasmonate, a plant signal transducer, increased the production to mg l − . fig. chemical structure of taxol. the dotted section corresponds to the molecule n-benzophenyl isoserine side chain as stated above, taxol has also been found to be a fungal metabolite (stierle et al. ; jiang et al. ). fungi such as colletotrichum gloeosporoides, colletotrichum capsici, fusarium maire, nodulisporium sylviforme, pestalotiopsis microspora, pestalotiopsis versicolor, phyllosticta citricarpa, taxomyces andreanae, and tubercularia sp. are taxol producers (stierle et al. ; flores-bustamante et al. ; gangadevi and muthumary ; kumaran et al. kumaran et al. , li et al. ; wang et al. ; xu et al. ; zhao et al. ]. c. gloeosporoides produced µg l − of taxol (gangadevi and muthumary ) , and the endophyte f. maire made lg l − (xu et al. ) . the production by p. citricarpa amounted to lg l − (kumaran et al. ) and was reported at lg l − by submerged fermentation with an engineered strain of the endophytic fungus ozonium sp. (efy- ). the transformed strain overproduced the rate-limiting enzyme of taxol biosynthesis and taxadiene synthase (wei et al. ). the endophyte p. versicolor, from the plant taxus caspodata, produced µg l − (kumaran et al. ) . c. capsici from capsicum annuum made lg l − (kumaran et al. ) . another endophytic fungus, phoma betae, isolated from the medicinal tree ginkgo biloba produced taxol at lg l − (kumaran et al. ) . colletotrichum annutum from capsium annuum cladosporium cladosporoides, an endophyte of the taxus media tree, produced lg l − of taxol (zhang et al. ). metarhizium anisopiliae h- , isolated from the tree taxus chinensis, yielded lg l − (liu et al. ). although a review of taxol production by endophytic fungi indicated that strain improvement had resulted in levels of only . - . mg l − (zhou et al. ) , it was reported that another fungus, alternaria alternate var. monosporus, from the bark of taxus yunanensis, after ultraviolet and nitrosoguanidine mutagenesis, could produce taxol at mg l − (duan et al. ) . another important antitumor agent is camptothecin (fig. ) , a modified monoterpene indole alkaloid produced by certain plants (angiosperms) and by the endophytic fungus, entrophospora infrequens. the fungus was isolated from the plant nathapodytes foetida (wall and wani ) . recently, it was found that trichoderma atroviridi strain ly , an endophytic fungus from c. acuminata, was an improved producer of camptothecin. the endophytic fungus produced µg l − of camptothecin in the presence of the elicitor methyljasmonate and xad adsorbent resin (pu et al. ) . in view of the low concentration of camptothecin in tree roots and poor yield from chemical synthesis, the fungal fermentation is very promising for industrial production of camptothecin. it is used for recurrent colon cancer and has unusual activity against lung, ovarian, and uterine (amna et al. ) . colon cancer is the second-leading cause of cancer fatalities in the usa and the third most common cancer among the us citizens. camptothecin is known commercially as camptosar and campto and achieved sales of $ billion in (lorence and nessler ) . camptothecin's water-soluble derivatives irinotecan and topotecan have been approved and are used clinically. metastatic colorectal cancer is treated by irinotecan, whereas topotecan has use for ovarian cancer, cervical cancer, and small-cell lung cancer. a review of the activities of camptothecin and its many small and macromolecular derivatives has been published by venditto and simanek ( ) . the cellular target of camptothecin is type i dna topoisomerase. when patients become resistant to irinotecan, its use can be prolonged by combining it with the monoclonal antibody erbitux (cetuximab). erbitux blocks a protein that stimulates tumor growth, and the combination helps metastatic colorectal cancer patients expressing epidermal growth factor receptor (egfr). this protein is expressed in % of advanced metastatic colorectal cancers. the drug combination reduces invasion of normal tissues by tumor cells and the spread of tumors to new areas. angiogenesis, the recruitment of new blood vessels, is necessary for tumors to obtain oxygen and nutrients. tumors actively secrete growth factors that trigger angiogenesis. anti-angiogenesis therapy is now known as one of the four cancer treatments; the other three are surgery, radiotherapy, and chemotherapy. by the end of , anti-angiogenesis drugs were in phase iii clinical trials and more than were in phase ii. fumagillin, a secondary metabolite of aspergillus fumigatus, was one of the first agents found to act as an anti-angiogenesis compound. next to come along were its oxidation product ovalacin and the fumagillin analog tnp- (=agm- ). tnp- binds to and inhibits type methionine aminopeptidase. this interferes with amino-terminal processing of methionine, which may lead to inactivation of enzymes essential for the growth of endothelial cells. in animal models, tnp- effectively treated many types of tumors and metastases. inhibitors of farnesyltransferase (ftis) have anticancer activity because farnesylation is required for the activation of ras, a necessary step in cancer progression. they also induce apoptosis in cancer cells. the fungus phoma sp. fl- produces an fti known as tan- (bernardes et al. ) . an individual's immune system is capable of distinguishing between native and foreign antigens and to mount a response only against the latter. suppressor cells are critical in the regulation of the normal immune response. the suppression of the immune response, either by drugs or radiation, in order to prevent the rejection of grafts or transplants or to control autoimmune diseases, is called immunosuppression. microbial compounds capable of suppressing the immune response have been discovered as fungal secondary metabolites. cyclosporin a was originally discovered in the s as a narrow-spectrum antifungal peptide produced by the mold, tolypocladium nivenum (previously tolypocladium inflatum) in an aerobic fermentation (borel et al. ). cyclosporins (fig. ) are a family of neutral, highly lipophilic, cyclic undecapeptides containing some unusual amino acids, synthesized by a non-ribosomal peptide synthetase, cyclosporin synthetase. discovery of the immunosuppressive activity of this secondary metabolite led to use in heart, liver, and kidney transplants and to the overwhelming success of the organ transplant field (borel ) . cyclosporin was approved for use in . it is thought to bind to the cytosolic protein cyclophilin (immunophilin) of immunocompetent lymphocytes, especially t lymphocytes. this complex of cyclosporin and cyclophilin inhibits calcineurin, which under normal circumstances is responsible for activating the transcription of interleukin- . it also inhibits lymphokine production and interleukin release and therefore leads to a reduced function of effector t cells. annual world sales of cyclosporin a are approximately $ billion. cyclosporin a also has activity against corona viruses (de wilde et al. ) . studies on the mode of action of cyclosporin and the later-developed immunosuppressants from actinomycetes, such as sirolimus (a rapamycin) and fk- (tacrolimus), have markedly expanded current knowledge of t cell activation and proliferation. these agents act by interacting with an intracellular protein (an immunophilin), thus forming a novel complex that selectively disrupts the signal transduction events of lymphocyte activation. their targets are inhibitors of signal transduction cascades in microbes and humans. in humans, the signal transduction pathway is required for the activation of t cells. pleuromutilin, a tricyclic terpenoid inhibitor of protein synthesis, was originally isolated in from the basidiomycete pleurotis sp. (kirst ) . although it was rapidly metabolized and had unfavorable pharmacokinetics, its semi-synthetic derivatives tiamalin and valnemulin have been successful for control and treatment of swine and poultry diseases. also, retapamulin (altabax ® ) was approved for topical treatment of human skin diseases. a very old broad-spectrum antibiotic, actually the first antibiotic ever discovered, is mycophenolic acid, which has an interesting history. bartolomeo gosio ( gosio ( - , an italian physician, discovered the compound in (bentley ) . gosio isolated a fungus from spoiled corn, which he named penicillium glaucum, which was later reclassified as p. brevicompactum. he isolated the crystals of the compound from culture filtrates in and found it to inhibit the growth of bacillus anthracis. this was the first time an antibiotic had been crystallized and the first time that a pure compound had ever been shown to have antibiotic activity. the work was forgotten, but fortunately the compound was rediscovered by alsberg and black ( ) and given the name mycophenolic acid. they used a strain originally isolated from spoiled corn in italy called penicillium stoloniferum, a synonym of p. brevi-compactum. the chemical structure was elucidated many years later ( ) by birkinshaw et al. ( ) in england. mycophenolic acid has antibacterial, antifungal, antiviral, antitumor, antipsoriasis, and immunosuppressive activities. its antiviral activity is exerted against yellow fever, dengue virus, and japanese encephalitis virus (sebastian et al. ) . it was never commercialized as an antibiotic because of its toxicity, but its -morpholinoethylester was approved as a new immunosuppressant for kidney transplantation in and for heart transplants in (lee et al. ). the ester is called mycophenolate mofetil (cellcept) and is a prodrug that is hydrolyzed to mycophenolic acid in the body. it is sometimes used along with cyclosporin in kidney, liver, and heart transplants. mycophenolic acid also appears to have anti-angiogenic activity (chong et al. ). only about % of cholesterol in humans comes from the diet. the rest is synthesized by the body, predominantly in the liver. many people cannot control their level of cholesterol at a healthy level by diet alone and require hypocholesterolemic agents. high blood cholesterol leads to atherosclerosis, which is a chronic, progressive disease characterized by continuous accumulation of atheromatous plaque within the arterial wall, causing stenosis and ischemia. atherosclerosis is a leading cause of human death. the last two decades have witnessed the introduction of a variety of anti-atherosclerotic therapies. the statins form a class of hypo-lipidemic drugs, formed as secondary metabolites by fungi, and used to lower cholesterol by inhibiting the rate-limiting enzyme of the mevalonate pathway of cholesterol biosynthesis, i.e., -hydroxymethyl glutaryl-coa (hmg-coa) reductase. inhibition of this enzyme in the liver stimulates low-density lipoprotein (ldl) receptors, resulting in an increased clearance of ldl from the bloodstream and a decrease in blood cholesterol levels. they can reduce total plasma cholesterol by - %. through their cholesterol-lowering effect, they reduce the risk of cardiovascular disease, prevent stroke, and reduce development of peripheral vascular disease (nicholls et al. ) . currently, there are a number of statins in clinical use. they reached an annual market of nearly $ billion before one became a generic pharmaceutical. the history of the statins has been described by akira endo, the discoverer of the first statin, compactin (mevastatin; ml- b) (endo ) . this first member of the group was isolated as an antibiotic product of p. brevicompactum (brown et al. ). at about the same time, it was found by endo and coworkers as a cholesterolemic product of penicillium citrinum (endo et al. ). although compactin was not of commercial importance, its derivatives achieved strong medical and commercial success. lovastatin (monacolin k; mevinolin; mevacor tm) was isolated in broths of monascus rubra and aspergillus terreus (alberts et al. ; endo and monacolin ) . lovastatin, developed by merck & co. and approved by the us food and drug administration (fda) in , was the first commercially marketed statin. in its chemical structure, lovastatin has a hexahydronaphthalene skeleton substituted with a p -hydroxy-lactone moiety (fig. ) . a semisynthetic derivative of lovastatin is zocor ® (simvastatin), one of the main hypocholesterolemic drugs, sold for $ billion per year before becoming generic. an unexpected effect of simvastatin is its beneficial activity on pulmonary artery hypertension (liu et al. ) . another surprising effect is its antiviral activity (bader et al. ) . simvastatin is active against rna viruses and acts as monotherapy against chronic hepatitis c virus in humans. it has been shown to act in vitro against hepatitis b virus (hbv). this virus infects million people and is the most common infectious disease agent in the world. the virus causes hepatocellular cancer, which is the leading cause of cancer. fungi produce poisons called mycotoxins, which, strangely enough, have been harnessed as medically useful agents. these agents (e.g., ergot alkaloids) caused fatal poisoning of humans and animals (ergotism) for centuries by the consumption of bread made from grain contaminated with species of the fungus claviceps. however, mycotoxins later were found useful for angina pectoris, hypertonia, serotonin-related disturbances, inhibition of protein release in agalactorrhea, reduction in bleeding after childbirth, and prevention of implantation in early pregnancy (bentley ; vining and taber ) . their physiological activities include the inhibition of action of adrenalin, noradrenalin, and serotonin, as well as the contraction of smooth muscles of the uterus. antibiotic activity is also possessed by some ergot alkaloids. members of the genus gibberella produce zearelanone and gibberellins. zearelanone (fig. ) is an estrogen made by gibbberella zeae (syn. fusarium graminearum) (hidy et al. ) . its reduced derivative zeranol is used as an anabolic agent in sheep and cattle, which increases growth and feed efficiency. gibberellic acid, a member of the mycotoxin group known as gibberellins, is a product of gibberella fujikuroi and causes "foolish rice seedling" disease in rice (jefferys ) . gibberellins are employed to speed up the malting of barley, improve the quality of malt, increase the yield of vegetables, and cut the time in half for obtaining lettuce and sugar beet seed crops. they are isoprenoid growth regulators, controlling flowering, seed germination, and stem elongation (tudzinski ) . more than tons are produced annually with a market of over $ billion. enzyme inhibitors have received increased attention as useful tools, not only for the study of enzyme structures and reaction mechanisms, but also for potential utilization in medicine and agriculture. several enzyme inhibitors with various industrial uses have been isolated from microbes (umezawa ) . among the most important are the statins and hypocholesterolemic drugs discussed previously. fungal products are also used as enzyme inhibitors against cancer, diabetes, poisoning, and alzheimer's disease. the enzymes inhibited include acetylcholinesterase, protein kinase, tyrosine kinase, glycosidases, and others (paterson ). since ad, monascus purpurea has been grown on rice to prepare koji or angkak (red rice), which is used as a traditional chinese food and medicine (ma et al. ) . monascorubramine and rubropunctatin are water-soluble red pigments fig. chemical structure of zearalenone formed upon the reaction of the orange pigments monascorubrin and rubropunctatin with amino acids in fermentation media (juzlova et al. ) . the fungus is used to prepare red rice, wine, soybean cheese, meat, and fish. it is authorized in japan and china for food use. there are known monascus pigments. they have an amazing number of activities: antimicrobial, anticancer, anti-mutagenesis, anti-diabetes, anti-obesity, anti-inflammatory, cholesterol-lowering, immunosuppressive, and hypotensive (feng et al. ; lee and pan ) . nutritional control of the formation of the red pigments has been described in a series of publications by lin and demain ( , , . carotenoids are tetra-terpenoid pigments which are excellent anti-oxidants. they are used as nutritional supplements, animal feeds, food additives, pharmaceuticals, food coloring agents, and in cosmetics. they are composed of hydrocarbons (carotenes and lycopene) and oxygenated derivatives (xanthophylls) and are used for protection against cancer, age-related muscular degeneration, and cardiovascular diseases (roukas ) . beta-carotene and lycopene are highly unsaturated isoprene derivatives which stimulate the immune system and prevent degenerative diseases and cancer. some are made microbiologically. they had a market of $ . billion, and their market is growing by . % per year. adaptive laboratory evolution was used to increase the microbial production of carotenoids in a genetically engineered s. cerevisiae strain. it was carried out by using a periodic hydrogen peroxide shocking strategy. the improved production was due to up-regulation of genes related to biosynthesis of lipid and mevalonate (reyes et al. ) . the production amounted to mg g − dry cell weight. beta-carotene, a precursor of vitamin a, has a market of $ million. although most is made chemically, it can be made by blakeslea trispora at g l − (vachali et al. ) . lycopene is another carotenoid. phaffia rhodozyma (xanthophyllomyces dendrorhous) is a heterobasidiomycetous yeast that has become the most important microbial source for the preparation of the carotenoid astaxanthin (andrewes et al. ; rodríguez-saiz et al. ). this oxygenated carotenoid pigment (fig. ) is used in the feed, food, pharmaceutical, nutraceutical, and cosmetic industries. it is responsible for the orange to pink color of salmonid flesh and the reddish color of boiled crustacean shells. feeding of penreared salmonids with a diet containing this yeast induces pigmentation of the white muscle (johnson et al. ) . it is a very good antioxidant, times more active than beta-carotene and times more than alpha-tocopherol. it is the second most important carotenoid. astaxanthin enhances the immune system and protects skin from radiation injury and cancer. it can be produced synthetically as hydroxyl-astaxanthin from petrochemicals with a selling price of $ per kg. however, the natural product is favored because the synthetic product is a mixture of stereoisomers. x. dendror-hous produces astaxanthin at mg l − . natural astaxanthin is more stable than the synthetic version and more bioavailable. the natural product is present in algae and fish as mono-and diesters of fatty acids. however, it is difficult to hydrolyze the esters from algae, which limits its usage to trout and salmon. the yeast product is better since it is the % free, non-esterified ( r, 'r) stereoisomer. the natural product is more expensive ($ per kg) than synthetic astaxanthin ($ per kg). the astaxanthin market was $ million in with % being synthetic. most of the production processes with the yeast yield levels of astaxanthin are lower than mg l − . however, white light improved production to mg l − (de la fuente et al. ) and mutant strain ubv-ax can make mg l − (jacobson et al. ) . thaumatin, a protein produced by the plant thaumatococcus danielli, can also be produced by p. roqueforti and aspergillus niger var. awamori (faus ) . thaumatin is intensely sweet (i.e., times sweeter than sucrose) and is approved as a foodgrade ingredient. the production by a. niger var. awamori was improved from mg l − up to mg l − by increasing gene dosage and use of a strong promoter (moralejo et al. ). the sweetener xylitol, normally produced by pichia stipitis, can be produced by recombinant s. cerevisiae in higher concentrations by transforming the xyl gene of p. stipitis into s. cerevisiae. the gene encodes a xylose reductase (hallborn et al. ). industrial enzymes include detergent enzymes, technical enzymes, food enzymes, and feed enzymes (hellmuth and bring ) . technical enzymes include those used for textiles, leather, pulp and paper, and fuel ethanol. the largest group is the food enzymes which include amylases, xylanases, glucose oxidase, hexose oxidase, pectinases, glucanase, invertase, glucose isomerase, protease, lipase, phosphorylase, lactase, milk-clotting enzymes, animal rennet, microbial rennet, and chymosin. fungal producers are a major source, and the main ones are a. niger and klyveromyces lactis. advances in the production of biopharmaceutical proteins by metabolic engineering have been reviewed by nielsen ( ) . yeasts are used to produce fig. chemical structure of astaxanthin recombinant proteins (celik and calik ) . they rapidly reach high levels of growth, produce high amounts of recombinant proteins, and do not contain pyrogens, pathogens, or viral inclusions. about % of the biopharmaceuticals on the market are made by s. cerevisiae. they include more than different recombinant proteins. this yeast is important for production of fda-approved insulin and its analogs, hepatitis b surface antigen, urate oxidase, glucagons, granulocyte-macrophage colony stimulating factor (gm-csf), hirudin and platelet-derived growth factor. the insulin market was $ billion in and is still on the increase. human serum albumin, used as a plasma expander in surgery, is produced by s. cerevisiae at g l − , and human transferrin, used for anemia, is produced at . g l − . yeasts also are used to make human serum albumin, hepatitis vaccines, and virus-like particles used for vaccination against human papilloma virus. s. cerevisiae carries out folding of many human proteins, secretes the proteins, and posttranslational modifications, e.g., proteolytic processing of signal peptides, disulfide bond formation, subunit assembly, acylation, and glycosylation. however, s. cerevisiae is not favored today because of plasmid instability, low levels of produced proteins, lack of secretion due to retention in the proteins in the periplasm, and hyper-glycosylation of the recombinant proteins including the high-mannose type of n-glycosylation which shortens the in vivo half-life, reduces efficacy, and elicits an immunogenic response to the non-human carbohydrate moiety. the yeasts that are used, having been engineered for more human-type n-glycosylation, include pichia pastoris, hansenula polymorpha, yarrowia lipolytica, and schizosaccharomyces pombe. titers of p. pastoris have reached - g l − , and it can secrete the proteins. p. pastoris has been engineered to produce human-like n-glycosylation that includes terminal addition of sialic acid to the glycoprotein. p. pastoris produces ecallantide, which was approved by fda in for hereditary angioedema. it also produces plant-derived hydroxynitrile lyase at over g l − (hasslacher et al. ) . h. polymorpha has been used for the production of hepatitis b vaccine, interferon alpha- a, hirudin, insulin, phytase, lipase, hexose oxidase, interleukin- , serum albumin, glucose oxidase, glycolate oxidase, and catalase; the first four are on the market. this yeast reaches high growth density, secretes proteins as large as kda, and is highly productive. for example, it produces . g l − of recombinant phytase. other useful yeasts include k. lactis for the production of bovine chymosin (rennin), glucoamylase, human serum albumin, interleukin- and interleukin- beta, and many other recombinant proteins. s. pombe has been used to produce human lipocortin i, human papillomavirus type vaccine, and many others. the beauty of these yeasts is their ability to perform post-translational modifications similar to those of higher eukaryotes, e.g., correct folding, disulfide bond formation, n-and o-linked glycosylation, and proteolytic processing of signal sequences. about % of all therapeutic proteins are glycoproteins. the production of recombinant microbial enzymes by fungi has been reviewed by liu et al. ( a, b) . aspergillus and pichia species has been reviewed by caspeta and nielsen ( ) . ethanol can be used as a fuel by itself or in combination with gasoline (e , e , and e ). it is mainly made in the usa (over billion gallons from corn) and in brazil. however, corn can only yield billion gallons, and corn prices are rising. cellulose is a possible source of ethanol but instead of containing only glucose, cellulose also contains c sugars such as xylose and arabinose. the best c utilizer is p. stipitis which can produce ethanol and clean up concentrated toxins liberated from lignocellulose degradation. its production of ethanol has been reviewed by agbogo and coward-kelly ( ) . it can produce ethanol from pretreated sources of biomass such as red oaks, wheat straw, sugarcane bagasse, rice straw, corn cobs, corn stover, aspen wood, pinewood, and poplar wood. from aspen wood such as orpinomyces defined medium, g l − can be made (slininger et al. ). attributes of p. stipitis include consumption of acetic acid, reduction in the furan ring toxins in hmf, and furfural present in cellulosic biomass conversions. the production of ethanol via biomass saccharification using fungi has been discussed by zhang ( ) . saccharification of biomass involves pretreatment, fractionation, and enzymatic hydrolysis. pretreatment may be the most expensive step, amounting to % of total processing costs. biodelignification of lignocellulose has been carried out by ascomycetes including trichoderma reesei, basidiomycetes such as the white rot fungus phaenerochaete sp. (chandel et al. ) . the key enzyme in delignification is manganese peroxidase. biodelignification is the most expensive step in the conversion of biomass to ethanol mainly due to its slow rate of action. protein engineering must be applied to make the delignification enzymes better suited to the temperature, ph, and reaction conditions of the industrial process. hydrolysis by cellulase is another expensive step costing cents to $ /gallon of produced ethanol. nearly - g of cellulase is used per gallon of ethanol produced, where specific activities of fungal cellulases are . - . filter paper units per mg of cellulase. filamentous fungi can produce native cellulases at levels of more than g l − (cherry and fidanstsel ) . novozymes, genencor, and iogen produce cellulase from trichoderma, whereas dyadic uses chrysosporium lucknowense. these commercial fungal fermentations produce over g crude cellulase per liter of broth, much higher than that produced by bacteria. an important move is to decrease the amount of cellulase used to produce ethanol. the overall action of t. reesei cellulase on cellulosic biomass is limited by a low content of beta-glucosidase. the result is an accumulation of cellobiose which limits further breakdown. by expressing the beta-glucosidase gene of pericona sp. in t. reesei, (dashtban and qin ) were able to increase the level of beta-glucosidase, the overall cellulase activity, and the action on biomass residues. during pretreatment of biomass, inhibitors are released such as furfural. tolerance to this inhibitor can be achieved by over-expression of s. cerevisiae genes encoding (a) yeast transcription activator msn (sasano et al. ), (b) zwf of the pentose phosphate pathway , (c) adh encoding alcohol dehydrogenase , and (d) tal encoding transaldolase (hasunama et al. ) . regulation of cellulolytic and hemi-cellulolytic enzyme production by filamentous fungi involves regulatory transcription factors such as xlnr from aspergillus which is involved in d-xylose induction of cellulolytic and xylanolytic enzymes (tani et al. ) . others include c r- from neurospora, manr, mcma, and c br from aspergillus, and bg r from trichoderma which regulate cellulolytic and/or hemi-cellulolytic enzyme production. s. cerevisiae is well known for its ability to produce ethanol. cassava mash-containing sludge was converted to ethanol at g l − by the s. cerevisiae ssf process, employing continuous fermentation (moon et al. ) . volumetric productivity was . g l − , and the percent yield was %. when immobilized on corn stalks, s. cerevsiae can produce g l − of ethanol from food waste (yan et al. ) . alcohol tolerance in this yeast is increased by adding potassium and raising the ph of the fermentation with koh (lam et al. ) . under these conditions, g l − was produced. using cell cycling of this yeast in very high-gravity fermentations led to an ethanol titer of g l − with a productivity of . g l − h − . the strain used (pe- ) was obtained from a distillery in brazil producing ethanol from sugarcane (pereira et al. ) . one hundred billion liters of ethanol are produced each year from sugar cane and corn starch by s. cerevisiae. production at high temperature (ca °c) reduces cooling costs, lowers the effects of contamination, and enables more efficient hydrolysis of feedstocks. this improves the productivity in the simultaneous saccharification and fermentation process. caspeta et al. ( ) , using adaptive laboratory evolution, isolated s. cerevisiae strains with improved growth and ethanol production at °c. these strains grew . times faster and excreted ethanol . times faster than the parent strain. they noted a change in sterol composition from ergosterol to fenosterol due to mutation in the c- sterol desaturase gene and increased expression of sterol biosynthesis genes. sterols contribute to membrane fluidity. the thermo-tolerant strains were improved in glucose consumption rate which increased by % at °c and by % at °c. jerusalem artichokes produce high levels of biomass, grow rapidly, need only little pesticide, fertilizer, and water, and can grow on marginal land. it could be a good substrate for the production of important products (li et al. ) . product titers achieved by fungi growing on jerusalem artichokes include g l − of ethanol by a mixed culture of s. cerevisiae and a. niger, and g l − by s. cerevisiae alone. biodiesel is a monoalkyl ester of long-chain fatty acids made by transesterification of feedstocks such as waste animal fats or vegetable oils, e.g., soybean oil. it is a very good fuel, contains less sulfur than conventional fuel, can be used in diesel engines without modification, and can be blended in any ratio with petroleum diesel. it is biodegradable and non-toxic (lin et al. ) . the four different methods of biodiesel production include transesterification, blending, microemulsions, and pyrolysis. transesterification is the method of choice, the catalyst being chemical (acid or base) or an enzyme. favored is transesterification via enzymes, i.e., lipases. microbial lipases are excellent since they are stable in organic solvents, do not need cofactors, have broad substrate specificity and high enantiospecificity. candida antartica is a favored lipase producer. yields of enzymic transesterification can reach %. maximum enzyme-catalyzed transesterification occurs at °c. the cost of lipase is high, but it can be lowered by the use of enzyme immobilization and recycling of the immobilized enzyme. adsorption is the best immobilization procedure due to its simplicity, ease, use of mild conditions, and low cost. genetic engineering has been used to convert s. cerevisiae into a biodiesel producer, i.e., one that is oleaginous, supplying fatty acids and alcohols, and converting them to biodiesel. production of intracellular lipids by yeasts growing on alkali-treated corn stover revealed that cryptococcus humicola produces g l − lipids in a total biomass weight of g l − (sitepu et al. ). , -butanediol is a fuel with a high heating value ( , j/g) and is used as a liquid fuel or fuel additive. when compared to acetone, alpha-pinene, -butanol, isobutanol, isopropanol, and fatty alcohols, , -butanediol shows lower toxicity. it also is used in the preparation of solvents, anti-freeze agents, synthetic rubber, and plastics. an engineered stain of s. cerevisiae can produce it at g l − ). metabolic engineering has improved yeasts as producers of important metabolites (liu et al. a, b) . important productivities include y. lipolytica, producing g l − erythritol, g l − citric acid from glycerol, g l − succinic acid, and g l − mannitol. s. cerevisiae produces malic acid at g l − , , -butanediol at g l − , and the artemisinin precursor amorpha- , -diene at g l − . l-lactic acid is made by candida boidini at g l − . p. pastoris can covert methanol to formaldehyde in a process responsible for the production of tons per year of formaldehyde (caspeta and nielsen ) . erythritol can be produced from glycerol by y. lipolytica at g l − (khanna et al. ) . mannitol is produced from glycerol at g l − by candida magnolia. alpha-ketoglutaric acid was produced at g l − by y. lipolytica (candida lipolytica) with a yield of . g g − of substrate when grown on n-paraffins (weissbrodt et al. ). this acid is used industrially in chemical synthesis of heterocycles or elastomers, as a dietary supplement and as an enhancer of wound healing. production of itaconic acid at g l − was achieved by a. terreus with a yield of . g g − glucose and a productivity of . g l − h − (kuenz et al. ) . microbial formation of this compound is more productive than by chemical processes. increasing ph during the production phase was found to increase production (hevekerl et al. ) . a titer of g l − was reached by raising ph from to or by raising it to after . days of cultivation. itaconic acid is used in the production of polymers, coatings, adhesives and textiles. about , tons are made each year with a selling price of $ kg − . citric acid production began in england in by john and edward sturge of the city of selby. it was made from italian citrus fruits at that time. in , the german microbiologist carl wehmer discovered that sugar-growing fungi secreted citric acid. after world war i, the fermentation became the method of choice. john n. currie had found that a. niger was an excellent producer of citric acid and, as a result, the pfizer company in new york began large-scale fermentation production in . worldwide production is . million tons per year. about % is used in the food industry. other uses include chemicals, medicinal, textiles, and metallurgy. chemicals include surfactants and synthetic detergents (morgunov et al. ). in addition to a. niger, another producer is y. lipolytica. production by the latter is favored by limitation of cell growth via limiting levels of nitrogen, phosphorus, or sulfur with nitrogen limitation being the most useful. this yeast produces high levels of both citric and isocitric acids from rapeseed oil . fumaric acid is used as a food acidulent, a beverage ingredient, and an antibacterial agent in the feed industry (xu et al. ) . its other uses are for the preparation of biodegradable polymers, plasticizers, polyester resins, and as an animal feed supplement to reduce methane emissions (thakker et al. ) . rhizopus arrhizus has been used by pfizer to produce it at tons per year (roa-engel et al. ) . other species are also good producers, e.g., rhizopus nigricans, rhizopus formosa, and rhizopus oryzae. r. nigricans produced g l − with a productivity of g l − h − and a yield of . (ling and ng ) . dupont patented a process using r. arrhizus nrrl- with limited dissolved oxygen to produce g l − . glycolic acid can be produced by s. cerevisiae and k. lactis (koivistoinen et al. ) , although it is currently made chemically. engineered s. cerevisiae made only g l − but engineered k. lactis produced g l − from ethanol plus d-xylose. it is polymerized to polyglycolic acid which is an excellent packaging material. glycolic acid can also be used with lactic acid to make a copolymer (plga) for medical application in drug delivery. the market for glycolic acid was $ million for the million kg produced. glycolic acid is also employed in the textile industry as a tanning and dyeing agent. gluconic acid is used in the construction and in the preparation of chemicals, pharmaceuticals, foods, beverages, textiles and leather. it is also used to chelate divalent and trivalent metal ions. about , - , tons are made annually using glucose as substrate. the price varies from $ . to $ . kg − . usually glucose or sucrose is used as fermentation substrate. golden syrup, a by-product of the process refining sugar cane juice into sugar, or by treating sugar with acid, can be used for fermentation by a. niger (purane et al. ) . about g l − was produced in h with a productivity of . g l − h − . previous workers had obtained g l − at . g l − h − with a. niger immobilized on cellulose microfibers (sankpal and kulkami ) . also, sankpal et al. ( ) reached g l − with a productivity of . g l − h − using immobilization on cellulose fibers and surface culture. about - g l − was obtained using immobilization on waste paper with a productivity of . g l − h − (singh and kumar ) . brown et al. ( ) described metabolic engineering of aspergillus oryzae nrrl to produce malic acid at g l − . the result was achieved by overexpressing (a) the c- -dicarboxylate transporter and (b) the cytosolic alleles of pyruvate carboxylase and malate dehydrogenase. the rate was . g l − h − , and the yield on glucose was . mol mol − . penicillium viticola produced g l − of calcium malate in a medium containing corn steep liquor (khan et al. ). the yield was . g g − glucose and productivity was g l − h − . malic acid is a c dicarboxylic acid produced at , tons per year. it is used in the food and beverage industry as an acidulent and taste enhancer/modifier in combination with artificial sweeteners. additional uses are for the preparation of polyester resins and coatings, in foods and feed, and in the pharmaceutical industry. it is sold for $ - kg − (thakker et al. ) . torulopsis glabrata (also called candida glabrata) can produce pyruvic acid at g l − on glucose with a yield of . g g − glucose, a high productivity of . g l − h − and high glucose tolerance (liu et al. (liu et al. , . the organism is an osmotolerant mutant. production is increased by the use of urea as nitrogen source (yang et al. ). this yeast is used for commercial production of pyruvic acid. the process was industrialized in by toray industries at tons per year. erythritol, a polyhydric alcohol, has - % of the sweetness of sucrose and is used to combat obesity. it is non-carcinogenic and non-caloric since it is not digested by humans and cannot be fermented by bacteria to cause dental caries. repeated batch cultures of y. lipolytica on crude glycerol yielded g l − with a yield of . g g − glycerol used and a productivity of . g l − h − (mironczuk and furgala ) . bioconversion of xylose to xylitol by debaryomyces hansenii amounted to g l − from g l − xylose (misra and raghuwanshi ) . the yield was . g g − . this sugar alcohol is used in food production, has high activity as a sweetener, is non-cariogenic, and has insulin-independent metabolism properties. it is commercially produced by chemical reduction of d-xylose, but this is an expensive process. its global market is over , tons per year. the bioconversion would probably be less expensive than the chemical procedure. xylitol is an excellent antioxidant. it can be made from lignocellulosic waste (lima de albuquerque et al. ) . it is used as a sucrose replacement for cakes, cookies, chocolate, and chewing gum and in pharmaceuticals to reduce tooth decay. it acts against oral biofilms produced by bacteria. it is also a contributor to tooth calcification and is active against diabetes, anemia, acute otitis media, and osteoporosis. candida athensensis converts vegetable waste containing g l − xylose to g l − xylitol with a yield of . g g − and a productivity of . g l − h − (zhang et al. ) . coenzyme q (ubiquinone) is an essential part of the respiratory chain producing atp. it is composed of a quinonoid nucleus and a side chain of isoprenoids. best producers include fungi such as species of candida, saitoella, trichosporon, and sporobolomyces. production of useful products by basidiomycetes includes carotenoids, fragrances, enzymes, astaxanthin, erythritol, lipids, and oils (johnson ) . trichosporon sp. produces lipids and is being considered for biodiesel production. pseudozyma (candida) antartica produces lipase for industrial use and is another biodiesel possibility. it also produces g l − of itaconic acid. sporobolomyces carnicolor accumulates % of its biomass as intracellular lipids. cryptococcus species make unique carotenoids such as the xanthophyll plectaniaxanthin. some cryptococci utilize glycerol and accumulate % of their biomass as triacylglycerols. fungi produce long-chain polyunsaturated fatty acids (pufas) (ratledge ) . they include (a) gamma linoleic acid (gla; : omega- ) from mucor circinelloides, (b) docohexaenoic acid (dha; : omega- ) from crypthecodinium cohnii spp, (c) arachidonic acid (ara; : omega- ) from mortierella alpine, and (d) eicosapentaenoic acid (epa) from genetically modified y. lipolytica (xue et al. ) . the oil produced has much higher levels of epa than natural oils. epa is important for the anti-inflammatory activity of fish oils, thus contributing to cardiovascular and joint health. the product is being commercialized by dsm. the yeast was engineered by transformation with heterologous genes encoding five different activities. pufas represent a multibillion dollar industry, mainly ara and dha for infant formulas. they are major components of phospholipids in cell membranes. they regulate cell fluidity, attachment of specific enzymes to cell membranes, and mediate signal transduction and other metabolic processes. they are used for the biosynthesis of eicosanoids, leukotrienes, prostaglandins, and resolvins, which function as anti-inflammatory, anti-arrhythmic, and anti-aggregatory effectors. many improve cardiovascular health, and certain of them improve eye function and memory in newborn infants and in adults. microbial oils are produced by - species of yeast and also by molds. the producers are known as oleaginous microbes. fungi can accumulate % of their biomass as oils. dha is produced at tonnes per year and has a market of $ million. ara is blended with dha and used in infant formulas. epa plus dha can be used to prevent cardiac problems. prebiotics have been reviewed by panesar et al. ( ) . they include fructo-oligosaccharide produced at g l − from sucrose by beta-fructofuranosidase from aspergillus japonicas. prebiotics are used in the nutraceutical, pharmaceutical, animal feed, and aquaculture areas. they stimulate the growth of beneficial intestinal bacteria and maintain health of humans by suppression of potentially harmful bacteria, improvement of defecation, eliminating ammonia, preventing colon cancer, stimulating mineral adsorption, and lowering cholesterol and lipids. pullulan is produced at g l − by the yeast aureobasidium pullulans strain rbf a (sharma et al. ) . it is an exopolysaccharide which has potential application in industries such as medical, food, pharmaceutical, cosmetic, and agriculture. some vitamins are produced by fungi (ledesmo-amaro et al. ) . although vitamin d is derived chemically from cholesterol and ergosterol, it can be made by s. cerevisiae, saccharomyces uvarum, and candida utilis at mg g − of dry cells. riboflavin (vitamin b ) is made by ashbya gossypii, eremothecium ashbyii, candida flaeri, and candida famata. a. gossypii produces g l − of riboflavin. the increase in production by a. gossypii as compared to wild-type strain atcc is due to (a) a nine percent increase in flux to pentose- -phosphate via the pentose phosphate pathway (ppp) and (b) a -fold increase in the flux from purine to riboflavin (jeong et al. ) . this is due to increased guanosine triphosphate flux through the ppp and the purine synthesis pathway. resveratrol (trans- , , '-trihydroxystilbene) is a polyphenol found in wine, grapes, berries, and peanuts which can be made by some fungi it is a phytoalexin, i.e., a low molecular weight secondary metabolite. it has beneficial effects against inflammation, carcinogenesis, oxidation, aging, diabetes, and neurodegenerative disease. recombinant s. cerevisiae can produce it at . mg l − upon feeding of coumaric acid or l-tyrosine (shin et al. ) . alternaria sp. , isolated from merlot cobs, produces µg l − (shi et al. ). an improved process for making the anti-malarial compound artemisinin using s. cerevisiae was devised by paddon et al. ( ) . the process applies synthetic biology to a previous s. cerevisiae process and improves the production of artemisinic acid which is then chemically converted to artemisinin. whereas the previous process yielded only . g l − of artemisinic acid, the new process reaches g l − . genome sequencing of an organism reveals many secondary metabolic pathways that are usually silent. aspergillus nidulans was found to have nearly such loci encoding polyketide synthases (pks) or non-ribosomal protein synthases (nrps). using various types of nutritional limitation in continuous chemostat cultures of a. nidulans, (sarkar et al. ) obtained expression of two pks genes encoding synthases of seven phenolic compounds which were not observed previously under normal growth conditions. the soil fungus aspergillus versicolor produces aspergillomarasmine (ama) which turns off a bacterial gene that normally leads to antibiotic resistance (king et al. ). the gene encodes new delhi metallo-beta-lactamase (ndm- ). together with a carbapenem antibiotic, ama inactivates the gene in escherichia coli, acinetobacter, and pseudomonas. ndm- requires zinc, and ama removes zinc from the enzymes. the combination of ama and the carbapenem has shown its beneficial effect in mice and human cell culture. trichoderma species make many valuable secondary metabolites (keswani et al. ) polyketide gliotoxin, an anti-malarial agent and immune system suppressor, ( ) harzianolide, an antifungal agent and plant growth promoter, ( ) koninginins, which are antifungals and plant growth regulators, ( ) -pentyl- h-pyran- -one, a plant growth promoter, and coconut aroma used commercially in confectionary products, ( ) trichokonins, broad-spectrum antifungals and plant defense inducers, ( ) viridofungins, potential anticancer agents, and bacteriocides, ( ) viridian, a broad-spectrum antifungal agent, anti-neoplastic, and anti-atherosclerosis agent, and ( ) viridiol, a herbicidal and anti-aging agent. activation of "silent" gene clusters by genome mining in a. nidulans has revealed many new secondary metabolites (yaegashi et al. ). the a. nidulans genome contains potential secondary metabolism core genes including polyketide synthase (pks) genes, two pks-like genes, non-ribosomal peptide synthetase (nrps) genes, nrps-like genes, and one hybrid nrps-pks gene. microorganisms have greatly contributed for about years to the development of medicine and agriculture. however, due to different situations, pathogenic microbes have become resistant to many antibiotics creating a dangerous situation and therefore the need for new antibiotics is imperative. unfortunately, most of the large pharmaceutical companies have abandoned the search for new antimicrobial compounds. due to economics, they have concluded that drugs directed against chronic diseases offer a better revenue stream than do antimicrobial agents, for which the length of treatment is short and government restriction is likely. some small pharmaceutical and biotechnology companies are still developing antibiotics but most depend on venture capital rather than sales income, and with the present regulations, face huge barriers to enter into the market. these barriers were raised with the best intentions of ensuring public safety but they are having the opposite effect, i.e., termination of antibiotic development while resistance continues to increase (livermore ) . however, there are some new bright possibilities. one of the more promising is the utilization of uncultivated microorganisms. considering that % of bacteria and % of fungi have not yet been cultivated in the laboratory, efforts to find means to grow such uncultured microorganisms are proceeding and succeeding (kaeberlein et al. ) . furthermore, researchers are now extracting bacterial dna from soil samples, cloning large fragments into, for example, bacterial artificial chromosomes, expressing them in a host bacterium and screening the library for new antibiotics. this metagenomic effort could open up the exciting possibility of a large untapped pool from which new natural products could be discovered (clardy et al. ) . another exciting possibility is that of genome mining (scheffler et al. ) . in addition to these relatively new techniques, chemical and biological modification of old antibiotics could still supply new and powerful drugs. these comments also apply to non-antibiotics such as antitumor agents and other microbial products. in addition, natural products must continue to be tested for desirable therapeutic activities. i believe that significant progress in identifying new antibiotics, oncology therapeutics, and other useful medicines will be made, probably not by the big pharmaceutical companies, but by biotechnology companies and small research groups from institutes and universities. cellulosic ethanol production using the naturally occurring xylose-fermenting yeast, pichia stipitis mevinolin, a highly potent competitive inhibitor of hydroxylmethylglutarylcoenzyme a reductase and a cholesterol-lowering agent usda bur plant ind, bull no. . government printing office bioreactor studies on the endophytic fungus entrophospora for the production of an anticancer alkaloid camptothecin accurate prediction of secondary metabolite gene clusters in filamentous fungi carotenoids of phaffia rhodozyma, a red-pigmented fermenting yeast fluvastatin inhibits hepatitis c replication in humans bartolomeo gosio, - : an appreciation microbial secondary metabolites play important roles in medicine: prospects to discovery of new drugs bioactive microbial metabolites. a personal view current state and recent advances in biopharmaceutical production in escherichia coli, yeasts and mammalian cells microbial-based therapy of cancer. current progress and future prospects molecular aspects of fungal bioactive polyketides studies in the biochemistry of microorganisms. the molecular constitution of mycophenolic acid, a metabolic product of penicillium brevicompactum dierckx. part . further observations on the structural formula for mycophenolic acid biological effects of cyclosporine a: a new antilymphocytic agent history of the discovery of cyclosporin and of its early pharmacological development regulation of fungal secondary metabolism fungal secondary metabolites. strategies to activate silent gene clusters crystal and molecular structure of compactin: a new antifungal metabolite from penicillium brevicompactum pharmaceutical and biotech firms taking on drug-resistant microbes metabolic engineering of aspergillus oryzae nrrl for increased production of l-malic acid signal-transduction cascades as targets for therapeutic intervention by natural products toward systems metabolic engineering of aspergillus and pichia species for the production of chemicals and biofuels production of recombinant proteins by yeast cells biodelignification of lignocellulose substrates: an intrinsic and sustainable pretreatment strategy for clean energy production directed evolution of industrial enzymes: an update identification of type inosine monophosphate dehydrogenase as an antiangiogenic drug target new antibiotics from bacterial natural products high-titer production of astaxanthin by the semi-industrial fermentation of xanthophyllomyces dendrorhous overexpression of an exotic thermotolerant b-glucosidase in trichoderma reesei and its significant increase in cellulolytic activity and saccharification of barley straw cyclosporin a inhibits the replication of diverse coronaviruses industrial mycology: past, present, and future. in: an z (ed) handbook of industrial mycology the regulatory factor pcrfx controls the expression of the three genes of b-lactam biosynthesis in penicillium chrysogenum screening the high-yield paclitaxel producing strain alternaria alternate var. monosporus ml- b and ml- c, new inhibitors of cholesterolgenesis produced by penicillium citrinin a new hypocholesterolemic agent produced by monascus species a historical perspective on the discovery of statins recent developments in the characterization and biotechnological production of sweet tasting proteins microbial paclitaxel: advances and perspectives isolation of colletotrichum gloeosporiodes, a novel endophytic taxol-producing fungus from the leaves of a medicinal plant to market, to market- tolerance to furfural-induced stress is associated with pentose phosphate pathway genes zwf , gnd , rpe , and tkl in saccharomyces cerevisiae xylitol production by recombinant saccharomyces cerevisiae the re-emergence of natural products for drug discovery in the genomics era high-level intracellular expression of hydroxynitrile lyase from the tropical rubber tree hevea brasiliensis in microbial hosts co-expression of tal and adh in recombinant xylose-fermenting saccharomyces cerevisiae improves ethanol production from lignocellulosic hydrolysates in the presence of furfural hidden biosynthetic treasures brought to light influence of the ph on the itaconic acid production with aspergillus terreus zearelanone and some derivatives: production and biological activities biotechnological advantages of laboratory-scale solid-state fermentation with fungi microbial production of enzymes used in food applications astaxanthin over-producing strains of phaffia rhodozyma. method for their cultivation and their use in animal feeds. us patent , , comparative metabolic flux analysis of an ashbya gossypii wild type strain and a high riboflavin-producing mutant strain downstream reactions and engineering in the microbially reconstituted pathway for taxol the gibberellin fermentation biotechnology of non-saccharomyces yeasts-the basidiomycetes phaffia rhodozyma as an astaxanthin source in animal diets secondary metabolites of the fungus monascus: a review isolating "uncultivable" microorganisms in pure culture in a simulated natural environment isocitric acid production from rapeseed oil by yarrowia lipolytica yeast unraveling the efficient applications of secondary metabolites of various trichoderma spp calcium malate overproduction by penicillium viticola using the medium containing corn steep liquor microbial conversion of glycerol: present status and future prospects production of , -butanediol by engineered saccharomyces cerevisiae aspergillomarasmine a overcomes metallo-b lactamase antibiotic resistance recent derivatives from smaller classes of fermentation-derived antibacterials glycolic acid production in the engineered yeasts saccharomyces cerevisiae and kluyveromyces lactis thiolactomycin and related analogues as novel anti-mycobacterial agents targeting kasa and kasb condensing enzymes in mycobacterium tuberculosis microbial production of itaconic acid: developing a stable platform for high product concentrations isolation of taxol, an anticancer drug produced by the endophytic fungus, phoma betae in vitro screening of taxol, an anticancer drug produced by the fungus colletotricum capsici taxol promising fungal endophyte, pestalotiopsis species isolated from taxus cuspidata taxol from phyllosticta citricarpa, a leaf spot fungus of the angiosperm citrus medica engineering alcohol tolerance in yeast microbial production of food ingredients, enzymes and nutriceuticals benefit of monascus-fermented products for hypertension prevention: a review bioavailability improvement of mycophenolic acid through amino ester derivatization endophytic taxol-producing fungi from bald cypress biorefinery products from the inulin-containing crop jerusalem artichoke pyruvate production in candida glabrata: manipulation and optimization of physiological function biotechnological production of xylitol from lignocellulosic wastes: a review genetic engineering of microorganisms for biodiesel production effect of nutrition of monascus on formation of red pigments leucine interference in the production of water-soluble red monascus pigments negative effect of ammonium nitrate as nitrogen source on the production of water-soluble red pigments by monascus sp resting cell studies on formation of water-soluble red pigments by monascus sp fermentation process for carboxylic acids enhancement of pyruvate production by osmotic-tolerant mutant of torulopsis glabrata isolation and characterization of endophytic taxol-producing fungi from taxus chinensis how to achieve high-level expression of microbial enzymes: strategies and perspectives frontiers of yeast metabolic engineering: diversifying beyond ethanol and saccharomyces simvastatin has beneficial effect on pulmonary artery hypertension by inhibiting nf-jb expression the need for new antibiotics camptothecin, over four decades of surprising findings constituents of red yeast rice, a traditional chinese food and medicine the inducers , -diaminopropane and spermidine produce a drastic increase in the expression of the penicillin biosynthetic genes for prolonged time, mediated by the laea regulator nutritional and engineering aspects of microbial process development enhanced production of erythritol by yarrowia lipolytica on glycerol in repeated batch cultures fermentation behavior of an osmotolerant yeast d. hansenii for xylitol production simultaneous saccharification and continuous fermentation of sludge-containing mash for bioethanol production by saccharomyces cerevisiae thaumatin production in aspergillus awamori by use of expression cassettes with strong fungal promoters and high gene dosage the citric acid production from raw glycerol by yarrowia lipolytica yeast and its regulation natural products as sources of new drugs over the last years statins, high-density lipoprotein cholesterol, and regression of coronary atherosclerosis production of biopharmaceutical proteins by yeast high-level semi-synthetic production of the potent antimalarial artemisinin biotechnological approaches for the production of prebiotics and their potential applications fermentation of xylose and cellobiose by pichia stipitis and brettanomyces clausenii fungal enzyme inhibitors as pharmaceuticals, toxins and scourge of pcr cell recycling during very high gravity bio-ethanol fermentations using the industrial saccharomyces cerevisiae strain pe- camptothecin-producing endophytic fungus trichoderma atroviride ly : isolation, identification, and fermentation conditions optimization for camptothecin production gluconic acid production from golden syrup by aspergillus niger strain using semiautomatic stirred-tank fermenter microbial production of food ingredients, enzymes and nutriceuticals improving carotenoids production in yeast via adaptive laboratory evolution fumaric acid production by fermentation xanthophyllomyces dendrorhous for the industrial production of astaxanthin the role of oxidative stress on carotene production by blakeslea trispora in submerged fermentation in vitro culture of taxus sp.: strategies to increase cell growth and taxoid production optimization of fermentation conditions for gluconic acid production using aspergillus niger immobilized on cellulose microfibrils continuous production of gluconic acid by aspergillus niger immobilized on a cellulosic support: study of low ph fermentative behaviour of aspergillus niger differential expression of silent polyketide biosynthesis gene clusters in chemostat cultures of aspergillus nidulans overexpression of the yeast transcription activator msn confers furfural resistance and increases the initial fermentation rate in ethanol production mycophenolic acid inhibits replication of japanese encephalitis virus antimicrobials, drug discovery, and genome mining microbial production of food ingredients, enzymes and nutriceuticals activities of tmc , rifampin, and pyrazinamide against mycobacterium tuberculosis infection in guinea pigs utilization of corn steep liquor for biosynthesis of pullulan, an important exopolysaccharide alternaria sp. mg , a resveratrol-producing fungus: isolation, identification, and optimal cultivation conditions for resveratrol production production of resveratrol from tyrosine in metabolically engineered saccharomyces cerevisiae biotechnological production of gluconic acid: future implications identification of oleaginous yeast strains able to accumulate high intracellular lipids when cultivated in alkaline pretreated corn stover nitrogen source and mineral optimization enhance d-xylose conversion to ethanol by the yeast pichia stipitis nrrl-y- taxol and taxane production by taxomyces andreanae, an endophytic fungus of pacific yew industrial antibiotics: today and the future complex regulation of hydrolytic enzyme genes for cellulosic biomass degradation in filamentous fungi metabolic engineering of carbon and redox flow in the production of small organic acids from flavors and pharmaceuticals to advanced biofuels: production of isoprenoids in saccharomyces cerevisiae biosynthesis of gibberellins in gibberella fujikuroi: biomolecular aspects enzyme inhibitors of microbial origin inhibition of fatty acid synthetases by the antibiotic cerulenin cancer therapies utilizing the camptothecins: a review of the in vivo literature the combinatorial chemistry of nature ergot alkaloids. in: rose ah (ed) secondary products of metabolism camptothecin and taxol: from discovery to clinic taxol from tubercularia sp. strain tf , an endophytic fungus of taxus mairei engineering taxol biosynthetic pathway for improving taxol yield in taxol-producing endophytic fungus efy- (ozonium sp.) production of -oxoglutaric acid by yeasts strain improvement and optimization of the media of taxol-producing fungus fusarium maire key technologies for the industrial production of fumaric acid by fermentation production of omega- eicosapentaenoic acid by metabolic engineering of yarrowia lipolytica recent advances in genome mining of secondary metabolite biosynthetic gene clusters and the development of heterologous expression systems in aspergillus nidulans ethanol production from concentrated food waste hydrolysates with yeast cells immobilized on corn stalk urea enhances cell growth and pyruvate production in torulopsis glabrata a novel impeller configuration to improve fungal physiology performance and energy conservation for cephalosporin c production effects of lignin-derived phenolic compounds on xylitol production and key enzyme activities by a xylose utilizing yeast candida athensensis sb an endophytic taxol-producing fungus from taxus media, cladosporium cladosporoides md what is vital (and not vital) to advance economically-competitive biofuels production study on the preparation and regeneration of protoplast from taxol-producing fungus nodulisporium sylviforme a review: recent advances and future prospects of taxol-producing endophytic fungi key: cord- -r w jbpg authors: tennant, bud c.; hornbuckle, william e. title: gastrointestinal function date: - - journal: clinical biochemistry of domestic animals doi: . /b - - - - . - sha: doc_id: cord_uid: r w jbpg this chapter discusses the functions of gastrointestinal tract. the principal functions of the gastrointestinal tract are assimilation of nutrients and excretion of the waste products of digestion. within the gastrointestinal tract, these substances are solubilized and degraded enzymatically to simple molecules, sufficiently small in size and in a form that permits absorption across the mucosal epithelium. the distribution of the different types of secretory cells in the salivary glands varies among species. the mandibular and sublingual glands are mixed salivary glands containing both mucous and serous types of cells, and produce a viscous secretion that contains large amounts of mucus. the cytoplasm of the secretory cells contains numerous zymogen granules that vary in size and number depending on the activity of the gland. these granules contain the precursors of the hydrolytic enzymes responsible for digestion of the major dietary components. the cells of the terminal ducts probably secrete the bicarbonate ion responsible for neutralizing hydrochloric acid that enters the duodenum from the stomach. the digestive system is composed of the gastrointestinal tract or alimentary canal, salivary glands, liver, and exocrine pancreas. the principal functions of the gastrointesti nal tract are assimilation of nutrients and excretion of the waste products of digestion. most nutrients are ingested in a form which is either too complex or insoluble for absorp tion. within the gastrointestinal tract, these substances are solubilized and degraded enzymatically to simple molecules, sufficiently small in size and in a form which permits absorption across the mucosal epithelium. in the following section, the normal biochemi- cal processes of intestinal secretion, digestion, and absorption are described. with these in perspective, we then discuss the mechanisms involved in the pathogenesis of the most important gastrointestinal diseases and the biochemical basis for diagnosis and treatment. a. saliva saliva is produced by three major pairs of salivary glands and by small glands distrib uted throughout the buccal mucosa and submucosa. two types of secretory cells are found in the acinar portions of the salivary glands: ( ) the mucous cells, which contain droplets of mucus, and ( ) the serous cells, which contain multiple secretory granules. in those species which produce salivary amylase, the secretory granules are the zymögen precur sors of this enzyme. a third cell type is found lining the striated ducts. the striations along the basal borders of these cells are caused by vertical infoldings of the cell membrane, a characteristic of epithelial cells involved in rapid movement of water and electrolytes. the primary secretion of the acinar cells is modified by active transport processes of the ductal epithelium. the distribution of the different types of secretory cells in the salivary glands varies among species. the parotid glands of most animals are serous glands which produce a secretion of low specific gravity and osmolarity, containing electrolytes and proteins including certain hydrolytic enzymes. the mandibular (submaxillary) and sublingual glands are mixed salivary glands containing both mucous and serous types of cells and produce a viscous secretion which contains large amounts of mucus (dukes, ) . a. mucus. mucus is an aqueous mixture of protein-poly saccharide complexes and glycoproteins (gottschalk, ) , which have relatively large amounts of carbohydrate bound to protein. the protein-poly saccharide complexes have long polysaccharide chains containing repeating units bound to a protein core. the glycoproteins contain numerous oligosaccharide residues distributed along the polypeptide chain. one of the most completely studied glycoproteins is mucin from the submaxillary glands of ruminants. the carbohydrate portion is a disaccharide of yv-acetylneuraminic acid (a sialic acid) and v-acetylgalactosamine. approximately such disaccharide molecules are present per molecule of mucin (bhavanandan et al., ; bertolini and pigman, ). an enzyme capable of linking protein with hexosamine was demonstrated in sheep submaxillary glands (mcguire and roseman, ) . the physiological functions of mucin are closely related to its high viscosity. n-acetylneuraminic acid is the component responsible for the formation of viscous aque ous solutions. at physiological ph, it causes expansion and stiffening of the mucin molecule (gottschalk and thomas, ) . the resistance of mucin to enzymatic break down is also due to the presence of disaccharide residues. removal of the terminal yv-acetylneuraminic acid residues by neuraminidase significantly increases the susceptibil ity of peptide bonds to trypsin (gottschalk and fazekas de st. groth, ) . b. amylase. the saliva of most species contains the α-amylase ptyalin. this enzyme is said to be absent, however, in the saliva of dogs, cats, and horses (dukes, ) . salivary amylase splits the a- , -glucosidic bonds of various polysaccharides. the sali vary enzyme is similar in all major respects to pancreatic α-amylase, which is described below (section ii,d). salivary amylase initiates digestion of starch and glycogen in the mouth of those species which secrete the enzyme. the optimal ph for amylase activity is approximately , and activity therefore terminates when the enzyme mixes with acidic gastric contents. saliva bathes the oral cavity continuously, serving to protect the surface epithelium. ingested food is moistened and lubricated by saliva, facilitating mastication and swallow ing. the teeth also are protected from decay by saliva, which washes food particles from the surfaces of the teeth and, because of its buffering capacity, neutralizes the organic acids produced by bacteria normally present in the mouth. ruminants produce much greater quantities of saliva than simple-stomached animals, and the saliva has a higher ph and bicarbonate ion concentration. in ruminants, saliva serves several unique functions (phillipson, ) . it is required for maintenance of the composition of the contents of the rumen. the great buffering capacity is necessary to neutralize the large amounts of short-chain fatty acids which are the major end products of rumen fermentation. the urea in saliva can be utilized by rumen bacteria for protein synthesis. protein synthesized in the rumen is then used to meet dietary protein require ments. in this way, urea nitrogen can be "recycled" through the amino acid pool of the body and in ruminants need not be considered an end stage in protein catabolism. the ability to reutilize urea has also been demonstrated in the horse and may be of particular benefit during periods of protein deficiency (houpt and houpt, ; prior et al., ) . the stomach is divided into two main regions on the basis of secretory function (grossman, ) . the oxyntic gland area corresponds approximately to the body of the stomach in most species of domestic animals and also to the fundus in the dog and cat. the oxyntic glands contain ( ) oxyntic or parietal cells, which are responsible for hydro chloric acid production, (b) peptic (zymogenic, chief) cells, which produce pepsinogen, and (c) mucous cells. the pyloric gland area contains the pyloric glands, which are slightly alkaline, and, in addition to mucus, contains the polypeptide hormone gastrin. a variety of stimuli can initiate gastric secretion. the sight or smell of food or the presence of food within the mouth causes gastric secretion by a reflex mechanism involv ing the vagus nerve. the presence of certain foods within the stomach or distention of the stomach alone also can initiate both intrinsic and vagai nerve reflexes which cause secre tion of gastric juice. in addition to neural reflexes, these stimuli cause release of the polypeptide hormone gastrin from the pyloric gland area, which enters the bloodstream, stimulating gastric secretion. the release of gastrin from the specific g cells responsible for synthesis is inhibited by excess hydrogen ion, and this negative feedback mechanism is fig. . amino acid sequence of porcine gastrin i (gregory, ) . gastrin ii differs from gastrin i by the presence of a sulfate ester group on the single tyrosyl residue. believed to be of physiological importance in the control of hydrochloric acid production. gastrin has been isolated in pure form from the antral mucosa of swine gregory and tracy, ; . when administered intraven ously, the purified hormone causes the secretion of hydrochloric acid and pepsin. it also stimulates gastrointestinal motility and causes pancreatic secretion. two separate peptides have been obtained from porcine gastric mucosa and have been designated gastrin i and gastrin ii. the structure of gastrin has been determined and has been confirmed by synthesis (anderson et al., ) . it is a heptadecapeptide amide, with a pyroglutamyl n-terminal residue and the amide of phenylalanine as the c-terminal residue (fig. ). in the center of the molecule is a sequence of five glutamyl residues, which give the molecule its acidic properties. gastrin ii differs from gastrin i only in the presence of a sulfate ester group linked to the single tyrosyl residue. the c-terminal tetrapeptide amide, trp-met-asp-phe-nh , is identical in all species so far studied (gregory, ) . the tetrapeptide has all of the activities of the natural hormone. it is not as potent as the parent molecule, but activity can be increased by lengthening the peptide chain. gastrin is the only hormone known to stimulate hc secretion (walsh and grossman, ) . as indicated above, gastrin is released in response to vagai stimulation by distention of the pyloric antrum and by direct luminal contact with food, particularly partially hydrolyzed protein (walsh and grossman, ) . the exact mechanism of action is not known, but studies using isolated preparations of isolated parietal cells suggest that the effects of gastrin are not mediated by cyclic amp (soil, ) . some of the other factors which are important in regulation of hc secretion are summarized in fig. after dousa and dozois ( ) . there is little doubt that histamine secreted locally within the mucosa has a major effect on the function of parietal cells (soil dousa and dozois, .) and grossman, ) . histamine has been recognized as a potent stimulant of hc production for many years (code, ) . this effect, however, was not inhibited by traditional antihistaminic drugs (hj antagonists), and, until the demonstration of h recep tors in the stomach (the atrium and uterus) by black et al. ( ) , the physiological role of histamine in hc secretion was controversial. specific h antagonists (burimamide, cimetidine, metiamide) now have been shown to inhibit the secretory response not only to histamine, but also to gastrin, to cholinergic stimuli, and to food (grossman and konturek, ) . although there has been significant conflict in the published literature, current evidence suggests that histamine activates the adenylate cyclase of parietal cells (dousa and dozois, ) , resulting in synthesis of cyclic amp and ultimately in hc secretion (fig. ). the controversy with regard to the role of cyclic amp as a mediator of histamine action has come from observations that prostaglandins and secretin, both potent inhibitors of gastric hc secretion, also stimulate adenylate cyclase (thompson et al., ) . it is now believed that prostaglandins, in addition to inhibiting hc secretion, act on a mucosal cell population which is different from parietal cells and that these cells secrete cytoprotective substances (mucin, glycosaminoglycans). the ulcerogenic effects of prostaglandin inhibitors (indomethacin, acetylsalicylic acid) apparently result from inhibition of this protective effect of endogenous prostaglandins. a. basal versus stimulated secretion. gastric juice is composed of two compo nents. one is secreted continuously by the surface epithelial cells and other mucusproducing cells. the other component is produced by the oxyntic glands in response to various stimuli. the basal component is neutral or slightly alkaline. the electrolyte composition is similar to that of an ultrafiltrate of plasma (table i) and contains large amounts of mucus, which protects the epithelium. the secretory component produced by the oxyntic glands in response to stimulation contains free hydrochloric acid and pepsinogen, the principal enzyme of gastric digestion. the composition of gastric juice depends on the relative amounts of the two secretory components present, which in turn is a function of flow rate. in the dog, gastric juice is produced in the resting state at a rate of approximately ml/hour (gray and bûcher, ) , and the composition is similar to that of the basal component, containing practi cally no peptic activity or hydrochloric acid. when the flow of gastric juice is stimulated maximally, the dog may produce ml or more per hour (gray and bûcher, ) , and this secretion contains large amounts of peptic activity and hydrochloric acid. sodium, which is the principal cation in the basal secretion, is replaced to a large extent by hydrogen ion. the concentration of potassium is similar in both basal and stimulated secretions and therefore remains relatively constant at various rates of flow. hydrochloric acid and pepsinogen are secreted by separate mechanisms, but these appear to be closely linked under physiological conditions. stimulation of the vagus nerve (bachrach, ; hirschowitz and sachs, ) or intravenous injection of gastrin (hirschowitz, ) increases pepsinogen and hydrochloric acid levels together. other stimuli may affect the two processes differently. in the dog, for example, histamine infusion stimulates hydrochloric acid production maximally but inhibits pepsinogen secre tion (abrams and brooks, ; hirschowitz, ; ernas and grossman, ) . inhibitor ( n · leu · leu · · · leu · glu ( mol. wt.) pepsinogen is the zymögen, or inactive precursor, of pepsin, the principal proteolytic enzyme of gastric juice. pepsinogen was first crystallized from the gastric mucosa of swine (herriott, ) , and several pepsinogens have been separated by ryle ( ) , ryle and porter ( ) , and ryle and hamilton ( ) . porcine pepsinogen has a molecular weight of approximately , and is composed of the pepsin molecule and several smaller peptides (fig. ) . one of these peptides has a molecular weight of and is an inhibitor of peptic activity (herriott, ) . activation of pepsin from pepsinogen occurs by selective cleav age of this small basic peptide from the parent pepsinogen (neurath and walsh, ) . autocatalytic conversion begins below ph .o. at ph . , the inhibitor peptide dis sociates from the parent molecule, and, at ph . - . , the inhibitor is completely digested by pepsin (taylor, ) . pepsin has a very acidic isoelectric point, being stable in acidic solution below ph . but irreversibly denatured at ph . or above. in contrast, pepsinogen is stable in neutral or slightly alkaline solution. the optimal ph for peptic activity is generally between . and . , but the effect of ph may vary with the substrate. pepsin is capable of hydrolyzing peptide bonds of most proteins, mucin being one important exception. pepsin splits bonds involving phenylalanine, tyrosine, and leucine most readily but can hydrolyze almost all other peptide bonds. c. rennin. rennin is another proteolytic enzyme produced by the gastric mucosa and has some characteristics which are similar to those of pepsin. it has been separated from pepsin in preparations from the stomachs of newborn calves. rennin splits a mucopeptide from casein to form paracasein, which then reacts with calcium ion to form an insoluble coagulum. the coagulated milk protein probably delays gastric emptying and increases the efficiency of protein digestion in young calves. d. hydrochloric acid. hydrochloric acid is produced by the oxyntic cells. when the normal mucosa is stimulated, both chloride and hydrogen ions are secreted together, but current evidence suggests that h + and cl" are secreted by separate, closely coupled pump mechanisms. small amounts of cl~ are secreted continuously by the unstimulated parietal cells in the absence of h + secretion, and this mechanism is responsible for the relative negative charge of the resting mucosal surface. hydrogen ion and clsecretory systems may also be differentiated in vitro by the demonstration of hydrogen ion secretion in the absence of cl~. a scheme for the secretion of hydrochloric acid is presented in fig. . for every h + secreted, an electron is removed. the electron ultimately is accepted by oxygen to form oh -, which is neutralized within the cell by h + from carbonic acid. the bicar bonate ion produced enters the venous blood, and this explains why the ph of gastric venous blood frequently is greater than that of arterial blood during hydrochloric acid secretion (davenport, ) . conversion of carbon dioxide and water to carbonic acid is catalyzed by carbonic anhydrase, which is present in high concentration within parietal cells. when the rate of acid secretion is high, this enzyme contributes to the secretory mechanism by maintaining normal intracellular ph. carbonic anhydrase inhibitors, such as acetazolamide, interfere with hydrochloric acid production in high concentrations and when the rate of acid secretion is high (janowitz et al., ) . bile is secreted continuously by the hepatocytes into the bile canaliculi and is trans ported through a system of ducts to the gallbladder, where it is modified, concentrated, and stored. during digestion, bile is discharged into the lumen of the duodenum, where it aids in emulsification, hydrolysis, and solubilization of dietary lipids. the digestive functions of bile are accomplished almost exclusively by the detergent action of its major components, the bile salts and phospholipids. the primary bile acids are c carboxylic acids synthesized by the liver from choles terol. bile acid formation represents the major pathway for cholesterol metabolism (danielsson, ) . cholic acid ( a, a, a-trihydroxy- / -cholanoic acid) and chenodeoxycholic acid ( a, a-dihydroxy- / -cholanoic acid) are the primary bile acids formed by most species of domestic animals. in swine, chenodeoxycholic acid is hydroxylated at the a position by the liver to yield hyocholic acid, which is a major primary bile acid in this species (haslewood, ) . bile acids are secreted as amino acid conjugates of either glycine or taurine. taurine conjugates predominate in the dog, cat, and rat. in the rabbit, the conjugating enzyme system appears to be almost completely specific for glycine (bremer, ) . both taurine and glycine conjugates are present in ruminants. in the newborn lamb, % of the bile acids are conjugated with taurine. as the lamb matures, glycine conjugates increase, accounting for one-third of the total in mature sheep (peric-golia and socie, ) . under normal conditions, only conjugated bile acids are present in the bile and in the contents of the proximal small intestine. in the large intestine, the conjugated bile acids are hydrolyzed rapidly by bacterial enzymes so that, in the contents of the large intestine and in the feces, free or unconjugated bile acids predominate. several genera of intestinal bacteria, including clostridium, enterococcus, bacteroides, and lactobacillus (midtvedt and norman, ) , are capable of splitting the amide bonds of conjugated bile acids. intestinal bacteria also modify the basic structure of the bile acids. one such reaction is the removal of the a-hydroxyl group at the position of cholic acid or chenodeoxycholic acid. these bacterial reactions yield the secondary bile acids, deoxycholic acid, and lithocholic acid, respectively (gustafsson et al., ) . lithocholic acid is relatively insoluble and is not reabsorbed to any great extent (gustafsson and norman, ) . deoxycholic acid is reabsorbed from the large intestine in significant quantities and is either rehydroxylated by the liver to cholic acid and excreted (lindstedt and samuelsson, ) or excreted as conjugated deoxycholic acid. the extent to which bacteria transform the primary bile acids depends on the nature of the diet, the composition of the intestinal microflora, and the influences which these and other factors have on intestinal motility (gustafsson et al., ; gustafsson and norman, a,b) . the carboxyl group of the bile acids is completely ionized at the ph of bile and is neutralized by sodium ion, resulting in the formation of bile salts. the bile salts are effective detergents. they are amphipathic molecules, which have both hydrophobic and hydrophilic regions. in low concentrations, bile salts form molecular or ideal solutions, but, when their concentration increases above a certain critical level, they form polymolecular aggregates known as micelles. the concentration at which these molecules aggregate is called the critical micellar concentration (cmc). bile salt micelles are spherical and consist of a central nonpolar core and an external polar region. fatty acids, monoglycerides, and other lipids are solubilized when they enter the central core of the micelle and are covered by the outside polar coat. solubilization occurs only when the cmc is reached. for the bile salt-monoglyceride-fatty acid-water system present during normal fat digestion, the cmc is approximately mm, which is ordinarily exceeded both in bile and in the contents of the upper small intestine (hofmann, ) . phospholipids, principally lecithin, are also major components of bile. in the lumen of the small intestine, pancreatic phospholipase catalyzes the hydrolysis of lecithin, forming free fatty acid and lysolecithin. the latter compound also is a potent detergent which acts with the bile salts to disperse and solubilize lipids in the aqueous micellar phase. the enterohepatic circulation begins as conjugated bile acids near the duodenum and mix with the intestinal contents, forming emulsions and micellar solutions. the bile acids are not absorbed in significant amounts from the lumen of the proximal small intestine. absorption occurs primarily in the ileum (lack and weiner, weiner, , weiner and lack, ) , where an active transport process has been demonstrated . the conjugated bile acids pass unaltered into the portal circulation (playoust and isselbacher, ) and return to the liver, where the cycle begins again. this arrangement provides optimal concentrations of bile acids in the proximal small intestine, where fat digestion and absorption occur, and then efficient absorption after these functions have been accomplished. absorption of unconjugated bile acids from the large intestine ac counts for - % of the total enterohepatic circulation (weiner and lack, ) . in dogs, the total bile acid pool was estimated to be . - . gm. the half-life of the bile acids in the pool ranged between . and . days, and the rate of hepatic synthesis was . - . gm/day (wollenweber et al., ) . the daily requirement for bile acids greatly exceeds the normal synthetic rate. this necessitates repeated reutilization of the bile acids, which is accomplished by means of the enterohepatic circulation. under steady-state conditions, the entire bile acid pool passes through the enterohepatic circulation approxi mately ten times each day (hofmann, ) . the size of the bile acid pool is dependent upon diet, the rate of hepatic synthesis, and the efficiency of the enterohepatic circulation. surgical removal of the ileum in dogs interrupts the enterohepatic circulation, causing an increase in bile acid turnover rate and a reduction in the size of the bile acid pool (playoust et al., ) . in diseases of the ileum, there may be defective bile salt absorption and bile salt deficiency. if severe, impaired utilization of dietary fat may occur, resulting in steatorrhea and impaired absorption of the fat-soluble vitamins. the exocrine pancreas is an acinous gland with the same general structure as the salivary glands. the cytoplasm of the secretory cells contains numerous zymogen granules, which vary in size and number depending on the activity of the gland. these granules contain the precursors of the hydrolytic enzymes responsible for digestion of the major dietary components. the cells of the terminal ducts probably secrete the bicarbonate ion responsible for neutralizing hydrochloric acid which enters the duodenum from the stomach. /. composition a. electrolyte composition. the cation content of pancreatic secretion is similar to that of plasma. sodium is the predominant cation, with smaller concentrations of potas sium and calcium being present. a unique characteristic of pancreatic juice is its high bicarbonate ion concentration and alkaline ph. in the dog, the ph ranges from . to . , depending on hc ~ content. the volume of pancreatic juice is directly related to hc ~ content and ph increase and the cl~ concentration decreases. the sodium and potassium ion concentrations and osmolarity appear to be independent of secretory rate (fig. ) . b. a-amy läse. the amylase produced by the pancreas catalyzes the specific hy drolysis of a-l, -glucosidic bonds, which are present in starch and glycogen (a- , glycan- -glycan hydrolase). pancreatic amylase appears to be essentially identical to the amylase of saliva. it is a calcium-containing metalloenzyme (vallee et al., ) . re moval of calcium by dialysis inactivates the enzyme and markedly reduces the stability of the apoenzyme. pancreatic amylase has an optimal ph for activity of . - . and is activated by chloride ion. synthesis of pancreatic α-amylase occurs in the ribosomes. the enzyme is transferred -rasmussen et al., .) from the endoplasmic reticulum to cytoplasmic zymogen granules for storage (redman et al., ) . it is secreted in active form upon stimulation of the acinar cells. newborn calves (huber et al., ) and pigs (walker, ) secrete amylase at a significantly lower rate than mature animals. the rate of synthesis is also influenced by diet. animals fed a high-carbohydrate diet synthesize amylase at several times the rate of animals on a high-protein diet (ben abdeljlil and desnuelle, ) . unbranched a- , -glucosidic chains, such as those found in amylase, are hydrolyzed in two steps. the first is rapid and results in formation of the disaccaride maltose and maltotriose. the second step is slower and involves hydrolysis of maltotriose with forma tion of glucose and maltose. polysaccharides such as amylopectin and glycogen contain branched chains with both a-\ , -and a-\ , -glucosidic linkages. when α-amylase attacks these compounds, the principal products are maltose (a-l, -glycosidic bond), isomaltose (a-l, -glucosidic bond), and small amounts of glucose. final hydrolysis of the maltose and isomaltose occurs at the surface of the mucosal cell, where the enzymes maltase and isomaltase are integral parts of the microvillous membrane. c. proteolytic enzymes. the proteolytic enzymes of the pancreas are responsible for the major portion of protein hydrolysis, which occurs within the lumen of the gastrointes tinal tract. two types of peptidases are secreted by the pancreas. trypsin, chymotrypsin, and elastase are endopeptidases, which attack peptide bonds along the polypeptide chain, producing smaller peptides. the exopeptidases attack either the carboxy-terminal or amino-terminal peptide bonds, releasing single amino acids. the principal exopeptidases secreted by the pancreas are carboxypeptidases a and b. the endopeptidases and exopep- (table ii) , producing free amino acids, which are absorbed directly, or small peptides, which are further hydrolyzed by the aminopeptidases of the intestinal mucosa (see section iii,c). the pancreatic peptidases are secreted as inactive proenzymes or zymogens termed trypsinogen, chymotrypsinogen, and procarboxypeptidase a and b. trypsinogen is con verted to active trypsin in two ways. at alkaline ph, trypsinogen can be converted autocatalytically to trypsin, the activated enzyme converting more zymögen to active enzyme. trypsinogen can also be activated by the enzyme enter okinase, which is pro duced by the duodenal mucosa. the latter reaction appears to be highly specific in that enterokinase will not activate chymotrypsinogen. chymotrypsinogen, proelastase, and the procarboxypeptidases a and b are converted to active enzymes by the action of trypsin. the amino acid sequences and other structural characteristics of bovine trypsinogen and chymotrypsinogen have been determined (hartley et al., ; hartley and kauffman, ; brown and hartley, ) . the polypeptide chain of trypsinogen contains amino acid residues. activation of the proenzyme occurs with hydrolysis of a single peptide bond located in the position between lysine and isoleucine. the c-terminal hexapeptide is released as enzyme activity appears. there is also substantial change in the helical structure of the parent molecule (davie and neurath, ; neurath et al., ) . chymotrypsinogen a is composed of amino acid residues and has numerous structural similarities to trypsinogen. activation of the chymotrypsinogen also occurs with cleavage of a single peptide bond. for a complete discussion of this subject, see the review by keller ( ) . d. lipase. the pancreas produces several lipolytic enzymes with different substrate specificities. the most important of these from a nutritional viewpoint is the lipase re sponsible for hydrolysis of dietary triglycéride. this enzyme has the unique property of requiring an oil-water interface for activity so that only emulsions can be effectively at tacked (sarda and desnuelle, ) . the principal products of lipolysis are glycerol, monoglycerides, and fatty acids. the monoglycerides and fatty acids accumulate at the oil-water interface and can inhibit enzyme activity. their transfer from the interface to the aqueous phase is favored by the presence of sodium bicarbonate also secreted by the pancreas and by bile salts. mattson and volpenhein ( ) described two other carboxylic ester hydrolases in pancreatic juice. both enzymes have an absolute requirement for bile salts, in contrast to glycerol ester hydrolase, which is actually inhibited by bile salts at ph . one of these enzymes is a sterol ester hydrolase responsible for hydrolysis of cholesterol esters. the other enzyme hydrolyzes various water-soluble esters. the two enzyme activities have been differentiated on the basis of stability and optimal ph. the pancreas secretes a third lipolytic enzyme which hydrolyzes phospholids. phospholipase a converts lecithin which is present in bile to lysolecithin, an effective deter gent which aids in emulsification of dietary fat. pancreatic secretion is controlled and coordinated by neural and endocrine mechanisms. when ingesta or hydrochloric acid enters the duodenum, the hormone secretin is released into the circulation by the duodenal mucosa. secretin increases the volume, ph, and hc ~ concentration of the pancreatic secretion. secretin is a polypeptide hormone which contains amino acid residues. all amino acids are required to maintain the helical structure of the molecule and its activity (bodanszky et al., ) . the c-terminal amide is a property of other polypeptide hormones, such as gastrin and vasopressin, which act on the flow of water in biological systems (mutt and jorpes, ) . in addition to its effects on the pancreas, secretin increases the rate of bile formation (wheeler and mancusi-ungaro, ) . the pancreatic juice which results from stimulation by secretin is large in volume and has high bicarbonate concentration but is low in enzyme activity. stimulation of the vagus nerve causes a significant rise in enzyme concentration. this type of response also is produced by pancreozymin, another polypeptide hormone secreted by the duodenal mu cosa. pancreozymin is now believed to be identical to cholecystokinin, an intestinal hormone which causes contraction of the gallbladder (thompson, ) . the c-terminal pentapeptide of pancreozymin-cholecystokinin is exactly the same as that of gastrin. this fascinating relationship suggests that gastrin and pancreozymin-cholecystokinin may par ticipate in some unified but as yet poorly understood system of digestive control (thompson, ) . during the past several years, a large number of papers have been published on the endocrine function of the gastrointestinal mucosa and on several new polypeptides which are being classified as gut hormones (table iii) . many of these new substances have not met the rigid physiological requirements for true hormone status, including ( ) biological action in very small concentration, ( ) release into the bloodstream, and ( ) normal serum levels comparable to those provided experimentally by exogenous administration. these criteria probably will be modified, particularly with regard to requirements for transport in the vascular system. a large class of peptides are under investigation which have paracrine rather than endocrine activities; that is, their actions are on cells and tissues in the immediate vicinity of the cells of origin. motilin is a polypeptide containing amino acids that was originally isolated from porcine duodenal mucosa (brown et al., ) . the amino acid composition and se quence have been described (brown et al., (brown et al., , . immunoreactive motilin has been found in the enterochromaffin cells of the duodenum and jejunum of several species (polak et al., ) and, by means of radioimmunoassay, motilin has been identified in the plasma of dogs (dryburgh and brown, ) . motilin has been shown to stimulate pepsin output and motor activity of the stomach (brown et al., ) and to induce lower esophageal sphincter contractions (jennewein et al., ) . studies by itoh et al., ( ) suggest that motilin plays an important role in initiating interdigestive gastrointestinal contractions. somatostatin, which is named for its growth hormone release-inhibiting activity, was first purified from bovine hypothalamus (brazlua and guilleman, ) . somatostatin also has been demonstrated in the stomach, pancreas, and intestinal mucosa in concen trations higher than in the brain (pearse et al., ) . somatostatin is a potent inhibitor of insulin and glucagon release. it also inhibits gastrin release and gastric acid secretion (barros d'sa et al., ; bloom et al., ) , apparently acting independently on parietal cells and on g cells. these and a variety of other physiological effects suggest that somatostatin has important gastrointestinal regulatory functions (pearse et al., ) . enteroglucagon is the hyperglycémie, glycogenolytic factor isolated from the intestinal mucosa. it occurs primarily in the distal small intestine and colon in at least two forms, one with a molecular mass of dal tons and the other somewhat larger (val verde et al., ) . enteroglucagon differs from pancreatic glucagon biochemically, immunologically, and in its mode of release. the physiological function of enteroglucagon is not known, but its release from the mucosa following a meal and the associated increase in circulating blood levels have suggested a regulatory role on bowel function (pearse et al., ) . enteroglucagon also differs significantly from glucagon produced by the a cells of the gastric mucosa of the dog (sasaki et al., ) . canine gastric glucagon is biologi cally and immunochemically identical to pancreatic glucagon. gastric glucagon appears to be unique to the dog, similar activity not being observed in the stomach of the pig or the abomasum of cattle and sheep (sutherland and de du ve, ) . the microvillous membrane of the intestinal mucosa, like other cell membranes, is a lipid structure which acts as a barrier to water and water-soluble substances. water and polar solutes penetrate in one of two ways. ( ) they may pass through pores in the membrane, which are believed to be aqueous channels connecting the luminal surface of the cell with the apical cytoplasm. the "effective" diameter of jejunal pores has been estimated to be approximately . nm (lindemann and solomon, ) . ( ) they may attach to membrane carriers, which facilitate passage through the lipid phase of the membrane. transport of water and water-soluble compounds is influenced by the permeability characteristics of the limiting membrane and by the nature of the driving forces which provide energy for transport. passive movement occurs either by simple diffusion or as a result of gradients in concentration (activity), ph, osmotic pressure, or electrical potential which may be present across the membrane. the passive movement of an ion in the direction of an electrochemical gradient is referred to as single-file diffusion (hladky, ) . when a substance moves in a direction opposite that of an established electrochem ical gradient, an active transport process is said to be responsible. most water-soluble compounds, such as monosaccharides and amino acids, cannot diffuse across the intestinal mucosal membrane at rates which are adequate to meet nutritional requirements. transport of these substances is believed to be by means of membrane carriers. the nature of thse carriers is not well understood, but they are believed to be an integral part of the membrane and responsible for binding the transported substance in a rather specific way. their existence is based primarily on kinetic evidence. carrier-mediated transport systems can be saturated and are competitively inhibited by related compounds. three types of carrier transport mechanisms are recognized (curran and schultz, ). ( ) active transport, as stated previously, involves movement of electrolytes against an electrochemical gradient. in the case of nonelectrolytes, such as glucose, active transport is defined as movement against a concentration gradient. active transport requires metabolic energy and is inhibited by various metabolic blocking agents or by low tempera ture. ( ) facilitated diffusion occurs when the passive movement of a substance is more rapid then can be accounted for by simple diffusion. facilitated diffusion systems can increase the rate of movement across the membrane by two or three orders of magnitude. the carrier mechanism is similar to that involved in active transport in that it displays saturation kinetics, may be inhibited competitively, and is temperature dependent. how ever, transport does not occur against concentration or electrochemical gradients, and direct expenditure of energy is not required. ( ) exchange diffusion is a transfer mechanism similar to facilitated diffusion. it was postulated originally by ussing ( ) to explain the rapid transfer of radioactive na + across cell membranes. the mechanism does not give rise to net transport but contributes in a major way to unidirectional flux rates, which are measured with isotopie tracers. in the intestine, net water absorption is the result of bulk flow through pores in the membrane. diffusion in the usual sense plays no important role in a water movement (section iii,a, ). when bulk flow occurs, it is possible for solutes to move across the membrane in the direction of flow by a phenomenon called solvent drag. the effect of solvent drag on the transport of a given solute depends on the rate of volume flow and upon the reflection coefficient, which is an expression of the relationship between the pore radius and the radius of solute molecule being transported. a solute such as urea can be transported by the intestine against a concentration gradient by means of solvent drag (hakim and lifson, ) . studies with isotopie tracers have shown that transport of water and electrolytes by the intestinal mucosa is a dynamic process, with rapid unidirectional fluxes of the substances occurring continuously in both directions. net absorption occurs when the flow from lumen to plasma exceeds that in the opposite direction (code et al., ; berger et al., ; hindle and code, ) . active transport of na + can occur along the entire length of the intestine, but the rate of absorption is greatest in the ileum and colon, where most net sodium and water absorption occurs. sodium transport is believed to be accomplished by an energy-requiring "sodium pump. " the characteristics of this pump are not completely understood, but skou ( ) presented evidence that the pump is intimately related to the activity of a na + -in dependent adenosine triphosphatase located within the cell membrane. this enzyme is inhibited by cardiac glycosides, such as oubain, which also are effective inhibitors of na + transport, and it has been suggested that this enzyme system may actually be the pump. in the jejunum, net absorption of sodium occurs slowly unless nonelectrolytes, such as glucose or amino acids, are absorbed simultaneously. in in vivo studies by fordtran et al. ( ) , jejunal absorption of sodium appeared to be explained, in part, by solvent drag which was associated with active glucose transport. in the ileum, na + absorption was independent of glucose absorption. water absorption in the jejunum also appears to be almost entirely dependent upon the absorption of glucose, while absorption from the ileum is unaffected by glucose (barry et al., ) . the differential effect of glucose on absorption from the jejunum and ileum appears to be the result of fundamental metabolic differences between these two areas of the intestine (curran, ; gilman and koelle, ) . as sodium is transported across the mucosal membrane, an equivalent amount of anion must be transported simultaneously to maintain electrical neutrality. a significant amount of chloride ion absorption can be accounted for on this basis. it is generally agreed that chloride transport in the intestine is a passive process (clarkson et al., ) , although active secretion by the gastric mucosa seems well established. the intestinal mucosa can, under certain circumstances, absorb cl" independently of cation absorption and maintain electrical neutrality by exchange secretion of bicarbonate into the lumen (ingraham and visscher, ). dietary potassium is absorbed almost entirely in the proximal small intestine. absorp tion appears to be a passive process since movement across the mucosa occurs down a concentration gradient (high luminal concentration to a low concentration in plasma). the fluid which reaches the ileum from the jejunum has a potassium concentration and a sodium/potassium ratio which is similar to that of plasma. in the ileum and colon, the rate of sodium absorption is much greater than that of potassium so that, under normal conditions, the sodium/potassium ratio in the feces is much lower than that of plasma, approaching a ratio of . the absorption of water has been one of the most extensively studied aspects of intestinal transport. it is now generally agreed that water movement is the result of bulk flow through membranous pores and that simple diffusion plays only a minor role. the question of whether water is actively or passively transported has been the subject of considerable controversy, and the controversy itself points to the fundamental difficulties which arise in trying to establish a definition of active transport. hypertonie saline so lutions can be absorbed from canine intestine in vivo (grim, ) and from canine and rat (parsons and wingate, ) intestine in vitro. these observations indicate that water absorption can occur against an activity gradient and that the process is dependent upon metabolic energy. this would suggest that an active transport process is involved. curran ( ) , however, presents an alternate interpretation which is now generally accepted. this view is that water transport occurs secondarily to active solute transport and is the result of local gradients established within the mucosal membrane. water transport is then coupled to the energy-dependent process responsible for solute transport but is one step removed from it. in the dog and probably other carnivores, the ileum is the main site of net sodium and water absorption. the colon accounts for no more than perhaps % of the total. in the case of herbivorous animals in which the large intestine is developed extensively, net secretion of water may occur in the ileum so that all net absorption of water must take place in the cecum and colon (powell et al., ; argenzio, ) . carbohydrate is present in the diet primarily in the form of polysaccharides of glucose. the most common polysaccharides are starch, glycogen, and cellulose. starch and glycogen are composed of long chains of glucose molecules linked together by repeating a- , -glucosidic bonds. branching chains are linked by a- , -glucosidic bonds. in those species which secrete salivary amylase, digestion of starch and glycogen begins in the mouth when this enzyme mixes with food. the action of salivary amylase is interrupted in the stomach, however, because of the low ph of the gastric secretion. starch digestion begins again in the proximal small intestine with the action of pancrea tic amylase. this enzyme catalyzes a series of stepwise hydrolytic reactions, resulting in formation of the principle end products of starch digestion, the disaccharides maltose and isomaltose, and small amounts of glucose. glucose is absorbed directly by the intestinal mucosa and transported to the portal vein. the disaccharides are broken down further by hydrolytic enzymes of the brush border. b. cellulose. cellulose, like starch, is a polysaccharide of glucose but differs from starch in that the glucose molecules are linked by ß- , -glucosidic bonds. starch can be utilized by all species, but cellulose is utilized as a source of energy only by animals which have extensive bacterial fermentation within the gastrointestinal tract. ruminant species digest cellulose most efficiently, but other animals in which the large intestine is well developed also can utilized cellulose to some degree. in ruminants, hydrolysis of cellulose is accomplished by cellulytic bacteria, which are part of the complex rumen microflora. the end products of cellulose fermentation are short-chain fatty acids-acetic, propionic, and butyric acids. these are absorbed directly from the rumen and serve as the major source of energy for ruminants. propionic acid is the major precursor for synthesis of carbohydrate. maltose and isomaltose are the disaccharides (glucose-glucose) produced as end prod ucts of starch digestion. the diet also contains lactose (galactose-glucose) and sucrose (fructose-glucose). it once was believed that disaccharides were hydrolyzed within the ( ) malathi ( ) malathi ( ) eichholz ( ), forstner et al. ( ) eichholz ( ), forstner et al. ( ) lumen of the intestine by enzymes secreted by the mucosa. there is now general agree ment, however, that disaccharide digestion is completed at the surface of the cell by disaccharidases (gray, ) , which are components of the brush border (table iv) . this is considered a form of intracellular digestion (ugolev, ) . the disaccharidases have been solubilized from the brush border and partially purified. two separate maltases have been isolated (auricchio et al., ) . isomaltase and sucrase have been separated and purified together as a two-enzyme complex (kolinskâ and semenza, ) . the mucosa also contains two enzymes with lactase activity. one of these is a nonspecific / -galactosidase which hydrolyzes synthetic / -galactosides effec tively but which hydrolyzes lactose at a slow rate. this enzyme has an optimal ph of and is associated with the lysozomal fraction of the cell. the other lactase hydrolyzes lactose readily. it is associated with the brush border fraction of the cell and is the enzyme which is important in the digestive process (alpers, ) . maltase, isomaltase, and sucrase are almost completely absent from the intestine in newborn pigs dahlqvist, ) and calves (huber et al., ) . the activity of these disaccharidases increases after birth and reaches adult levels during the first months of life. lactase activity is highest at birth and decreases gradually during the neonatal period. the relatively high lactose activity seems to be an advantage to the newborn in utilizing the large quantities of lactose present in the diet. by water and demonstrated lactase deficiency following acute enteric infections and suggested that lactose utilization may be decreased in such cases. a. specificity of monosaccharide transport. regardless of whether monosaccharides originate in the lumen of the intestine or are formed at the surface of the mucosal cell, transport across the mucosa involves processes which have a high degree of chemical specificity. glucose and galactose are absorbed from the intestine more rapidly than other monosaccharides. fructose is absorbed at approximately one-half of the rate of glucose, and mannose is absorbed at less than one-tenth the rate of glucose (kohn et al., ) . glucose and galactose can be absorbed against concentration gradients and are said, by definition, to be actively transported. active absorption requires metabolic energy and can be inhibited by a variety of substances which block oxidative phosphorylation. the monosaccharides that are transported most efficiently against concentration gradients have certain common structural characteristics, which were summarized by wilson ( ) . these include ( ) the presence of a pyranose ring, ( ) a carbon atom attached to c- , and ( ) a hydroxyl group at c- with the same stereoconfiguration as d-glucose. these features once were believed to be necessary for active monosaccharide transport, but recent observations suggest that they are not absolute requirements. both d-xylose, which has no substituted carbon atom at c- , and d-mannose, which lacks the appropriate hydroxyl configuration at c- , can be transported against concentration gradients under proper experimental conditions (csâky and lassen, ; csâky and ho, ; alvarado, b) . most current concepts imply that, during the initial phase of monosaccharide absorption, the monosaccharide molecule attaches to a mobile carrier located within the cell membrane . the evidence for such membrane carriers comes from kinetic studies of the overall transport process. the rate of glucose absorption is independent of luminal concentration over a rather wide range, but a maximal rate of absorption can be demonstrated at very high concentrations. this limitation of transport is believed to be due to saturation of binding sites on the membrane carrier. glucose transport is competitively inhibited by galactose (cori, ; fisher and par sons, ) and by a variety of substituted hexoses, which compete with glucose for carrier binding sites. the glucoside phlorizin is a very potent inhibitor (parsons et al., ; alvarado and crane, ) . phlorizin also competes for binding sites but has a much higher affinity for these sites than does glucose. the absorptive surface of the mucosal cell is the microvillous membrane, or brush border (figs. a and b). it is through this part of the plasma membrane that glucose must pass during the initial phase of mucosal transport. techniques have been developed for isolating highly purified preparations of microvillous membranes from mucosal homogenates forstner et al., ) . faust et al. ( ) studied the binding of various sugars to these isolated membrane fractions. they found that d-glucose was bound by the membrane preferentially to l-glucose or to d-mannose and that glucose binding was completely inhibited by . mm phlorizin. the specificity of their observa tions suggested that binding represented an initial step in glucose transport, namely, attachment to a membrane carrier. c. sodium requirement. the absorption of glucose and other monosaccharides is influenced significantly by sodium ion (schultz and curran, ; kimmich, ) . when sodium is present in the solution bathing the intestinal mucosa, glucose is absorbed rapidly, but, when sodium is removed and replaced by equimolar amounts of other cations, glucose absorption virtually stops (riklis and quastel, ; csâky, ; bihler and crane, ; bihler et al., ) . glucose abosrption is inhibited by oubain, digitalis, and other cardiac glycosides which are also inhibitors of na-k-dependent adenosine triphosphatase activity and sodium transport (csâky and hara, ; schultz and zalusky, ) . these observations suggest a close relationship between the transport of glucose and sodium. on the basis of their own observations, crane and co-workers ( ) sug gested that sodium ion acts directly upon the membrane carrier to increase affinity of the carrier for glucose. csâky ( ) interprets the apparent coupling of sodium transport to the transport of various nonelectrolytes as being due to the need to maintain a critical intracellular sodium concentration, which, in turn, is essential for conversion of metabolic energy (atp, etc.) to energy for transport. the initial step in protein digestion is enzymatic hydrolysis of peptide bonds with formation of smaller peptides and amino acids. the endopeptidases (proteases) hydrolyze peptide bonds within the protein molecule and also hydrolyze certain model peptides. exopeptidases hydrolyze either the carboxy-terminal (carboxypeptidase) or the aminoterminal (aminopeptidase) amino acids of peptides and certain proteins. dietary proteins first come in contact with proteolytic enzymes in the stomach. the best known of the gastric proteases is the family of pepsins (samloff, ) , which attack most proteins with the exception of keratins, protamines, and mucins. pepsins are relatively nonspecific endopeptidases and split peptide bonds involving many amino acids. the most readily hydrolyzed peptide bonds are those of leucine, phenylalanine, tyrosine, and glutamic acid (ryle, ; ryle and hamilton, ; meyer and kelly, ) . the extent of proteolysis in the stomach depends on the nature of the dietary protein and the length of time spent in the stomach. the food bolus mixed with saliva has a neutral or slightly alkaline ph as it enters the stomach, and a certain period of time is necessary for it to mix with gastric secretions and become acidified. proteolytic digestion begins when the ph of the gastric contents approaches and occurs optimally in two ph ranges, . - . and . - . (taylor, a,b) . because of the relative lack of specificity of the pepsins, some peptide bonds of almost all dietary proteins are split during passage through the stomach. the gastric phase of protein digestion appears to have only a minor and probably dispensable role in overall protein assimilation (freeman and kim, ) . the reservoir function of the stomach, however, contributes to the gradual release of nutrients, insuring more efficient utilization in the small intestine. partially digested protein passes from the stomach to the duodenum, where the acidic contents are neutralized by sodium bicarbonate secreted in the bile and pancreatic juices. peptic activity persists in the duodenum only during the period required to raise the ph above . . the major peptidase activity in the lumen of the small intestine comes from the pancreatic enzymes trypsin, chymotrypsin, elastase, and carboxypeptidases a and b. the action of these enzymes is integrated so that the endopeptidases produce peptides with c-terminal amino acids which are appropriate substrates for the exopeptidases. trypsin produces peptides with basic c-terminal amino acids which are particularly suited for the action of carboxypeptidase b. chymotrypsin produces peptides with aromatic amino acids in the c-terminal position, and elastase produces peptides with c-terminal amino acids which are nonpolar. carboxypeptidase a hydrolyze both types of c-terminal peptide bonds (table ii) . the intestinal mucosa contains a broad range of aminopeptidases which complete the process of protein digestion (heizer and laster, ) . most of the aminopeptidase activity is found in the soluble fraction of the cell (newey and smyth, ), but a small fraction is tightly bound to the microvillous membrane and appears to serve a digestive function at the cell surface similar to that described for the disaccaridases (rhodes et al., ). an endopeptidase from the intestinal mucosa was studied by hsu and tappel ( ) using hemoglobin as substrate. over % of the activity was located in the particulate fraction of the cell. the association with other acid hydrolases suggests that this is a lysozomal enzyme, and its relationship to the normal process of protein digestion is not known. despite the long interest in and controversy regarding the subject of this section, the relative amounts of the various types of protein digestion products, i.e., peptides and amino acids, which are actually absorbed by intestinal mucosal cells during normal digestion are still not known. it is a difficult process to investigate from a kinetic standpoint because the products of proteolysis are absorbed rapidly after they are formed. studies of luminal contents, therefore, give only an estimate of the overall rate of protein digestion. in addition, dietary protein is continually mixed with endogenous protein in the form of digestive secretions and extruded mucosal cells. endogenous protein is hydrolyzed and the amino acids absorbed in a manner similar to that of dietary protein, and the two processes occur simultaneously. endogenous protein accounts for a significant part of the amino acids of the intestinal contents (nässet and ju, ) . even when the dietary protein is labeled with a radioactive tracer, there is such rapid utilization that the tracer soon reenters the lumen in the form of endogenous protein secretion. in adult mammals, protein is not absorbed from the intestine in quantities of nutritional significance without previous hydrolysis. most neonatal animals absorb significant amounts of immunoglubin and other colostral protein, but this capacity is lost soon after birth (see section iii,a, below). the intestinal mucosa is not totally impermeable to large polypeptide molecules, however. the absorption of insulin (mw ) (laskowski et al., ; danforth and moore, ) , ribonuclease (mw , ) (alpers and isselbacher, ), territin (bockman and winborn, ) , and horseradish peroxidase (cor nell et al., ) has been demonstrated. the intestine produces a part of the plasma / -globulin. this is believed to be the result of de novo synthesis of protein, however, presumably from individual amino acid precursors. during the digestion of protein, the amino acid content of the portal blood increases rapidly. attempts to demonstrate parallel increases in the level of peptides in the portal blood have not been successful (levenson et al., ) . this has sometimes been taken as evidence that only amino acids can be absorbed by the intestinal mucosa and that the absorption of peptides does not occur. while it seems clear that a significant part of the dietary protein is absorbed in the form of free amino acids, peptides also may be taken up by the mucosal cell. evidence of the mucosal uptake of peptides came originally from experiments with isolated loops of intestine (wiggans and johnston, ; newey and smyth, ) . various peptides were placed in solutions bathing the mucosa and analyses made sub sequently of the serosal fluid. with the exception of small amounts of glycylglycine, peptides were never found on the serosal side, but free amino acids were found in significant quantities. the final steps to peptide digestion appear to be associated with mucosal epithelial cells. almost all of the aminopeptidase activity is associated with the mucosa, and very little activity is present in luminal contents (lindberg, ) . as described above, mucosal aminopeptidase activity is located in the cytosol and in the brush border mem brane fractions of the epithelial cell (heizer and laster, ; kim et al., ) . these physically separate enzymes have remarkably different substrate specificities (kim et al., ) . the brush border enzyme has more than % of the activity for tripeptides, yet less than % of the total activity for dipeptides relative to the cytosolic enzyme(s) (peters, ; kim et al., ) . almost all activity for tetrapeptides is present in the brush border (freeman and kim, ) . proline-containing peptides are hydrolyzed almost exclusively by cytosolic peptidases, whereas leucine aminopeptidase activity is located primarily in the brush border. from these studies, it appears that, in the intact animal, peptides are absorbed in physiologically important quantities by intestinal mucosal cells and hydrolyzed either at the cell surface or intracellularly to constituent amino acids. the individual amino acids then are transported to the apical part of the cell and finally enter the portal circulation. amino acids, like glucose and certain other monosaccharides, are absorbed and trans ferred to the portal circulation by active transport processes. the same type of saturation kinetics observed in studies of monosaccharide absorption are observed with amino acids, suggesting carrier transport mechanisms. certain monosaccharides inhibit amino acid transport (saunders and isselbacher, ; newey and smyth, ) . inhibition generally has been of the noncompetitive type, but alvarado ( a) demonstrated competitive inhibition between galactose and cycloleucine, suggesting that some form of common carrier may be involved. most amino acids are transported against concentration and electrochemical gradients, and the overall transport process requires metabolic energy. the chemical specificity of these transport mechanisms is demonstrated by the observation that the natural / forms of various amino acids are absorbed more rapidly than the corresponding d forms, and only the /-amino acids appear to be actively transported. sodium ion is necessary for absorp tion of amino acids as it is for a variety of other nonelectrolyte substances (schultz and curran, ; gray and cooper, ) . separate transport systems appear to exist for different groups of amino acids. each member of a group inhibits the transport of other members competitively, suggesting that they share the same binding site. there is some overlap between groups, indicating that, in the overall transport process, certain steps may be common to all amino acids and other steps more specific (saunders and isselbacher, ; matthews and laster, ; wise man, ) . these groups are the following: . monoaminomonocarboxylic (neutral) amino acids, including histidine. these amino acids show mutual competition for transport and have the greatest requirement for na + . . monoaminodicarboxylic amino acids. aspartic and glutamin acids are not trans ported against concentration gradients. following uptake, they are transaminated, and, under physiological conditions, almost all of the aspartic and glutamic acid enters the portal blood as alanine. . dibasic amino acids, including lysine, arginine, ornithine, and the neutral amino acid cystine. these amino acids are apparently transported by the same transport system. . proline, hydroxyproline, the n-substituted glycine derivatives n-methylglycine (sarcosine), and n-dimethylglycine, and betaine. proline and hydroxyproline also can be transported by the first mechanism but the affinity of both amino acids for the nadependent pathway is low. the γ-glutamyl cycle has been proposed as a possible transport system for amino acids (meister and tate, ) . γ-glutamyltransferase (ggt) is a membrane-bound en zyme which is present in a number of mammalian tissues and catalyzes the initial step in glutathione degradation. the γ-glutamyl moiety of glutathione is transferred to amino acid (or peptide) receptors with the production of cysteinylglycine: glutathione + amino acid ^τ γ-glutamyl-amino acid + cys-gly the highest ggt activity is present in tissues which are known to transport amino acids actively, e.g., the jejunal villus and the proximal convoluted tubule of the kidney. meister and his colleagues ( ) have suggested that ggt may function in translocation by interaction with extracellular amino acids and with intracellular glutathione. the hypothet ical mechanism involves the noncovalent binding of extracellular amino acids to the plasma membrane, while intracellular glutathione interacts with ggt to yield a γ-glutamyl enzyme. when the γ-glutamyl moiety is transferred to the membrane-bound amino acid, a γ-glutamyl-amino acid complex is formed and, when released from the membrane binding site, moves into the cell. the γ-glutamyl-amino acid complex is split by the action of γ-glutamylcyclotransferase, an enzyme appropriately located in the cytosol. glutathione is regenerated by means of the γ-glutamyl cycle, which are good substrates for ggt (thompson and meister, ) . the γ-glutamyl cycle does not require sodium, and the previously demonstrated sodium dependence for amino acid transport would not be explained by the cycle. the cycle is not considered to be the only amino acid transport system, and its quantitative significance in individual tissues is unknown. certain nutrient cell types which are deficient in ggt have been shown to transport amino acids normally. at birth most domestic species, including the calf, foal, lamb, pig, kitten, pup, and infant, absorb significant quantities of colostral protein from the small intestine (brambell, ; walker and isselbacher, ) . γ-globulin either is absent in the serum of these species at birth, or is at a low level. within a few hours after ingestion of colostrum, the serum γ-globulin level rises. this is the principal mechanism by which the young of the above-listed species acquire maternal immunity. under normal environmental con ditions, ingestion of colostrum is an absolute requirement for the health of these species during the neonatal period (fig. ) . in the neonatal calf, immunoglobulin deficiency has a role in the pathogenesis of gram-negative bacterial septicemia (smith, ; gay, ; roberts et al, ) . most calves deprived of colostrum develop septicemia early in life but may develop diarrhea before death (smith, ; roberts et al., ; wood, ; tennant et al, ) . hypogammaglobulinemia is almost always demonstrable in calves dying of gramnegative bacterial septicemia (fey, ) , and hypogammaglobulinemia is believed to be due to insufficient immunoglobulin intake or to insufficient intestinal absorption. the factor in colostrum that protects against systemic infections is the igm fraction (penhaie et al, ) . serum immunoglobulin values of neonatal calves vary, and a % incidence of hypogammaglobulinemia may occur in clinically normal calves (tennant et al, a; house and baker, ; smith et al, ; thornton et al, ; braun et al, ) . most hypogammaglobulinemic individuals probably had insufficient colostrum intake. even when calves were given the opportunity to ingest colostrum, a surprising number were hypogammaglobulinemic. some of the reasons for varying gammaglobulinemia values are recognized, but the relative importance of each reason is not known. the concentration of lactoglobulin, the volume consumed (bush et al, ; selman et al, ) , the time elapsed from birth to ingestion of colostrum , and the method of ingestion (natural suckling versus bucket feeding) may have an important influence on the serum γ-globulin (smith et al, ; mcbeath et al, ) . calves that suckle their dams usually attain serum γ-globulin concentrations that are higher than those attained by calves given colostrum from a bucket. the frequency of hypogamma globulinemia may be influenced by seasons (gay et al., b; mcewan et al., a) , although this relationship has not always been observed (smith et al., ; thornton et al., ) . familial factors also influence hypogammaglobulinemia (tennant et al., a) . regardless of cause, the mortality of hypogammaglobulinemic calves is higher than that of calves with normal serum γ-globulin values (gay, ; house and baker, ; thornton et al., ; mcewan et al., a; boyd, ; naylor et al., ) . in addition to having more septicémie infections (smith, ; gay, a; roberts et al., ; wood, ; fey, ; mcewan et al., a) , hypogammaglobulinemic calves have a greater prevalence of acute diarrheal disease (boyd, ; naylor et al., ; penhale et al., ; gay et al., ) ; the local protective effects of immunoglobulin in the intestine apparently are important (fisher et al., ; . the prevalence of hypogammaglobulinemia and the high mortality associated with it has led to the development of several rapid tests for identification of hypogamma globulinemic calves (mcbeath et al., ; aschaffenburg, ; fisher and mcewan, a; patterson, ; stone and gitter, ) . the zinc sulfate turbidity test (kunkel, ) was the first to be used for determination of serum immunoglobulin concentrations of neonatal calves (mcewan et al., ) . a close correlation has been established between test results and the amount of serum igg and igm (fisher and mcewan, a,b; mcewan et al., b; penhale et al., ) . the sodium sulfite turbidity test is similar to the zinc sulfate test and also has been used to identify hypogammaglobulinemic calves (stone et al., ; pfeiffer and mcguire, ) . failure of turbidity to develop when serum is added to a saturated solution of sodium sulfite indicates immunoglobulin deficiency, and semiquantitative assessment of the immunoglobulin concentration may be made by grading the degree of turbidity (stone and gitter, ) . the refractometer is used as a rapid test for immunoglobulin deficiency (mcbeath et al., ; boyd, ) . the close relationship between the concentration of γ-globulin and that of total serum protein in neonatal calves was described previously (tennant et al., a) , and the wide variation in total protein concentration was due to differences in γ-globulin concentration. direct linear correlation between the serum protein concentra tion (refractive index) and the immunoglobulin concentration also has been described (mcbeath et al., ) . the equation for the regression line in that report was virtually identical to that observed recently (tennant, et al., ) . the y intercepts in our study and in that previously reported were identical ( gm/dl). the refractometer has value as a rapid field instrument for the assessment of immunoglobulin status, but in cases of hemoconcentration it has limitations (boyd, ) . the glutaraldehyde coagulation test was used originally for the detection of hypergammaglobulinemia in cattle, using whole blood (sandholm, ) . glutaraldehyde reagent also has been used in a semiquantitative test to evaluate γ-globulin in canine (sandholm and kivisto, ) and human serum (sandholm, ) . we modified this procedure to detect hypogammaglobulinemic calves ( table v) . calves that had a negative test result (serum γ-globulin ^ . gm/dl) had markedly higher mortality than did calves with posi tive results (table vi) (tennant, et al., ) , findings similar to those obtained by using the zinc sulfate turbidity test (gay et al., a; mcewan et al., a) . many tests can be initiated at one time using the glutaraldehyde coagulation test, and all results can be evaluated rapidly without instrumentation (tables v and vi) . protein enters the absorptive cell by pinocytosis and passes across the cell to the lymphatics. the process is not selective because many proteins other than the immune globulins can be absorbed (payne and marsh, a,b) . the ability to absorb intact protein is lost by domestic species within or days following birth. in rodents, protein absorption normally continues for approximately weeks. the mechanism of intestinal "closure" was studied by lecce and co-workers ( ; lecce, ; lecce and morgan, a samples of serum were obtained at birth, but no follow-up of calves was made. the death rate of calves that were test negative was significantly (p < . ) greater than that of testpositive calves, using t test for significance of differences between two percentages. ). they found that complete starvation of pigs lengthened the period of protein absorption to - days, whereas early feeding shortened the period. feeding different fractions of colostrum including lactose and galactose resulted in loss of protein absorptive capacity. the route of feeding may not be the critical factor, however. calves prevented from eating but which receive nutrients parenterally lose the ability to absorb protein at the same time as control calves (deutsch and smith, ). a. luminal phase. the fat present in the diet is primarily in the form of triglycérides of long-chain fatty acids. the initial step in utilization of triglycérides occurs in the lumen of the proximal small intestine, where hydrolysis is catalyzed by pancreatic lipase. this enzyme, which is secreted in active form, requires an oil-water interface for activity so that only emulsions are attacked (sarda and desnuelle, ) . enzyme activity is directly related to the surface area of the emulsion. the smaller the emulsion particle, the greater the total surface area of a given quantity of triglycéride and the greater the rate of hydrolysis (benzonana and desnuelle, ) . bile salts are not an absolute requirement, but they favor hydrolysis ( ) by their detergent action, which causes formation of emul sions with small particle sizes, and ( ) by stimulating lipase activity within the physiologi cal ph range of the duodenum (borgström, (borgström, , a . a colipase is present in the pancreatic secretion which facilitates the interaction of lipase with its triglycéride substrate and protects lipase from inactivation (borgström and erlanson, ) . pancreatic lipase splits the ester bonds of triglycérides preferentially at the and positions (sari et al., ) , so that the major end products of hydrolysis are -monoglycerides and nonesterified fatty acids (mattson et al., ; volpenhein, , ) . both compounds are relatively insoluble in water but are brought rapidly into micellar solution by the detergent action of bile salts. the mixed micelles so formed have a diameter of approximately . nm (borgström, b; laurent and persson, ) and are believed to be the form in which the products of fat digestion are actually taken up by the mucosal cell (hofmann and small, ) . the intraluminal events which occur in fat absorption are schematically summarized in fig. . b. mucosal phase. the initial step in fat transport is the uptake of fatty acids and monoglycerides by the mucosal cell from micellar solution. just how this occurs is not completely clear, but present evidence suggests that the lipid contents of the micelle are somehow discharged at the cell surface so that they enter the cell in molecular rather than micellar form . the net effect is the absorption of the end products of lipolysis with the exclusion of bile salts, which are absorbed farther down the intestine, primarily in the ileum (lack and weiner, ) . uptake of fatty acids appears to be a passive process having no requirement for metabolic energy (johnston and borgström, ; strauss, ) . within the mucosal cell, the fatty acids are transported by a soluble binding protein to the endoplasmic reticulum, where the fatty acids and monoglycerides are rapidly reesterified to triglycéride (ockner and manning, ; ockner and isselbacher, ) . the two biochemical pathways for triglycéride biosynthesis in the intestine are summarized in fig. . direct acylation of monoglyceride occurs in the intestine (senior and isselbacher, ) and probably is the major pathway for lipogenesis in the intestine during normal fat absorption (kern and borgström, ; mattson and volpenhein, ) . the initial step in this series of reactions involves activation of fatty acids by acyl-coa synthetase, a reaction which requires mg + , atp, and coa (dawson and isselbacher, ; clark and hübscher, , ; brindley and hübscher, ) and which has a marked specificity for long-chain fatty acids (dawson and isselbacher, ; brindley and hübscher, ) . this specificity appears to explain the observation by bloom et al. ( ) that mediumand short-chain fatty acids are not incorporated into triglycérides during intestinal trans port but enter the portal circulation as nonesterified fatty acids. the activated fatty acids (from isselbacher, .) then react sequentially with mono-and diglycerides to form triglycérides in steps catalyzed by mono-and diglyceride transacylases (ailhaud et al., ) . the enzymes responsible for this series of reactions were partially purified by rao and johnston ( ) from the microsomal fraction of the cell. they observed that purification of the separate enzyme activities occurred simultaneously, suggesting that these enzymes occur together in the endoplasmic reticulum as a "triglyceride-synthetase" complex. an alternate route which is available for fatty acid esterification involves la-glycerophosphate, which may be derived from glucose or from dietary glycerol by the action of intestinal glycerokinase (haessler and isselbacher, ; clark and hübscher, ) . activated fatty acid coa derivatives react with l-a-glycerophosphate to form lysophosphatidic acid (monoglyceride phosphate), which by a second acylation forms phosphatidic acid (diglyceride phosphate). phosphatidic acid phosphatase then hydrolyzes the phosphate ester bond, forming diglyceride, and by means of a transacylase step similar to that described in the previous paragraph, triglycéride can then be formed. although this pathway appears to be one of minor importance for triglycéride synthesis in the intestine, johnston ( ) pointed out the importance of certain of the intermediates in this sequence of reactions in the synthesis of phospholipids which are necessary for stabilization of the chylomicron. the next step in fat transport is formation of chylomicrons within the endoplasmic reticulum. the chylomicron is composed primarily of triglycéride and has an outer mem branous coating of cholesterol, phospholipid, and protein (zilversmit, ) . the js-lipoprotein component of the chylomicron is synthesized by the intestinal mucosal cell (isselbacher and budz, ; hatch et al., ; windmueller and levy, ) . inhibi tion of protein synthesis by puromycin or acetoxycycloheximide interferes with chylomic ron formation and significantly reduces fat transport (sabesin and isselbacher, ) . the final step in fat absorption is extrusion of the chylomicron into the intercellular space opposite the basal lateral portion of the absorptive cell. this is accomplished by a process which is essentially the reverse of pinocytosis (palay and karlin, ) . from the intercellular space the chylomicron passes through the basement membrane and enters the lacteals through small pores. the chylomicron passes from the lacteal into lymph ducts and ultimately reaches the general circulation, having bypassed the liver completely during the initial phase of absorption. a. cholesterol. dietary cholesterol is present in both free and esterified forms, but only nonesterified cholesterol is absorbed (vahouny and treadwell, ) . cholesterol esters are hydrolyzed within the lumen of the intestine by sterol esterase secreted by the pancreas. bile salts are required both for the action of this enzyme (vahouny et al., ) and for the absorption of nonesterified cholesterol. in the mucosal cell, cholesterol is reesterified and transferred by way of the lymph to the general circulation. the type of triglycéride present in the diet significantly affects the absorption of cholesterol and its distribution in lymph lipids . b. vitamin a. the diet contains vitamin a activity in two principal forms: ( ) as esters of preformed vitamin a alcohol (retinol) and fatty acids and ( ) as provitamin a, primarily in the form of jÖ-carotene. vitamin a ester is hydrolyzed by a pancreatic esterase within the lumen (murthy and ganguly, ) , and the free alcohol is absorbed in the upper small intestine by a process which apparently requires metabolic energy (skala and hrubâ, ) . vitamin a alcohol is reesterified in the mucosa utilizing primarily palmitic acid (mahadevan et al., ) . the vitamin a ester is absorbed by way of the lymph. after reaching the general circulation, it is rapidly cleared from the plasma and stored in the liver. in the postabsorptive state, vitamin a circulates as the free alcohol. this is also the form released from the liver as needed by the action of a specific hepatic retinylpalmitase esterase (mahadevan et al., ) . the blood level of vitamin a is independent of the liver reserve, and, as long as a small amount of vitamin a is present in the liver, the blood level remains normal (dowling and wald, ) . in diets which lack animal fat, the carotenes, mainly / -carotene, serve as the major vitamin a precursors. the intestinal mucosa plays the primary role in conversion of provitamin a to the active vitamin, although conversion can occur to a limited degree in other tissues (bieri and pollard, ; zachman and olson, ) . the exact mechanism involved in the conversion of / -carotene to vitamin a is not completely established, but studies by olson ( ) suggest that there is central cleavage of/ -carotene into two active vitamin a alcohol molecules, which are subsequently esterified and transported by the lymphatics as with the preformed vitamin. bile salts are required for the mucosal uptake of / -carotene and for the conversion of ß-carotene to vitamin a. uptake of carotene and release of vitamin a ester into the lymph appear to be rate-limiting steps. cattle also absorb substantial amounts of carotene without prior conversion to vitamin a, and these pigments are responsible for much of the yellow color of the plasma. most other species have no carotene in the plasma, and it has been suggested that extraintestinal conversion may be more efficient in these species than in cattle . c. vitamin d. vitamin d, like cholesterol, is a sterol which is absorbed from the intestine by way of the lymph (schachter et al., ) . intestinal absorption differs, however, in that vitamin d is transported to the lymph in nonesterified form (bell, ) . the uptake of vitamin d by the mucosal cell is favored by the presence of bile salts. simultaneous absorption of fat from micellar solutions increases transport out of the cell into the lymph, a step which appears to be rate limiting (thompson et al., ) . one of the major actions of vitamin d is to enhance the intestinal absorption of calcium ion. the mechanism of action of vitamin d has been described by wasserman and co-workers ( ; wasserman and taylor, , ) . they have shown that vitamin d causes synthesis of a calcium-binding protein present in the soluble fraction of the intesti nal mucosal cell. they have accumulated a substantial amount of evidence which suggests that this protein plays a central role in the active transport of calcium. vomiting is a coordinated reflex act which results in rapid, forceful expulsion of gastric contents through the mouth. the reflex may be initiated by ( ) local gastric irritation caused by a variety of toxic irritants or infectious agents, ( ) foreign bodies, ( ) gastric tumors, ( ) obstruction of the pyloric canal or of the small intestine, or ( ) drugs, such as apomorphine, or other toxic substances which act centrally on the "vomiting center" located in the medulla. severe vomiting produces loss of large quantities of water and of h + and cl~ ions. these losses cause dehydration, metabolic alkalosis with elevated plasma bicarbonate concentration, and hypochloremia. chronic vomiting may also be associated with loss of tissue k + and hypokalemia. the k + deficit is caused primarily by increased urinary excretion, which is the result of the existing alkalosis (leaf and santos, ) . gastric secretions contain significant quantities of k + (section ii,b), and losses in the vomitus also contribute to the k + deficiency. potassium deficiency, which develops initially because of alkalosis, ultimately may perpetuate the alkalotic state by interfering with the ability of the kidney to conserve h + (koch et al., ; darrow, ) . both potassium deficiency and the hypovolemia caused by dehydration may result in renal tubular damage and ultimately in renal failure (haden and orr, , ) . vomiting occurs frequently in the dog, cat, and pig but is an unusual sign in the horse, which has anatomical restrictions of the esophagus that interfere with expulsion of gastric contents. in cattle, sheep, and goats, the physiological process of rumination utilizes neuromuscular mechanisms similar to those involved in vomiting. uncontrolled expulsion of ruminai contents is, however, an uncommon sign, occurring most frequently after ingestion of toxic materials. the contents of the abomasum are not expelled directly even when the pyloric canal is obstructed. a syndrome does occur in cattle with pyloric obstruction, however, which is similar metabolically to that observed in nonruminants. the syndrome has been observed in right-sided displacement of the abomasum with torsion (espersen, ; boucher and abt, ) . we have also observed the syndrome in cows with functional pyloric obstruction, the result of reticuloperitonitis (a variety of "vagai indigestion"). when the pylorus is obstructed, abomasal contents are retained, causing distention of the abomasum, which in turn stimulates further secretion and reten tion. retained abomasal contents may be regurgitated into the large reservoir of the rumen and there are sequestered from other fluid compartments of the body. the net result is loss of h + and cl" ions and development of metabolic alkalosis, hypochloremia, and hypokalernia (espersen and simesen, ; svendsen, ) . chronic hypertrophie gastritis has been demonstrated in the dog (van der gagg et al., ; happe and van der gagg, ; kipnis, ) which resembles menetrier's disease in man. van kruiningen's series of cases were basenjis which had concomitant lymphocytic-plasmocytic enteritis. three unpublished cases were studied at the new york state college of veterinary medicine. signs of illness usually involved chronic vomiting, weight loss, and occasionally diarrhea. hypoalbuminemia was documented in most of these cases. in man, hyperchlorhydria or achlorhydria can occur. the morphological changes in the stomach wall (hypertrophie rugae) as well as some of the clinical features help to differentiate this disease from gastric neoplasia and canine zollinger-ellison syndrome. canine zollinger-ellison syndrome was reported in four dogs (straus et al., ; van der gagg et al., ) . vomiting, diarrhea, inappetance, and weight loss were reported. all of the dogs had pancreatic non-js islet cell tumors, resulting in hypergastrinemia, hyperchlorhydria, hypertrophie gastritis, peptic esophagitis, and duodenal ulcers. the term "diarrhea" is used loosely to describe the passage of abnormally fluid feces with increased frequency and/or with increased volume. the significance of diarrhea depends primarily on the underlying cause and on the secondary nutritional and metabolic disturbances which are caused by excessive fecal losses. there are theoretically three factors which could act independently or in combination to produce diarrhea: ( ) increased rate of intestinal transit, ( ) decreased intestinal absorptive capacity, and ( ) increased secretion into the intestinal lumen. an increase in the rate of intestinal transit has been considered to be important in various functional disorders of the gastrointestinal tract in which "hypermotility" has been considered the primary cause. although increased intestinal motility may be a factor in certain types of diarrheal disease when the direction of motility has been investigated, diarrhea has actually been associated with decreased motility (christiansen, ) . decreased intestinal assimilation of nutrients may result from either ( ) decreased intraluminal hydrolysis of nutrients, e.g., maldigestion (kaiser, ) , due to pancreatic exocrine insufficiency or to bile salt deficiency or ( ) defective mucosal transport of nutrients, malabsorption, which may be the result of various types of inflammatory bowel disease, intestinal lymphoma, or intrinsic biochemical defects in the mucosal cell which interfere with normal digestion and absorption. the role of increased intestinal secretion in the pathogenesis of certain types of acute diarrhea is now recognized. enteropathogenic strains of escherichia coli have been shown to produce soluble enterotoxins (smith and halls, ; köhler, ; moon, ) , which alter bidirec tional sodium and water flux (fig. ) . rapid advances in understanding the pathogenesis of enterotoxin-induced diarrhea and the molecular basis of enterotoxin action have been made. the most extensively studied enterotoxin is that produced by vibrio cholerae. this bacterium produces a large molecular weight heat-labile toxin (ct), one subunit of which has properties similar to those of heat-labile (lt) enterotoxin produced by certain strains of e. coli (richards and douglas, ) . the mechanism of action of ct is believed to moon, .) involve the activation of adenylate cyclase. this membrane-bound enzyme converts atp to cyclic ', '-adenosine monophosphate (camp), which is then responsible for the greatly increased secretion of water and electrolytes by the intestinal mucosa (moon, ) . although species differences have been observed (hamilton et al., a,b; forsyth et al., ) , this mechanism appears to be important in the mode of action of e. coli lt as well (richards and douglas, ) . additional extensive studies have centered on the molecular mechanism of action of ct. under physiological conditions, adenylate cyclase is activated by the binding of guanosine triphosphate to the inactive enzyme. an associated gtpase inactivates the enzyme by converting enzyme-bound gtp to gdp and inorganic phosphate. this gtp-gdp system apparently plays a critical role in the regulation of adenylate cyclase. cholera toxin is believed to bind to the adenyl cyclase in a way which inhibits hydrolysis of gtp, thereby maintaining the enzyme in an activated state (levinson and blume, ; johnson et al., ; cassel and pfeuffer, ) (fig. ) . certain enteropathogenic strains of e. coli produce a low molecular weight heat-stable toxin (st) alone or in addition to lt (richards and douglas, ; moon, ; hamil ton et al., a) . in most epidemiological studies of neonatal diarrheal diseases of calves, isolated strains of e. coli produce only st (moon et al., ; braaten and myer, ; lari vier et al., ) . in contrast to lt and ct, which induce intestinal sodium and . proposed mechanism of action of cholera toxin, which inhibits hydrolysis of gtp, thereby increas ing adenylate cyclase activity. (after cassel and selinger, .) water secretion only after a lag phase of several hours, st increases intestinal secretion at once. recent evidence suggests that st induces intestinal secretion by activating guanylate cyclase and that the mediator of intestinal secretion induced by st is cyclic ', 'guanosine monophosphate field et al., ) . such advances in our fundamental knowledge of the pathogenesis of enterotoxininduced diarrheal disease have opened several avenues of investigation which may lead to pharmacological modification of intestinal secretion as a mode of therapy or prophylaxis. enterotoxin-induced intestinal secretion has been shown to be effectively blocked by cycloheximide, inhibitor of protein synthesis (serebro et al., ) . the lack of speci ficity and the toxicity of cycloheximide precluded its clinical use, but acetazolamide has been shown to inhibit intestinal fluid secretion (norris et al., ; moore et al., ) , and ethacrynic acid, another potent diuretic, has been shown to inhibit enterotoxininduced fluid secretion (carpenter et al., ) . unfortunately, the diuretic effects of these drugs preclude their clinical use, but an "intestinal-specific" derivative would have significant therapeutic potential. adenosine analogues also have been shown in prelimi nary studies to inhibit cholera toxin-stimulated intestinal adenylate cyclase, but their potential as prophylactic or therapeutic agents is not known. prostaglandin e t and ct have a similar effect on electrolyte transport in rabbit ileum. application of either to the mucosa inhibits sodium absorption and stimulates chloride secretion. one possible explanation for the effects of ct is that it stimulated release of prostaglandin, which then acted on adenylate cyclase, producing camp. to test this hypothesis, the effects of inhibitors of prostaglandin release on enterotoxin-stimulated intestinal secretion were investigated. both indomethacin (gots et al., ) and acetylsalicylic acid (farris et al., ) were shown to be potent inhibitors of enterotoxininduced intestinal secretion using laboratory animal models. current evidence does not support the hypothesis that prostaglandins play a primary role in the pathogenesis of cholera or other enterotoxin-induced diarrheal diseases (schwartz et al., ) , but the effects of these known prostaglandin inhibitors and other drugs on the intestinal secretory process warrant their evaluation as possible prophylactic and therapeutic agents. in pre liminary studies, jones and his colleagues demonstrated a positive therapeutic response to a new prostaglandin inhibitor (jones et al., ) . the autonomie nervous system has important effects on intestinal ion transport and water absorption (tapper et al., ) . catecholamines stimulate formation of camp in a variety of mammalian cells (sutherland and rail, ; schultz et al., ) , apparently by activating the gtp-gdp system described above (cassel and selinger, ; ciment and devellis, ) . adrenergic blocking agents, such as chlorpromazine (holmgren et al., ) and propranolol (donowitz et al., ) , have been shown to have significant inhibitory effects on enterotoxin-induced intestinal secretion. although the mechanism of action of these two adrenergic blockers is not known, they represent still another class of drugs which may be of therapeutic benefit. the intestinal "adsorbent" drug pepto bismol, a patented medication containing bis muth subsalicylate, and attapulgite, a heat-treated silicate, have been shown to have antienterotoxic effects (drucker et al., ; ericsson et al., ; gyles and zigler, ) . controlled therapeutic trials with bismuth subsalicylate have demonstrated signifi cant therapeutic benefit in certain large-volume diarrheal diseases in man suspected of being enterotoxigenic in origin (portnoy et al., ; dupont et al., ; dupont, ) . the mechanism of action in inhibiting intestinal secretion has not been determined, but the chemical relation of bismuth subsalicylate to other known prostaglandin inhibitors is recognized. it is possible that such drugs, by decreasing endogenous production of prostaglandin, decrease the basal level of cyclic nucleotides, which in turn causes an increase in the threshold of response to enterotoxin. recent evidence suggests that salicylates also may stimulate sodium chloride absorption (powell et al., ) . these observa tions taken collectively suggest that new, innovative methods for therapy and control of acute clinical diarrheal disease may be developed in the not too distant future. acute diarrhea represents the leading cause of morbidity and mortality in neonatal calves and pigs. the pathogenesis of the neonatal enteric infection is complex, often involving nutritional or environmental factors as well as infectious agents, such as enteropathogenic strains of e. coli, the transmissible gastroenteritis virus (tge), rota viruses, and other bacterial and viral pathogens. the severe clinical signs and frequently fatal outcome of acute diarrheal disease are often directly related to dehydration and to associated hydrogen ion and electrolyte disturbances (dalton et al., ; fisher and mcewan, b; tennant et al, tennant et al, , . in acute diarrhea with watery stools of large volume, the fecal fluid originates primarily in the small intestine. the electrolyte composition of the stool in such cases is similar to that of the fluid found normally in the lumen of the small intestine, which in turn is similar to that of an ultrafiltrate of the plasma. the rapid dehydration which accompanies acute enteritis in the newborn soon produces hemoconcentration and ultimately hypovolemic shock. such cases are characterized by metabolic acidosis (dalton et al., ; phillips and knox, ) caused by decreased excretion of h + due to renal failure and by in creased production of organic acids, the result of decreased tissue oxygénation, which leads to excessive anaerobic glycolysis. hyperkalemia also is observed characteristically in young, severely dehydrated animals. hyperkalemia in such cases is the result of increased movement of cellular potassium into the extracellular fluid and to decreased renal excretion. cardiac irregularities caused by hyperkalemia can be demonstrated with the electrocardiogram, and cardiac arrest related to hyperkalemia is believed to be a direct cause of death in calves with acute diarrhea fisher and mcewan, b) . marked hypoglycemia also has been observed occasionally prior to death in calves with acute enteric infections. hypoglycemia is believed to be due to decreased gluconeogenesis and increased anaerobic glycolysis, the result of hypovolemic shock (tennant et al., ) . the sequence of metabolic changes which occur during acute neonatal diarrhea is summarized in fig. . in chronic forms of diarrheal disease, excessive fecal losses of electrolyte and fluid may be compensated in part by renal conservation mechanisms and by oral ingestion. if water is consumed without adequate ingestion of electrolytes, hyponatremia and hypokalemia may develop (tasker, ; patterson et al., ) . in such cases, the osmolarity of the plasma is significantly decreased and hypotonie dehydration occurs. in longer-standing cases of chronic diarrhea, the plasma k + concentration may become dangerously low. it is imperative, in this situation, that intravenous fluids contain sufficient k + to prevent further reduction in plasma concentration. if they do not, additional cardiac irregularities or cardiac arrest may result. decreased assimilation of nutrients may occur either as a result of defective intraluminal digestion (maldigestion) (kaiser, ) , or because of defects in mucosal transport (jeffries, et al., ; floch, ; wilson and dietchy, (from tennant et al., .) or the malabsorption syndrome is observed in several types of intestinal disease, including chronic intestinal granulomatous diseases such as johne's disease, intestinal parasitic infections, and lymphoma of the intestine. primary clinical signs include persistent or recurrent diarrhea, nutrient loss in the feces (e.g., steatorrhea), and weight loss. mucosal cell-enzymatic defects may be accompanied by chronic inflammation, villous atrophy, or cellular infiltrations of the lamina propria of the intestine. early reports of primary or idiopathic intestinal malabsorption in dogs (miller, ; vernon, ; kaneko et al., ) were compared to nontropical sprue (adult celiac disease, gluten induced enteropathy) of man, but no convincing association to with gluten sensitivity was demonstrated. subsequent reports of malabsorption syndromes in the dog have described a variety of causes (van kruiningen, ; ewing, ; van kruiningen andhayden, ; hill, ; hill and kelly, ; schall, ; anderson, anderson, , burrows et al., ) , which must be distinguished from the maldigestion caused by pancreatic insufficiency (anderson and low, a,b) (juvenile pancreatic atrophy, chronic pancreatitis) and from certain forms of hepatic or gastric disease. intestinal malab sorption can occur with protozoal enteritis (giardiasis, coccidiosis), lactase deficiency, eosinophilic gastroenteritis, lymphangiectasis, villus atrophy, lymphocytic-plasmacytic enteritis, histoplasmosis, chronic "bacterial" enteritis, malignant lymphoma, and intesti nal amyloidosis of the bowel. some authors (anderson, ; hay den and van kruiningen, ; arrick and kleine, ) described malabsorption and pseudoobstruc tion secondary to hypoplasia of the tunica muscularis of the jejunum in a dog. intestinal malabsorption is reported less frequently in the cat than in the dog (theran and carpenter, ; wilkinson, ) .malabsorption syndromes similar to those recog nized in dogs are being recognized with increased frequency in farm animals (blood et al., ) . meuten et al. ( ) , cimprich ( ), and merritt et al., ( ) have reported malabsorption in the horse secondary to chronic granulomatous enteritis and specific amino acid malabsorption has been reported in johne's disease (patterson and berret, ) . steatorrhea, the presence of excessive amounts of fat in the feces, is a prominent sign of intestinal malabsorption in dogs. the stools are bulky, gray or tan, and, grossly, may have an oily appearance. the normal dog excretes - gm of fat in the stool each day. this level of fecal fat is quite constant and is independent of dietary fat intake over a wide range of to gm/day (heersma and annegers, ) . in intestinal malabsorption, the ability to absorb fat is decreased and fecal fat excretion increases significantly. under these conditions, the amount of fecal fat excreted becomes proportional to dietary intake. merritt et al. ( ) reported that body weight is an important factor in fat output. small dogs (i.e., less than - kg body weight) with intestinal malabsorption had fecal fat outputs lower than or equal to published normal values. fecal fat excretion for normal dogs was . ± . gm/kg body weight per day. steatorrhea can be documented qualitatively by staining the fresh stool with a lipophilic stain, such as sudan iii, and observing increased numbers of oil droplets under the light microscope. in experienced hands, this method is a reliable diagnostic procedure (drummey et al., ) . the following methods can be used to demonstrate neutral and split fats. for neutral fat, two drops of water are added to a stool sample on a glass slide and mixed. two drops of % ethanol are then added and mixed followed by several drops of a saturated solution of sudan iii in % ethanol. a coverslip is applied to the mixture, which is then examined for yellow or pale orange refractile globules of fat, particularly at the edges of the coverslip. normally, two or three fat droplets per high-power field are present. a large number of neutral fat droplets suggests a lack of pancreatic lipase activity, i.e., exocrine pancreatic insufficiency. ¥ or free fatty acids, several drops of % acetic acid are added to a stool sample on a glass slide and mixed. several drops of sudan iii solution are then added and mixed. a coverslip is applied, and the slide gently heated over an alcohol burner until it begins to boil. the slide is air-cooled and then quickly heated again, this procedure is repeated two or three times. the warm slide is examined for stained free fatty acid droplets, which, when warm, appear as deep orange fat droplets from which spicules and soaps, resem-bling the pinna of the ear, form as the preparation cools. normal stools may contain many tiny droplets of fatty acids (up to per high-power field). with increasing amounts of split fats, the droplets become larger and more numerous, which suggests an abnormality in fat absorption. quantitation of fecal fat is the most accurate method of assessing steatorrhea (burrows et al., ) with dietary fat balance being determined for a period of - hours. fecal fat is analyzed using a modification of the technique of van de kamer et al. ( ) , which employs ether extraction of fecal lipid and titration of fatty acids. the results are ex pressed as grams of neutral fat excreted per hours. merritt et al. ( ) have suggested that dogs be fed gm fat per kilogram per day for two to three days prior to fecal collection. analysis of a -hour collection of stool when this is done is believed to be as accurate as a -hour stool collection. results are expressed as fat excretion in grams per kilogram body weight. in addition to mal absorption of fat, the canine malabsorption syndrome is associated with decreased absorption of other nutrients. these defects in absorption are responsible for the progressive malnutrition which is a cardinal feature of the disease. there may be malabsorption of vitamin d and/or calcium, resulting in osteomalacia. the anemia some times observed may be the result of malabsorption of iron or of the b vitamins, which are required for normal erythropoiesis. malabsorption of vitamin k can result in hypoprothrombinemia. glucose malabsorption has been clearly documented by kaneko et al. ( ) , and it is likely that amino acids, which are absorbed at a similar level of the small intestine, are also malabsorbed. carbohydrate and fat malabsorption unquestionably con tributes to the calorie deficit which results in weight loss. amino acid malabsorption may contribute to the development of hypoproteinemia, although this is thought to be due primarily to increased intestinal loss of plasma protein (see section iv,c). the diagnosis of idiopathic canine malabsorption can be made only after appropriate diagnostic procedures have ruled out the presence of ( ) other primary inflammatory, neoplastic, or parasitic diseases of the intestine and ( ) the diseases of the pancreas, liver, or stomach which result in defective intraluminal digestion. the presence of parasitic infection is determined by examining the feces for parasite ova. other inflammatory or neoplastic diseases of the intestine may be suggested on the basis of clinical or radiologi cal examination, but a definitive diagnosis usually depends on histopathological examina tion of an intestinal biopsy specimen. both primary and secondary intestinal malabsorption must be differentiated from those diseases in which there is decreased intraluminal hydrolysis of nutrients. the latter are due most frequently to pancreatic exocrine insufficiency, the result of such diseases as chronic pancreatitis or juvenile atrophy. in these diseases, degradation of the major dietary con stituents is reduced because of a primary lack of pancreatic enzymes. intraluminal hy drolysis of fat may also be decreased because of a deficiency of bile salts caused either by decreased hepatic secretion or by bile duct obstruction. under certain experimental condi tions, diversion of bile flow in the dog actually has a quantitatively small effect on fat absorption (wells et al., has a quantitatively small effect on fat absorption (wells et al., ; hill and kidder, a ). the problems of pancreatic exocrine deficiency are discussed in detail elsewhere in this text (chapter ). the most simple and perhaps most widely used test to differentiate intestinal malabsorption from pancreatic exocrine insufficiency is that described by jasper ( ) . the test is employed to detect reduction in trypsin-like activity in the feces of dogs with decreased pancreatic exocrine secretion (grossman, ) . there is wide variation in normal activity, making interpretation of the test difficult (frankland, ; hill and kidder, ; burrows et al., ) . the test reveals only the presence or absence of hydrolysis of gelatin and does not differentiate between gelatinase activity produced by intestinal bacteria from that secreted by the pancreas. there is evidence in some species that trypsin is almost completely destroyed by bacteria during its passage through the intestine and that the proteinase activity of the feces is primarily of bacterial origin (borgström et al., ) . despite these theoretical objections, the test has been of clinical diagnostic value in our hands. fecal gelatinase activity has been detected consistently in cases of intestinal malabsorption and is almost always absent when severe pancreatic exocrine insufficiency is present. burrows et al. ( ) reported that the mean -hour trypsin output in dogs with pancreatic insufficiency was significantly lower, and in dogs with malabsorption significantly higher, than clinically normal dogs. an indirect method to test chymotrypsin activity has been described (strombeck, ) . a synthetic peptide, rc-benzoyltyrosine//?-aminobenzoic acid, is administered to test dogs orally. if chymotrypsin is present in the duodenum, hydrolysis of this peptide occurs and p-aminobenzoic acid (paba) is released in a free form, which is absorbed and subsequently excreted in the urine within hours. the urine is analyzed for paba. less than % paba excretion identifies dogs with suspected pancreatic exocrine insuffi ciency. a. oleic acid and triolein absorption. several tests have been developed for the clinical evaluation of intestinal absorptive capacity. the absorption of i-labeled oleic acid and i-labeled triolein has been studied extensively in normal dogs (turner, ; michaelson et al., ) , and kaneko et al. ( ) used this test to study dogs with in testinal malabsorption. the day before administration of the l i-labeled compound, a small amount of lugol 's iodine solution is administered to block thyroidal uptake of the isotope. tracer amounts of the test substances are mixed with nonradioactive carrier and are administered orally. absorption is determined by measuring the radioactivity of the plasma at intervals following administration can calculating the percentage of the dose absorbed based on plasma volume. it is possible to use the results of these two tests, when performed in sequence, to differentiate between steatorrhea caused by a deficiency of pancreatic enzymes and that caused by a primary defect in absorption (kallfelz et al., ) . if steatorrhea is caused by a lack of pancreatic lipase, oleic acid absorption will be normal, whereas that of triolein, which requires lipolysis for absorption, will be significantly reduced. the absorption of both compounds is reduced in intestinal malabsorption (fig. a,b) . the results of this test also may vary depending on the rate of intestinal motility (tennant et al., b) . kaneko et al., .) of vitamin a, mean serum vitamin a concentrations peak at - hours, with values ranging between three and five times fasting serum levels in normal dogs. breed dif ferences and delayed gastric emptying will alter results. c. glucose absorption. the absorption of glucose can be measured by means of an oral glucose tolerance test in which a test dose of glucose is given by mouth and the blood glucose level measured at intervals for - hours following administration. the test has been used in canine malabsorption in which the normal rise in blood glucose level is reduced (kaneko et al., ) . the test also has been reported for use in the horse (roberts and hill, ) . dogs with pancreatic exocrine deficiency may, however, have "diabetic" tolerance curves (hill and kidder, b) . the major disadvantage of relying on this test alone is that it does not differentiate between decreased intestinal absorption and increased tissue uptake following absorption. this problem can be minimized by comparing results of the oral glucose tolerance test with those obtained with the intraven ous tolerance test. the results of this test, however, must be interpreted carefully and in relation to other clinical and laboratory findings. hill and kidder ( ) reported that dogs on low-carbohydrate diets can have "diabetic" tolerance curves; test dogs should be on a high-carbohydrate diet - days before testing. the absorption of d-xylose also can be used to evaluate intestinal function. d-xylose is not metabolized by the body to any significant degree, and the problems of evaluating tissue utilization which occur with glucose are eliminated. because of the large amounts of d-xylose used in the test, absorption is independent of active transport processes, and the rate of absorption is proportional to luminal concentra tion. a d-xylose absorption test for dogs has been described by van kruiningen ( ) . in this procedure, a standard -gm dose of d-xylose is administered by stomach tube. during the -hour period following administration, the dog is confined in a metabolism cage, and urine is collected quantitatively. at the end of the -hour test period, the urine remaining in the bladder is removed by catheter, and the total quantity excreted in hours is determined. normal dogs excreted an average of . gm during the test period, with a range of . - . gm. the results obtained by this method are dependent not only on the rate of intestinal absorption, but also on the rate of renal excretion, and it is necessary, therefore, to know that kidney function is normal. the oral xylose tolerance test has received most clinical use (hill et al., ; hayden and van kruiningen, ) . dogs are fasted overnight, a blood sample is obtained, and d-xylose is administered by stomach tube at the rate of . gm/kg. a control test is performed on a normal dog simultaneously with each dog with signs of intestinal malab sorption. the first blood sample is obtained one-half hour after administration. the second sample is obtained hour following administration, and additional samples are taken at hourly intervals for hours. the xylose concentration in the blood is determined by the method of roe and rice ( ) . maximal blood levels almost always are reached at hour after administration of the test dose; hill expects a xylose level of at least mg/dl within - minutes in a normal dog. in preliminary studies of four dogs with the malabsorption syndrome, maximal blood xylose levels averaged % of corresponding control values. in dogs with pancreatic exocrine insufficiency with normal intestinal mucosa, there should be a normal xylose response test. the d-xylose absorption test also has been described for use in differential diagnosis of equine diarrheal diseases (roberts, ) . bolton et al. ( ) reported that a dosage of . gm xylose per kilogram body weight was useful in detecting horses that absorbed the pentose abnormally. gastrointesti nal lesions associated with abnormal results were classified as ( ) villous atrophy, ( ) edema of the lamina propria, or ( ) necrosis of the lamina propria. at this dosage in normal horses, the mean peak plasma concentration is less than one-third that seen in normal dogs given xylose (normal dogs: - mg % at minutes). albumin, γ-globulin, and other plasma proteins are present in normal gastrointestinal secretions. because protein usually undergoes complete degradation within the intestinal lumen, it has been suggested that the gastrointestinal tract must have a physiological role in the catabolism of plasma proteins. the relative significance of this pathway, however, has been the subject of considerable controversy. some investigators have concluded that as much as % or more of the normal catabolism of albumin (glenert et al., campbell et al., ; wetterfors, wetterfors, , wetterfors et al., ) and γ-globulin occurs in the gastrointestinal tract. others believed that the physiological role of the intestine in plasma protein catabolism is far less significant, accounting for only about % of the total catabolism (waldmann et al., (waldmann et al., , katz et al., ; franks et al., a,b) . regardless of the questions concerning the relative importance of the gastrointestinal tract in plasma catabolism, it is well established that normal intestinal losses are increased significantly in a variety of gastrointestinal diseases, which are referred to collectively as protein-losing enteropathies. the increased loss causes hypoproteinemia (especially hypoalbuminemia), which may be observed in various types of chronic enteric diseases. the excessive losses are produced by ulcérations or other mucosal changes which alter permeability or by obstruction of lymphatic drainage from the intestine. if severe, hypoalbuminemia may result in retention of fluid with development of ascites and subcutaneous edema of pendant areas. excessive plasma protein loss has now been demonstrated in swine with chronic ileitis (nielsen, ) , in calves with acute enteric infections (marsh et al., ) , in cattle with parasitic or other inflammatory abomasal disease (nielsen and nansen, ; halliday et al., ; murray, ) , and in johne's disease (patterson et al., ; nielsen and andersen, ; patterson and berret, ) . in addition to the classic mucosal and submucosal lesions of johne's disease, nielsen and andersen ( ) demonstrated the presence of secondary intestinal lymphangiectasia. meuten et al., ( ) described protein-losing granulomatous enteritis in two horses and discussed a comparative over view of diseases causing mal absorption in the horse, cow, dog, pig, and man. protein-losing enteropathy has been seen with some frequency in the dog (campbell et al., ; farrow and penny, ; hill, ; fineo et al, ; hayden and van kruiningen, ; mattheeus, et al; hill and kelly, ; milstein and sanford, ; barton et al, ; olson and zimmer, ) . intestinal lymphangiectasia was com monly reported. the dog described by milstein and sanford ( ) was not hypoproteinemic because the rate of albumin synthesis by the liver was greater than protein loss into the intestine. protein loss has also been documented in dogs with chronic hypertrophic gastritis (section iv,a). increased intestinal protein loss is the most likely explanation for the hypoalbuminemia associated with certain other enteric diseases, including intestinal mal absorption and lymphoma of the intestine. munro ( ) demonstrated that protein loss in dogs with experimentally induced protein-losing gastropathy occurs by an intercellular route. isotope-labeled polyvinylpyrolidine ( i-pvp), cr-labeled ceruloplasmin, and crlabeled albumin have been used to evaluate enteric protein loss in the dog (fineo et al, ; barton et al, ; hill and kelly, ; van der gagg et al, ; olson and zimmer, ) . canine ulcerative colitis was described originally in the report of cello ( ). since that time, ulcerative colitis and its variant form, granulomatous colitis of boxer dogs, has been reported by several investigators kennedy and cello, ; koch and skelley, ; sander and langham, ; ewing and gomez, ; gomez et al, ; russell et al, ). the etiology is generally unknown. ewing and aldrete ( ) reported a case of canine giardiasis presenting as chronic ulcerative colitis and cases of ulcerative colitis in dogs have been attributed to trichuriasis, balantidiasis, protothecosis, histoplasmosis, eosinophilic ulcerative colitis, or neoplasia (lorenz, ) . rarely, severe ulcerative colitis is seen in the cat. in some of these cases, feline leukemia virus is demonstrated. shindel et al ( ) described colonie lesions in cats caused by feline panleukopenia. histopathologically, periodic acid-schiff-positive macrophages are pathognomonic for the granulomatous colitis of boxer dogs. the disease causes chronic, intractable diarrhea, which is often hemorrhagic. in addition, afflicted dogs may vomit and are often emaciated. fever is usually not present. biochemical manifestations of ulcerative colitis depend upon duration and severity of ewing and gomez ( ) . thirty-six observations on affected dogs. illness, degree of colorectal involvement, and the presence of systemic complications. in severe cases of long duration with extensive colorectal involvement, hypoalbuminemia and hypergammaglobulinemia (table vii) are sometimes observed. the pathogenesis of hypoalbuminemia probably involves increased loss of plasma through the denuded and inflammed colorectal mucosa. hypergammaglobulinemia is probably an associated re sponse to chronic inflammation. the digestive process of ruminants differs from that of other animals because of microbial digestion and metabolism in the rumen which occurs prior to other normal digestive processes. the short-chain fatty acids (acetic, propionic, and butyric acids) are the pri mary end products of rumen fermentation and represent the chief dietary source of energy for ruminants (hungate et al., ) . the polysaccharide cellulose, which undergoes only very limited digestion in most simple-stomached animals, is readily utilized by ruminants because of the activity of cellulytic bacteria. significant quantities of nonprotein nitrogen also can be utilized by ruminai bacteria for protein synthesis, and this bacterial protein subsequently can be utilized to meet the protein requirements of the animal. bacterial production of vitamins may also meet essentially all the requirements of ruminants. maintenance of bacterial fermentation within the rumen also presents certain unusual hazards to ruminant animals. when rapid changes in dietary intake occur, the products of fermentation can be released more rapidly than they can be removed. acute rumen tympany, acute indigestion of d-lacticacidosis, and urea poisoning are diseases which result from such abrupt changes in diet (hungate, (hungate, , . acute rumen indigestion occurs in sheep or cattle on a high-roughage diet when they inadvertently are allowed access to large amounts of readily fermentable carbohydrate, e.g. grain and apples (dunlop, ) . streptococcus bovis is the rumen microorganism believed to be chiefly responsible for rapid fermentation and for production of large quantities of lactic acid (hungate et al., ; krogh, a,b) . as lactic acid accumulates more rapidly than absorption, the rumen ph falls and rumen atony results. rumen bacteria produce a racemic mixture of lactic acid. some l-lactate may be metabolized by the liver and other tissues, but d-lactate cannot be and contributes significantly to the acid load of the body. the excessive lactic acid production results in metabolic acidosis, which is characterized by reduced blood ph and bicarbonate concen tration and by a fall in urine ph from a normal value of . - . to as low as . . fluid accumulates in the rumen because of increased osmolarity of its contents, causing hemoconcentration, which may lead to hypovolemic shock and death (hyldgaard-jensen and simesen, ) . if affected animals survive the initial period of explosive fermenta tion, a chemical rumenitis, caused by lactic acid, may develop. secondary mycotic rumenitis may also occur and be fatal. hepatic abscesses also may result from severe rumenitis. the rumen of mature cattle can produce . - . liters gas per minute (hungate et al., ) . the gas is composed primarily of carbon dioxide and methane, which are products of rumen fermentation. carbon dioxide is also released when salivary bicarbonate is acted upon by organic acids within the rumen. under normal conditions, these large amounts of gas are continually removed by eructation. any factor which interferes with eructation can produce acute tympany of the rumen (bloat), leading to rapid death. interruption of the normal eructation reflex or mechanical obstruction of the esophagus typically results in free-gas bloat. the most important form of bloat, however, is seen in cattle consuming large quantities of legumes or in feedlot cattle on high-concentrate diets. the primary factor in these more common types of bloat is a change in the ruminai contents to a foamy or frothy character. because of altered surface tension, gas is trapped in small bubbles with the rumen and cannot be eliminated by eructation (clarke and reid, ) . the chemical changes which cause foam to form within the rumen are not completely clear. some reports (nichols, ; nichols and deese, ) suggest that plant pectin and pectin methyl esterase, an enzyme system also from plants, are critical factors. the enzyme acts on pectin to release pectic and galacturonic acids, which greatly increase the viscosity of the rumen fluid, resulting in formation of a highly stable foam. slimeproducing bacteria also have been incriminated in the pathogenesis of frothy bloat. these microorganisms produce an extracellular polysaccharide, which results in stable foam formation. effective medical treatment and control are directed toward decreasing or preventing foam formation. this has been accomplished with certain nonionic detergents with surfac tant properties which break up or prevent formation of foam within the rumen (bartley, ) . another approach has been the prophylactic administration of sodium alkyl sulfonate, which inhibits pectin methyl esterase activity, preventing foam formation by eliminating the products of this enzyme reaction (nichols, ) . much effort is now being directed toward genetic selection of cattle which are less susceptible to rumen tympany and to varieties of legumes which are less likely to produce bloat (howarm, ) . unlike monogastric species, ruminants can effectively use nonprotein nitrogen to meet dietary protein requirements. urea, biuret (oltjen et al., ) and ammonium salts (webb et al., ) all can serve as dietary supplements. urea, which is the most frequently used, is hydrolyzed by bacterial urease within the rumen and the free ammonia formed is incorporated into amino acids by microorganisms within the rumen. the bacte rial protein so produced is digested and absorbed in the small intestine along with protein from the diet. signs of urea poisoning typically develop within minutes after consumption of food containing toxic amounts of urea. clinical manifestations are the result of excessive ammonia production (word et al., ; elmer and barclay, ) and are due to the encephalotoxic effects of free ammonia absorbed from the rumen. tolerance to urea may be significantly increased by gradually elevating the amounts of urea in the diet or by adding readily fermentable carbohydrate. it has actually been possible for ruminants to adapt and thrive on a diet in which urea was the sole source of dietary nitrogen. however, if urea is fed at a level of more than % in the diet of unadapted animals, toxic effects are likely. poisoning may occur when, by accident, animals obtain access to large amounts of urea-containing dietary supplement or in animals receiving bulk feed when there has been an error in formulation or when the urea-containing additive is incompletely mixed. oral administration of acetic acid has been shown to reduce acute urea toxicity, apparently by decreasing absorption of free ammonia from the rumen. acetic acid also has been used clinically for the treatment of urea poisoning but under experimental conditions it has more value prophylactically than in animals with frank signs of poisoning (word et al., ) . textbook of veterinary internal medicine-diseases of the dog and cat current veterinary therapy veterinary medicine cornell vet. , vet. ree. , nature (london) , handbook of physiology a guide to learning fluid therapy physiology of the digestive tract proc nati. acad. sci. u.s.a. , . drucker the physiology of domestic animals current veterinary therapy proc. nati. acad. sci the vitamins vet. ree. , glycoproteins: composition, structure and function handbook of physiology the rumen and its microbes handbook of physiology nord. veterinaermed. , handbook of physiology handbook of physiology acta vet. scand. , . krogh, n proc. soc. exp. biol. med. , . lack textbook of veterinary internal medicine-diseases of the dog and cat nature (london) , vet. ree. , . penhale current veterinary therapy handbook of physiology proc. nati. acad. sci nature (london) , diabetes , . van de kamer current veterinary therapy gastroenterology , handbook of physiology intestinal absorption handbook of physiology key: cord- -y vg frb authors: montané, xavier; kowalczyk, oliwia; reig-vano, belen; bajek, anna; roszkowski, krzysztof; tomczyk, remigiusz; pawliszak, wojciech; giamberini, marta; mocek-płóciniak, agnieszka; tylkowski, bartosz title: current perspectives of the applications of polyphenols and flavonoids in cancer therapy date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: y vg frb the development of anticancer therapies that involve natural drugs has undergone exponential growth in recent years. among the natural compounds that produce beneficial effects on human health, polyphenols have shown potential therapeutic applications in cancer due to their protective functions in plants, their use as food additives, and their excellent antioxidant properties. the possibility of combining conventional drugs—which are usually more aggressive than natural compounds—with polyphenols offers very valuable advantages such as the building of more efficient anticancer therapies with less side effects on human health. this review shows a wide range of trials in which polyphenolic compounds play a crucial role as anticancer medicines alone or in combination with other drugs at different stages of cancer: cancer initiation, promotion, and growth or progression. moreover, the future directions in applications of various polyphenols in cancer therapy are emphasized. the appearance of the severe acute respiratory syndrome coronavirus (sars-cov- ) in december last year and its very rapid spread around the world in early , known to cause covid- disease, has evidenced, among other things, the importance of investing in research to improve the people's quality of life or eradicate diseases that still do not have an effective treatment. as observed in figure , there has been an exponential increase of research and publications related to the possible use of polyphenolic compounds in cancer therapy [ ] . the fact that polyphenols can be extracted using simple and green techniques-such as ultrasound-assisted extraction, and that after being sterilized, polyphenols preserve most of their properties intact-will contribute to the study of these compounds as potential anticancer drugs [ , ] . as observed in figure , there has been an exponential increase of research and publications related to the possible use of polyphenolic compounds in cancer therapy [ ] . the fact that polyphenols can be extracted using simple and green techniques-such as ultrasound-assisted extraction, and that after being sterilized, polyphenols preserve most of their properties intact-will contribute to the study of these compounds as potential anticancer drugs [ , ] . stilbenes or stilbenoids are hydroxylated derivatives of stilbene with a c -c -c chemical structure. these kinds of compounds are produced in various plants such as strawberries, grapes, peanuts, and cannabis [ ] . furthermore, various trees synthesize stilbenes as secondary products of heartwood that can act as antimicrobial and antioxidative substances. stilbenes share most of their biosynthesis pathway with chalcones, which is a class of flavonoids. the most representative compound of the stilbene family that has many health benefits is resveratrol [ ] . resveratrol ( , , ′-trihydroxy-trans-stilbene) is a natural polyphenol of the stilbene family. resveratrol is produced by several plants (grapes, almonds, beans, blueberries, raspberries, mulberries, peanuts, etc.) in response to infections and injuries or as a defense against different kinds of pathogens attacks, such as fungi or bacteria [ ] . furthermore, red wine also contains significant amounts of resveratrol. in , jang et al. were the first researchers that reported the inhibition of skin cancer development in mice by using resveratrol [ ] . since then, many investigations have suggested that resveratrol is able to prevent cancer or delay its onset [ ] . in point of fact, studies demonstrated that resveratrol has in vitro effects against a large range of human tumors: breast, skin, ovary, stomach, prostate, colon, liver, pancreas, cervix, thyroid carcinoma cells, lymphoid, and myeloid cancer cells [ ] . it has been proven that resveratrol shows beneficial effects at different stages of cancer (initiation, promotion, and progression of cancer). for example, resveratrol protects dna from reactive oxygen species (ros) and traps hydroxyls, superoxides, and free radicals produced in cellsevents that are usually related to the initiation of tumors [ ] . in another study, yin et al. demonstrated that the application of resveratrol inhibits the promotion and progression of a lung cancer cells in nude mice. however, the authors mentioned that further studies should be performed in order to evaluate other parameters, such as the applied dose of resveratrol [ ] . besides, clinical trials on humans have been performed with the use of resveratrol, obtaining satisfactory results [ ] [ ] [ ] . stilbenes or stilbenoids are hydroxylated derivatives of stilbene with a c -c -c chemical structure. these kinds of compounds are produced in various plants such as strawberries, grapes, peanuts, and cannabis [ ] . furthermore, various trees synthesize stilbenes as secondary products of heartwood that can act as antimicrobial and antioxidative substances. stilbenes share most of their biosynthesis pathway with chalcones, which is a class of flavonoids. the most representative compound of the stilbene family that has many health benefits is resveratrol [ ] . resveratrol ( , , -trihydroxy-trans-stilbene) is a natural polyphenol of the stilbene family. resveratrol is produced by several plants (grapes, almonds, beans, blueberries, raspberries, mulberries, peanuts, etc.) in response to infections and injuries or as a defense against different kinds of pathogens attacks, such as fungi or bacteria [ ] . furthermore, red wine also contains significant amounts of resveratrol. in , jang et al. were the first researchers that reported the inhibition of skin cancer development in mice by using resveratrol [ ] . since then, many investigations have suggested that resveratrol is able to prevent cancer or delay its onset [ ] . in point of fact, studies demonstrated that resveratrol has in vitro effects against a large range of human tumors: breast, skin, ovary, stomach, prostate, colon, liver, pancreas, cervix, thyroid carcinoma cells, lymphoid, and myeloid cancer cells [ ] . it has been proven that resveratrol shows beneficial effects at different stages of cancer (initiation, promotion, and progression of cancer). for example, resveratrol protects dna from reactive oxygen species (ros) and traps hydroxyls, superoxides, and free radicals produced in cells-events that are usually related to the initiation of tumors [ ] . in another study, yin et al. demonstrated that the application of resveratrol inhibits the promotion and progression of a lung cancer cells in nude mice. however, the authors mentioned that further studies should be performed in order to evaluate other parameters, such as the applied dose of resveratrol [ ] . besides, clinical trials on humans have been performed with the use of resveratrol, obtaining satisfactory results [ ] [ ] [ ] . curcuminoids are natural polyphenols that contain two phenol units joined through a linear diarylheptanoid. the presence of curcuminoids gives a yellow color to plants that contain these kinds of natural structures. the phenolic rings of curcuminoids are chemically modified with other chemical groups with the aim of overcoming some drawbacks of natural curcuminoids in clinical applications such as their poor solubility, low absorption, and bioavailability [ ] . among the curcuminoids, curcumin is one of the most known and studied structures with a high potential as medicine to treat different cancers, apart from also being useful in treating other types of diseases. nonetheless, the poor solubility of curcumin in water of acidic and physiological ph requires the use of diverse alternatives to avoid losing the effectiveness of curcumin as a medicine, such as the synthesis of other curcumin derivatives or the combination of curcuminoids with surfactants or co-surfactants. curcumin is a natural compound and the principal curcuminoid of turmeric plants, which is responsible for turmeric's yellow color [ ] . in addition to its applications in medicine, the use of curcumin has reached other fields. in the food industry, it has been used as a dietary supplement (it is sold as herbal supplement) or a food additive. additionally, it is used in cosmetics and other products. curcumin is commonly used in cancer therapies of different types of cancer: lung, cervix, prostate, breast, bone, and liver [ ] . nevertheless, the administration of free curcumin presents some drawbacks: poor solubility in water, instability in aqueous conditions, low bioavailability, and poor cellular uptake. to overcome these problems, two different solutions were attempted: - the synthesis of curcumin derivatives [ ] , and - the encapsulation of curcumin in different nanostructures ranging from liposomes to natural biopolymeric nanoparticles [ , ] . one of the curcumin derivatives used in breast and renal cancer therapies is dimethoxy curcumin. chen et al. recently proved that this curcumin derivative can be effective in the therapy of colon cancer cells due to causing the reduction of survivin expression and the enhancement of e-cadherin, a cell adhesion molecule, whose loss contributes to the formation of epithelial types of cancers such as carcinomas [ ] . recently, various research groups have reported that the combination of both curcumin and resveratrol can reduce the incidence of lung and prostate cancer [ , ] . lignans are diphenolic compounds found in a wide variety of plants including broccoli, beans, soybeans, rye, sesame seeds, pumpkin seeds, flax seeds, and some berries in very small amounts (µg of lignans per g of dry product) [ ] . their structure consists of two c -c units linked by β,β' bonds. lignans are one of the two main groups of phytoestrogens, which are well known for their good antioxidant properties. in fact, some antioxidant phytochemical compounds could be used as anticancer drugs as they are mimicking the functions of human hormones. some studies on rats showed that lignans prevent the growth of breast and prostate tumors [ , ] . numerous lignans could be considered as possible anticancer medicines due to their large pharmacologically valuable properties. among all of them, arctigenin, magnolol, and honokiol are the main lignans investigated in medicine. nonetheless, etoposide is a commercial lignin belonging to the podophilotoxin subfamily that is used in the treatment of different types of cancer such as lung cancer and breast cancer [ , ] . however, etoposide chemotherapy presents several side effects: low blood cell counts, vomiting, diarrhea, fever, loss of appetite, and alopecia. certain plants belonging to the family known as compositae produce arctigenin, especially the seeds of greater burdock (arctium lappa). some studies revealed that arctigenin inhibits the growth of various cancer cells: stomach, lung, liver, and colon, as well as leukocytes [ ] . at the same time, the addition of arctigenin intensifies the activity of caspase- , which is a protein that plays a crucial role in the death of carcinogenic cells. as a matter of fact, huang et al. demonstrated that the treatment of ovcar and skov ovarian cancers with arctigenin causes the apoptosis of cancer cells in vitro [ ] . one of the most used conventional anticancer drugs is doxorubicin, which is a medicine that belongs to the anthracycline family applied in the treatment of, among other cancers, bladder, stomach, ovaries, lung and thyroid cancers. however, doxorubicin exhibits side effects among which the most frequent are severe nauseas, vomiting, and alopecia [ ] . studies were conducted by lee et al. on adding natural products such as arctigenin to doxorubicin and determining the efficiency of both drugs in improving breast cancer treatment and reducing the side effects provoked by doxorubicin [ ] . the work concludes that the combination of arctigenin and doxorubicin induced the apoptosis of mda-mb- human breast cancer cells in vitro. the addition of arctigenin ameliorates the cellular uptake of doxorubicin, which causes the death of carcinogenic cells. another lignan that was tested in some studies on cancer therapy is magnolol. as its name indicates, magnolol is an isomer of honokiol found in magnolia bark [ ] . since ancient times, extracts from the bark of magnolia have been used in traditional chinese, korean, and japanese medicine. in the last decades, the research on the use of natural products in various cancer treatments has been focused on attempts of understanding mechanisms that induce the antitumor agents' response in the tumor cells [ ] . this year, su and co-workers elucidated the mechanism that reduces the endogen activity of nuclear factor kappa-light-chain-enhancer of activated b cells (nf-κb), which is a protein complex that controls dna transcription and cell survival. therefore, the cells that do not have regulated nf-κb can contribute to the onset and growth of various types of cancers. moreover, magnolol used in the treatment of colorectal cancer reduces the phosphorylation of protein kinase c delta type (pkcδ) and nf-κb, which are two proteins that are involved in tumour progression in vitro and in vivo [ ] . following the methodology used with other drugs, magnolol was co-encapsulated with trastuzumab, an anticancer drug commonly used in stomach or throat cancer therapies, and gold nanoparticles, building a nanocarrier cluster. the synthesized nanocarriers induced a specific photothermal near-ir response combined with targeted anticancer activity resulting in an improvement of magnolol cytotoxicity to breast cancer cells [ ] . as mentioned before, honokiol (also known as houpa or hnk) is a lignan isolated from the bark, seed cones, and leaves of trees belonging to the genus of magnolias, which includes around species. honokiol, which has been used in traditional eastern herbal medicines as an analgesic and together with magnolol, obovatol, and -o-methylhonokiol in the treatment of anxiety and mood disorders, has a spicy odor [ ] . honokiol is most frequently taken orally. in nature, honokiol and magnolol isomers are found together. usually, the separation and purification of both compounds had always been complexed, and it is commonly limited to hplc. in , amblard and co-workers developed a method in which the authors protect the near hydroxyl groups in magnolol to produce a magnolol acetonide that can be simply separated from honokiol via flash chromatography over silica [ ] . recent studies suggest that honokiol could be an effective agent in cancer treatment due to its physical properties-honokiol's ability to easily cross the blood-brain barrier and the bloodcerebrospinal fluid barrier-and its high bioavailability. many research studies have shown that honokiol can kill carcinogenic cells in melanoma, sarcoma, myeloma, and leukemia, as well as in bladder, lung, prostate, and colon cancers [ , ] . besides, honokiol enhances the apoptotic effects of some etoposides, such as doxorubicin. for instance, micelles with encapsulated doxorubicin and honokiol allow a controlled drug co-delivery that inhibits the progression of breast cancer tumors and reduces the doxorubicin side effects when compared with the micelles without honokiol [ ] . on the other hand, the effectiveness of honokiol in the fight with typically drug-resistant multiple myelomas and chronic b-cell leukemia has been proved by various authors [ , ] . ishitsuka and co-workers certified that honokiol presents the ability to kill drug-resistant multiple myeloma carcinogenic cells by varied mechanisms [ ] . another subgroup of polyphenols that can be found in several plants, especially in dried fruit, are phenolic acids. these compounds are characterized by containing a phenolic ring and an organic carboxylic acid function (c -c skeleton) [ ] . phenolic acids are divided in two classes: -derivatives of benzoic acid, and -derivatives of cinnamic acid. in general, derivatives of cinnamic acid are more common in plants than the derivatives of benzoic acid. despite that, some red fruit, onions, and black radish contain significant amounts of benzoic acid derivatives [ ] . to date, the phenolic acid that exhibited medicinal properties that turn it into a plausible candidate for cancer treatment is p-coumaric acid. p-coumaric acid p-coumaric acid (or -hydroxycinnamic acid) is an organic compound derived from cinnamic acid that can be found in a wide variety of edible plants (tomatoes, carrots, garlic, mushrooms, white beans, and others). moreover, p-coumaric acid found in pollen is a constituent of honey [ ] . additionally, p-coumaric can be synthesized from cinnamic acid or l-tyrosine by the action of -cinnamic acid hydroxylase (c h) or tyrosine ammonia lyase (tal) enzymes, respectively. during the last decade, few studies that evidenced the anticancer activity of p-coumaric acid in colon and gastric cancer cells have been published [ , ] . lately, jang et al. have shown that p-coumaric acid suppresses the growth of snu- gastric cancer cells [ ] . the most important group of polyphenols are flavonoids. the chemical structure of flavonoids is composed of carbon atoms comprising cycles of six carbon atoms linked by a -carbon chain (rings a and b, in figure ). the flavonoids family consists of over molecules that have been identified and isolated, but there are undoubtedly many more flavonoid structures to discover [ ] . honokiol can kill carcinogenic cells in melanoma, sarcoma, myeloma, and leukemia, as well as in bladder, lung, prostate, and colon cancers [ , ] . besides, honokiol enhances the apoptotic effects of some etoposides, such as doxorubicin. for instance, micelles with encapsulated doxorubicin and honokiol allow a controlled drug co-delivery that inhibits the progression of breast cancer tumors and reduces the doxorubicin side effects when compared with the micelles without honokiol [ ] . on the other hand, the effectiveness of honokiol in the fight with typically drug-resistant multiple myelomas and chronic b-cell leukemia has been proved by various authors [ , ] . ishitsuka and co-workers certified that honokiol presents the ability to kill drug-resistant multiple myeloma carcinogenic cells by varied mechanisms [ ] . another subgroup of polyphenols that can be found in several plants, especially in dried fruit, are phenolic acids. these compounds are characterized by containing a phenolic ring and an organic carboxylic acid function (c -c skeleton) [ ] . phenolic acids are divided in two classes: -derivatives of benzoic acid, and -derivatives of cinnamic acid. in general, derivatives of cinnamic acid are more common in plants than the derivatives of benzoic acid. despite that, some red fruit, onions, and black radish contain significant amounts of benzoic acid derivatives [ ] . to date, the phenolic acid that exhibited medicinal properties that turn it into a plausible candidate for cancer treatment is p-coumaric acid. p-coumaric acid p-coumaric acid (or -hydroxycinnamic acid) is an organic compound derived from cinnamic acid that can be found in a wide variety of edible plants (tomatoes, carrots, garlic, mushrooms, white beans, and others). moreover, p-coumaric acid found in pollen is a constituent of honey [ ] . additionally, p-coumaric can be synthesized from cinnamic acid or l-tyrosine by the action of -cinnamic acid hydroxylase (c h) or tyrosine ammonia lyase (tal) enzymes, respectively. during the last decade, few studies that evidenced the anticancer activity of p-coumaric acid in colon and gastric cancer cells have been published [ , ] . lately, jang et al. have shown that pcoumaric acid suppresses the growth of snu- gastric cancer cells [ ] . the most important group of polyphenols are flavonoids. the chemical structure of flavonoids is composed of carbon atoms comprising cycles of six carbon atoms linked by a -carbon chain (rings a and b, in figure ). the flavonoids family consists of over molecules that have been identified and isolated, but there are undoubtedly many more flavonoid structures to discover [ ] . flavonoids are found in abundance in colored vegetables (spinach) and fruit such as berries, blueberries, apples, grapes, oranges, strawberries, plums, and in some foods and beverages widely used in the human diet, including dark chocolate, nuts, red wine, tea, soy, and soy derivatives. flavonoids are found in abundance in colored vegetables (spinach) and fruit such as berries, blueberries, apples, grapes, oranges, strawberries, plums, and in some foods and beverages widely used in the human diet, including dark chocolate, nuts, red wine, tea, soy, and soy derivatives. flavonoids have a wide spectrum of functions in plants: -flavonoids attract pollinating insects through the color or smell that they give to the plant or its flowers, -filtration of uv light, -protection against herbivorous predators, -protection against fungi, -they are involved in the hormone auxin transport, -regulation of the cell cycle, -pigmented blue colors given by anthocyanins are responsible for the resistance of plants to the photooxidation of uv light from the sun, and - in carnivorous plants, they attract prey. usually, two criteria are used to classify flavonoids: -the chemical structure of the c heterocycle (if it is present), and -to which carbon of the c ring the b ring is attached (c and c in figure ). according to these two factors, seven groups of flavonoids can be distinguished: flavonols, flavones, flavanones, flavan- -ols, isoflavones, chalcones, and anthocyanidins ( figure ). the chemical structures of these groups are shown in figure . usually, two criteria are used to classify flavonoids: -the chemical structure of the c heterocycle (if it is present), and -to which carbon of the c ring the b ring is attached (c and c ′ in figure ). according to these two factors, seven groups of flavonoids can be distinguished: flavonols, flavones, flavanones, flavan- -ols, isoflavones, chalcones, and anthocyanidins ( figure ). the chemical structures of these groups are shown in figure . flavonols are a class of flavonoids based on the backbone -hydroxyflavone. there is a wide variety of flavonols, which depend on positions that can be hydroxylated ( figure ). many fruits (apples, peaches, oranges, blackberries, raspberries), vegetables (onions, broccoli, kale, brussels sprouts, cucumbers, lettuce, tomatoes, potatoes, spinach), leaves (aloe vera, rosemary, soybean, pinus sylvestris, holly, endive), seeds (grapes), and grains (several cereals including quinoa, buckwheat, barley, and oat) are rich sources of flavonols [ ] . flavonols are responsible for the color of flowers in some plants as well as protecting them from uv light and ros [ ] . furthermore, flavonols are bioactive polyphenols that are widely used due to their excellent antioxidant properties [ ] : -in medicine: antimicrobial, anti-inflammatory, antiaging, anticancer, or insecticidal agents. -in agriculture: as pesticides. kaempferol and quercetin are the main flavonols studied in medicine. nevertheless, other flavonols such as herbacetin, myricetin, and fisetin have also been investigated as anticancer drugs [ , ] . kaempferol is a flavonol that is found in plants, plant-derived foods, and traditional medicines, including in tea, kale, beans, spinach, and broccoli [ ] . once isolated, kaempferol is a yellow crystalline solid of poor solubility. one study reported by liu suggested that kaempferol intake contributes to approximately % of the total average intake of flavonols and flavones in a normal diet [ ] . during the last few years, numerous investigations provided new evidence of the anticancer mechanisms of kaempferol both in vitro and in vivo. discovering such mechanisms has enabled the analysis and understanding of kaempferol's role as an anticancer drug and afterwards may lead to an improvement of applied techniques and methods, such as the development of kaempferol-loaded targeted drug delivery systems [ ] . one of the cancers in which the effect of kaempferol has been studied the most is breast cancer [ ] . several research groups have proved the cytotoxicity of kaempferol against breast cancer cells both in vitro and in vivo: -by inhibiting the growth of cancer cells, flavonols are a class of flavonoids based on the backbone -hydroxyflavone. there is a wide variety of flavonols, which depend on positions that can be hydroxylated ( figure ). many fruits (apples, peaches, oranges, blackberries, raspberries), vegetables (onions, broccoli, kale, brussels sprouts, cucumbers, lettuce, tomatoes, potatoes, spinach), leaves (aloe vera, rosemary, soybean, pinus sylvestris, holly, endive), seeds (grapes), and grains (several cereals including quinoa, buckwheat, barley, and oat) are rich sources of flavonols [ ] . flavonols are responsible for the color of flowers in some plants as well as protecting them from uv light and ros [ ] . furthermore, flavonols are bioactive polyphenols that are widely used due to their excellent antioxidant properties [ ] : in medicine: antimicrobial, anti-inflammatory, antiaging, anticancer, or insecticidal agents. - in agriculture: as pesticides. kaempferol and quercetin are the main flavonols studied in medicine. nevertheless, other flavonols such as herbacetin, myricetin, and fisetin have also been investigated as anticancer drugs [ , ] . kaempferol is a flavonol that is found in plants, plant-derived foods, and traditional medicines, including in tea, kale, beans, spinach, and broccoli [ ] . once isolated, kaempferol is a yellow crystalline solid of poor solubility. one study reported by liu suggested that kaempferol intake contributes to approximately % of the total average intake of flavonols and flavones in a normal diet [ ] . during the last few years, numerous investigations provided new evidence of the anticancer mechanisms of kaempferol both in vitro and in vivo. discovering such mechanisms has enabled the analysis and understanding of kaempferol's role as an anticancer drug and afterwards may lead to an improvement of applied techniques and methods, such as the development of kaempferol-loaded targeted drug delivery systems [ ] . one of the cancers in which the effect of kaempferol has been studied the most is breast cancer [ ] . several research groups have proved the cytotoxicity of kaempferol against breast cancer cells both in vitro and in vivo: -by inhibiting the growth of cancer cells, -by stopping the progression and proliferation of cancer cells, and -by inducing cancer cells apoptosis. one of the latest investigations to clarify the mechanism of kaempferol as an anticancer drug against breast tumors was carried out by zhu et al. the authors mentioned that kaempferol induced apoptosis and dna damage in mda-mb- cancer cells by the upregulation of the phosphorylated form of the h a histone family member x (γh ax), caspase , caspase , and the protein serine/threonine kinase (p-atm) [ ] . da and co-workers tested kaempferol in prostate cancer cells [ ] . the authors concluded that the use of kaempferol against lncap prostate cancer cell lines led to cancer cells death and impeded cancer cell proliferation and invasion in a dose-dependent manner. quercetin is the most common flavonoid in human diet with an average daily consumption of - milligrams [ ] . quercetin is mainly found in red onions, kale, apples, grapes, broccoli, and tea. in red onions, quercetin represents around % of its dry weight. various in vitro and in vivo studies showed that quercetin is one of the most potent antioxidants of the flavonoid family [ ] , which makes it an ideal candidate for an anticancer drug. indeed, quercetin is the active ingredient of yang-yin-qing-fei-tang, which is a traditional chinese medicine. furthermore, quercetin exhibited cytotoxicity in various tumor cells, in breast, cervical, colon, liver, lung, gastric, prostate cancers, and in leukemia [ , ] . making use of the anticancer effects of quercetin, the most recent studies combined quercetin with other anticancer drugs with the aim of increasing the efficiency of cancer therapies. some examples are summarized below. one of the natural compounds that lately has been combined with quercetin in cancer therapy studies is curcumin. srivastavaa et al. showed that the mixture of quercetin and curcumin improved the inhibition of cancer cell proliferation by regulating the wnt/β-catenin signaling and promoting the carcinogenic cells death by distinct pathways [ ] . furthermore, sunoqrot and co-workers combined both curcumin and quercetin by preparing nanoparticles with encapsulated curcumin and a shell of quercetin covalently bonded with polyethylene glycol (peg) prepared in a one-pot procedure [ ] . once tested in vivo, these nanocarriers exhibited a controlled drug delivery of curcumin in physiological conditions, which makes it a potentially powerful tool in cancer therapy. it has also been observed that the addition of quercetin to docetaxel therapy in prostate cancer reduces the docetaxel resistance of carcinogenic cells. that increases the efficacy of cancer therapy resulting from an intensification of the apoptosis of cancer cells and the reduction of tumor proliferation and migration [ ] . flavones are a class of flavonoids with a chemical structure very similar to flavonols, from which they only differ in the non-hydroxyl substitution at the carbon -position of flavones ( figure ). flavones are basically found in herbs (parsley, thyme, chamomile, mint, chrysanthemum flowers) and red or purple plants and vegetables (apple skins, broccoli, cabbages, celery, onion leaves, carrots, and red peppers) [ ] . in plants, flavones usually act as defense mechanisms against diseases originated by pathogens. some of the flavones have been in use for many years. the most representative example is luteolin, which since ancient times has been used as yellow dye. apigenin has also been used to dye wool. moreover, wogonin is well known because it is one of the active ingredients of sho-saiko-to, which is a japanese herbal supplement [ ] . however, the interest in using this family of flavonoids in medicine has been growing because they demonstrate efficient antimicrobial, antioxidant, antifungal, anti-inflammatory, antimutagenic, and anticancer activity [ ] . inside the flavones family, the anticancer properties of apigenin and luteolin are widely investigated. apigenin, which is a yellow crystalline solid, is one of the flavones most commonly found in nature. many fruits and vegetables, such as parsley, celery, celeriac, carrot, oregano, and chamomile tea contain apigenin. in the particular case of chamomile tea, apigenin constitutes % of the total flavonoids content [ ] . for many centuries, apigenin has been widely used as a traditional medicine [ , ] . the excellent properties of this natural compound have prompted the study of its application as an anticancer drug [ , ] . in fact, various positive effects of apigenin administration, alone or in combination with other chemotherapeutic agents, in different types of cancer treatments were reported in the literature [ ] . the following aspects were mentioned: -inducing the death of cancer cell lines, -triggering both autophagy and apoptosis, -suppressing cancer cell migration and invasion, and -inducing the cancer cells cycle arrest. one of the recently carried out investigations mentions that apigenin promotes pancreatic cells death by increasing intracellular ros [ ] . in this work, montani et al. tried to understand the mechanism happening in cancer cells in which apigenin was applied. in fact, they suggested a biological mechanism occurring between heat shock protein (hsp ), a protein that stabilizes proteins involved in the growth of cancer cells, and tp gene mutations that reduce the cytotoxic effect of the chemotherapy with apigenin. the targeting of these molecules is an important anticancer strategy that has been extensively explored. on the other hand, liu et al. evaluated the synergistic effect in cancer therapy involving apigenin combined with metal ions [ ] . in this work, the authors examined the thermal stability of two flavones (apigenin and luteolin) when combined with ferrous or cupric ions, which negatively affects the anticancer activities of both flavones against human cervical cancer hela cells. luteolin is usually found in the leaves and bark of some plants. the major natural sources of luteolin are celery, thyme, dandelion, clover flower, ragweed pollen, chamomile, and perilla [ ] . due to its beneficial effects on the human body (antioxidative and anti-inflammatory properties, being a free radicals scavenger, promoting carbohydrate metabolism, and modulating the immune system), it is assumed that luteolin could perform an important role in cancer therapy [ , ] . some of the flavones have been in use for many years. the most representative example is luteolin, which since ancient times has been used as yellow dye. apigenin has also been used to dye wool. moreover, wogonin is well known because it is one of the active ingredients of sho-saiko-to, which is a japanese herbal supplement [ ] . however, the interest in using this family of flavonoids in medicine has been growing because they demonstrate efficient antimicrobial, antioxidant, antifungal, anti-inflammatory, antimutagenic, and anticancer activity [ ] . inside the flavones family, the anticancer properties of apigenin and luteolin are widely investigated. apigenin, which is a yellow crystalline solid, is one of the flavones most commonly found in nature. many fruits and vegetables, such as parsley, celery, celeriac, carrot, oregano, and chamomile tea contain apigenin. in the particular case of chamomile tea, apigenin constitutes % of the total flavonoids content [ ] . for many centuries, apigenin has been widely used as a traditional medicine [ , ] . the excellent properties of this natural compound have prompted the study of its application as an anticancer drug [ , ] . in fact, various positive effects of apigenin administration, alone or in combination with other chemotherapeutic agents, in different types of cancer treatments were reported in the literature [ ] . the following aspects were mentioned: -inducing the death of cancer cell lines, -triggering both autophagy and apoptosis, -suppressing cancer cell migration and invasion, and -inducing the cancer cells cycle arrest. one of the recently carried out investigations mentions that apigenin promotes pancreatic cells death by increasing intracellular ros [ ] . in this work, montani et al. tried to understand the mechanism happening in cancer cells in which apigenin was applied. in fact, they suggested a biological mechanism occurring between heat shock protein (hsp ), a protein that stabilizes proteins involved in the growth of cancer cells, and tp gene mutations that reduce the cytotoxic effect of the chemotherapy with apigenin. the targeting of these molecules is an important anticancer strategy that has been extensively explored. on the other hand, liu et al. evaluated the synergistic effect in cancer therapy involving apigenin combined with metal ions [ ] . in this work, the authors examined the thermal stability of two flavones (apigenin and luteolin) when combined with ferrous or cupric ions, which negatively affects the anticancer activities of both flavones against human cervical cancer hela cells. luteolin is usually found in the leaves and bark of some plants. the major natural sources of luteolin are celery, thyme, dandelion, clover flower, ragweed pollen, chamomile, and perilla [ ] . due to its beneficial effects on the human body (antioxidative and anti-inflammatory properties, being a free radicals scavenger, promoting carbohydrate metabolism, and modulating the immune system), it is assumed that luteolin could perform an important role in cancer therapy [ , ] . to enhance the anticancer effects of luteolin, the flavone is usually used together with other anticancer drugs. ren and co-workers demonstrated that the application of luteolin in combination with oxalipatlin, a conventional anticancer drug used to inhibit the development of cancer cells, stopped the proliferation of gastric cancer cells in vitro by the upregulation of the activity of caspase- and bax proteins [ ] . the construction of nanocarriers containing anticancer drugs allows obtaining controlled drug delivery systems. by the encapsulation of luteolin in polymeric micelles, hu et al. developed a thermosensitive nanocarrier that demonstrated an improved apoptosis of colorectal cancer cells compared to the administration of free luteolin [ ] . flavanones are colorless ketones derived from flavone. flavanones are found in a wide variety of foods included in our daily diet and in herbs [ , ] in citrus fruits, flavanones are usually glycosylated by a disaccharide in position ( figure ). they present different functions in plants: taste-modifying properties (eriodictyol, homoeriodictyol and sterubin), and -they are responsible for the bitter taste in citrus fruits (naringin). in the last decades, flavanones have gained a lot of importance in medicine for their antioxidant activity, radical scavenging, cardiovascular, anti-inflammatory, antiviral, and anticancer effects [ ] . naringenin and hesperetin are the most often investigated for being anticancer drugs. nevertheless, some tests were carried out using other flavanones such as didymin and alpinetin [ , ] . naringenin is a flavanone predominating in oranges and grapefruits. it is also found in bergamot, sour orange, tomatoes, cocoa, water mint, beans, etc. [ , ] . in some of these fruits, narigenin is present in its glycosidic form: naringin (which has attached a disaccharide neohesperidose via a glycosidic linkage at carbon ). as it has been proven in several studies, naringenin induces cytotoxicity in various carcinogenic cells of breast, stomach, liver, cervix, pancreas, colon cancers, and in leukemia [ ] . nevertheless, its poor solubility and instability in physiological medium limits the medical applications of naringenin. to solve these drawbacks, akhter et al. reported the encapsulation of naringenin in plga (poly(lactide-co-glycolid acid)) nanoparticles. moreover, they suggested that the encapsulated naringenin showed higher cytotoxicity when compared with free naringenin due to a more controlled drug release [ ] . another option that could enhance the anticancer properties of naringenin involves the synthesis of naringenin derivatives [ ] . an alternative recent study demonstrated naringenin's effectivity as an anticancer drug in breast cancer treatment is due to the activation of the caspase- protein and caspase- enzymes [ ] , while kumar and co-workers showed in vivo that naringenin showed antitumor effects on skin cancer [ ] . hesperetin and hesperetin's -o-glycoside (also known as hesperidin) are the main flavonoids found in lemons and sweet oranges [ ] . hesperetin's anticancer properties against specific tumors are well documented in numerous research publications: -it inhibits glucose uptake in various cancer cell lines [ , ] , -reduces the nf-κb activity, which leads to a decrease in tumor progression [ ] , and -upgrades the apoptosis via the induction of intracellular ros formation [ ] . in a more recent study, the addition of hesperetin improves the activity of cisplatin, which is an anticancer drug that is commonly used to treat lung cancer [ ] . it was observed that hesperetin inhibits mdr protein (multidrug resistance protein ), which is associated with the resistance to cisplatin developed in a great number of patients subjected to cancer therapy. curiously, the administration of both naringenin and hesperetin were tested in vitro and in vivo trials to analyze the anticancer effects in human pancreatic cancer [ ] . for the first time, the authors reported that the combination of both naringenin and hesperetin could be used as a potential non-toxic cancer therapy system that stops pancreatic cancer development. flavanols or flavan- -ols are another group of monomeric flavonoids. catechin and its derivatives are included in this group. natural sources of flavan- -ols are mainly the "tea plant" (camellia sinensis), and some cocoas. therefore, they are highly present in the human diet in both beverages (tea) and solid foods (chocolates) [ , ] . since studies of flavanols have started in the course of the last century, it has been found that these compounds provide resistance against dangerous trespassers, including microbes, fungi, insects, and herbivorous animals [ ] . thereby, the flavanols' health benefits have been broadly studied in humans. some investigations suggest that the intake of cocoa flavanols could help in the prevention of cardiovascular and metabolic diseases. indeed, the european food safety authority approved cocoa products containing mg of flavanols because they "help to maintain the elasticity of blood vessels, which contributes to normal blood flow" [ ] . epigallocatechin gallate (epigallocatechin- -gallate or egcg) is a catechin that is mostly found in tea and one of the polyphenolic compounds most commonly found in nature; it is also the ester of epigallocatechin and gallic acid [ ] . the objective of finding a correlation between green tea intake and the risk of cancer onset has been a well-studied topic [ ] . as an obvious example, the study presented by guo et al. [ ] validated that the consumption of green tea-and therefore catechins-up to seven cups a day provided a small reduction in the prostate cancer risk. moreover, egcg has been tested against certain cancer cell lines. in ht- colorectal cell lines, egcg upregulated the activity of tfr (transferrin receptor), which is a carrier protein for transferrin, and inhibited the activity of the ferritin-h protein via the iron chelation activity in ht- colorectal cancer cells [ ] . in another example, the synergistic effect of egcg and trail (tumor necrosis factor (tnf)-related apoptosis-inducing ligand), a protein that causes cell death, intensifies the activity of both caspase and the death receptor , causing the death of sw and hct colon cancer cells [ ] . despite the fact that egcg is commonly found in nature, this flavanol shows some drawbacks that limit its applications in cancer therapy (poor stability, low absorption, and hepatotoxicity) [ ] . so, the encapsulation of egcg can be a promising solution to minimize the limitations of the egcg use [ ] . the (−)-epicatechin molecule is a flavonoid of which large quantities are found in cocoa [ ] . the use of epicatechin in cancer therapy has been emerging over the last decade in the attempt to overcome some of the drawbacks of egcg [ , ] . pereyra-vergara and co-workers studied the effects and mechanism of (−)-epicatechin in breast cancer cells [ ] . it was shown that the addition of (−)-epicatechin to carcinogenic cells results in the apoptosis of the two tested breast cancer cell lines (mda-mb- and mcf- ). moreover, the authors proved that (−)-epicatechin increased the intracellular ros production and intensified the activity of bcl associated agonist of cell death (bad) and bcl- -like protein (bax), proteins that are associated with cell apoptosis. isoflavones are another type of biological active flavonoids. isoflavones are mostly found in plants of the leguminosea family. this family includes many species that are of great importance in the human diet (peas, lentils, licorice, beans, chickpeas, and carob), in animal fodder (alfalfa, clover, and carob) and as ornamental plants (mimosa and false acacia) [ , ] . since isoflavones present estrogenic properties, plants use these kinds of compounds as part of their natural defense system against the overpopulation of herbivores by controlling their male fertility [ ] . moreover, these properties make isoflavones good complementary therapeutic options in treating menopause and its symptoms such as osteoporosis, anxiety, emotional instability, and headaches. genistein and daidzein are the most studied compounds of this subgroup in terms of medical applications. nevertheless, other isoflavones such as glabridin and alpinumisoflavone have raised interest as potential cancer medicines in various types of cancer such as breast, liver, or thyroid cancers [ , ] . the isoflavone most reported in medicine is genistein, which is a phytoestrogen compound produced in soybeans. genistein was for the first time isolated in . however, it was not until the end of the last century that researchers started to explore its potential beneficial effects on human health and its possible applications as a medical compound in a wide range of diseases, including cardiovascular diseases, osteoporosis prevention, diabetes, and some types of cancers [ ] . it has been proven that genistein is involved in the regulation of different genes that are associated with the onset of cancers by various mechanisms [ ] . in a recent research, hsiao et al. studied the effects and mechanisms of genistein against leukemia cell lines. in fact, the application of genistein to hl- leukemia cells revealed that this natural medicine kills the carcinogenic cells via two different pathways (endoplasmatic reticulum stress and mitochondria-dependent pathway) in vitro and in mouse xenograft models in vivo [ ] . furthermore, different authors studied the effects of genistein when it is combined with other anticancer drugs [ ] . in a recent investigation, liu et al. tested mixtures of genistein and cisplatin in varied concentrations as a plausible anticancer agent in the treatment of cervical cancer cells [ ] . the authors proved that the addition of genistein improved the chemotherapeutic activity of cisplatin, requiring a lower dose of the drug in cancer treatment, which led to a reduction in the therapy side effects. the second isoflavone most commonly found in nature, which similar to genistein is also isolated from soybeans, is daidzein [ ] . the chemical structure of daidzein is very similar to genistein, without the hydroxyl group at position (table ) . rigalli et al. studied in vitro the effects of daidzein use in breast cancer therapy [ ] . in one of those studies, they proved that daidzein downregulated the expression of multidrug resistance-associated protein (mrp ) in both michigan cancer foundation- (mcf- ) and mda-mb- breast cancer cell lines. the reduction of this protein's activity is very important because mrp is involved in transporting many of the chemotherapeutic drugs out of the cells (for example, doxorubicin or mitoxantrone). in another study in vivo, mice were inoculated with t breast cancer cells and then treated with daidzein administered orally for days. in this case, the highest dose of daidzein ( mg/kg) was required to observe a considerable decrease in tumor size. at the same time, the authors reported that the combination of daidzein with regular exercise promotes the breast cancer cells apoptosis via the fas/fasl-mediated mechanism [ ] . chalcones are a class of polyphenolic compounds that are characterized by the presence of an aromatic ketone and an enone in their central core. many fruits such as citrus and apples, vegetables such as tomatoes, potatoes, shallots, and bean sprouts, and some edible plants such as licorice contain chalcones [ ] . besides, chalcones can be synthesized in the form of base-catalyzed aldol condensation of benzaldehydes with acetophenones (for example, sodium hydroxide) [ ] . the most studied chalcone in the field of medicine is ellagic acid, which has been investigated as a potential antitumor agent [ , ] . ellagic acid is an antioxidant that is found in various natural resources: in oak species such as white oak (quercus alba) and european red oak (quercus robur) or in medicinal fungi (phellinus linteus). peaches, pomegranates, grapes, strawberries, raspberries, pecans, walnuts, and raw chestnuts also contain a considerable amount of ellagic acid [ ] . the anti-proliferative and antioxidative properties of ellagic acid have encouraged researchers to study the health benefits of this natural compound. for years, the effects of treating tumors with ellagic acid have been studied by the evaluation of various alternatives (chemical modifications of ellagic acid or its encapsulation among other options) [ ] . one of the last studies that examined the breast cancer treatment with ellagic acid was published by yousuf et al. [ ] . this work evaluated the capacity of numerous phytochemicals in addition to ellagic acid (capsaicin, tocopherol, rosmarinic acid, ursolic acid, limonene, caffeic acid, and ferulic acid) to inhibit the activity of cyclin-dependent kinase (cdk ), which is an important gene associated with cancer progression. among all the tested natural compounds, ellagic acid showed the highest binding affinity for cdk , decreasing the tumor proliferation. however, the encapsulation of ellagic acid to enhance its poor solubility combined with an improvement of its controlled delivery was attempted by some research groups [ , ] . in a recent work, pirzadeh-naeeni et al. reported the nanoencapsulation of ellagic acid in two different biopolymers (schizophyllan and chitin), which were then tested against mcf- breast cancer cells [ ] . in this case, the controlled release of ellagic acid improved the cytotoxicity when compared with non-encapsulated ellagic acid. it also reduced the progression of tumor cells. anthocyanidins are water-soluble pigments found in plants. they are responsible for leaves, flowers, and fruit colors. some fruits included in the human diet are rich in anthocyanins: blueberries, raspberries, black rice, and black soybeans (normally known as dark fruit). the term anthocyanin was coined in by ludwig clamor marquart, a german pharmacist, to denote the blue pigment of red cabbage (brassica oleracea) [ ] . table . summary of various polyphenols, their chemical structures, and their anticancer effects. resveratrol dna protection against reactive oxygen species (ros), trap the hydroxyl and superoxide groups and the free radicals produced into the cells. inhibition of a lung cancer cells with the activation of caspase- . u. s. department of health and human services public health service food and drug administration status: bulk ingredient for human prescription compounding. [ , ] other colored fruits and vegetables, is one of the most common anthocyanidins [ , ] . the antitumour activity of delphinidin has been demonstrated by numerous researchers. in , jeong et al. studied the effect of delphinidin in prostate cancer treatment. they found that delphinidin increased the activity of caspase- , - , and - , in effect causing the death of cancer cells. moreover, they demonstrated that delphinidin intensified the roles of genes that induce the apoptosis of cancer cells and decreased the activity of some genes that dissuade killing the cancer cells [ ] . alternatively, delphinidin obstructs the progression of skov ovarian cancer cells in vitro by decreasing the akt pathway (a signal transduction pathway) activation, which can in result activate numerous factors that play a critical role in cancer migration [ ] . the chemical structure of the polyphenolic compounds mentioned in this review, their anticancer effects, and the corresponding references are summarized in table . as indicated in the table, the administration of resveratrol and quercetin has been approved by the food and drug administration (fda). [ [ ] [ ] [ ] , jeong et al. studied the effect of delphinidin in prostate cancer treatment. they found that delphinidin increased the activity of caspase- , - , and - , in effect causing the death of cancer cells. moreover, they demonstrated that delphinidin intensified the roles of genes that induce the apoptosis of cancer cells and decreased the activity of some genes that dissuade killing the cancer cells [ ] . alternatively, delphinidin obstructs the progression of skov ovarian cancer cells in vitro by decreasing the akt pathway (a signal transduction pathway) activation, which can in result activate numerous factors that play a critical role in cancer migration [ ] . the chemical structure of the polyphenolic compounds mentioned in this review, their anticancer effects, and the corresponding references are summarized in table . as indicated in the table, the administration of resveratrol and quercetin has been approved by the food and drug administration (fda). improves the cellular uptake of doxorubicin and reduces its side effects. apoptosis of mda-mb- breast cancer cells. [ , ] of cancer cells and decreased the activity of some genes that dissuade killing the cancer cells [ ] . alternatively, delphinidin obstructs the progression of skov ovarian cancer cells in vitro by decreasing the akt pathway (a signal transduction pathway) activation, which can in result activate numerous factors that play a critical role in cancer migration [ ] . the chemical structure of the polyphenolic compounds mentioned in this review, their anticancer effects, and the corresponding references are summarized in table . as indicated in the table, the administration of resveratrol and quercetin has been approved by the food and drug administration (fda). kappa-light-chain-enhancer of activated b cells (nf-κb) signaling through protein kinase c delta type (pkcδ) inactivation. upgrades the cytotoxicity of trastuzumab and increases the specificity to breast cancer cells. synergistic effects of honokiol and doxorubicin in breast cancer by suppressing the metastasis of carcinogenic cells and apoptosis induction. apoptosis of multiple myeloma cancer cells. [ , ] p-coumaric acid apoptosis of hct- colon cancer cells through ros mitochondrial pathway. inhibits the growth of snu- gastric cancer cells. [ [ ] [ ] [ ] kaempferol induces the apoptosis and dna damage in mda-mb- breast cancer cells by the upregulation of h a histone family member x (γh ax), caspase , caspase , and the protein serine/threonine kinase (p-atm). induces the apoptosis of lncap prostate cancer cells. impedes the proliferation of cancer cells. luteolin synergistic effects of luteolin and oxaliplatin: stops the proliferation of gastric cancer cell. promotes apoptosis and stops the proliferation of colorectal cancer cells. [ , ] u. s. department of health and human services public health service food and drug administration status: drug for further processing. promotes apoptosis of pancreatic cancer cells by increasing intracellular ros. damages dna of hela cervical cancer cells. inhibits the growth of cancer cells and induces its apoptosis. [ , ] luteolin synergistic effects of luteolin and oxaliplatin: stops the proliferation of gastric cancer cell. promotes apoptosis and stops the proliferation of colorectal cancer cells. [ , ] naringenin [ , ] naringenin apoptosis of breast cancer cells by the increase of the activity of caspase- and caspase- . suppression of skin cancer cells. [ , ] molecules , in conclusion, the exceptional antioxidative properties make polyphenols strong candidates for agents used in various types of cancer treatments. actually, the anticancer effects of several polyphenolic compounds have been mainly studied in in vitro cancer cells and in preclinical animal models. nevertheless, there are very few clinical data on many of the polyphenols application as anticancer medicines (clinical studies on cancer therapy involve only the most common polyphenols such as resveratrol, curcumin, and quercetin). nowadays, the vast majority of these clinical studies are still in progress. the research on cancer therapies involving varied polyphenol families, and particularly flavonoids, has contributed to the development of natural medicines that are less aggressive than conventional anticancer drugs. in fact, various research works proved that polyphenols could be used anthocyanidins have varied functions in plants: attracting pollinating insects, preventing the freezing of fruits such as grapes, and protecting plants against harmful uv radiation [ ] . moreover, these kinds of compounds are widely used in the food industry (preparation of food coloring, a parameter for determining wine quality) and in medical industry (decreased risk of contracting various diseases such as obesity, improved memory and age-related deficiencies, or improvement of the immunological system) due to their chemical and physical properties [ ] . some anticancer properties of anthocyanidins extracted from the plant cyanomorium coccineum have been recently described by rescigno et al., which demonstrated the antiproliferative effect of anthocyanidins against different leukemia cell lines [ ] . delphinidin, which can be found in red cabbage, grapes, berries, and sweet potatoes among other colored fruits and vegetables, is one of the most common anthocyanidins [ , ] . the antitumour activity of delphinidin has been demonstrated by numerous researchers. in , jeong et al. studied the effect of delphinidin in prostate cancer treatment. they found that delphinidin increased the activity of caspase- , - , and - , in effect causing the death of cancer cells. moreover, they demonstrated that delphinidin intensified the roles of genes that induce the apoptosis of cancer cells and decreased the activity of some genes that dissuade killing the cancer cells [ ] . alternatively, delphinidin obstructs the progression of skov ovarian cancer cells in vitro by decreasing the akt pathway (a signal transduction pathway) activation, which can in result activate numerous factors that play a critical role in cancer migration [ ] . the chemical structure of the polyphenolic compounds mentioned in this review, their anticancer effects, and the corresponding references are summarized in table . as indicated in the table, the administration of resveratrol and quercetin has been approved by the food and drug administration (fda). in conclusion, the exceptional antioxidative properties make polyphenols strong candidates for agents used in various types of cancer treatments. actually, the anticancer effects of several polyphenolic compounds have been mainly studied in in vitro cancer cells and in preclinical animal models. nevertheless, there are very few clinical data on many of the polyphenols application as anticancer medicines (clinical studies on cancer therapy involve only the most common polyphenols such as resveratrol, curcumin, and quercetin). nowadays, the vast majority of these clinical studies are still in progress. the research on cancer therapies involving varied polyphenol families, and particularly flavonoids, has contributed to the development of natural medicines that are less aggressive than conventional anticancer drugs. in fact, various research works proved that polyphenols could be used as chemotherapy adjuvant agents in cancer therapies. however, the process of discovering the polyphenols' mechanisms of action as 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prodelphinidins from dietary sources influence of fruit juice processing on anthocyanin stability delphinidin induces apoptosis via cleaved hdac -mediated p acetylation and oligomerization in prostate cancer cells delphinidin inhibits bdnf-induced migration and invasion in skov ovarian cancer cells this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -do px gq authors: mucha, artur; drag, marcin; dalton, john p.; kafarski, paweł title: metallo-aminopeptidase inhibitors date: - - journal: biochimie doi: . /j.biochi. . . sha: doc_id: cord_uid: do px gq aminopeptidases are enzymes that selectively hydrolyze an amino acid residue from the n-terminus of proteins and peptides. they are important for the proper functioning of prokaryotic and eukaryotic cells, but very often are central players in the devastating human diseases like cancer, malaria and diabetes. the largest aminopeptidase group include enzymes containing metal ion(s) in their active centers, which often determines the type of inhibitors that are the most suitable for them. effective ligands mostly bind in a non-covalent mode by forming complexes with the metal ion(s). here, we present several approaches for the design of inhibitors for metallo-aminopeptidases. the optimized structures should be considered as potential leads in the drug discovery process against endogenous and infectious diseases. amino-terminal modifications of nascent polypeptides are the most common processing events, occurring on nearly all proteins. aminopeptidases are a class of enzymes that play a pivotal role in this processing. aminopeptidases (ec . . e hydrolases/peptidases/aminopeptidases, according to the classification of the international union of biochemistry and molecular biology) are proteolytic enzymes that hydrolyze peptide bonds from the amino termini of polypeptide chains. they may hydrolyze the first peptide bond in a polypeptide chain with the release of a single amino acid residue (aminopeptidases in a strict sense) or may remove dipeptides or tripeptides (dipeptidyl-and tripeptidylpeptidases) from polypeptide substrates. regarding catalytic mechanism, most of the aminopeptidases are metallo-enzymes but cysteine and serine peptidases are also included in this group. this review focuses on the strict metallo-aminopeptidases because they constitute the largest and the most homogenous class of these enzymes and use one or two metal ions in their active sites to specifically release the n-terminal amino acid residues of polypeptides and proteins. since over papers dealing with aminopeptidases have been published (medline database). aminopeptidases are ubiquitous enzymes widely distributed throughout the biological kingdoms and are found in many subcellular organelles, in cytoplasm, and as membrane components where they perform essential cellular functions. aminopeptidases act in concert with other peptidases to complete diverse proteolytic pathways. they play a vital role in a range of biological processes and disease situations. processes as distinct as angiogenesis, antigen presentation, neuropeptide and hormone processing, pregnancy and reproduction, protein turnover, memory, inflammation, tumor growth, cancer and metastasis, blood pressure and hypertension all involve one or more critical aminopeptidases. these enzymes can efficiently retrieve amino acids from dietary proteins and endogenous proteins degraded during protein turnover, thereby also covering a nutritional role. in addition to the book of hooper and lendeckel [ ] , which describes role of aminopeptidases in biology and medicine, several excellent reviews on various aspects of their biology and the application of their inhibitors have been published [ e ]. we therefore limit our review to selected recent achievements in this field, although we present this in a certain historical context. the classification of aminopeptidases has often been based on mechanism of catalysis, the structure of the active site, substrate specificity (broad or narrow) and molecular properties. the nomenclature of many peptidases has been determined by their preferences or requirements for a particular n-terminal amino acid. the rapidly accumulating data covering new representatives of proteolytic enzymes required the development of an integrated source of information [ ] . such a role was fulfilled by the merops database (http://merops.sanger.ac.uk/), which uses hierarchical, structure-based classification of these enzymes. this database relies on the fact that enzymes performing the same (or similar) chemical functions in different organisms generally turn out to have similar overall three-dimensional structures, and also show significant conservation of their amino acid sequences, polypeptide chain lengths and domain organization. in particular, in the regions of their active sites, a high degree of conservation of residue identities and structural positions is observed. in the merops peptidase information database each protease is assigned to a certain family on the basis of statistically significant similarities in amino acid sequence, and families that are thought to be homologous are grouped together into clans. clans consist of families of peptidases that are believed to share a single evolutionary origin, evidenced by similarities in their tertiary structures and/or their active site architectures. fifteen clans of metalloproteases have been identified, with metallo-aminopeptidases found in six which are designated as, ma (the largest one, containing over families), mf, mg, mh, mn and mq. the families in clan ma are united by the presence of an hexxh motif in which the two his residues are zinc ligands and the glu has a catalytic function. clans mf (two zinc ions in the active site), mg (with the pita-bread fold and containing two cobalt or two manganese ions in their active centers) and mq (typically with two zinc ions) consists of only one family of peptidases each (m , m and m , respectively). the mh clan forms the most heterogeneous group and contains a variety of zinc-dependent exopeptidases. their structures show similar protein folds and are co-catalytic zinc peptidases containing two atoms of zinc per molecule, which have five amino acid ligands. clanmn contains only one enzyme e damino acid-specific aminopeptidase from bacillus subtilis. although metallo-aminopeptidases occur in all types of organisms, the mammalian enzymes were amongst the first proteases discovered in tissues and therefore they have been most intensively studied. human enzymes are particularly increasing in interest since the alterations in their function and regulation underline many human diseases. for example, leucine aminopeptidases (laps, ec . . . ), belonging to m family, have been the most extensively studied because they play a key role in the metabolism of proteins and biologically active peptides. these enzymes, perhaps the first that were isolated, are cytosolic exopeptidases of broad substrate specificity that are ubiquitous in nature being present not only in animals, but also in plants and bacteria [ ] . they have medical and biological importance because of their functions in the metabolism of hormones and neurotransmission, cell maturation, and turnover of proteins, including utilization of exogenous proteins as nutrient substances and elimination of nonfunctional proteins. human lap is important in processing of antigenic peptides and in determination of immunodominance of various peptides [ e ] as well as in development of human eye lens cataract [ ] . the protease plays a vital role in progression of cancers [ ] . it may also have an important function in early events of hiv infection and thus serum activity of this enzyme may be useful as a surrogate marker for hiv infection and response to chemotherapy [ ] . another example of medically important enzyme is microsomal aminopeptidase (belonging to m family), known also as aminopeptidase n, cd or alanyl aminopeptidase (ec . . . ) [ ] . in biological systems, their primary peptide substrates include a wide variety of neuropeptides and hormones [ , ] . it has been established as a myelomonocytic marker in leukemia typing [ ] , as a receptor for human coronavirus e and cytomegalovirus [ , ] , as a mediator of both inflammation and cell invasion [ ] , as a regulator of blood pressure and the pathogenesis of hypertension [ ] , and as a regulator of analgesia via metabolism of endorphins and enkephalins [ ] . furthermore, it also regulates il- bioavailability in the endometrium and therefore may contribute to the process of angiogenesis [ ] . it also plays key roles in physiological and pathological processes, such as embryogenesis, immune responses, angiogenesis, tumor cell invasion, and metastasis [ ] . methionine aminopeptidases (aminopeptidase m, metaps, ec . . . ), belonging to m family, are an example of peptidases that exhibit narrow specificity [ ] . generally they are responsible for the removal of methionine from the amino-terminus of newly synthesized proteins. they maintain stringent specificity for the n-terminal methionine and accept no other natural amino acid residues. they also have a strong preference for small and uncharged second residues in peptide chains. since the mammalian enzymes play a critical role in the regulation of post-translational processing and protein synthesis they play an important role in the development and malignancy of different types of cancer [ e ]. human aminopeptidase m is also involved in neurofibromatosis, one of the most common tumor predisposition syndromes [ ] . although scarce, there are also reports on aminopeptidase isolation and characterization from other vertebrate species, as exemplified by recent findings in fishes (carp and red sea bream) [ , ] and birds (chicken) [ ] . far more information is known about insect aminopeptidase n, which is one of the membrane proteins identified as a receptor to cry proteins in various species [ e ] . cry proteins produced by bacillus thuringiensis are toxic to insects and thus this strain is exploited commercially as a bioinsecticide. aminopeptidases involved in the degradation of insect neuropeptides have been also studied in some respects [ ] . the other groups of metallo-aminopeptidases explored intensively are of bacterial origin. the first studies on these enzymes were carried out over years ago, and since then a large number of aminopeptidases of microbial origin have been characterized. they may be localized in cytoplasm, on membranes associated with the cell envelope or secreted into the extracellular media [ ] . the interest in these enzymes stems from their potential to act as targets to combat bacterial diseases. in this respect, a wide variety of structurally diverse aminopeptidases have been recently isolated and characterized from a range of bacterial species. these include: aminopeptidase p isolated from common strain of escherichia coli [ ] , aminopeptidase m from pathogenic mycobacterium tuberculosis [ ] and leucine aminopeptidase from helicobacter pylori [ ] , cold-active aminopeptidase from psychrotropic colwellia psychrerythraea [ ] , a thermophilic enzyme from geobacillus thermoleovorans [ ] , an extracellular zinc metalloprotease and putative virulence factor involved in pathogenicity of the fish pathogen vibrio anguillarum [ ] , and a lysine-specific enzyme from unusually resistant, hyperthermophilic archaeon pyrococcus furiosus [ ] . this clearly indicates that bacterial aminopeptidases are widely distributed and are of vital importance. in recent years there has been a considerable interest in aminopeptidases of parasitic protozoans, which cause important diseases in humans, animals and birds. the alanine and leucine aminopeptidases from plasmodium falciparum, a causative agent of malaria, the most significant parasitic disease of humans, are the most comprehensively studied [ , ] . these aminopeptidases are promising targets for designing anti-malarial drugs (for a recent review see [ ] ). another important enzyme of plasmodium e aspartyl aminopeptidase is being considered as an additional target for drug design [ , ] . intensive studies on the role and biochemistry of aminopeptidases isolated from other parasitic organisms, including legionella pneumophila (causative agent of legionnaires' disease) [ ] , eimeria tenella (causes hemorrhagic cecal coccidiosis in young poultry) [ ] , babesia gibsoni (parasite found in red blood cells and transmitted by ticks) [ ] and microsporidia (causing diseases in immunosuppressed patients) [ ] are ongoing. diseases caused also by trematodes, commonly known as bloodflukes, affect hundreds of million people in impoverished areas of africa, central and south america and east asia, with those caused by schistosoma spp. are considered by the world health organization as second in importance only to malaria. leucine aminopeptidase is thought to play a central role in hatching of the miracidium from the schistosome egg and therefore is intensively studied as candidate for drug design [ , ] . similar motivation has driven studies on the leucine aminopeptidase from paragonimus westermani, a tissue-invading trematode that causes inflammatory lung disease as well as systemic infections including cerebral invasion in carnivorous mammals [ ] . specific parasite aminopeptidases might also be considered as targets for vaccine design as shown for fasciola hepatica [ ] , a vector of an important freshwater snail-borne helminthiasis that produces a chronic liver infection of cattle and sheep. understanding the mechanism of action for each family of metallo-aminopeptidases is of a key importance to rational design of more potent and more specific inhibitors of these enzymes and, consequently, to obtain drugs of improved properties. therefore, a substantial effort has been made in studying the mode of binding of their substrates and transition state inhibitors in enzymatic binding sites, as well as in the elucidation of the three-dimensional structure of active sites and the detailed mechanisms of catalyzed reactions. a feature common to all metallo-aminopeptidase active sites is that the metal ion (in most cases zinc) is surrounded by a shell of hydrophilic groups that is embedded within a larger environment of hydrophobic groups. in addition, amino acid side chains serving as ligands usually form hydrogen bond contacts with neighboring residues, perhaps to preorder the metal ion binding site and to decrease its entropic cost of binding. the structures of active sites suggest that a number of reaction paths are possible. two catalytic roles of metal ions might be considered. first, they might stabilize a highly reactive hydroxide ion, thereby ensuring that an activated nucleophile is available for catalysis at physiological ph (mechanism a in fig. ) . second, the positively charged metal ion may serve as an electrophilic catalyst complexing an oxygen atom of the scissile peptide bond and facilitating the nucleophilic attack of water molecule (mechanism b in fig. ). there is also a possibility that the active site glutamate (especially in the case of m family of peptidases) acts as a nucleophile resulting in formation of a covalent enzymeeinhibitor complex followed by its fast hydrolysis by water (mechanism c in fig. ). the glutamate assisted process is being considered as a less probable mechanism than a and b. in the case of the enzymes containing binuclear metal centers the substrate carbonyl oxygen is coordinated to one of them and a hydroxide ion bridging two metal ions acts as the nucleophilic agent (mechanism d in fig. ). this may explain the fact that several dinuclear metallopeptidases retain some catalytic activity when converted into mononuclear . catalytic roles considered for the metal (zinc) ions in the mechanism of metalloaminopeptidases action: stabilization of a highly reactive hydroxide ion (mechanism a), complexation of the oxygen atom of the scissile peptide bond to facilitate the nucleophilic attack of a water molecule (mechanism b). fig. . alternatives of catalytic mechanisms considered for metallo-aminopeptidases: glutamate acting as a nucleophile e formation of a covalent enzymeeinhibitor complex and its fast hydrolysis (mechanism c), bridging of a hydroxide ion in binuclear metal centers (mechanism d). ones but typically exhibit faster rates with dinuclear active sites. finally, in all cases the additional role of a metal ion is to stabilize developing negative charge(s) in the transition state of the catalytic mechanism. there is no single method for the elucidation of the mechanism of certain enzymatic reaction. thus, a combination of several methods is required. enzyme kinetics cannot prove which modes of catalysis are used by an enzyme. however, some kinetic data can suggest possibilities to be examined by other techniques. the evaluation of the role of metal ion is usually studied by metal exchange technique. in particular, the role of each metal ion in the dinuclear sites of aminopeptidases from the m family towards peptide hydrolysis was studied by kinetic and spectroscopic methods after replacement of one of active site zinc ions by mn(ii), co(ii), ni(ii), zn(ii), and cd(ii) [ e ] . important data about detailed mechanisms of action of aminopeptidases are also gained by production of altered enzymes by means of site-directed mutagenesis [ e ] . good examples are the studies on the role of active site glutamic acid in various aminopeptidases of m family [ , ] . this was achieved by replacing glutamic acid either by structurally related aspartic acid (possessing an acidic side group), glutamine (lacking an acidic side group), or structurally different alanine, and studies of kinetic properties of the mutated enzyme. the method that provides the most important insights into domains organization and architecture of active sites of aminopeptidases, and thus into mechanisms of their action, is crystallography. in that respect crystal structures of native enzymes and those complexed with small ligands are extremely useful (for representative recent examples see [ e ]), especially those determined for enzymes bound with inhibitors being considered as transition state analogues [ , , e ] . there is also an emerging power in the application of computeraided methods as a mean to study mechanisms of enzymatic reactions. the calculations enable the researcher to choose one of the several possible reaction pathways (and thus to determine reaction mechanism) and to establish the structures of transition states. these methods are based on the knowledge of enzyme threedimensional structure available either by crystallographic methods or obtained by computations using homology approach techniques (for representative examples considering metallo-aminopeptidases see [ e ]). besides the elucidation of the functional roles of active-site residues, an estimation of the environment effects are also possible. in the case of metallo-aminopeptidases such popular techniques such as studies employing modified substrates [ ] , studies on isotopic effects [ ] or construction of the chemical models of enzyme active sites [ ] are quite scarce. due to their association in several medical disorders, metalloaminopeptidases are considered important targets for the design of inhibitors, which could potentially enter clinical trials as candidates for drugs. the presence of the metal ion(s) in the active center has determined a general strategy applied for the development of such synthetic ligands. these possess two fundamental structural features, a specific war-head portion dedicated to recognize the active site of the enzymes and specific functional groups that create appropriate complexes with the metal ions. a metal ligand fragment can be incorporated into a backbone containing optimized side chain(s) that are able to interact with the enzyme binding pocket(s). as the result, non-covalent inhibitors of the amino acid/ peptide structure (natural substrate, transition state or product analogues) have appeared the most suitable for this purpose. indeed, the majority of compounds designed to date have such characteristics. bidentate tetrahedral phosphonates and aldehydes (hydrated in the gem-diolate form), bidentate planar carboxylates and hydroxamates, as well as monodentate thiols, are classical examples. recently, a variety of promising heteroaromatic or miscellaneous (heteroaromatic based sulfonamides/carboxylates/amides) inhibitors of metallo-aminopeptidases have being identified from random screening of compound libraries. their structure usually involve a bidentate n,n, n,o or o,o donor system incorporated into a rigid hydrophobic environment, and thus such compounds also act in a non-covalent mode. finally, natural products are a also a source of the appropriate effectors of this group of peptidases, with bestatin being a prototypical representative. its peptide-like framework offers a choice of heteroatom groups that get involved in an active site metal ion complexation. other natural non-covalent heteroatom-rich systems are based on terpene or polyphenol scaffolds. fumagillin and ovalicin are quite unique examples of very specific inhibitors that possess an electrophilic moiety (an epoxide) capable of reacting with the nucleophilic his side chain in the active site. the proven medicinal importance of aminopeptidase n (apn, cd ), leucine aminopeptidase (lap) and methionine aminopeptidases (metaps) has resulted in the focus on design of inhibitors primarily for these three enzymes and extensive thematic reviews have been recently published [ , e ] . here, we present a selection of several recent and historic approaches for design and optimization of the inhibitors of metallo-aminopeptidases, which could serve as lead compounds in the future studies of this group of proteases. among de novo constructed targeted molecules, organophosphorus compounds, namely a-aminoalkanephosphonates (general formula , fig. ) and phosphorus containing pseudodipeptides (predominantly phosphinic, ), have probably contributed the most to the inhibition studies on the neutral aminopeptidases: apn (alanyl m ) and lap (leucine m ). although the phosphonate/phosphinate group is a rather weak zinc complexing moiety, it offers other advantageous structural and electronic features. similar to other amino acid and peptide mimetics used as protease inhibitors, this is the effect of the incorporation of a covalent or non-covalent binding group (here involved in coordination of a catalytic metal ion(s) in the enzyme active site) into a substrate structure. for the phosphorus modified compounds it is also uniquely combined with its tetrahedral shape that is considered to mimic the high energy transition state of the peptide bond hydrolysis. additionally, the p side chain of the aminophosphonic acid analogues (or more effectively, both p and p residues of the pseudopeptides phosphoryl moiety) gives further possibility of structural optimization of substituents interacting with the s and s binding pockets of the enzyme (fig. ) fig. ), appeared to be efficient inhibitors of lap with a k i ¼ . [ ] and . mm [ ] for the r (l) enantiomers. the d (s) antipodes were strongly discriminated, by e orders in magnitude for the given examples. non-coded arylalkyl derivatives, exemplified by phosphonic homophenylalanine ( ) and homotyrosine ( ), were bound preferentially to an even slightly greater extent (k i ¼ . and . mm, respectively, for the racemic mixture, fig. ) [ ] . promising affinity (k i . mm) was also found for extended linear homologues, namely -amino-nhexanephosphonic [ ] and -amino-n-octanephosphonic acid [ ] . these results indicate that the s binding pocket of the leucine aminopeptidase can accommodate hydrophobic ligands even bulkier than indicated by its substrate preferences (the hydrolase cleaves substrates with the broad band specificity, however, those of the hydrophobic character at the n-termini, like leucine, methionine, isoleucine, valine, etc., are processed noticeably faster [ , ] ). somewhat similar situation is observed for the alanyl aminopeptidase apn, both the mammalian one [ ] and the orthologue from a lower organism, the protozoan p. falciparum [ ] . for example, phosphonic amino acid analogues of strong hydrophobic character, such as phenylalkyl or (cycloalkyl)alkyl (compounds and , fig. ), inhibited the porcine kidney enzyme with the k i values at low micromolar range [ ] . an interesting attempt of mapping of the apn s binding pocket and rationalisation of these data in the context of the substrate-inhibitor structural relationship has been recently undertaken [ ] . the specificity of apn was determined using an extensive collection of fluorogenic substrates bearing both natural and non-natural p residues. then, the obtained kinetic parameters were correlated with the activity of the corresponding a-aminophosphonic inhibitors. surprisingly, not the turnover velocity (expressed by the k cat/ k m ) but only the strength of substrate binding (described by the k m value) predicted the most reliably structural features responsible for the inhibitory potency. thus, the appropriate p residue incorporated into the a-aminophosphonate core was proved to be indispensable for the tight binding of a ligand to apn. because of the resemblance in the substrate specificity, the two aminopeptidases (lap and apn) are frequently studied together to refer the selectivity of newly developed ligands [ e ]. in general, a-aminoalkanephosphinic acids are much more potent inhibitors of the leucine aminopeptidase. for example, hydrophobic aliphatic compounds, such as and ( fig. ) , expressed an affinity of more than two orders of magnitude higher for lap compared to apn as indicated by the appropriate k i values [ ] . the reason for this observation seems to be the presence of the two zinc ions in the binding site of the cytosolic aminopeptidase (lap contain two zinc ions while apn contains one). involvement of the necepo portion in interactions with both metal ions definitely results with a tighter binding. contrarily, the leucine aminopeptidase does not readily accept p substituents containing a nitrogen atom. thus, to achieve a high differentiating factor in favor of apn, bulky hydrophobic residues should be appropriately modified with a heteroatom. compounds and ( fig. ) , bearing an additional amino moiety, was obtained by aziridinephosphonate ring opening with an amine and are low micromolar inhibitors of apn but do not effect lap [ ] . it is worth noting that among the phosphorus analogues of amino acids studied so far, n -cyclhexyl- , -diaminoethanephosphonic acid ( ) appeared the most active towards apn. the availability of a broad collection of the applied a-aminophosphonates allowed a systematic structureeactivity relationship in this context of the s binding pocket preferences and the specificity of lap and apn. however, to achieve more significant inhibition these compounds needed to be extended to interact with the s pocket as well. three kinds of phosphonate elongation/modification were envisaged to provide such dipeptidic transition state mimetics. formally, their structure is the result of the replacement of the scissile amide bond by the phosphonodepsi, phosphonamidate or phosphinate moiety. the potential of all three variations for aminopeptidase inhibition was evaluated in detail for the leu-leu mimetics and lap as the target [ , ] . the synthesis of the phosphonate analogue (compound , x ¼ o, fig. ) was described in the early work of bartlett, however, the compound showed only moderate potency towards the enzyme studied [ ] . despite this, a systematic computer-aided approach was undertaken by grembecka et al. to design new generations of more active inhibitors. the methodology was preliminary validated to rationalize the structureeactivity relationship obtained for various phosphorus containing amino acid analogues [ , ] . then, when applied for pseudodipeptides, it positively confirmed the idea of addition of the p portion, albeit only via pen or pec bonding [ ] . pseudodepsidipeptide bond (peo) was found to be unfavourable because of entropic effects upon inhibitor binding and oxygeneoxygen electrostatic repulsions with the carbonyl of ala . the two other types of analogues ( , x ¼ nh and , x ¼ ch , fig. ) were synthesized and evaluated [ ] . disappointedly, the fully deprotected phosphonamidate appeared to be unstable at ph below . the hydrolysis of the pen bond that occurred, which effected two component amino acids, was clearly correlated with the presence of free neighboring amino group (from the other side, crucial for the effective binding) [ ] . thus, despite being the most promising compounds according to the computed predictions (compound was theoretically calculated to bind with the affinity of k i ¼ nm), phosphonamidates were excluded for practical reasons. in turn, phosphinic pseudodipeptides exhibited perfect hydrolytic stability and inhibition constants in nanomolar range, and were ranked among the most effective ligands of lap reported to date. the mixture of two diastereoisomers of the leu-leu analogue showed k i ¼ nm, similarly to the phosphinate hphe-phe and hphe-tyr mimetics, both containing arylalkyl p and p residues (compounds and , respectively, fig. , tested as the mixture of four diastereoisomers) [ ] . chiral chromatography performed for compound allowed separation and assignment of the activity of its individual stereoisomers. the final k i value for r,s- , counterpart of the l,l natural peptide configuration, was determined as nm [ ] . interestingly, compounds and , particularly the latter one, appeared also effective inhibitors of apn (k i ¼ nm and nm, respectively, for the mixture of four diastereoisomers) [ ] . clear preference for the tyr residue at the p position indicated the significance of the terminal phenolic oh group. this observation was explained by formation of a very specific hydrogen bond (to the carboxylate of glu of apn) with the use of a model homologous to the leukotriene a hydrolase structure. further exploration of the p structure and its termini, in the context of lap versus apn selectivity, was undertaken via a parallel n-alkylation strategy of appropriate amino acid building blocks. unfortunately, the final products appeared only moderate and poorly selective inhibitors (k i for both enzymes varied at . e mm range) [ ] . importantly, phosphinic pseudodipeptides were found to be excellent inhibitors of parasite counterparts of lap and apn and useful tools for their validation as potential targets in a novel treatment of malaria [ ] . p. falciparum m and m aminopeptidases are responsible for the cleavage of the neutral residues in the terminal stages of the host haemoglobin degradation. thus, being a limiting stage in generating amino acids essential to parasite growth and development, they represent an attractive target for the development of novel anti-malarial drugs. compounds and inhibited recombinant m enzyme with the potency superior to that observed for the mammalian peptidase (k i ¼ . and . nm, respectively) [ ] . the affinity of measured for the pfm was also elevated (k i ¼ . nm) when compared to porcine apn (k i ¼ nm). in addition, the phosphinates efficiently controlled the growth of p. falciparum in culture, including malaria cells lines that were resistant to the well-known anti-malarial chloroquine. finally, in vivo studies using a non-lethal plasmodium chabaudi murine malaria model demonstrated that treatment of mice with compound reduced infection by % compared with controls [ ] . recently, this compound was co-crystallized with pfm [ ] and pfm [ ] to give an insight into the active site architecture and the mechanism of action of both aminopeptidases which will greatly facilitate the design of new optimized ligands with potential as anti-parasite agents. appropriate phosphinic pseudodipeptide building blocks can be further elongated by means of solution or solid-phase peptide synthesis to produce tripeptide analogues that possess an additional p substituent. such optimized compounds were reported to be the most potent organophosphorus inhibitors of the alanyl (apn) and glutamyl (aminopeptidase a, apa, ec . . . ) metalloaminopeptidases reported to date. chen et al. described a series of nanomolar ligands of mammalian apn, exemplified by the ala-phe-phe analogue ( , fig. anti-nociceptive activity thanks to the dual action against apn and neprilysin which caused an analgesic response after administration in mice [ ] . compound inhibited equipotently both the mammalian alanyl aminopeptidase as well as its bacterial orthologue. it also served as a ligand to resolve the three-dimensional structure of the latter enzyme [ ] . related phosphinate tripeptidic analogues were used for co-crystallization with the leukotriene a hydrolase/aminopeptidase, a prototypic m family member. as the result, substrate and transition state binding details together with a presumed catalytic mechanism, scarce data for the m enzymes, were reported [ ] . a phosphinic pseudotripeptide, the glu-leu-ala analogue ( , fig. , tested as mixture of four diastereoisomers), showed high affinity towards the zinc glutamyl aminopeptidase (mice recombinant) expressed by its k i equal to . nm [ ] . within a series of compounds, n-terminal pseudoglutamyl residue was found to be crucial for the high potency and selectivity. for example, the differentiating factor between apa and apn (k i ¼ mm) exceeded four orders of magnitude in favor of the apa. modifications of the phosphinate metal binding group in order to increase the number of coordination to the ion present in the active site can represent another approach to improve the potency of aminopeptidases inhibitors. this effect can be achieved by the introduction of a neighboring group containing a heteroatom that is additionally involved in metal complexation. four types of such modifications have been proposed and evaluated recently for the alanyl aminopeptidase apn [ , ] . they involved the application of a-aminoalkane-a -hydroxyalkanephosphinic acids (general formula , fig. ), bis-a-aminoalkanephosphinic acids ( ) , carbamoylated and thiocarbamoylated a-aminoalkanephosphinic acids ( and ). the structural variant was previously described by bartlett for lap but the inhibition achieved for the leu-leu analogue (r ¼ i- [ ] . more interesting results were reported for apn. combination of the hydrophobic residues p (r ¼ i-pr, i-bu, n-bu, ph or (ch ) ph) and p (r ¼ ph, (ch ) ph or ch (p-ome-c h )) allowed for regulation of the enzyme activity with the ic value to . mm (this result corresponded to k i ¼ nm) [ , ] . interestingly, all four structural variations e produced comparable, low micromolar or sub-micromolar ic values of inhibition. originally isolated from streptomyces olivoreticuli (md -c ) more than years ago by umezawa and co-workers, bestatin (( s, r)- -amino- -hydroxy- -phenylbutanoyl-l-leucine e ubenimex d , , fig. ) was one of the first potent inhibitors of metalloaminopeptidases with broad spectrum of action [ ] . bestatin has been extensively investigated in biological systems both in vitro and in vivo, which resulted in discovery of several interesting properties of this compound such as ability to induce apoptosis in cancer cells, anti-angiogenic, anti-malarial or immunomodulatory effects [ ] . presently, bestatin is on the market in japan where it is applied for treatment of cancer and bacterial infections. examples of successful inhibition of aminopeptidases by bestatin include aminopeptidase n (cd ), leucine aminopeptidase (lap), aminopeptidase b (ec . . . ) or lta hydrolase [ e ]. bestatin can act as slow (lap) or fast (apn) binding, competitive inhibitor of aminopeptidases [ ] . it resembles a phe-leu dipeptide substrate, however its phe residue is b-amino-a-hydroxy amino acid [ ] . this a-hydroxy group together with the neighboring carbonyl group coordinate a zinc ion, which results in a competitive active site-directed inhibition. bestatin is weaker inhibitor of aminopeptidases containing one metal ion in the active center (apn, apb) and much stronger of enzymes with two metal ions (lap). this feature is explained by larger amount of interactions made by inhibitor with both enzyme metal ions in the active center along with additional contacts made by side chains of the inhibitor in s and s pockets of the enzyme. to date several stereoselective, synthetic methods leading to desired bestatin diastereomer have been described [ e ] (see recent reviews presenting available synthetic methods for bestatin and some modifications [ , ] ). absolute configuration is a key issue responsible for good inhibitory effect and discrimination of the appropriate binding partners (substrates and inhibitors) for most of the proteases. diastereomer of bestatin with inverse configuration at carbon atom with hydroxy group (( r, r)- -amino- -hydroxy- phenylbutanoyl-l-leucine) is known as epibestatin and is frequently used as negative control in biological experiments [ ] . the structure of bestatin has also been a subject of several modifications in hope to improve its inhibitory and pharmacological properties. examples of such derivatives are bestatin thioamide (compound , fig. ) [ ] , para-hydroxybestatin ( ) [ ] or -thiolbestatin ( ) [ ] . the scaffold of bestatin was also used for the design of activity [ , ] . these compounds are tri-and tetrapeptides and are better inhibitors of aminopeptidase n when compared to bestatin. this is due to an increased amount of contacts made with s , s , s and s pockets of the enzyme and side chains of the inhibitors, which in most cases have very hydrophobic character (phe, leu, val). hydroxamic acids (n-hydroxyamides) can be considered as analogues of carboxylic acids and amides that uniquely combine the features of both of these groups. simultaneously, the hydroxamate moiety is an effective planar bidentate o,o metal chelating system. this close structural similarity to the products/substrates of the peptide bond hydrolysis and metal-complexing properties make it an attractive war-head for constructions of inhibitors targeted towards metallo-dependent proteases. the synthesis is not problematic and usually involves a one-step transformation of an acid or an ester with the use of an appropriate hydroxylamine derivative. accordingly, one-side optimized hydroxamate analogues of acids, amino acids and peptides have found biomedical relevance. the most promising applications have been associated with inhibition of matrix metalloproteinases as perspective targets for anti-cancer therapy. matrix metalloproteinases, involved in normal and abnormal tissue remodeling, angiogenesis and tumor metastasis, have been potently regulated by hydroxamates (e.g. batimastat, marimastat) that reached advanced phases of the clinical trials. these finally failed because of side effects associated with cross interactions with other metal containing proteins [ , ] . fundamental work on the metallo-aminopeptidases inhibition by a-aminohydroxamates was performed by chan et al. and concerned the leucine aminopeptidase [ ] . the derivatives of hydrophobic amino acids (exemplified by l-leu-nhoh, compound , fig. ) regulated the enzyme activity with the k i values of micromolar range. other c-terminally modified compounds, such as the l-leu amide, alcohol, or hydrazide, together with the amino acid alone, were much less potent, typically at least by a -fold ratio. these observations were consistent with a subsequent study by a series of phenylalanine derivatives targeted towards apn. compound (fig. ) appeared a moderate inhibitor of the enzyme [ ] . both l-leu-nhoh and l-phe-nhoh were overpowered by the corresponding thiols which indicated a much tighter individual sulfurezinc interaction than the bidentate oxygenezinc binding. since then, a choice of hydroxamic acid inhibitors (not necessarily based on the amino acid skeleton) of different metallo-aminopeptidases have been reported in the literature. a representative selection from recent examples is presented in fig. . compound , bearing a hydrophobic substituent, was identified by screening of a natural and synthetic compound library and appeared equipotent to bestatin for apn [ ] . compound controlled the basic fibroblast growth-factorinduced invasion of endothelial cells at low micromolar range. the hydroxamate was highly selective as it exerted no action towards members of the matrix metalloprotease family. interesting hydroxamate based inhibitors were also developed for the methionine aminopeptidases from different organisms. hu et al. described the synthesis of compounds possessing an additional substitution at the hydroxamate nitrogen atom [ ] . these n-hydroxydipeptides, derivatives of met-gly (compound , fig. ), allowed for structural optimization of both sides of the molecule. the products inhibited the bacterial as well as both forms of human metap, with a slight preference to e. coli enzyme. a cooperative action of the n-terminal amino group and two hydroxamate oxygen atoms towards two catalytic metal ions in the active center was suggested as the binding pattern. the bacterial form of metap served also as a model for comparison of the activity of -aryl-furane- -carboxylic acids with other variants of the c-termini [ ] . such heteroaromatic hydroxamic acids (exemplified by , fig. ) were found to be superior inhibitors to corresponding acids, esters, amides, hydrazides, alcohols and nitriles towards distinct metalloforms of the ecmetap . certain metallo-aminopeptidases are promising targets in anticancer therapy in humans, but they can also be exploited in the development of antibacterial, antifungal and antiparasitic agents. the zinc p. falciparum m aminopeptidase is being actively explored in this context (as described in the section devoted to organophosphorus compounds). the dual function amide-hydroxamate template was also used to target the malaria enzyme [ ] . the compounds developed consisted of three portions: hydroxamic acid termini dedicated for zn(ii) complexation, a hydrophobic a-substituent and the amide function that served for extensive structural diversification. the use of bulky hydrophobic amines for the amide formation (as exemplified by , fig. ) yielded potent inhibitors of the parasite enzyme, with the ic values in low nanomolar range [ ] . importantly, consecutive iterative optimization gave derivatives that were characterized by spectacular selectivity versus the mammalian orthologue. compounds were active in parasite growth inhibition and displayed good pharmacokinetic properties [ ] . tosedostat (chr- , , fig. ) [ ] seems to be the most attractive novel pharmacologically active product among hydroxamic acid metallopeptidase inhibitors. this orally available cyclopentyl ester prodrug is converted in vivo into the intracellularly active acid metabolite. the latter is a potent inhibitor of a number of aminopeptidases, including leucine aminopeptidase, aminopeptidase n, puromycin sensitive aminopeptidase and leukotriene a hydrolase/aminopeptidase (with the ic in nanomolar range) [ ] . chr- exerted anti-proliferative effects against tumor cell lines in vitro and in vivo, and exhibited times more potency than bestatin. the proposed mechanism of tumor cell killing involves depletion of amino acids by blocking protein processing/recycling [ ] . chr- is well tolerated and can be safely administered at doses that result in the metabolite activity as evidenced in preclinical models [ ] . recently, tosedostat demonstrated promising efficacy in patients with acute myeloid leukemia and myelodysplastic syndrome in the phase ii of clinical trials [ ] . activation of the latter and its subsequent substitution with a sulfur containing nucleophile [ e ]. alternatively, the mitsunobu reaction is utilized as the oh replacement procedure [ , ] . as the optically active substrates are easily available and there is no risk of racemization, final products are obtained in the enantiomerically pure form. when the mode of binding to metalloaminopeptidases is considered, -aminothiols are typical analogues of the n-terminal portion of the peptide substrates that act in reversible competitive manner. the thiol group is a termini designed to interact non-covalently with the catalytic metal ion(s). except for the functional groups essential for the polar contacts, aminothiols contain a side chain that occupies the s pocket. despite a structural simplicity and low molecular weight, the potency of these compounds against metallo-aminopeptidases is very high. the reason for this is a strong affinity of the sulfur atom for divalent soft acid metal ions, such as zn(ii). consequently, aminothiols exhibit much higher activity than amino acid analogues of the corresponding complexity, but containing other metal chelating moieties, such as phosphonate or hydroxamate ones. frequently, these compounds are discriminated with an affinity more than -fold ratio lower in comparison to the analogous thiols. in general, -aminothiols are one-handed inhibitors directed specifically towards the s subsite part. the availability of extended ligands that possess an additional pn portion is limited because of a complex synthesis and diastereomeric purity. nevertheless, -aminothiols have found some fundamental and practical applications connected with metallo-aminopeptidase inhibition. the most spectacular achievements are associated with the regulation of action of aminopeptidases a and n, enzymes involved in the brain renineangiotensin cascade that represent perspective targets in a hypertension therapy. a series of representative -aminothiols, derived from the natural amino acids, is given in fig. . l-lysine thiol (the absolute configuration s, compound ) was shown to be an extremely potent inhibitor of arginyl aminopeptidase (aminopeptidase b, apb) with a subnanomolar k i value [ ] . l-leucine thiol (compound ) binds to the same target but with a lower activity. more interestingly, it exhibited a -fold ratio stereochemical preference towards the enzyme for the natural configuration (k i ¼ mm for the d (r) enantiomer). unexpectedly, compound poorly inhibited leucine aminopeptidase (lap), what was explained by the use of the zinc-magnesium hybrid enzyme in these experiments [ ] . according to an earlier study, l-leucine thiol ( ) was shown to be a potent competitive inhibitor of the microsomal aminopeptidase from porcine kidney (apn, k i ¼ nm) [ ] . this compound was then used as a lead structure to optimize the size of a neutral hydrophobic p residue, in the context of construction of potentially analgesic dual inhibitors of neprilysin (nep) and apn [ ] . a broad series of inhibitors was synthesized that showed high equipotent efficacy for the aminopeptidase n (k i ¼ e nm). among them, the methionine thiol (compound , fig. ) appeared the most active in vitro. -aminothiols were at least four orders in magnitude more effective than carboxylates, phosphonates and hydroxamates of the corresponding structure [ ] . despite such potency, the use of selected compounds in intravenous administration did not induce anti-nociceptive responses in mice on the hot plate test, evidently because of difficulties to cross the bloodebrain barrier. indeed, the oxidation to the disulfide form to increase the lipophilicity made them efficient prodrugs. much more significant response was stimulated by application of a mixed prodrug which structure was disulfide based combination of two thiol inhibitors, each directed towards a certain enzyme, either apn or nep [ ] . elucidation of an intrinsic function in the brain and, therefore, the medical potential of selected aminopeptidases in the renineangiotensin system stimulated continuation of studies on aminothiols. these two metallo-aminopeptidases, namely apn and glutamyl aminopeptidase (aminopeptidase a, apa), are supposed to participate in metabolism of brain angiotensin ii and iii (angii and angiii). glu-thiol (compound , fig. ) [ ] was described as first efficient inhibitor of apa, however it was equipotent to apn (k i ¼ versus nm, respectively). to study the physiological importance of these enzymes, specific agents, discriminating between the two targets to a high degree are indispensable. in a quest for a selective inhibition, a number of -aminothiol derivatives were synthesized and evaluated [ , ] . it appeared to be a relatively simple task to identify the p residue responsible for high nanomolar potency of an apn ligand. an extended p hydrophobic portion, favorably terminated with a heteroatom group (as exemplified by compounds and , fig. ), represented such an option. the affinity of these compounds towards apa was of -fold ratio lower. anyway, according to recent data l-methionine thiol developed earlier [ ] gave even more for apa, respectively [ ] ). such a convenient modification was not found for the opposite case. a compromise was achieved for short side chains with an acid function other than carboxyl at the terminus (such as , fig. ) [ , ] . the affinity of the sulfonic acid for apa was not elevated in comparison to the lead, but a drop in potency measured for apn was more significant and finally resulted in a -fold discrimination ratio. these parameters were furthermore improved by exploration of the sn subsite of the aminopeptidase a. the synthesis of tripeptidomimetics that contained an optimum p substituent, attached as the -amino- -mercaptocarboxyl to the n-termini of dipeptide libraries, allowed determination of the advantageous p and p side chains [ ] . the s binding pocket accommodated hydrophobic residues whereas the s pocket preferred negatively charged residues. as a result, an exceptionally potent compound ( , fig. ) characterized by a striking , fold selectivity ratio, was identified. interestingly, the most active diastereoisomer exhibited non-natural configuration r at the p portion which was explained by sterical constrains of the whole molecule [ ] . despite a remarkable activity (a first subnanomolar thiol inhibitor of apa), compound was poorly bioavailable. a limited p fragment optimization starting from the lead gave a much simpler molecule: ( s, s)- -amino- -mercapto- -phenylhexane- -sulfonic acid [ ] . the substitution of the c atom with a hydrophobic arylalkyl fragment resulted with a satisfactory potent apa inhibition (k i ¼ nm) (however, the kinetic data for apn were not reported). over-activity of the renineangiotensin system in the brain has been implicated in the development and maintenance of hypertension. using specific agents described above, it was demonstrated that apa and apn are involved in the metabolism of angiotensin ii and iii, respectively, within this system. angiii ( e ) is generated from angii by apa assisted cleavage of the n-terminal asp-arg bond, whereas apn functions in subsequent inactivation of angiii. apa specific sulfonate increased the half-life of angii in mice, blocked the angii to angiii conversion which in turn controlled vasopressin release [ , ] . the dose-dependent decrease of blood pressure was achieved only by intra-cerebroventricular injection. contrarily, selective inhibitors of apn ( and ), administered by the same route, caused elevated blood pressure. thus, the level of angiotensin ii and iii in the brain, in particular that controlled by the aminopeptidases, was suggested to be a potential target of hypertension treatment [ , ] . to this end, the disulfide dimer of the lead was reported as a first orally available candidate for the therapy based on aminopeptidase a inhibition [ , ] . the majority of inhibitors discovered for metallo-aminopeptidases are compounds which interact with enzyme in a non-covalent way. however, for some enzymes covalent inhibitors have also been found. fumagillin (( z, e, e, e) -oxodeca- , , , -tetraenoic acid, , fig. ) and ovalicin (( s, r, s, r, s)- -hydroxy- -methoxy- -[ -methyl- -( -methylbut- -enyl)oxiran- -yl]- -oxaspiro [ . ] octan- -one, ), which are fungal metabolites inhibit methionine aminopeptidase type (metap ) by the formation of a covalent, irreversible bond between a reactive epoxide and his- present in the active center of the enzyme [ , ] . these compounds are not effective towards other aminopeptidases. selectivity towards metap and complete lack of selectivity towards metap is explained by difference in only one amino acid (ala- in metap and threonine in metap ) around the active center of these enzymes as determined by comparison of crystal structures [ , ] . this approach revealed also that tight binding of fumagillin and derivatives to metap is also a combination of several other interactions made by the inhibitor components with the surface around the active center of the enzyme. in biological studies fumagillin and ovalicin have been found as potent inhibitors of angiogenesis due to their inhibition of endothelial cell proliferation. this resulted in extensive sar studies and synthesis of several derivatives of these compounds. compound tnp- ( , fig. ) has entered clinical trials and was evaluated for application in anti-angiogenic therapy [ , ] . numerous competitive inhibitors of metallo-aminopeptidases that have been recently developed, frequently starting from leads identified by random screening (e.g. high throughput screening utilizing fluorogenic [ , ] and chromogenic assays [ , ] or virtual screening [ , ] ), are based on a heteroatom-rich fragment. such an appropriate heteroaromatic (or rarely heterocyclic) scaffold contains a set of the nitrogen, oxygen and/or sulfur atoms involved in coordination of the catalytic metal ion(s). structural rigidness and constraint of this portion of a ligand is often privileged for formation of hydrophobic interactions, specifically pep stacking, with the neighboring residues of the active site. the synthetic procedures used for obtaining such compounds should allow the combination with reliable methods of the substituent(s) diversification. this can be performed prior to a ring formation/ aromatization or by parallel decoration of the target scaffold with the use of a simple chemistry, such as the amide bond formation. an extensive structural optimization of certain scaffolds has led to very potent inhibition of selected aminopeptidases by surprisingly low molecular weight compounds (even below ). methionine aminopeptidases (particularly human metap ), are representative examples of successful application of this strategy. , , -triazoles were originally described in the patent literature as potent reversible non-peptidic inhibitors of the methionine aminopeptidase type [ , ] . variously substituted compounds bound to the cobalt form of metap with the affinity expressed by fig. ) and their biological relevance was also subsequently presented [ ] . iterative refinement of the inhibitor lead structure allowed the identification of compounds with the potency against metap in the picomolar range. systematic modifications of the key structural fragments revealed certain tendencies responsible for elevation or decrease in their activity. the and substitution of the aniline phenyl ring with small residues was the most favorable (compounds and , fig. ). contrarily, any changes in the optimal benzylthio s residue structure were found deleterious, similarly to methylation of any of the triazole nitrogen atoms (to a relatively smaller extent for the n ). selected derivatives inhibited human endothelial cell proliferation and the growth of new blood vessels in a model of angiogenesis. low molecular weight -aryl substituted , , -triazoles were found to act as almost equipotent reversible inhibitors of the human metap [ ] as described for , , derivatives. diversification of the position was performed by the use of various aromatic aldehydes that were transformed into the appropriate alkyne substrates for the cycloaddition. several effective ligands of the cobalt-activated metap were identified, showing the k i,app values down to nm. for example, k i,app ¼ nm for given in fig. corresponded to k i ¼ . nm and ic ¼ nm in full kinetic analysis [ ] . sar studies performed for the phenyl ring excluded bulky and substitution showed that only small alkyl chains were tolerated whereas the halogens were favorable in these positions (see for example compound in fig. ). contrarily, a addition of a bulky residue, even a heteroaromatic one, was well accepted and indicated some additional room in the active site. replacement of the whole phenyl ring by a substituted pyridyl was also profitable. the use of the pyrazole based scaffolds instead of the triazole one, revealed the key role of n and n atoms in high affinity. regarding their biological activity, the compounds inhibited human and mouse endothelial cell growth. enzyme-ligand crystal structures accompanied studies on the triazoles [ , ] . they revealed, among others, the significance of the p aromatic ring of a certain size (substituted or not, present in potent metap inhibitors) that should occupy the hydrophobic pocket formed by phe , his , tyr and his , and form the appropriate pep stacking. the size of the pocket (narrower for metap ) was postulated to be a discrimination factor between the two forms of the methionine aminopeptidase. the difference in the binding affinity is usually e orders in magnitude in favor for metap for the triazoles studied (for example, k i,app ¼ . nm versus nm for , ic ¼ nm versus nm for , fig. ). the structural data provided a rationale for sar discussion, but first of all confirmed the significance of the key interactions between n and n nitrogen atoms of the inhibitors and the cobalt ions. the molecular mechanism of triazole action was elucidated originally using the crystal structures of the staphylococcus aureus orthologue of the metap enzyme complexed with , -disubstituted- , , triazoles (such as compound , fig. ) [ ] . according to these results, each of the triazole n and n nitrogen atoms interacts with one of the two cobalt ions such that the nen bond is nearly co-linear with the co to co. the distances between the nitrogen and the coordinated cobalt are very similar to each other and tight (close to Å) which indicates strong interactions [ ] . the triazole ring forms a stacking contact with the hydrophobic face of the imidazole side chain of h , a residue that is believed to be involved in the catalytic process. somewhat surprisingly, the substituent of the substrate-like methionine structure of docks to the s subsite, and not to the s site. in turn, its natural position is occupied by the thiobenzyl residue as described above. metap is believed to be a dinuclear cobalt dependent enzyme as co(ii) activates all its known forms, however, the identity of the metal cofactor is still ambiguous. triazoles that inhibited exclusively the cobalt-activated human metap even with high nanomolar potency, although failed in cell proliferation assays, were used to shed light on this matter [ , ] . such metal dependent action indicates that other divalent ions must be considered as the relevant cofactors, particularly in respect to the fact that the architecture of the enzyme active site does not depend on the identity of the bound metal. in this context, manganese was postulated as the physiologically relevant metal [ ] . indeed, the triazoles investigated were not effective towards such activated metap form. contrary to triazoles, , -thiazoles appeared selective inhibitors of the methionine aminopeptidase type of both bacterial and human origin. the essential function of metap in bacteria suggested that the enzyme could be a promising target for the development of novel broad spectrum antibiotic agents. accordingly, thiazole based active compounds were identified by screening of libraries towards the e. coli methionine aminopeptidase. it is worth noting that selected leads did not represent typical heteroaromatic scaffolds. these were not the central core of the molecule, but rather a proximity group linked with another hydrophobic portion (frequently heteroaromatic as well) by a carbonecarbon, amide or carbonyl based bonding. thus, the complexing mode of the enzyme catalytic ion(s) was not as characteristic as for triazoles and usually involved participation of two neighboring heteroatom groups. the random screening of compounds by nan and coworkers led to the amide of pyridine- -carboxylic acid and thiazol- -ylamine ( , fig. ) that inhibited the e. coli metap with the ic ¼ mm and served as a perspective lead compound [ ] . all primary chemical mutation in the basic structure, such as change in position of pyridine substitution or heteroatom replacement by an isosteric group, appeared deleterious for the potency [ , ] . most probably, the combination of three electronegative groups that are involved in the metal ions coordination, namely the aromatic nitrogen of the pyridine ring, the carbonyl moiety in position and a heteroatom of the thiazole, forms a system essential for the tight binding. nevertheless, substitution of the position in pyridine with an amide or ester group appeared profitable in terms of optimization of interactions with the binding pocket. the affinity was elevated to nanomolar range, as for example for compound , fig. [ ] . importantly, introduction of a bulky cyclic or aromatic residue at the proximity of the amide allowed for efficient discrimination between the bacterial enzymes from different sources. for x ¼ benzoyloamino or cyclohexanecarbamino, by substituting the structure the selectivity factor between e. coli and the saccharomyces cerevisiae exceeded -fold ratio (ic ¼ . and . mm for ecmetap , respectively and ic > mm for scmetap for both compounds). wide optimization of the x moiety demonstrated that introduction of an additional heteroatom to the extended amide structure was also well tolerated ( , fig. ) [ , ] . finally, replacement of the whole pyridine ring by another thiazole system gave compound (fig. ) with low nanomolar potency [ ] . interestingly, -substituted pyridine- -carboxylic acid thiazol- -ylamides selectively inhibited human metap versus metap , as exemplified by a -fold differentiating factor for compound (fig. ) [ ] . despite their moderate potency in vitro, the treatment of tumor cell lines with such specific agents suggested a role of metap in g /m phase transition. furthermore, this study confirmed the previously reported results on variations in the metal binding in the active site of the co(ii)-ecmetap in the presence of the thiazole based ligands [ ] . according to the appropriate crystal structures, these compounds seemed to be responsible for driving a third metal ion into the enzyme active site. the auxiliary cobalt atom was not present in the native enzyme, even when crystallized in the presence of high concentration of the metal salt. the additional cobalt interacts with a single histidine residue of the enzyme, three water molecules at the entrance of the active center and two nitrogen atoms of the ligand (those of the pyridine and the amide). inhibitor binding is stabilized by several contacts, mostly of the hydrophobic character. obviously, such an unusual situation must be taken into consideration when sar of novel metap effectors is discussed and the in vitro versus in vivo experiments data are correlated (see below for another example). closely related thiazole inhibitors of the bacterial methionine aminopeptidase were discovered by the high throughput screening of a ten times larger library [ ] . the main goal of the study was identification of potent but predominantly selective inhibitors that could distinguish between various metalloforms of e. coli metap. indeed, thiazole based compounds exemplified by compound (fig. ) strongly inhibited the cobalt dependent enzyme and only moderately the manganese, iron or nickel ones [ ] . the opposite pattern of activity was found for other heteroaromatic compounds. -arylfuran- -carboxylic acids and their derivatives exhibited preferences towards mn(ii)-ecmetap [ , ] , whereas the cathecholcontaining thiazoles preferred fe(ii)-ecmetap [ ] . however, these two groups of compounds did not follow the mode of binding typical for heteroaromatic inhibitors of the cobalted form of metap. they did not employ the heteroatom(s) of the ring but instead binding involved the two proximity oxygen atoms, either of the carboxylate [ , ] or the phenol moieties [ ] , respectively, for complexing to the metal ion(s). direct combinations of two heteroaromatic rings represent another approach to developing methionine aminopeptidase inhibitors. these compounds bind the driven active site auxiliary cobalt ion by employing nitrogen atoms from each of the rings that are placed in co-planar manner. thiabendazoles (benzimidazoles substituted in position with the thiazole ring) and their derivatives (compounds e , fig. ) regulated the co(ii)-ecmetap activity with k i value in sub-micromolar range, although they appeared ineffective in vivo [ ] . this was explained with the use of the metap-ligand crystal structures that revealed variations in the metal binding in the active site [ , ] as described above for pyridine- -carboxylic acid thiazol- -ylamides. a family of related -( -pipyridinyl)pyrimidines was identified upon screening of a , member library for inhibitors of human and p. falciparum methionine aminopeptidases [ , ] . interestingly, the compounds were virtually equipotent towards both mammalian types of the enzyme (for example see , fig. ). similarly as before, they complexed to the auxiliary co(ii) that is normally not involved in the catalytic process [ ] . nevertheless, they appeared to be novel potential anti-malarial agents. three active isoforms of pfmetap were obtained after cloning, expression and purification, and tested in vitro. the inhibitors were remarkably selective towards the b type, with the highest obtained potency with ic ¼ nm. the most active compound inhibited also the p. falciparum proliferation of both chloroquine sensitive and dependent erythrocyte cultures with ic ¼ . and . mm, respectively [ ] . consequently, this compound was used in murine malaria models and positively confirmed pfmetap b as a promising target for development of new anti-malarials. boronic acids as inhibitors of aminopeptidases were described first by baker et al. for aeromonas aminopeptidase (ec . . . ) [ , ] . in this report simple aliphatic derivatives were used as competitive, transition state analogues that bound to the active center of enzyme with good efficiency. among five tested derivatives -butaneboronic acid ( , fig. ) was the best inhibitor of the enzyme. in another approach, shenvi described a series of a-aminoboronic acids as effective inhibitors of human enkephalin degrading aminopeptidase (heda), microsomal leucine aminopeptidase and cytosolic leucine aminopeptidase [ ] . the advantage of these inhibitors over simple aliphatic derivatives was the presence of the free amine group at carbon a, a feature that is known to improve binding of the ligand to aminopeptidases. detailed analysis of kinetic data for cytosolic leucine aminopeptidase revealed biphasic slow-binding inhibition mechanism of a-aminoboronic acids. this suggested that slow-binding step is responsible for formation of tetrahedral boronate molecule from trigonal boronic acid. the inhibitory activity of the tested derivatives ( e ) also strongly correlated with the side chain type used in the study (fig. ). aldehyde derivatives of amino acids have been also described as inhibitors of aminopeptidases. andersson et al. first reported this group of compounds as very effective transition states analogue inhibitors (upon hydratation they formed a gem-diolate involved in zinc complexation) of porcine cytosolic and microsomal leucine aminopeptidases [ ] . the most effective inhibitor described in this report was l-leucinal (compound , fig. , k i ¼ nm for lap and k i ¼ nm for apn). importance of the aldehyde war-head was demonstrated in this report by comparison of the value of l-leucinal inhibitory constant with those found for simple amino acid l-leucine as well as its hydroxy derivative l-leucinol. these compounds were around e orders of magnitude less effective towards both aminopeptidases tested. additionally, glycine aldehyde derivative (glycinal, compound , fig. ) investigated in this study was also much less effective (k i ¼ . mm for lap). this result confirmed the importance of the side chain in binding efficiency of the designed inhibitors, but also proved that information from the substrate activity screening can be used for the design of the inhibitors. l-leucine p-nitroanilide substrate is much more efficiently processed by lap than analogous glycine derivative. another group of inhibitors for aminopeptidases with aldehyde scaffold were proposed by tarnus et al., -amino- -hydroxy-propionaldehyde and -amino- -hydroxypropan- -one derivatives ( and , respectively, fig. ) [ ] . these compounds designed as general inhibitors of metallo-aminopeptidases were micromolar competitive inhibitors of lap and apn. for some derivatives selective inhibition of apn over lap was observed. unfortunately, due to their susceptibility to oligomerization as well as very high reactivity aldehyde derivatives are not the best candidates for in vivo studies. however, they are an interesting alternative for design of inhibitors, which can be used for investigation of aminopeptidases in vitro. sulfonamides belong to a group of the most recognized compounds with biomedical relevance. easily obtained in the reaction of appropriate sulfonyl chlorides with amines, they offer a great potential of structural variations of both substrates. indeed, since the discovery of the antimicrobial properties, sulfonamides have found a vast number of other biological applications connected with regulation of enzymatic activity. in the context of proteases inhibition, a rationale for the sulfonamide moiety application is the isosteric and isoelectronic resemblance to the high energy tetrahedral transition state that is present in the amide bond hydrolysis (similarly to the phosphorus containing peptide analogues). surprisingly, there are not many recent examples of the use of this strategy towards metallo-aminopeptidase targets. what is more significant, the mode of sulfonamides action in those rare cases is miscellaneous and does not follow the theoretically considered pattern of transition state interactions. the title functional group plays more of a role as a linker between hydrophobic portions of the inhibitor, with another metal binding entity incorporated into one of them. alternatively, a cooperative action of two heteroatom-rich moieties (including the sulfonamide one) is observed. the most advanced studies on this topic seemed to be performed in abbott laboratories and concerned anthranilic acid sulfonamides as inhibitors of the human recombinant methionine aminopeptidase type (as a target for orally available drugs in an anti-cancer therapy) [ e ]. a series of sulfonamide compounds, such as (fig. ) , was identified using affinity selection by a mass spectroscopy screening method [ ] . they exhibited micromolar activity for the manganese form of the human metap and were moderately potent in a cell proliferation assay. thanks to promising pharmacokinetics and synthetic viability they were pointed out as novel attractive leads. consecutive x-ray studies allowed for the rational design of a new generation of ligands and tracking of the efficiency of structural optimizations. first iteration , ic enlargement of the aromatic portion of the anthranilic acid to a naphthalene or tetrahydronaphthalene system (compound , fig. ) allowed -fold improvement in activity of the starting molecule [ ] . as revealed by the crystal structure, tetrahydronaphthyl ring fitted tightly into a hydrophobic pocket of the active site. the carboxylate was an actual metal chelating group, whereas the sulfonamide moiety simply ensured the proper twist of the molecule to point the aromatic rings into a lipophilic environment. unfortunately, these inhibitors exhibited a limited cellular activity and extensive binding to human serum albumin. a positively charged ortho substitution in the arylsulfonamide ring was predicted to obey these drawbacks, in particular to disrupt interactions with albumin [ ] . a set of substituents to the structure based on complex amines and diamines was introduced at this position and positively validated the approach. the modified products (exemplified by , fig. ) showed potent activity for metap , associated with high selectivity versus related aminopeptidases ( -fold ratio less active for metap , for example) [ ] . importantly, their efficiency in cell proliferation assays was improved by a greater than -fold gain in potency and ranked in low nanomolar range. similar parameters were achieved for , disubstituted anthranilic acids containing and additional heteroatom group that was presumed to tighten interactions with manganese ions [ ] . since they exhibit strong anti-cancer activity, enhanced accessibility and oral available such sulfonamides could be considered as optimized for therapeutic use [ ] . a cooperative binding mode was observed for quinoline-based sulfonamides potent towards the e. coli metap . these inhibitors were discovered by screening of a , member small organic compounds library [ ] . selected hits were screened for different metalloforms of the enzyme, with the highest affinity towards the cobalted form displayed by compound (fig. ) . the sulfonamides behaved as typical competitive inhibitors; however, as disclosed by the x-ray structural studies, their mode of interactions was not typical. consistent with data described for the heteroaromatic ligands, the enzyme active site was loaded with three metal ions. the inhibitor bound as a bidentate sulfonamide/ quinoline n,n donor to the auxiliary manganese or cobalt atom [ ] . although the methanesufonate fragment was deeply buried, the molecule had no direct interactions with the catalytic metal ions. . tetralone derivatives first described in by schalk et al. derivatives of -amino- -tetralone were found to be nanomolar inhibitors of porcine kidney aminopeptidase n [ ] . these compounds ( and , fig. ) have non-peptidic character and are competitive inhibitors of the enzyme. the possible mechanism of their coordination with enzyme zinc ion is via carbonyl and amine groups located on the neighboring carbons in the cyclohexyl scaffold. interestingly, these compounds were almost completely inactive towards aspartate and arginine aminopeptidases and only slightly active towards lap. in another approach albrecht et al. synthesized various new derivatives of -amino- -tetralone [ ] . several methyl ketone, substituted oximes or hydroxamic acids, phosphinic acids and hydrazides derivatives (exemplified by compounds e , fig. ) were obtained and tested towards leucine aminopeptidase, aminopeptidase n, aeromonas proteolytica aminopeptidase, and leukotriene a hydrolase. even if theoretically equipped with better zinc chelating groups, these compounds were rather weak (up to low micromolar k i values) inhibitors of aminopeptidases with one active site zinc and very weak inhibitors of aminopeptidases with two zinc ions. inhibitors bearing gallic acid in the structure were designed based on the previously known biological properties of this compound as well as by assumption that methoxy (natural gallic acid has three free hydroxy groups) substituted hydrophobic ring of this compound will tightly bind in the s pocket of aminopeptidase n. several amino acids derivatives of this compound like -amino-l-proline, l-iso-glutamine and cyclo-l-iso-glutamine have been obtained [ e ] . among them gallolylamide derivatives based on proline scaffold (compounds and , fig. ) were extremely good inhibitors of aminopeptidase n. the best compounds had ic values in low nanomolar range [ ] . in another study l-iso-glutamine and cyclo-l-iso-glutamine derivatives (compounds and , fig. ) have been synthesized. these compounds were not as good inhibitors as proline derivatives and had ic [ ] . betulinic acid ( , fig. ) is a triterpene isolated from bark of several different plants (birch bark is rich source of this compound) [ ] . this compound has been found to be potent inducer of apoptosis in cancer cells and is currently in clinical trials [ ] . it is proposed that betulinic acid acts by increasing mitochondrial membrane permeability, which subsequently facilitates release of apoptosis stimulating proteins (cytochrome c). however, several others biological targets for this compound have been proposed and one of them is membrane aminopeptidase n (cd ). melzig et al. determined the ic for betulinic acid against aminopeptidase to be . mm [ ] . naturally occurring in plants polyphenolecurcumin ( , fig. ) has been also described as inhibitor of aminopeptidase n. curcumin is known as potent antitumor agent and currently its mechanism of action as well as biological targets are extensively investigated [ , ] . this compound interacted with cd in irreversible and non-competitive mode and strongly inhibited apn-positive tumor cell invasion as well as induced angiogenesis by basic fibroblast growth factor [ ] . interestingly, curcumin did not influence invasion of apn-negative cells, what further strengthened hypothesis that anti-invasive activity of this compound is a result of cd inhibition. aminopeptidases play pivotal roles in the turnover of proteins and the regulation of intracellular amino acids pools. they are essential to the metabolism, growth and development of all cells and tissues, and are part of the processes that regulate our immune and neurological systems. they also perform a broad spectrum of functions outside the cell, on the surface or even in the surrounding milieu (receptors, hormone processing and regulation etc). their involvement in the cause or maintenance of certain pathological diseases, particularly cancer, has focused our attention on their structure and function in the hope of developing new treatments. more recently, we have learned that aminopeptidases are also central to the cellular physiology of many parasites, including malaria, which has opened new avenues for development of antiinfectious disease drugs. crucial to the development of new drugs, however, is our detailed understanding of the mechanism of binding and interaction of inhibitory compounds to the active site of their targets. the present review highlights how this has been progressing well for several aminopeptidases, including leucine, alanine and methionine aminopeptidases, but also exposes our lack of information on a large number of aminopeptidase families. clearly, these gaps will be filled in time due to the improvements in inhibitor discovery and design (e.g. screening of chemical libraries followed by medicinal chemistry) and in methods for threedimensional structure determination. the challenge will be to discover inhibitors with selectivity not only for specific enzyme types so as to avoid off-target effects on other systems, but can be delivered to block specific functions or physiological roles of a particular aminopeptidase since each enzyme often performs a variety of defined and ancillary roles. aminopeptidases in biology and disease, proteases in biology and disease human aminopeptidases: a review of the literature industrial enzymes the properties and functions of bacterial aminopeptidases merops: the peptidase database leucine aminopeptidase as a target for inhibitor design. mini rev proteolysis and class i major histocompatibility complex antigen presentation leucine aminopeptidase is not essential for trimming peptides in the cytosol or generating epitopes for mhc class i antigen presentation interferon-gamma can stimulate postproteasomal trimming of the n terminus of an antigenic peptide by inducing leucine aminopeptidase identification and quantification of leucine aminopeptidase in aged normal and cataractous human lenses and ability of bovine lens lap to 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aminopeptidase- inhibitor on b cell differentiation ectopic expression of methionine aminopeptidase- causes cell transformation and stimulates proliferation map d, a novel methionine aminopeptidase family member is overexpressed in colon cancer roles of p /metap as a tumor suppressor cerebrospinal fluid proteomic analysis reveals dysregulation of methionine aminopeptidase- expression in human and mouse neurofibromatosis -associated glioma purification and characterization of a leucine aminopeptidase from the skeletal muscle of common carp (cyprinus carpio) leucine aminopeptidase from red sea bream (pagrus major) skeletal muscle: purification, characterization, cellular location, and tissue distribution purification and properties of aminopeptidase h from chicken skeletal muscle purification and properties of major midgut leucyl aminopeptidase of morimus funereus (coleoptera, cerambycidae) larvae purification and characterization of aminopeptidase n from spodoptera litura expressed in sf insect cells mutation of an aminopeptidase n gene is associated with helicoverpa armigera resistance to bacillus thuringiensis cry ac toxin identification and characterization of aedes aegypti aminopeptidase n as a putative receptor of bacillus thuringiensis cry a toxin neuropeptidases and the metabolic inactivation of insect neuropeptides complexes of mutants of escherichia coli aminopeptidase p and the tripeptide substrate valproleu expression and characterization of two functional methionine aminopeptidases from mycobacterium tuberculosis h rv the leucyl aminopeptidase from helicobacter pylori is an allosteric enzyme crystal structure of the cold-active aminopeptidase from colwellia psychrerythraea, a close structural homologue of the human bifunctional leukotriene a hydrolase purification and characterization of hyperthermotolerant leucine aminopeptidase from geobacillus thermoleovorans b mutational analysis of the zinc metalloprotease empa of vibrio anguillarum characterization 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leucine aminopeptidase from bacillus kaustophilus as evaluated by site-directed mutagenesis a functional comparison of the tet aminopeptidases of p. furiosus and b. subtilis with a proteinengineered variant recombining the former's structure with the latter's active site histidines and of bacillus stearothermophilus leucine aminopeptidase ii are essential for the catalytic activity of the enzyme analyzing the catalytic role of asp in the methionine aminopeptidase from escherichia coli kinetic and spectroscopic analysis of the catalytic role of h in the methionine aminopeptidase from escherichia coli kinetic, spectroscopic, and x-ray crystallographic characterization of the functional e h aminopeptidase from aeromonas proteolytica the catalytic role of glutamate in the leucine aminopeptidase from aeromonas proteolytica structure of a microsporidian methionine aminopeptidase type complexed with fumagillin and tnp- crystal structure of isoaspartyl aminopeptidase in complex with 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key: cord- - bhg t l authors: al-nour, mosab yahya; ibrahim, musab mohamed; elsaman, tilal title: ellagic acid, kaempferol, and quercetin from acacia nilotica: promising combined drug with multiple mechanisms of action date: - - journal: curr pharmacol rep doi: . /s - - -w sha: doc_id: cord_uid: bhg t l the pharmacological activity of acacia nilotica’s phytochemical constituents was confirmed with evidence-based studies, but the determination of exact targets that they bind and the mechanism of action were not done; consequently, we aim to identify the exact targets that are responsible for the pharmacological activity via the computational methods. furthermore, we aim to predict the pharmacokinetics (adme) properties and the safety profile in order to identify the best drug candidates. to achieve those goals, various computational methods were used including the ligand-based virtual screening and molecular docking. moreover, pkcsm and swissadme web servers were used for the prediction of pharmacokinetics and safety. the total number of the investigated compounds and targets was and , respectively. according to the results, the pharmacological activity was attributed to the interaction with essential targets. ellagic acid, kaempferol, and quercetin were the best a. nilotica’s phytochemical constituents that contribute to the therapeutic activities, were non-toxic as well as non-carcinogen. the administration of ellagic acid, kaempferol, and quercetin as combined drug via the novel drug delivery systems will be a valuable therapeutic choice for the treatment of recent diseases attacking the public health including cancer, multidrug-resistant bacterial infections, diabetes mellitus, and chronic inflammatory systemic disease. acacia nilotica is a tropical and sub-tropical medicinal plant belonging to the fabaceae family [ ] . no doubt, medicinal plants play a vital role in drug discovery, since they are affluent with bioactive phytochemical constituents that are valuable in the treatment of various diseases, particularly those causing recent threats attacking the public health including cancer, multidrug-resistant bacterial infections, diabetes mellitus, and chronic inflammatory systemic diseases [ , ] . the higher incidence of cancer and mortality rate [ ] , the emergence of bacterial resistance with the declining in the antibacterial research at several pharmaceutical companies [ ] , the huge prevalence and complications associated with diabetes mellitus [ ] as well as the long-term suffering associated with the chronic inflammatory systemic diseases such as rheumatoid arthritis and multiple sclerosis [ ] are leading forces that encourage us to participate in fighting against the probable threats. such an issue is attained via the discovery and development of efficient innovative anticancer, antibacterial, antidiabetic, and anti-inflammatory drugs. unfortunately, drug discovery is a time-consuming, costive, as well as difficult process [ , ] ; hence, it necessitated to involve sophisticated techniques in the drug discovery process in order to overcome those limitations. recently, one of the promising sophisticated techniques is the computational tools (computer-aided drug design) that have a valuable impact in the discovery and development of newer drugs with a reduction in time and cost [ ] . they include the ligand-based virtual screening that is based on the searching for the compounds having the highest probability in pharmacological activity [ ] and molecular docking that relies on the energy-based scoring function to identify ligand-target complex lowest energy [ ] . moreover, they involve the software of pharmacokinetics, toxicity, and the drug-likeness prediction that work by many algorithms [ ] including the graph-based signature [ ] . many studies concerning the application of the computational tools in the discovery of natural-derived drugs were conducted [ ] [ ] [ ] [ ] . a. nilotica is opulent of many phytochemical constituents including tannins, alkaloids, terpenoids, and flavonoids. many studies were conducted in it resulting in an evidence-based pharmacological data that revealed the potential pharmacological activities of the phytochemical compounds including anticancer, antibacterial, antidiabetic, anti-inflammatory, and other activity making the plant as a promising source for the development of innovative, safe, biodegradable drugs with great activity. the chemical structure of active a. nilotica's phytochemical constituents was elucidated, the correlation between the responsible phytochemical constituents for treatment and the diseases were conducted [ ] , but the determination of exact targets that phytochemical constituents bind and the mechanism of action were not performed; consequently, based on established literature and studies, we aim to identify the exact targets that phytochemical constituents bind to exert the pharmacological activity by utilizing the computational methods as a tool for the study so as to understand the mechanism of action. within the current drug design pipeline, drug target identification is a very important step in the understanding of the probable mechanism of action, increasing the confidence and reducing the attrition in clinical trials [ ] . furthermore, we aim to predict the pharmacokinetics (adme) properties and safety profile with the intention of identifying the best drug candidates. the qsar-based virtual screening is characterized by great and fast throughput with respectable hit rank [ ] . molecular docking is valuable to predict the stability of the ligand-target complex that reflects the biological activity [ ] . the pharmacokinetics, toxicity, and drug-likeness prediction are helpful to identify the best drug candidates [ , ] . to our knowledge, such a study was not conducted before. the chemical structure of the reported a. nilotica's phytochemical constituents ( compounds) [ ] was drawn via marven sketch software version . [ ] (fig. ) . the d structure was generated in a mol format with open babel software [ ] , minimized and optimized with cresset flare software [ ] at the accurate type calculation method. the screening for the exact target that the phytochemical constituents bind was performed via similarity ensemble search tool [ ] and targetnet web servers [ ] . the compound structures were submitted in smile format. the targets with higher probability score were selected for further validation via molecular docking study ( targets) . the linkage between predicted targets with the diseases was attained via uniprot [ ] , pharos [ ] , and therapeutic target databases [ ] . the results are listed in tables , , , , , and . the d structure of selected targets from virtual screening was obtained from the rcsb protein data bank [ ] . the structure with better resolution and validation scores was selected for the study. in order to validate the docking results, multiple d x-ray crystallographic structures for the same target were downloaded in pdb format. for the structures that have no practically determined d structure, phyre [ ] , swiss-model web server [ ] , and raptorx [ ] web servers were used for d structure modeling, then downloaded in pdb format. the target preparation was carried out in cresset flare software [ ] according to the default settings. after preparation, the targets d structures were minimized via cresset flare software [ ] at the normal type calculation method. the targets were input to the software in pdb format. the preparation of reported a. nilotica's phytochemical constituents for molecular docking study was carried out as described above. the docking calculations were carried out in cresset flare software [ ] in normal mode and default settings. the grid box was defined according to the co-crystallized ligands, but in the absence of co-crystallized ligands, the grid box was defined via picking of active site amino acids. beside the a. nilotica's phytochemical constituents, drugs that are well known to bind with the predicted targets (selected randomly from therapeutic target [ ] and pharos [ ] databases) and the co-crystallized ligands were used as positive controls. the compounds and the targets were input in mol and pdb format, respectively. the results are listed in tables , , , , , , and and figs. , , , , and . the intestinal absorption, volume of distribution, bloodbrain barrier, p-glycoprotein and cytochrome-p enzymes inhibition, the renal oct substrate probability, and total clearance were predicted via pkcsm [ ] and swissadme web servers [ ] . moreover, the hepatotoxicity, skin sensitization, the herg potassium channel inhibition, ames toxicity, human maximum tolerated dose, carcinogenicity, oral rate acute, and chronic toxicity were predicted via pkcsm web server [ ] at the default settings via submitting of the chemical structures in smile format. the results of pharmacokinetics are listed in tables and and toxicity in table . the probability of a. nilotica's phytochemical constituents to be as drug candidates was carried via applying of lipinski, ghose, veber, egan, and muegge filters. in addition, lead likeness and synthetic accessibility were used to predict medicinal chemistry friendliness. the prediction was carried out via swissadme web server via submitting of the chemical structures in smile format [ ] . the results are listed in table . the consistency and the reproducibility of the used tools including the molecular docking were validated by the resubmission of the compounds for many times. the anticancer activity of a. nilotica's was attributed to the suppression of the oncogenic transformations, progression, and development, dna replication, and transcription. moreover, the prevention of cancer cells proliferation, invasion, angiogenesis as well as the suppression of drug resistance and the induction of apoptosis. the anti-breast cancer activity was due to the inhibition of the aromatase enzyme and estrogen receptor beta. in contrast, the anti-prostate cancer activity is due to the control of metastatic behavior of prostate cancer via the interaction with nuclear receptor ror-alpha and the inhibition of steroid alpha-hydroxylase (table and fig. ) . . the antibacterial activity of a. nilotica was attributed to the prevention of fatty acids, peptidoglycans biosynthesis as well as the prevention of bacterial resistance to the beta-lactam antibiotics. the fatty acid biosynthesis inhibitory activity was against different types of bacteria including mycobacterium, pseudomonas aeruginosa, and vibrio cholera. the antiviral activity was attributed to the action on toll-like receptor . the anti-hiv activity is due to the inhibition of hiv integrase enzyme. the anti-coronavirus activity is due to coronavirus replicase polyprotein ab enzyme. the antiplasmodial activity was attributed to the inhibition of enzymes mo -related protein kinase pfmrk and m aspartyl aminopeptidase as well as the prevention of fatty acid biosynthesis via inhibition of the enzymes: β-hydroxy acyl-acp dehydratase fabz and hydroxyacyl-[acyl-carrier-protein] dehydratase (table ). the antidiabetic activity was attributed to the interaction with the insulin receptor, glycogen phosphorylase enzyme, sodium/glucose co-transporter as well as the aldose reductase enzyme ( table ). the anti-inflammatory activity was attributed to the inhibition of enzymes: arachidonate -lipoxygenase, cyclooxygenase- (cox- ), phospholipase a , receptor-interacting serine/ threonine protein kinase , and xanthine dehydrogenase/ oxidase as well as the interaction with macrophage migration inhibitory factor ( table ). the antidiarrheal, anti-platelets, and anticholinesterase targets the antidiarrheal activity was attributed to the interaction with the opioid receptors mu-type delta-type. the anti-platelets activity was attributed to the interaction with the p y receptor. the inhibition of the enzyme acetylcholinesterase is contributed to the anticholinesterase activity. according to the results, acacetin, γ-sitosterol, kaempferol, flavone, lupenone, lupeol, niloctane, and quercetin had the highest gastrointestinal absorption, tissue distribution (vd), and respectable total clearance. moreover, flavone, nilobamate, and niloctane were permeable to the blood-brain barrier (bbb). besides, acanilol- , acanilol- , γ-sitosterol, flavone, lupenone, and lupeol were found to be subjected to the metabolism via cyp a enzyme (table ) . moreover, (+)-mollisacacidin, catechin, chalconaringnen- -o-beta-glucopyranoside, epicatechin, niloticane, kaempferol- glucoside, leucocyanidin, and nilobamate were free from drugdrug interaction via the inhibition of cytochrome-p (cyp) or pglycoprotein (p-gp) i and ii enzymes (table ). w z* aua* -acacetin --− . -cyclin-dependent kinase enzyme bit is involved in regulation of transcription^ [ ] bcg* gzh* -acacetin . -death-associated protein kinase bit regulates type i apoptotic, type ii autophagic cell deaths^ [ ] auv* auu* -quercetin . -focal adhesion kinase enzyme bit is essential in angiogenesis, cell migration and apoptosis^ [ ] k y* d * -ellagic acid cell survival, and proliferation^ [ ] ayd binvolved in tumor transformation, progression, survival, angiogenesis and metastasis^ [ ] esm* cuh* -quercetin --− . -nuclear receptor ror-alpha bit is involved in cell growth, differentiation, and control of metastatic behavior of androgen-independent prostate cancer^ [ ] n * b w* -acacetin bit regulates centrosome separation, bipolar spindle formation in cell mitosis^ [ ] xnn* wqo* -quercetin . -p-glycoprotein and bthey involved in multi-drug resistance^ [ ] xwk* cbz* -acacetin . -platelet-derived growth factor receptor bit has a pro-angiogenic action [ in compounds, the numbers , , , … indicate a. nilotica's phytochemical constituents, letters a, b, … indicates positive controls, • indicates the cocrystallized ligands, and the italic emphasis indicates compounds with the higher scores. at ligand-based virtual screening score (lbvs sco.), en dash (-) means that the compound was not screened. asterisk (*) indicates the pdb id. swiss means that the d structure of the target was modeled using swiss-model web server [ ] the predicted toxicity according to the results, , -di-o-galloyl-beta-d-glucose, ellagic acid, kaempferol, and quercetin were non-toxic as well as non-carcinogen (table ) . according to the results, (+)-mollisacacidin, acacetin, catechin, epicatechin, kaempferol, naringenin, niloctane, and quercetin were found to be the best lead and drug candidates with good synthetic accessibility, followed by digallic acid, ellagic acid, leucocyanidin, and melacacidin (table ). despite the enormous conducted studies on the pharmacology activity of a. nilotica's [ ] , the determination of the target that contribute to its activity and the understanding of the mechanism of action as well as to assess the pharmacokinetics, safety, and the drug-likeness probability are important issues that were not conducted yet. such studies are required to bring the plant in the drug discovery pipeline so as to design a novel drug with broad-spectrum of therapeutic activity and safety. to identify the targets, targetnet web servers that utilize a qsar model based on the chemogenomic data as a predictive algorithm [ ] and similarity ensemble search tool [ ] were used. to validate the predicted target from the web servers, a molecular docking study was performed using cresset flare software [ ] that uses the lead finder program [ ] for bit is an essential bacterial enzyme in peptidoglycan biosynthesis^ [ ] dgi* c p* r * -quercetin bit is responsible for hydrolysis of beta-lactams, with substrate specificity toward cephalosporins, has an important role in cephalosporins resistance^ [ ] hdq* pu * r w in compounds, the numbers , , , … indicate a. nilotica's phytochemical constituents, letters a, b, … indicate positive controls, • indicates the cocrystallized ligands, and the italic emphasis indicates compounds with the higher scores. at ligand-based virtual screening score (lbvs sco.), en dash (-) means that the compound was not screened. asterisk (*) indicates the pdb id. phyre and raptor x means the d structure of target was modeled by phyre [ ] and raptorx [ ] web servers, respectively docking calculation. moreover, pkcsm [ ] and swissadme web servers [ ] were used to predict the pharmacokinetics (adme: absorption, distribution, metabolism, and elimination), toxicity, and the drug-likeness probability. the total predicted targets form the virtual screening with the highest probability that was validated by the molecular docking were targets. the interaction of acacetin with the cell division control protein homolog (cdc ) will prevent the oncogenic transformations. the inhibition of enzymes-anaplastic lymphoma kinase by quercetin, cyclin-dependent kinases , , and by ellagic acid and acacetin, aurora a and b by ellagic acid and quercetin, serine/threonine protein kinase nek by quercetin, proto-oncogene tyrosine-protein kinase src by ellagic acid, tankyrase and by acacetin as well as m phase inducer phosphatase by digallic acid, epicatechin, and kaempferol-will prevent the cancer progression and development. moreover, the inhibition of the enzymes-cell division cycle- -related protein kinase by ellagic acid, serine/ threonine-protein kinase pim- by quercetin, ellagic acid, and kaempferol as well as dna topoisomerase by in compounds, the numbers , , , … indicate a. nilotica's phytochemical constituents, letters a, b, … indicate positive controls, • indicates the cocrystallized ligands, and the italic emphasis indicates compounds with the higher scores. at ligand-based virtual screening score (lbvs sco.), en dash (-) means that the compound was not screened. asterisk (*) indicates the pdb id bit is a pro-inflammatory cytokine counteracts the anti-inflammatory activity of glucocorticoids^ [ ] . bit is responsible for the release of the arachidonic acid from arachidonyl phospholipids, thereby involved in the initiation of the inflammatory response^ [ ] . in compounds, the numbers , , , … indicate a. nilotica's phytochemical constituents, letters a, b, … indicate positive controls, • indicates the cocrystallized ligands, and the italic emphasis indicates compounds with the higher scores. at ligand-based virtual screening score (lbvs sco.), en dash (-) means that the compound was not screened. asterisk (*) indicates the pdb id kaempferol- -glucoside and , -di-o-galloyl-beta-d-glucose-will suppress the dna replication; the inhibition of enzymes-cyclin-dependent kinase by acacetin and telomerase reverse transcriptase by leucocyanidin, quercetin, ellagic acid, and kaempferol-will suppress the transcription; the inhibition of tyrosine-protein kinase lyn enzyme by ellagic acid will prevent the cancer cells proliferation; as well as the inhibition of angiopoietin- receptor, proto-oncogene tyrosine-protein kinase src by ellagic acid, and protein kinase c epsilon by kaempferol and naringnen will suppress the cancer cells invasion. furthermore, the inhibition of ephrin type b receptor and platelet-derived growth factor receptor by ellagic acid, vascular endothelial growth factor receptor by quercetin, as well as focal adhesion kinase enzyme ellagic acid and quercetin will suppress the angiogenesis. the inhibition of p-glycoprotein , transporters by kaempferol- -glucoside, chalconaringnen- -o-betaglucopyranoside, kaempferol, and quercetin as well as atp binding cassette sub-family g member by (+)-catechin- , digallate, chalconaringnen- -o-beta-glucopyranoside, in compounds, the numbers , , , … indicate a. nilotica's phytochemical constituents, letters a, b, … indicates positive controls, • indicates the cocrystallized ligands, and the italic emphasis indicates compounds with the higher scores. at ligand-based virtual screening score (lbvs sco.), en dash (-) means that the compound was not screened. asterisk (*) indicates the pdb id leucocyanidin, quercetin, ellagic acid, and kaempferol will suppress the cancer cells resistance. the interaction of lupeol, quercetin, ellagic acid, and kaempferol with the enzyme inducible nitric oxide synthase on macrophage will promote a tumoricidal action. the inhibition of the enzymes-bcl- -related protein a by leucocyanidin, quercetin, ellagic acid, and kaempferol, the induced myeloid leukemia differentiation protein mcl- by acacetin, quercetin, ellagic acid, and kaempferol as well as the interaction with enzymes: caspase by (−)-epigallocatechin- -gallate, quercetin, ellagic acid, and kaempferol, death-associated protein kinase by kaempferol and quercetin-will induce cancer cell apoptosis. consequently, those compounds show substantial anticancer activity ( table ) . the interaction of kaempferol and quercetin with the enzymes -oxyacyl-[acyl carrier protein] reductase fabg and the interaction of (−)-epigallocatechin- -gallate, (+)-catechin- , -digallate, and quercetin with enoyl-acyl carrier protein reductase will inhibit the bacterial fatty acids biosynthesis that is essential in the formation of bacterial membrane phospholipids [ ] leading to ban impairment in the cellular envelope structure and function, the ability to form biofilms as well as increasing the susceptibility to the environmental stress^ [ ] . moreover, the inhibition of the enzyme d-alanine d-alanine ligase by quercetin will suppress the peptidoglycans biosynthesis that is vital in bacterial cell structure causing loss of bacterial cell integrity [ ] . therefore, those compounds have significant antibacterial activity ( table ) . the interaction of leucocyanidin, ellagic acid, kaempferol, and quercetin with toll-like receptor will activate this innate immune receptor that helps in the recognition of microbial dna [ ] . the interaction of digallic acid, the italic emphasis indicates desirable prosperity bbb blood-brain barrier, vd volume of distribution, renal oct human organic cation transporter [ ] acacetin, ellagic acid, kaempferol, and quercetin with hiv integrase enzyme will inhibit the viral dna integration into host dna leading to the suppression of replication cycle [ ] . the interaction of quercetin with coronavirus replicase polyprotein ab will inhibit the transcription and replication of viral rnas [ ] . thus, those a. nilotica's phytochemical constituents exhibit considerable antiviral activity ( table ). the interaction of acacetin with the enzyme mo -related protein kinase pfmrk will disrupt the regulation of plasmodial cell cycle [ ] , and the interaction of quercetin and (+)-mollisacacidin with the enzyme m aspartyl aminopeptidase will prevent the invasion in host erythrocyte and the degradation of host hemoglobin [ ] . furthermore, the interaction of quercetin with the plasmodial enzymes β-hydroxy acyl-acp dehydratase fabz and hydroxyacyl-[acyl-carrier-protein] dehydratase will inhibit the fatty acid biosynthesis [ , ] that are important for plasmodial membrane [ ] . subsequently, those a. nilotica's phytochemical constituents show considerable antiplasmodial activity ( table ) . the interaction of ellagic acid with the insulin receptor will promote glucose uptake that lowers the blood glucose level [ ] . the interaction of quercetin with the enzyme glycogen phosphorylase will inhibit the glycogenolysis that reduces the hyperglycemia [ ] . moreover, the interaction of , -di-ogalloyl-beta-d-glucose and chalconaringnen- -o-betaglucopyranoside with the sodium/glucose co-transporter will inhibit the renal glucose reabsorption leading to a reduction in plasma glucose level [ ] , the interaction of dicatechin, kaempferol- -glucoside, leucocyanidin, ellagic acid, kaempferol, and quercetin with the aldose reductase fig. the d interaction between the best compounds with some of their predicted anticancer targets. a quercetin (violet) with anaplastic lymphoma kinase enzyme. staurosporine (turquoise) as control. b ellagic acid (dark yellow) with angiopoietin receptor. cabozantinib (turquoise) as control. c ellagic acid (dark yellow), kaempferol (pink), and quercetin (violet) with the aromatase enzyme. anastrozole (teal) and the co-crystallized ligand a asd (turquoise) as a control. d ellagic acid (dark yellow) and quercetin (violet) with aurora a kinase enzyme. the cocrystallized ligand a adp (turquoise) as a control. e ellagic acid (dark yellow), kaempferol (pink), and quercetin (violet) with caspase enzyme. the isatin sulfonamide (turquoise) as a control. ellagic acid (dark yellow), kaempferol (pink), and quercetin (violet) with steroid alpha-hydroxylase enzyme. galeterone (turquoise) as a control enzyme will suppress the development of the secondary diabetic complications [ ] , as well as the interaction of ellagic acid and quercetin with the beta-secretase enzyme will upregulate the insulin receptors in the liver [ ] . successively, those a. nilotica's phytochemical constituents have significant antidiabetic activity ( table ). the interaction of (−)-epigallocatechin- -gallate, ellagic acid, kaempferol, and quercetin with the enzyme arachidonate -lipoxygenase and the interaction of digallic acid, acacetin, ellagic acid, kaempferol, and quercetin with the receptor-interacting serine/threonine protein kinase will interrupt the inflammatory responses [ , ] . the interaction of (+)-mollisacacidin, naringnen, e l l a g i c a c i d , k a e m p f e r o l , a n d q u e r c e t i n w i t h cycloxygenase- enzyme (cox- ) will prevent the formation of inflammatory mediators prostaglandins [ ] , and the interaction of digallic acid, kaempferol, and quercetin with phospholipase a enzyme will prevent the initiation of the inflammatory response [ ] . moreover, the interaction of , -di-o-galloyl-beta-d-glucose, digallic acid, acacetin, ellagic acid, kaempferol, and quercetin with the xanthine dehydrogenase/oxidase enzyme will inhibit the formation of uric acid and reactive oxygen species [ ] . thus, those of a. nilotica's phytochemical constituents exhibit considerable antiinflammatory activity ( table ). the interaction of dicatechin with the mu and delta opioid receptors will lead to antisecretory and anti-transit action that will inhibit diarrhea [ ] . the interaction of , -di-o-galloylbeta-d-glucose with p y receptor will inhibit the platelet activation [ ] ; consequently, they have considerable antidiarrheal and anti-platelets activity, respectively (table ). the chemogenomic-based qsar models of targetnet web server were strictly evaluated and validated leading to respected screening results [ ] . furthermore, the lead finder program [ ] on cresset flare software [ ] is characterized by the combination between the genetic algorithm and different optimization strategies leading to great efficiency, robustness, accuracy, and speed of calculations [ ] . musab ibrahim et al. [ ] found the results of a molecular docking study about novel synthesized cox enzyme inhibitors conducted in cresset flare software were aligned with results of the conducted in vivo study. depending on that, the obtained results of the predicted targets could be with considerable accuracy. since the pharmacological activity does not depend only on the pharmacodynamic properties, but also on the pharmacokinetic properties. moreover, as the drug safety, the assessment of drug-likeness probability, and the synthetic accessibility are important issues [ ] , the identification of the best a. nilotica's phytochemical constituents will be attained by the assessment of those issues collectively. the pharmacokinetics is concerning the study of the entrance, movement, changing, and leaving of the drug to the body [ ] . the higher absorption from the gastrointestinal tract leads to higher drug concentration on the blood, the higher volume of distribution provides higher supply to the body tissues, and the adequate metabolism and elimination prevent the accumulation of the drug in the body, hence reduce the toxicity [ ] . consequently, the consideration of the pharmacokinetics in drug design is an essential task [ ] . fig. the d interaction between the best compounds with some of their predicted antiplasmodial and antidiabetic targets. a kaempferol (pink), and quercetin (violet) with β-hydroxy acyl-acp dehydratase fabz. the co-crystallized ligand b km (turquoise) as a control. b quercetin (violet) with hydroxyacyl-[acyl-carrier-protein] dehydratase. the cocrystallized ligand a emo (turquoise) as a control. c (+)-mollisacacidin (green), epicatechin (teal), and quercetin (violet) with m aspartyl aminopeptidase enzyme. d ellagic acid (dark yellow) with insulin receptor. ceritinib (turquoise) as a control. e quercetin (violet) with glycogen phosphorylase (muscle). the co-crystallized ligand avf (turquoise) as a control. f , -di-o-galloyl-beta-d-glucose (green) with sodium /glucose cotransporter . canagliflozin (turquoise) as a control for instance, lupenone is highly lipophilic; hence, it has higher absorption percent ( %); in contrast, the hydrophilic groups of ellagic acid reduce it absorption percent to . %, however, still it as a high absorption percent. the higher absorption will make lupenone is highly bioavailable. niloctane is permeable to bbb; therefore, its concentration that reaches the brain targets is more than ellagic acid that is not permeable to the bbb. the predicted volume of distribution (vd) of melacacidin ( . l/kg) is the highest one, meaning that it has the highest distribution in body tissues. in contrary, nilobamate has the highest predicted total clearance, meaning that it is the fastest one that eliminated from the body (table ) . moreover, the inhibition of cytochrome-p enzyme cyp a by ellagic acid will decrease the biotransformation of drugs that metabolized by it leading to increase in the concentration of them that may increase the side effects; consequently, the drug-drug interaction must be in consideration. the binding of dicatechin with the p-glycoprotein may decrease the transportation of drugs transported by this transporter and may involve in the drug resistance by the pumping out mechanism (table ) . furthermore, the predicted ames toxicity of epicatechin will lead to genotoxicity and mutagenicity [ ] , the predicted herg ii potassium channel inhibitory effect of acacetin bprolongs the qt interval in ecg that increases the risk for potentially fatal ventricular arrhythmias^ [ ] ; subsequently, such drugs will not be considered as drug-likeness candidate (table ) . besides, quercetin has no violation in lipinski rule of five; hence, it will a good candidate as an orally active drug as well as it has no violation in ghose, veber, and egan filters; therefore, it will be a good lead-likeness candidate [ ] (table ) . the d interaction between the best compounds with some of their predicted antiinflammatory, antidiarrheal, and anti-platelets targets and acetyl cholinesterase enzyme. a ellagic acid (dark yellow), kaempferol (pink), and quercetin (violet) with arachidonate -lipoxygenase enzyme. the co-crystallized ligand a c e (turquoise) as a control. b ellagic acid (dark yellow), kaempferol (pink), and quercetin (violet) with cyclooxygenase- enzyme. indomethacin (turquoise) as a control. c dicatechin with mu-type opioid receptor. eluxadoline (turquoise) as a control. d dicatechin with delta-type opioid receptor. eluxadoline (turquoise) as a control. e , -di-o-galloyl-beta-dglucose (yellow) with p y receptor. clopidogrel (turquoise) as a control. f digallic acid (yellow) and leucocyanidin (blue) with acetylcholinesterase enzyme. neostigmine (turquoise) as a control according to the results of pharmacodynamics, pharmacokinetics, safety, and drug-likeness predictions collectively, ellagic acid, kaempferol, and quercetin were the best a. nilotica's phytochemical constituents that contribute to the therapeutic activities. the d interaction with their predicted targets demonstrates marked ligand superimposing with the control compounds (e.g., figs. a, b, e, and b) ; however, it may at the same active site without ligand superimposing (e.g., figs. c and c). ellagic acid interacts with aurora a kinase enzyme with two binding modes (fig. d) . ellagic acid, kaempferol, and quercetin interact with steroid alpha-hydroxylase enzyme at a binding mode that differs from the binding mode of control galeterone (fig. f) . they were followed by (+)-mollisacacidin, epicatechin, and melacacidin, those of their predicted ames toxicity decreased their rank. the predicted herg ii potassium channel inhibitory effect of acacetin decreased its rank; however, it has good pharmacodynamics and pharmacokinetics profile. despite the efficient pharmacodynamics and the respectable safety profile of ellagic acid, kaempferol, and quercetin, practically, each compound suffers from the low bioavailability [ ] [ ] [ ] , albeit the predicted intestinal absorption of them is high ( table ) . the reduced bioavailability of ellagic acid is attributed to the poor absorption and rapid elimination from the body [ ] (the predicted total clearance of ellagic acid is high). the higher topological polar surface area (tpsa) ( table ) contributes to the poor absorption. the reduced absorption of kaempferol is attributed to the larger particle size and poor water solubility [ ] . the reduced bioavailability of quercetin is attributed to bthe poor solubility and crystalline form at body temperature^ [ ] . moreover, ellagic acid, kaempferol, and quercetin have many polar phenolic hydroxyl groups (structure l, o, and x); consequently, they are subjected to direct glucuronide conjugation with as phase ii metabolism. bkaempferol and quercetin are rapidly excreted in urine as glucuronides mainly^ [ ] . the reduced bioavailability affects pharmacological activity. hence, to maintain the pharmacological activity, the bioavailability must be enhanced. the nano-suspension form of kaempferol is increased its absorption and bioavailability [ ] . the administration of isoquercetin (quercetin- -glucoside) increases the absorption and bioavailability of quercetin [ ] . the bioavailability ellagic acid, kaempferol, and quercetin is increased bypassing the entero-hepatic phase ii conjugation (e.g., formation of ester derivatives) and by using novel drug delivery systems as the liposomes. furthermore, the co-administration of ginkgo biloba extract with kaempferol and quercetin increased the bioavailability of them [ ] . consequently, the combination of ellagic acid, kaempferol, and quercetin will be optimum treatment choice that maximizes the therapeutic activity and the safety profile as well as overwhelms the limits in the bioavailability. as they naturally are available in one plant, the combination of them at the therapeutic doses will be additive and will not induce drugdrug interactions. the design of multi-target drug is an effective promising approach for the treatment of complex disease [ ] . the computational methods including the virtual screening are not to substitute the in vitro and in vivo methods, however, to reduce the time, cost, and the difficultness in the drug target identification [ ] . therefore, this study is an attempt to identify the best a. nilotica's phytochemical constituents that contribute to its pharmacological activity as well as their targets. it is not meaning that this study alone will be sufficient to judge about the result; however, experimental studies are required to validate the results. according to the results of pharmacodynamics, pharmacokinetics, safety, and drug-likeness predictions collectively, ellagic acid, kaempferol, and quercetin were the best a. nilotica's phytochemical constituents that contribute to the therapeutic activities; consequently, we recommend the use of ellagic acid, kaempferol, and quercetin as a combined drug via the novel drug delivery systems for the treatment of recent diseases attacking the public health including cancer, multidrug-resistant bacterial infections, diabetes mellitus, and chronic inflammatory systemic diseases. moreover, we recommend wet lab studies to validate the results. of ligand-based virtual screening web tools and screening the synthetic accessibility is from (very easy) to (very difficult). the bold indicates desirable prosperity, the italic indicates undesirable prosperity as well as the bold-italic indicates the best compounds mw molecular weight, rotatb. bonds rotatable bonds, m r molar refractivity, h-don hydrogen bond donors, h-acc hydrogen bond acceptors, tpsa topological polar surface area a lipinski rule of five, ghose, veber, egan, and muegge describe the relationship between the pharmacokinetic and physiochemical parameters. the parameters including molecular weight, number of rotatable bonds, molar refractivity, number of hydrogen bond donors and acceptors, as well as the topological polar surface area. each parameter has a specific range that the structure must not be under or above it to be free from violation [ ] acacia nilotica (l.): a review of its traditional uses, phytochemistry, and pharmacology medicinal plants: future source of new drugs bioactive compounds from medicinal plants and their importance in drug discovery in pakistan cancer statistics. www.cancer.gov. accessed antibacterial drug discovery and structure-based design association. statistics about diabetes. www.diabetes.org chronic inflammatory systemic diseases an evolutionary trade-off between acutely beneficial but chronically harmful programs advances in molecular modeling and docking as a tool for modern drug discovery from drug target to leads-sketching a physico-chemical pathway for lead molecule design in silico a comparison of various optimization algorithms of protein-ligand docking programs by fitness accuracy swissadme: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules pkcsm: predicting smallmolecule pharmacokinetic and toxicity properties using graphbased signatures computational approaches for therapeutic application of natural products in alzheimer's disease. neuromethods combining ligand-and structure-based in silico methods for the identification of natural product-based inhibitors of akt computational methodologies in the exploration of the marine natural product leads computeraided drug design of bioactive natural products an industry perspective on drug target validation molecular docking: a powerful approach for structure-based drug discovery prediction and optimization of pharmacokinetic and toxicity properties of the ligand open babel: an open chemical toolbox molecular field extrema as descriptors of biological activity: definition and validation relating protein pharmacology by ligand chemistry targetnet: a web service for predicting potential drug-target interaction profiling via multi-target sar models the universal protein resource (uniprot) pharos: collating protein information to shed light on the druggable genome therapeutic target database update : enrich resource for facilitating bench-to-clinic research of targeted therapeutics bcl family proteins in carcinogenesis and the treatment of cancer caspase- as a therapeutic target for treating cancer cdc in oncogenic transformation, invasion, and tumorigenesis targeting cell division cycle kinase: a new approach for cancer therapy targeting cyclindependent kinase (cdk ) but not cdk / or cdk is selectively lethal to myc-dependent human breast cancer cells dna topoisomerases in cancer therapy estrogen receptor beta in cancer: an attractive target for therapy inhibition of glycogen synthase kinase- beta induces apoptosis and mitotic catastrophe by disrupting centrosome regulation in cancer cells gelatinase b/mmp- in tumour pathogenesis and progression 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intestinal glucose transport from bench to beside role of aldose reductase and oxidative damage in diabetes and the consequent potential for therapeutic options the beta-secretase bace regulates the expression of the insulin receptor in the liver in vivo inhibition of ripk kinase alleviates inflammatory disease antidiarrheal properties of supraspinal mu and delta and kappa opioid receptors: inhibition of diarrhea without constipation central role of the p y receptor in platelet activation acetylcholinesterase inhibitors: pharmacology and toxicology the protein data bank substratedependent inhibition of the human organic cation transporter oct : a comparison of metformin with experimental substrates lead finder: an approach to improve the accuracy of protein-ligand docking, binding energy estimation, and virtual screening bacterial fatty acids synthesis and its relationships with polyketide synthetic pathways impact of membrane phospholipid alterations in escherichia coli on cellular 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potassium channel assays in drug development production, characterization, and evaluation of kaemferol nanosuspension for improving oral bioavailability bioavailability of ellagic acid in pomegranate (punica granum l.) juice bioavailability of quercetin bioavailability of ellagic acid after single dose administration using hplc disposition of quercetin and kaempferol in a human following an oral administration of ginkgo biloba flavonoid bioavailability and attempts for bioavailability enhancement comparative pharmacokinetics and bioavailability studies of quercetin, kaempferol, and isorhamnetin after oral administration of ginkgo biloba extracts, ginkgo biloba extract phospholipid complexes and ginkgo biloba extract solid dispersions in rats advancement of multi-target drug discoveries and promising applications in the field of alzheimer's disease qsar-based virtual screening: advances and applications in drug discovery conflict of interest the authors declare that having no conflict of interest. the italic emphasis indicates desirable prosperity, the bold emphasis indicates undesirable prosperity ames tox. ames toxicity key: cord- -g qe ml authors: di sotto, antonella; vitalone, annabella; di giacomo, silvia title: plant-derived nutraceuticals and immune system modulation: an evidence-based overview date: - - journal: vaccines (basel) doi: . /vaccines sha: doc_id: cord_uid: g qe ml immunomodulators are agents able to affect the immune system, by boosting the immune defences to improve the body reaction against infectious or exogenous injuries, or suppressing the abnormal immune response occurring in immune disorders. moreover, immunoadjuvants can support immune system acting on nonimmune targets, thus improving the immune response. the modulation of inflammatory pathways and microbiome can also contribute to control the immune function. some plant-based nutraceuticals have been studied as possible immunomodulating agents due to their multiple and pleiotropic effects. being usually more tolerable than pharmacological treatments, their adjuvant contribution is approached as a desirable nutraceutical strategy. in the present review, the up to date knowledge about the immunomodulating properties of polysaccharides, fatty acids and labdane diterpenes have been analyzed, in order to give scientific basic and clinical evidence to support their practical use. since promising evidence in preclinical studies, limited and sometimes confusing results have been highlighted in clinical trials, likely due to low methodological quality and lacking standardization. more investigations of high quality and specificity are required to describe in depth the usefulness of these plant-derived nutraceuticals in the immune system modulation, for health promoting and disease preventing purposes. immunomodulators are defined as agents able to affect the immune response, which represents the set of reactions activated to protect the organism against infective agents, environmental injuries and illness; moreover, immune response can counteract the invasion of harmful native cells, such as precancerous and cancerous ones [ ] . immune response is mediated by a first line of defence, namely innate immunity (figure ), which is characterized by physical and biochemical barriers, alongside a non-specific cell-mediated immune response, including granulocytes, macrophages, natural killer cells and humoral elements, which cooperate to counteract pathogen infection and malignant transformation [ ] . moreover, an adaptive immunity is activated as a second defense line after a macrophage-mediated presentation of antigens to b lymphocytes, with the help of t lymphocytes; then, b cells can mediate the humoral immunity through the production of high-affinity antibodies and establish immunological memory [ ] . moreover, t lymphocytes can mediate cellular immunity after activation by cytokines released from helper t cells [ ] . responses involved in innate and adaptive immunity. fast and nonspecific responses, occurring against all factor identified as nonself, are involved in innate immunity; conversely, adaptive immunity is a highly specific, complex and slow response mediated by t and b lymphocytes, which release antigen-specific antibodies and cytokines. immunomodulators can directly affect innate and adaptive response or the factors involved, thus leading to immunostimulant or immunosuppressive effects. a further group of immunomodulators is represented by immunoadjuvants, able to enhance immune response to vaccines without producing specific antigenic effects, and more recently approached as adjuvant pharmacological treatments, especially for viral infections and cancers [ ] [ ] [ ] [ ] [ ] . immune-associated disorders, including autoimmune diseases, viral or bacterial infections, and chronic diseases, are usually associated with acute inflammation, which represents a key component for the activation of immune response [ ] . on the other hand, the chronicity of inflammatory response can negatively influence the immune function, affecting both innate immune cells and t and b lymphocytes, thus suggesting a possible usefulness of anti-inflammatory immunomodulators [ ] . accordingly, immunomodulatory agents, with antioxidant and anti-inflammatory activity, have attracted great attention as possible chemopreventive agents, due to their ability to counteract chronic inflammation, which provides favorable conditions for the transition from normal to cancer cell [ ] . responses involved in innate and adaptive immunity. fast and nonspecific responses, occurring against all factor identified as nonself, are involved in innate immunity; conversely, adaptive immunity is a highly specific, complex and slow response mediated by t and b lymphocytes, which release antigen-specific antibodies and cytokines. immunomodulators can directly affect innate and adaptive response or the factors involved, thus leading to immunostimulant or immunosuppressive effects. immunomodulating agents can affect immunity in a negative or positive manner, thus being categorized as suppressing or stimulant [ ] . particularly, immunosuppressors inhibit the activation of immune response or decrease the activity of its components, thus restoring normalcy. they are of interest in organ transplantations and in autoimmune disorders, wherein the immune system mistakenly activates an immune response against the own body tissues, leading to their destruction [ ] . for instance, vitamin d has been shown to counteract the aberrant immune responses of systemic lupus erythematosus, without compromising the physiological defences and to produce benefits in atopic dermatitis too [ , ] . conversely, immunostimulants boost the endogenous immune defences, thus allowing one to restore or maintain the body homeostasis [ ] . they can be usefully exploited as immunotherapeutic agents by individuals with immunocompromised conditions; however, they can represent suitable prophylactic strategies for healthy individuals or more susceptible subjects against viral infections [ ] . in support, during the current sars-cov- pandemic, the trained immunity by vaccines, which induce heterologous protection, have been proposed as a rational strategy to boost antiviral defences and reduce susceptibility to infection [ ] . a further group of immunomodulators is represented by immunoadjuvants, able to enhance immune response to vaccines without producing specific antigenic effects, and more recently approached as adjuvant pharmacological treatments, especially for viral infections and cancers [ ] [ ] [ ] [ ] [ ] . immune-associated disorders, including autoimmune diseases, viral or bacterial infections, and chronic diseases, are usually associated with acute inflammation, which represents a key component for the activation of immune response [ ] . on the other hand, the chronicity of inflammatory response can negatively influence the immune function, affecting both innate immune cells and t and b lymphocytes, thus suggesting a possible usefulness of anti-inflammatory immunomodulators [ ] . accordingly, immunomodulatory agents, with antioxidant and anti-inflammatory activity, have attracted great attention as possible chemopreventive agents, due to their ability to counteract chronic inflammation, which provides favorable conditions for the transition from normal to cancer cell [ ] . furthermore, immunostimulants can act as adjuvant anticancer treatments, to counteract their immunosuppressive side-effects [ ] . particularly, aristolochic acid, an alkaloid from aristolochia clematitis l., showed immunostimulatory properties, by enhancing the phagocytic activity of peritoneal macrophages and leukocytes; however, its potential cannot be exploited, because of its carcinogenic risk [ ] . likewise, vincristine and staurosporine act as immunostimulants at low doses, while as immunosuppressors particularly, aristolochic acid, an alkaloid from aristolochia clematitis l., showed immunostimulatory properties, by enhancing the phagocytic activity of peritoneal macrophages and leukocytes; however, its potential cannot be exploited, because of its carcinogenic risk [ ] . likewise, vincristine and staurosporine act as immunostimulants at low doses, while as immunosuppressors at higher doses [ ] . among polyphenols, resveratrol stimulated both cellular and humoral immunity in preclinical models, thus preventing pathogen replication and inflammation, and promoted antitumor immune response too [ ] . furthermore, cichoric acid from echinacea promoted phagocytic activity, both in vitro and in vivo [ ] . anti-inflammatory and immune-modulatory effects has been highlighted for curcumin too, although the poor bioavailability limits its clinical application [ ] . in the present review, up to date knowledge on the scientific basis for the immunomodulatory activity and clinical relevance of some emerging classes of plant-derived nutraceuticals, including polysaccharides, fatty acids and labdane diterpenes, has been reported. a comprehensive search was made using pubmed and scopus electronic databases and selecting english as the preferred language, although no language limitations nor filters were applied. for more specific requirements, google scholar and clinicaltrials.gov were considered too. the following searching keywords and their combinations through the boolean logical operators were used: "herbal immunomodulators", "phytochemicals", "immune system", "nutraceuticals", "medicinal plants", "immunomodulation", "immune system boosters", "immunosuppressors", "immunoadjuvants", "gut microbiome", "natural occurrence", "chemical features", "preclinical studies", "clinical trials", "polysaccharides", "echinacea", "astragalus", "β-glucan", "fatty acids", "pufa", "oleic acid", "punicic acid", "γ-linolenic acid", "linoleic acid", "evening primrose oil", "borage oil", "flaxseed oils", "labdane diterpenes" and "andrographolide". this overview allows one to identify novel immune system modulators to be usefully exploited for health promoting and disease preventing purposes. polysaccharides are carbohydrate macromolecules containing at least monosaccharide units, joined by glycosidic linkages to form long-chain molecules, which can be both linear and highly branched. they are called homopolysaccharides when constituted of the same monosaccharide unit, while heteropolysaccharides if different units are present. some of them are also referred to as dietary fibres, meaning that these macromolecules are neither digested nor absorbed in the human small intestine [ ] . several polysaccharides have been found to modulate both innate and adaptive immune responses, among which, glucans, mannans, pectins, fucoidans, galactans, fructans, and xylans are the most studied ( figure ) [ ] . chemical structure, molecular weight, conformation, the presence of functional groups (i.e., acetyl and sulfate groups), and branching have been identified as structural features for the immunostimulatory properties of polysaccharides. the chemical structures of the polysaccharides associated with immunomodulatory properties are displayed in figure . glucans are based on the d-glucopyranosyl unit (homoglucans); the different glycosidic bonds, namely (β → ), (β → ), and (β → ) or (α → ), (α → ), and (α → ), allow the production of linear and branched glucans. it seems that (β → )-d-glucan moiety, triple helix conformations, sulfation and carboxymethylation of (β → )-d-glucans, and chain acetylation are involved in glucan immunostimulatory activity. regarding (α → ) (α → )-d-glucans, their structure activity relationship is less characterized [ ] . while heteropolysaccharides if different units are present. some of them are also referred to as dietary fibres, meaning that these macromolecules are neither digested nor absorbed in the human small intestine [ ] . several polysaccharides have been found to modulate both innate and adaptive immune responses, among which, glucans, mannans, pectins, fucoidans, galactans, fructans, and xylans are the most studied ( figure ) [ ] . chemical structure, molecular weight, conformation, the presence of functional groups (i.e., acetyl and sulfate groups), and branching have been identified as structural features for the immunostimulatory properties of polysaccharides. the chemical structures of the polysaccharides associated with immunomodulatory properties are displayed in figure . glucans are based on the d-glucopyranosyl unit (homoglucans); the different glycosidic bonds, namely (β → ), (β → ), and (β → ) or (α → ), (α → ), and (α → ), allow the production of linear and branched glucans. it seems that (β → )-d-glucan moiety, triple helix conformations, sulfation and carboxymethylation of (β → )-d-glucans, and chain acetylation are involved in glucan immunostimulatory activity. regarding (α → ) (α → )-d-glucans, their structure activity relationship is less characterized [ ] . mannans consist of a d-mannose backbone, linked mainly by β → bonds, which can be ramified with other monosaccharide, so originating glucomannan, galactomannan, and galactoglucomannan [ ] . furthermore, (β → )-or (α → )-, (β → )-, and (β → )-or (α → )-dmannosidic bonds are reported [ ] . the (β → )-d-mannan moiety (e.g., galctoglucomannans), acetyl and sulfate group presence, and this kind of branching seems to confer a high immunostimulatory activity [ ] [ ] [ ] . pectins are complex polysaccharides which contain a common galactopyranosyluronic acid. homogalacturonans, xylogalacturonan, apiogalacturonan, rhamnogalacturonan, type i and ii arabinogalactans belong to this class. particularly, type i arabinogalactans (ag-i) possess an α- arabinofuranosyl and β-d-galactopyranosyl units linked via position at the main chain, while type ii arabinogalactans (ag-ii) comprise highly branched polysaccharides with ramified chains of (β → )-and (β → )-d-galactopyranosyl units [ , ] , to which the arabinosyl units might be attached. the degree of branching, methyl esterification, acetylation, and the type of branched chains and mannans consist of a d-mannose backbone, linked mainly by β → bonds, which can be ramified with other monosaccharide, so originating glucomannan, galactomannan, and galactoglucomannan [ ] . furthermore, (β → )-or (α → )-, (β → )-, and (β → )-or (α → )-d-mannosidic bonds are reported [ ] . the (β → )-d-mannan moiety (e.g., galctoglucomannans), acetyl and sulfate group presence, and this kind of branching seems to confer a high immunostimulatory activity [ ] [ ] [ ] . pectins are complex polysaccharides which contain a common galactopyranosyluronic acid. homogalacturonans, xylogalacturonan, apiogalacturonan, rhamnogalacturonan, type i and ii arabinogalactans belong to this class. particularly, type i arabinogalactans (ag-i) possess an α- -arabinofuranosyl and β-d-galactopyranosyl units linked via position at the main chain, while type ii arabinogalactans (ag-ii) comprise highly branched polysaccharides with ramified chains of (β → )-and (β → )-d-galactopyranosyl units [ , ] , to which the arabinosyl units might be attached. the degree of branching, methyl esterification, acetylation, and the type of branched chains and molecular weight determine the structural diversity [ ] . moreover, flexible chain conformation and branched regions are the main ones responsible for the immunomodulatory properties [ , ] . galactans are polysaccharides rich in galactose and include, beside type i and ii arabinogalactans, carrageenans, chemically characterized by repeating disaccharide units of sulfated or unsulfated d-galactose, that are linked by (β → )-and (α → )-bonds. low molecular weight (< kda) and a high degree of sulfation have been reported as features that high influence their immunomodulatory properties [ , ] . fucoidans are heteropolysaccharides rich in l-fucopyranosyl sulfated units linked by (α → ), (α → ) or (α → ) bonds. other monosaccharides can be present, such as galactopyranosyl, mannopyranosyl, xylosepyranosyl and uronic acids [ ] . the naturally higher content of sulfate groups and the presence of acetyl groups are associated with a higher stimulatory activity [ ] . fructans are polysaccharides which constitute up to fructose units with a sucrolose terminal molecule. they are classified in inulin with a (β → )-d-fructofuranosyl, levan with a (β → )-d-fructofuranosyl, and mixed type, with both (β → )-and (β → )-linked d-fructofuranosyl moieties. a helical conformation has been associated with the modulatory activity on the immune system [ , ] . at last, xylans are polysaccharides containing predominantly a backbone of (β → )-dxylosepyranosyl units. other monomers attached to their backbone include α-dglucopyranosyl a units (glucuronoxylans) and α-l-arabinofuranosyl units (arabinoxylans). a correlation between their structure and activity has not been elucidated yet [ , ] . polysaccharides are naturally occuring in animal body fluids, cell walls, bacteria, yeast and fungi, extra cellular fluids, and in plant seeds, stems and leaves, which represent the focus of the present review. the main advantage of plant polysaccharides seems to be the low toxicity with respect to immunomodulatory bacterial polysaccharides and synthetic compounds [ ] . thus, they represent an ideal alternative for immune modulation. a variety of polysaccharides with immunomodulatory properties have been discovered in different species of plants (table ). among the most studied, there are type i and ii arabinogalactans from astragalus membranaceus (fisch.) bge., fructans from allium sativum l. [ ] , fucogalactoxyloglucan and type ii acidic arabinogalactan from echinacea purpurea l. (moench), ginsan and panaxanes from panax ginseng c.a. meyer, acemannan and aloeride from aloe vera l. [ ] , and glucomannan from amorphallus konjac koch [ ] . several studies have shown that polysaccharides from plants can modulate both innate and acquired intestinal immunity, by direct and indirect mechanisms. the former include the activation of immune cells (e.g., macrophages, dendritic cells, natural killer cells, t cells, b lymphocytes), while the latter the short-chain fatty acid (scfa) formation ( figure ). of immune cells (e.g., macrophages, dendritic cells, natural killer cells, t cells, b lymphocytes), while the latter the short-chain fatty acid (scfa) formation ( figure ). the immunomodulatory effects of plant polysaccharides on macrophages are mainly achieved through the generation of reactive oxygen and nitrogen species (ros and nos), and the stimulation of cytokines secretion, cell proliferation, and macrophage phagocytic activity [ ] . for example, a. membranaceus polysaccharides have been shown to promote nitric oxide (no) synthesis in macrophages, by inducing the gene expression of inducible nitric oxide synthase (inos), through the activation of nuclear factor kappa-b (nf-κb)/rel [ , ] . moreover, they were also able to increase the macrophage phagocytic activity, by enhancing their secretion of release factor and intracellular ca + concentration [ , ] . pectic polysaccharides from citrus unshiu marc. have been shown to simultaneously regulate the expression of pro-and anti-inflammatory cytokines. particularly, they increased the production of the pro-inflammatory cytokines tumor necrosis factor (tnf)-α and interleukin (il)- and the antiinflammatory cytokine il- in macrophage raw . , so showing a regulatory mechanism to maintain an equilibrium state [ ] . arabinogalactan from e. purpurea has been reported to increase macrophages activation and il- , tnf-α and interferon (ifn)-β production [ ] . the activation of macrophages by plant polysaccharides seems to be due to specific receptors present on their surface, which initiates the immune response, and exerts an immunomodulatory effect. these receptors are called pattern recognition molecules, and include: toll-like receptor (tlr ), cd , complement receptor (cr ), scavenger receptor (sr), mannose receptor (mr), and dectin- . their activation determines a series of intracellular signaling cascades, leading to the transcriptional activation and production of inflammation-related cytokines [ ] . immunity modulation by plant polysaccharides can be achieved also by modulating the cytokine release from intestinal dendritic cells. indeed, pectin has been shown to reduce il- and il- release induced by the synthetic lipopeptide p csk [ ] . moreover, inulin, pectin, arabinoxylan and β-glucan have been found to elevate il- /il- ratio and to reduce the release of ifn-γ, il- , il- , il- , il- , monocyte chemoattractant protein (mcp)- , macrophage inflammatory proteins the immunomodulatory effects of plant polysaccharides on macrophages are mainly achieved through the generation of reactive oxygen and nitrogen species (ros and nos), and the stimulation of cytokines secretion, cell proliferation, and macrophage phagocytic activity [ ] . for example, a. membranaceus polysaccharides have been shown to promote nitric oxide (no) synthesis in macrophages, by inducing the gene expression of inducible nitric oxide synthase (inos), through the activation of nuclear factor kappa-b (nf-κb)/rel [ , ] . moreover, they were also able to increase the macrophage phagocytic activity, by enhancing their secretion of release factor and intracellular ca + concentration [ , ] . pectic polysaccharides from citrus unshiu marc. have been shown to simultaneously regulate the expression of pro-and anti-inflammatory cytokines. particularly, they increased the production of the pro-inflammatory cytokines tumor necrosis factor (tnf)-α and interleukin (il)- and the anti-inflammatory cytokine il- in macrophage raw . , so showing a regulatory mechanism to maintain an equilibrium state [ ] . arabinogalactan from e. purpurea has been reported to increase macrophages activation and il- , tnf-α and interferon (ifn)-β production [ ] . the activation of macrophages by plant polysaccharides seems to be due to specific receptors present on their surface, which initiates the immune response, and exerts an immunomodulatory effect. these receptors are called pattern recognition molecules, and include: toll-like receptor (tlr ), cd , complement receptor (cr ), scavenger receptor (sr), mannose receptor (mr), and dectin- . their activation determines a series of intracellular signaling cascades, leading to the transcriptional activation and production of inflammation-related cytokines [ ] . immunity modulation by plant polysaccharides can be achieved also by modulating the cytokine release from intestinal dendritic cells. indeed, pectin has been shown to reduce il- and il- release induced by the synthetic lipopeptide p csk [ ] . moreover, inulin, pectin, arabinoxylan and β-glucan have been found to elevate il- /il- ratio and to reduce the release of ifn-γ, il- , il- , il- , il- , monocyte chemoattractant protein (mcp)- , macrophage inflammatory proteins (mip)- α, rantes and tnf-α by dendritic cells [ ] . polysaccharide enriched extracts of e. purpurea have been found to promote the phenotypic and functional maturation of dendritic cells by modulating c-jun n-terminal kinase (jnk), p mitogen-activated protein kinase (mapk) and nf-κb pathways [ ] . the activation of natural killer (nk) cells also contributes to the immunity modulation by polysaccharides. indeed, it has been shown that a. membranaceus polysaccharides can enhance the activity and killing effects of nk cells and promote their proliferation in rats with gastric cancer [ ] . moreover, they were able to increase cd -cd -cd + nks in peripheral blood lymphocytes [ ] . nks activation is probably due to the polysaccharides interaction with the killer cell lectin-like receptor k (klrk ) of nks [ ] . arabinoxylans extracted from wheat bran have been shown to inhibit the growth of transplantable tumors, and to promote the nk cell activity in s tumor-bearing mice [ ] . moreover, the mgn- rice bran arabinoxylan showed to enhance natural killer (nk) cell activity in aged c bl/ and c h mice upon its intraperitoneal injection [ ] . adaptive immunity is also modulated by plant polysaccharides. particularly, fan et al. have shown that polysaccharides from a. membranaceus significantly up-regulated the proliferation of b lymphocytes, probably through the interaction with immunoglobulin on the surface of b cells [ ] [ ] [ ] . a. membranaceus polysaccharides were also able to increase the number of cd +cd +cd + memory t helper (th) cells and cd +cd -cd + cytotoxic t cells [ ] . moreover, they also enhance the cd +/cd + t cell ratio [ ] . furthermore, arabinoxylan were found to increase the activation of tand b-cells and humoral and cell-mediated immunity in tumor bearing mice [ ] . at last, β-glucan microparticles enhanced t-cell activation and proliferation in vitro [ ] . their ability to affect the immune system by inducing th and/or th type immune response makes polysaccharides suitable adjuvants of the vaccine. among them, inulin, chitosan, glucans and mannans have been most extensively studied. particularly, the gamma and delta forms of inulin fructan have shown adjuvant activity against infectious pathogens by stimulating both th and th responses without inducing immunoglobulin e (ige) production [ ] . moreover, advax, a polysaccharide derived from delta inulin, has demonstrated to increase the immunization derived from influenza vaccine in mice. particularly, an induction of neutralizing antibody and memory b-cell against influenza, an increase in cd and cd t-cell proliferation, and enhanced levels of il- , ifn-γ, il- , il- were highlighted [ ] . advax also enhanced the immunogenicity of hepatitis b surface antigen (hbs) in mice and guinea pigs, by increasing both anti-hbs antibody titers and anti-hbs cd and cd t-cells. th , th and th responses were increased too [ ] . astragalus polysaccharides were also used as adjuvants of hepatitis b virus dna vaccine in a mice model, showing increased hbsag-specific antibody levels, higher activity of t cells, the production of il- , il- and ifn-γ by cd + t cells, and ifn-γ expression of cd + t cells. moreover, a stimulation of cytotoxic lymphocytes and dendritic cells maturation, and a reduction in the frequency of regulatory t cells were observed [ ] . mannans and fructooligosaccharide have also been shown to possess adjuvanticity [ , ] . furthermore, indirect effects are involved in the immunomodulatory properties of polysaccharides. in particular, dietary fibers (e.g., inulin, mannan, β-glucan, pectin) are metabolized by intestinal bacteria in the anaerobic environment of the cecum and colon, so generating scfa, such as acetate, propionate and butyrate [ ] . these molecules are able to cross the gut epithelium and interact with surface receptors on the immune cells, such as the g-protein coupled receptors (gprs) and [ ] . the activation of gprs by scfa modulates inflammatory signalling pathways, such as nf-κb, erk and p mapk [ , ] . moreover, it has been highlighted that scfa can reach t lymphocyte nucleus, so modulating several functions through a histone deacetylase (hdac) inhibition. recently, scfa have been reported able to induce t cells metabolic alterations by enhancing the mtor complex activity. particularly, after absorption into t cells, scfa can stimulate the activity of mtor complex, so increasing the conversion of pyruvate into acetyl-coa. moreover, the acetyl groups from scfa can be link to coa and enter the tricarboxylic acid cycle. the increased levels of citrate are exported from mitochondria into the cytoplasm, where the enzyme atp citrate lyase converts it into acetyl-coa, then used by histone acetyltransferases (hats) for histone acetylation and the regulation of cytokine gene expression [ ] . some clinical studies have been carried out on the potential immunomodulatory properties of polysaccharides, and a. membranaceus, e. purpurea and β-glucan have been most investigated. in a clinical trial on a. membranaceus by jiang et al. [ ] , twenthy-eight stable continuous ambulatory peritoneal dialysis patients were treated with peritoneal dialysis fluid containing astragalus ( ml/ l) for one week. an increase in the macrophage phagocytic capacity, no and tnf-α contents were observed in patients compared to those before the treatment [ ] . furthermore, ji et al. [ ] investigated the effect of astragalus pre-operative treatment of colorectal cancer patients (n = ) on immune function. results showed that astragalus pre-operative treatment promoted the nk cell activity in postoperative patients. in addition, the possible immunomodulatory activity of astragalus in patients with acute exacerbations of bronchial asthma (n = ) has been investigated [ ] . particularly, it was observed that the combination of conventional therapy with astragalus injection for days improved the effects of routine treatment, by enhancing t lymphocyte and nk-cells immune function. results of clinical trials on the immune system modulation by e. purpurea are controversial. particularly, a randomized blinded trial carried out on patients revealed that there was no significant difference in the incidence and severity of colds and respiratory infection between echinacea treatment ( weeks) and placebo groups. however, a small decrease of total lymphocyte counts was observed [ ] . another randomized, placebo controlled, double-blind clinical trial investigated the effect of different echinacea preparations, namely echinaforce ® (e. purpurea preparation from % herba and % radix), e. purpurea concentrate (same preparation at times higher concentration), special e. purpurea radix preparation (totally different from that of echinaforce ® ) on the reduction of the complaint index, defined by symptoms in healthy, adult volunteers who caught a common cold. the treatment continued until the enrolled patients felt healthy again, but not longer than days. the supplementation with echinaforce ® and its concentrated preparation showed to be significantly more effective than the special echinacea extract or placebo. moreover, all treatments were well tolerated [ ] . furthermore, prevention trials have been carried out, showing that echinacea products slightly reduce the risk of getting a cold in healthy individuals [ ] . however, the heterogeneity (e.g., different species and part used) of preparations used in the trials makes the conclusions on the potential immunomodulatory properties of echinacea difficult. clinical trials concerning the β-glucan immunomodulatory properties have also been carried out, although in some cases, yeast-derived-glucan were used. particularly, three randomized, double-blind, placebo-controlled studies have evaluated the effects of short-term β-glucan supplementation on children with chronic respiratory problems. after days' treatment, significant improvements in immunoglobulin, lysozyme, exhaled nitric oxide, and calprotectin production were found [ ] [ ] [ ] . furthermore, the combination of resveratrol plus carboxymethyl-β-glucan as a solution for aerosol has been tested in clinical trials. particularly, the ability of the combination to prevent or treat recurrent respiratory infections in children was studied [ , ] . in both cases, resveratrol plus carboxymethyl-β-glucan had a positive impact on children clinical conditions. indeed, nasal obstruction, rhinorrhea, sneezing, cough, fever, medication use, medical visits, and school absence were significantly reduced. moreover, resveratrol plus carboxymethyl-β-glucan have also been shown to relief nasal symptoms in children with allergic rhinitis, due to pollen allergy [ ] . at last, mannans should be mentioned. they have been reported to possess adjuvant-vaccine properties in clinical studies, probably mediated by its interaction with mannose receptors. particularly, it has been shown that oxidized mannan-mucin can be useful as an adjuvant in the breast cancer immunotherapy. indeed, a - years follow-up has highlighted that it decreases the cancer recurrence rate and prolongs recurrence time, without inducing toxicity or adverse reactions [ ] . fatty acids (fa) are a large group of lipids, characterized by a different number of carbons, arranged in a linear carbon chain skeleton of variable length with a terminal carboxylic group [ ] . based on the number of carbons in the chain, fatty acids can be classified as shortchain fatty acids (scfa; aliphatic tails up to a maximum of six carbons), medium-chain fatty acids (mcfa; aliphatic tails of - carbons), long-chain fatty acids (lcfa; aliphatic tails of to carbons) and very long-chain fatty acids (vlcfa; aliphatic tails of and more carbons) [ ] . among them, scfa, such as acetate, propionate, and butyrate, are produced by gut microbiota enzymes (i.e., propionate-coa transferase and propionaldehyde dehydratase) during the metabolism of carbohydrates and peptides containing branched-chain amino acids [ ] . bacteroidetes are reported to be mainly responsible for the production of acetate and propionate, while firmicutes are the primary contributors of butyrate; however, other bacteria such as lactobacillus and bifidobacterium spp. are involved too [ ] . based on the presence of different double bonds in this structure, fatty acids can be distinguished in saturated fatty acids (sfa), lacking double bonds in their carbon backbone, and unsaturated fa (ufa), which may contain one or more double bonds, thus leading to monounsaturated (mufa) and polyunsaturated fa (pufa) [ ] . sfa include palmitic acid (c : ), lauric acid (c : ), myristic acid (c : ), and stearic acid (c : ), whereas n- oleic acid (c : ) is an example of mufa. furthermore, pufa class includes fatty acids such as α-linolenic acid (ala; c : ), linoleic acid (la; c : ) and further long-chain metabolites [ ] . the number of carbon atoms and unsaturated bond position are used for the systematic nomenclature of fa. moreover, the greek letters omega (ω) and delta (∆) are included, to indicate how far a double bond is from the terminal methyl carbon and the presence and position of one or more double or triple bonds in the carbon backbone, respectively [ ] . a further "ω" or "n" classification designates the position of the first double bond in the skeleton from the end opposite to the carboxy group. accordingly, oleic acid is classified as a ω- (or n- ) fatty acid, while linoleic acid and α-linolenic acid are ω- (or n- ) and ω- (or n- ) fatty acids, as they contain the double bond nine, six and three carbons from the methyl end [ ] . nomenclature of the major representative fatty acids in the different fa classes is displayed in table . unsaturated fatty acids can be characterized on the basis of the cisor transorientation of the double bonds. usually, natural fatty acids carry a cisconfiguration, although some trans-fatty acids can also occur in foods as a consequence of the hydrogenation process, which can move double bonds from their naturally occurring position to a trans-configuration [ ] . trans-fatty acids are considered undesirable compounds in foods, as their intake is associated with an increased risk of cardiovascular and metabolic diseases [ ] . pufa can be further classified depending on the relative positions of the double bonds, as conjugated (double-bonded carbon atoms alternate with single bonds) and unconjugated (double bonds separated by one or more single bonds) [ ] . unconjugated pufa, especially ω- , ω- , and ω- series, are the most occurring in nature. the most common conjugated pufa are trienes, such as octadecatrienoic acids (e.g., punic acid, calendic acid). fatty acids within the series are biosynthetically related, being synthesized through enzymatic processes of desaturation, chain elongation, and chain shortening [ ] . particularly, the biosynthesis of ω- and ω- pufa starts from α-linolenic acid (ala or linolenate; , , - : ) and linoleic acid (la or linoleate; , - : ), respectively ( figure ). these precursors cannot be synthetized by mammals, which lack the ∆ and ∆ desaturases responsible for the convertion of : ω- fa to : ω- and : ω- pufa, and must be supplied by the diet, thus being considered as essential fatty acids. the initial rate-limiting step for the biosynthesis of ω and ω fatty acids is the insertion of a further double bond at the ∆ carbon into the carbon chain of ala and la, through the help of a ∆ desaturase enzyme: stearidonic acid (sa; , , , - : ) and γ-linolenic acid (gla; , , - : ) are formed, respectively. these compounds are converted to eicosatetraenoic acid (eta; , , , - : ) and dihomo γ-linolenic acid (dgla; , , - : ) by the elongase , being further converted to eicosapentaenoic acid (epa; , , , , - : ) and arachidonic acid (aa; , , , - : ) , by the addition of a double bond at the ∆ position, through a ∆ desaturase. further elongations convert epa and aa to docosapentaenoic acid ( , , , , - : ) and adrenic acid ( , , , - : ) ; then, a desaturation by ∆ desaturase generates docosahexaenoic acid (dha; , , , , , - : ) [ ] . both series of fatty acids can be further metabolized by cyclooxygenase and lipoxygenase enzymes, to obtain eicosanoids, including prostaglandins, thromboxanes and leukotrienes, acting as central modulators of the inflammatory process [ ] . the byosynthetic pathways of ω and ω fatty acids are interconnected; indeed, it is known that long-chain derivatives from linolenic acid are accumulated in tissue only slightly when competing ω analogues exceed their amounts. therefore, suitable levels can be reasonably obtained through diet and when an optimum ratio of ω and ω series is maintained. both series of fatty acids can be further metabolized by cyclooxygenase and lipoxygenase enzymes, to obtain eicosanoids, including prostaglandins, thromboxanes and leukotrienes, acting as central modulators of the inflammatory process [ ] . the byosynthetic pathways of ω and ω fatty acids are interconnected; indeed, it is known that long-chain derivatives from linolenic acid are accumulated in tissue only slightly when competing ω analogues exceed their amounts. therefore, suitable levels can be reasonably obtained through diet and when an optimum ratio of ω and ω series is maintained. figure . biosynthetic pathways of ω- and ω- fatty acids. fatty acids occur widely in nature, being identified in both animal tissue and plants. particularly, short-chain saturated acids are components of milk fats: in bovine milk, butanoic acid along with other scfa and mcfa have been reported [ ] . likewise, the mcfa lauric acid and myristic acid are the major components of the oils obtained from some lauraceae and myristiceae species [ ] . moreover, palmitic acid is the most representative sfa in vegetable oils, such as palm oil [ ] . fatty acids with immune modulating properties mainly belong to the long-chain classes; among them, punicic acid is a peculiar conjugated triene, found to be a unique component of pomegranate seed oil [ ] , while oleic acid is one of the most widely distributed fatty acids: it represents % to % of total fa in olive oil, although it does occur in high amounts in other oils, such as those from grape seeds, canola and sufflower [ , ] . moreover, ω and ω fatty acids have been highlighted in several natural sources, wherein both series co-occur (table ) , although in different amounts [ ] . table . major natural sources of long-chain monounsaturated (mufa) and polyunsaturated fatty acids (pufa) associated with immune system modulating activities and relative amounts. some vegetable oils, including rapseed, hemp seed, and sunflower oils, contain higher levels of la (essential ω pufa), while ala (essential ω pufa) is in lower proportion; a similar trend has also been reported for soybean, corn, and for dried black walnuts and brazilnuts; conversely, higher amounts of ala respect to la are reported in flaxseed oil and in the seeds of chia and perilla [ ] . likewise, green leafy vegetables seem to be an interesting source of ala [ ] . fish oils are also sources of both epa and dha (ω pufa), with lower amounts of dpa (ω pufa) [ ] . wild marine species showed to contain higher ω pufa levels compared to farmed ones, likely due to the feed composition [ ] . some vegetables can supply both the essential pufa and some derivative fatty acids. particularly, the oils obtained from the seeds of borago spp., echium spp., ranunculus spp. and oenothera biennis l. have been reported to contain high levels of both la and γ-linolenic acid (gla) [ , ] . fatty acids play energetic, metabolic, and structural functions, being the main component of phospholipids, triglycerides, diglycerides and monoglycerides. a separate category is represented by scfa, which act as metabolites of carbohydrates, produced by gut microbiota: their role in the modulation of immune function is described in section . . long-chain fatty acids have been found involved in immune modulation, being able to affect both innate and adaptive response. although specific profiles characterize each class of fatty acids, these effects are mainly ascribed to their ability to target the cell membrane, where they can be incorporated, thus changing membrane composition and fluidity and modulating membrane-protein interaction and signal transduction. furthermore, a role in the control of inflammation has been reported. epithelial growth factor receptors (a critical crossroad of multiple receptor pathways which is potentially implicated in the regulation of proliferation and possibly involved in atherogenesis) are considered possible targets for unsaturated fatty acids [ ] . mufa, especially oleic acid, have attracted great attention in the years as possible immunomodulating nutrients. preclinical studies demonstrated the ability of oleic acid to modulate the immune system, through affecting both innate and adaptive immunity response [ ] . indeed, it diminished nk cell activity [ ] and the expression of the leucocyte adhesion molecules, which have shown to be implicated in some pathophysiological conditions, such as rheumatoid arthritis [ ] . furthermore, it enhanced neutrophil aggregation and neutrophil-endothelial cell attachment, phagocytic and candidacidal capacities [ , ] . in regard to adaptive response, it inhibited the proliferation of immune cells, such as jurkat t cells and lymphocytes, likely through the regulation of the cell cycle, although the true mechanisms remain to be clarified [ ] . similar suppressive effects were also highlighted for its synthetic analogue minerval and confirmed in animal models [ , ] . furthermore, the treatment with oleic acid and minerval induced proapoptotic effects in jurkat (t lymphocyte) and raji (b lymphocyte) cells, likely due to mitochondrial depolarization and ros production [ ] [ ] [ ] . recently, oleate has been reported to be able to protect macrophages from palmitate-induced lipotoxicity; moreover, it has been associated with an increase in the regulatory phenotype of the myeloid msc- suppressor cells and suppression of activated t cells [ , ] . in the skin, oleic acid, along with other unsaturated fa, seems to be incorporated into the lipid moiety of staphylococcus aureus lpp, inducing an immune response against the pathogen [ ] . regarding conjugated pufa, punicic acid has been shown to improve the immune system development, stimulate the cd + and cd + lymphocyte-mediated immunity and increase the immune response against viruses [ ] . these immune boosting effects are due to nuclear peroxisome proliferator-activated receptor (ppar)γ-and δ-dependent mechanisms, as punicic acid is able to act as an agonist of these receptors; in support, the loss of pparγ in immune cells impaired its effects [ , ] . moreover, punicic acid inhibited the tnf-α-induced priming of ros production by inhibiting the ser -p phox phosphorylation and upstreaming kinase p mapk; likewise, it blocked the tnf-α-induced release of myeloperoxidase from neutrophils, and decreased neutrophil-activation and ros/mpo-mediated tissue damage in vivo [ ] . antinflammatory properties were found to be related to the activation of pparγ and the suppressed expression of inflammatory genes (encoding cytokines, chemokines, cyclooxygenase, no synthase, and metalloproteinases) [ ] . immunomodulatory properties of ω- and ω- pufa have been highlighted in different preclinical models and have been associated with their ability to modulate the inflammatory process [ ] . these fatty acids share common biosynthetic enzymes which mediate the production of different series of eicosanoids, starting from typical precursors, including dihomo-γ-linoleic acid (dgla), arachidonic acid (aa) and eicosapentaenoic acid (epa). among prostanoids, three types of prostaglandins (pg), including pg , pg and pg , can be obtained. pg is associated with beneficial effects and lower inflammation, thus being considered as an antinflammatory prostanoid; conversely, pg has opposite behaviour, increasing inflammation, vasoconstriction and blood clotting. pg acts through a mixture of functions and is able to reduce the pg -mediated inflammation [ ] . starting from dgla, both anti-inflammatory pg and pro-inflammatory pg , through the conversion into arachidonic acid, can be produced (figure ) . effects and lower inflammation, thus being considered as an antinflammatory prostanoid; conversely, pg has opposite behaviour, increasing inflammation, vasoconstriction and blood clotting. pg acts through a mixture of functions and is able to reduce the pg -mediated inflammation [ ] . starting from dgla, both anti-inflammatory pg and pro-inflammatory pg , through the conversion into arachidonic acid, can be produced (figure ) . this synthesis is controlled by the activity of Δ -desaturase and Δ -desaturase enzymes, which are often compromised during inflammatory conditions and diseases. it has been found that diets enriched in ω- fatty acids are able to activate the conversion of dgla into pg , whereas low ω- intake induces the conversion in aa with the synthesis of proinflammatory prostanoids [ ] . aa can also be released by the cell membranes through the action of phospholipase a during cell injuries or changes in biomembrane composition, thus representing a physiological activator of inflammation as a defence response. aa and eicosapentaenoic acid (epa) compete for the synthesis of different series of pg, mediated by cyclooxigenase, while -lipooxygenase (lox) is involved in their conversion into thromboxanes and leukotrienes. particularly, tromboxane a and leukotriene b are produced from aa, while tromboxane a and leukotriene b from epa. epa and dha are also precursors of lipoxins, resolvins and protectins, which produced anti-inflammatory effects and regulate vascular tone and blood pressure [ ] . like punicic acid, the anti-inflammatory effects of epa and dha are mediated by the activation of the pparα/γ [ ] . despite the antinflammatory role of ω- and ω- -based diets, aa increases the plasmatic levels of proinflammatory eicosanoids, associated with an increased incidence of allergic and inflammatory disorders and with excessive cell proliferation. anyhow, it is not clear the usefulness to select ω- with respect to ω- in the diet: a balance between ω- and ω- pufa seems to be essential for mantaining ahealth status. the ability of fatty acids to be incorporated in the cell membrane seems to represent a key mechanism accounting for the immunomodulating properties of ω- and ω- pufa. indeed, immune cells (i.e., t cells and neutrophils) can incorporate exogenous fatty acids into membrane with a lateration in the function of cell surface pattern recognition receptors [ ] . dietary ω- pufa has been shown able to modulate the macrophage function, through the activation of g protein coupled receptors (gpr) and to induce a shift to an anti-inflammatory phenotype [ ] . modulating signalings through the gpr receptor activation can also affect leukocyte function. likewise, an inhibition of the pro-inflammatory phenotype of dendritic cells and of the t cell responses has been reported [ ] . they are also able to inhibit neutrophil and monocyte adhesion, depending on the activation of ppar-α [ ] . conversely, ω- pufas seem to promote inflammation, associated with incresead ros levels, in neutrophils [ ] . particularly, linoleic acid increased the marginated pool of neutrophils in tissues by the induced expression of adhesion molecules;it also complexed with the anti-inflammatory this synthesis is controlled by the activity of ∆ -desaturase and ∆ -desaturase enzymes, which are often compromised during inflammatory conditions and diseases. it has been found that diets enriched in ω- fatty acids are able to activate the conversion of dgla into pg , whereas low ω- intake induces the conversion in aa with the synthesis of proinflammatory prostanoids [ ] . aa can also be released by the cell membranes through the action of phospholipase a during cell injuries or changes in biomembrane composition, thus representing a physiological activator of inflammation as a defence response. aa and eicosapentaenoic acid (epa) compete for the synthesis of different series of pg, mediated by cyclooxigenase, while -lipooxygenase (lox) is involved in their conversion into thromboxanes and leukotrienes. particularly, tromboxane a and leukotriene b are produced from aa, while tromboxane a and leukotriene b from epa. epa and dha are also precursors of lipoxins, resolvins and protectins, which produced anti-inflammatory effects and regulate vascular tone and blood pressure [ ] . like punicic acid, the anti-inflammatory effects of epa and dha are mediated by the activation of the pparα/γ [ ] . despite the antinflammatory role of ω- and ω- -based diets, aa increases the plasmatic levels of proinflammatory eicosanoids, associated with an increased incidence of allergic and inflammatory disorders and with excessive cell proliferation. anyhow, it is not clear the usefulness to select ω- with respect to ω- in the diet: a balance between ω- and ω- pufa seems to be essential for mantaining ahealth status. the ability of fatty acids to be incorporated in the cell membrane seems to represent a key mechanism accounting for the immunomodulating properties of ω- and ω- pufa. indeed, immune cells (i.e., t cells and neutrophils) can incorporate exogenous fatty acids into membrane with a lateration in the function of cell surface pattern recognition receptors [ ] . dietary ω- pufa has been shown able to modulate the macrophage function, through the activation of g protein coupled receptors (gpr) and to induce a shift to an anti-inflammatory phenotype [ ] . modulating signalings through the gpr receptor activation can also affect leukocyte function. likewise, an inhibition of the pro-inflammatory phenotype of dendritic cells and of the t cell responses has been reported [ ] . they are also able to inhibit neutrophil and monocyte adhesion, depending on the activation of ppar-α [ ] . conversely, ω- pufas seem to promote inflammation, associated with incresead ros levels, in neutrophils [ ] . particularly, linoleic acid increased the marginated pool of neutrophils in tissues by the induced expression of adhesion molecules; it also complexed with the anti-inflammatory molecule -antitrypsin, thus reducing lps-induced il- secretion in neutrophils [ ] . on the whole, preclinical evidence highlighted that these fatty acids could increase neutrophil function, thus promoting innate immunity. regarding adaptive immunity, ω- pufa have been reported able to improve the mitogen-mediated activation of immune cells and to promote the development of a th -type immune response [ ] . moreover, an increased production of associated anti-inflammatory cytokines like il- , in spite of a reduction of pro-inflammatory tnf-α, was found [ ] . similar effects were highlighted with both fish oil-enriched diets and the purified epa and dha [ ] . the beneficial influence of ω- pufa has been highlighted also on epithelial cells during inflammation, being able to restore impaired barrier function and reduce the production of pro-inflammatory mediators [ ] . moreover, a strictly interplay between omega- fatty acids, immunity and gut microbiota has been reported and seems to be an essential factor to maintain the intestinal wall integrity. these effects have been ascribed to the ability of ω- pufa to positively affect the microbiota composition and increase the production of anti-inflammatory compounds, like short-chain fatty acids [ ] . although a major interest over the years has been focused on marine sources of ω- -enriched oils or on pure compounds, some plant species have been studied for their immunomodulating and anti-inflammatory properties, likely ascribable to ω- and/or ω- pufa, although the major evidence has been highlighted for linum usitatissimun l., oenothera biennis l. and borago officinalis l. [ , , ] . the seed oil from l. usitatissimum, also known as flaxseed oil, has been reported to induce immunomodulating effects, likely through suppressing cell mediated immunity, without the involvement of humoral immunity. being a rich source of ala, its effects are mainly ascribed to this compound, although further studies suggested a possible contribution of bioactive phenolics [ ] . flaxseed oil was found to be effective in reducing skin inflammatory responses, although with a lower immunosuppressive power with respect to fish oil [ ] . moreover, it improved systemic and gut immunity, in a piglet model with intrauterine growth retardation: increased plasma concentration of immunoglobulin g, decreased cd +cd + t lymphocytes, and the downregulation of genes expression for proinflammatory factors have been reported [ ] . regarding o. biennis, the administration of the seed oil (namely, evening primrose oil) in animal models enhanced pge synthesis in peritoneal macrophages, decreased pge amounts in granulocytes, and suppressed the natural killer (nk) cell activity and lymphocyte proliferation; moreover, it decreased the serum levels of interferon γ (ifn-γ) and mcp- , while stimulating tnf-α [ ] [ ] [ ] [ ] [ ] . furthermore, anti-inflammatory effects have been found to be involved in the immunomodulation by evening primrose oil [ ] . these effects were ascribed to the content of gla, whose t-regulatory cell activity in autoimmune disease models was highlighted [ ] . however, a contribution of la to the antinflammatory effects seems to be likely; indeed, la can itself modulate inflammation as it is metabolized by lox to hydroxyoctadecadienoic acids (hodes) and oxo-hodes, characterized by antinflammatory properties [ ] . similarly, the seed oil from b. officinalis seeds produced immonomodulating and antinflammatory effects, likely through its gla content [ ] . a chemotactic migration of monocytes to necrotic site that differentiate into macrophages is associated with the administration of this product. moreover, it is known to reduce the levels of proinflammatory cytokines, such as tnf-α, and to promote pge generation; a reduced expression of inflammatory genes, especially those of macrophages involved in atherosclerosis, has been reported too [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . clinical studies mainly focused on the effects of fatty acid-enriched diet on inflammation, although specific immune-based pathological conditions associated with inflammation were assessed too. regarding mufa, few studies are available, and results differ from those in animal models. indeed, a mufa-rich diet (with highly refined olive oil for weeks) did not alter the immune function in healthy subjects; such effects could be due to the high amounts administered in animal models [ ] . conversely, clinical evidence about the immunomodulatory power of punicic acid in healthy or sick subjects is lacking [ ] . the ω- pufa series and the relative enriched fish oils have been mainly evaluated for their immunomodulating and antinflammatory effects in humans. although preclinical evidence highlighted their ability to influence both innate and adaptive immunity, the clinical relevance of these results remains to be clarified, due to lacking or inconclusive data [ ] . inadequacy of clinical results should be due to the different doses used in preclinical studies, wherein often high fatty acids levels were administered; moreover, other factors such as genetic and epigenetic heterogeneity of the recruited subjects, diet diversity, nutritional habits and microbiome can be considered as additional confounding factors [ ] . although limitations of clinical studies require further confirmation, ω- pufa intake produced significant clinical benefits and reduction of the symptoms in patients with autoimmune disorders, especially rheumatic diseases and systemic lupus erythematosus [ ] [ ] [ ] . in support, low levels of pufas have been found in the serum of patients with rheumatic diseases [ ] . conversely, inconsistent results are reported for multiple sclerosis, thus the possible usefulness of these fatty acids as supportive therapy requires more clinical trials [ ] . regarding ω- pufa, although they are associated with possible increased inflammatory conditions, being arachidonic acid a presursor of proinflammatory prostanoids, such a risk is not confirmed by clinical evidence [ ] . indeed, studies in healthy human adults highlighted that an increased intake of these fatty acids did not induce inflammation; conversely, epidemiological evidence reported reduced inflammatory conditions [ ] . the antinflammatory effects of la have been reported too [ ] . similarly, increased la intake was found to be not related to increased amounts of ara and proinflammatory factors; however, an inverse correlation with epa and dha was reported [ ] . this suggests that the interaction between ω-e and ω- series is regulated by complex mechanisms that requires further clarifications. major clinical studies have been performed using evening primrose oil (from the seeds of o. biennis), as a source of la and gla, in inflammatory diseases associated with immune system disorders, including atopic dermatitis, psoriasis, multiple sclerosis and rheumatoid arthritis. standardized oils for the content in la and ala (for instance, efamol is titred to contain % la and % gla) were usually used [ ] . the treatment with evening primrose oil ( and weeks) produced clinical improvements in patients with atopic dermatitis, as revealed by measuring the scoring atopic dermatitis [ ] . some beneficial effects were also reported in multiple schlerosis patients, although the few available studies limited the evidence in this disorder. conversely, evening primrose oil in combination with fish oil and vitamin e (efamol marine) failed to improve the symptoms of psoriasiac patients but produced antinflammatory effects [ ] . similarly, in association with ω- fatty acids, it did not induce improvements in patients with rheumatoid arthritis [ ] . a cochrane revision highlighted moderate evidence for oils containing gla (i.e., evening primrose, borage, or blackcurrant seed oil) to produce benefit in rheumatoid arthritis [ , ] . evening primrose oil along with borage oil were not effective to treat eczema too [ ] . highly variable results were also obtained for borage oil in the treatment of atopic dermatitis, although, in all the studies, a moderate efficacy degree was displayed [ ] . regarding flaxseed oils, some clinical trials higlighted a significant improvement of inflammatory parameters in subjects with cardiovascular diseases non-associated with the immune system [ ] . reported studies, although performed in pathological conditions associated with immune system disfunction, did not give a direct measure of the immunomodulatory effects of the treatments. furthermore, specific and high-quality studies are required for better characterizing the possible usefuleness of these pufa-enriched oils as anti-inflammatory and immunomodulating treatments. labdane compounds have a molecular formula c h with an average mass of . da ( figure ). labdane-related molecules have a hydrocarbon skeleton, originated from dual biosynthetic cyclization and/or rearrangement reactions, produced through the biosynthetic pathway of gibberellin phytohormones by the diterpene cyclases. the labane diterpenoids belong to a superfamily of natural products, in which the hydrocarbon skeleton might serve as privileged scaffolds for their biological activity [ ] . labdane compounds have a molecular formula c h with an average mass of . da ( figure ). labdane-related molecules have a hydrocarbon skeleton, originated from dual biosynthetic cyclization and/or rearrangement reactions, produced through the biosynthetic pathway of gibberellin phytohormones by the diterpene cyclases. the labane diterpenoids belong to a superfamily of natural products, in which the hydrocarbon skeleton might serve as privileged scaffolds for their biological activity [ ] . labdane diterpenes have been found in the various matrix of vegetal origin (leaves, rizomes, fruits, etc.) of different plants. in table , some of them (where diterpenes have been found), their botanical family (in parentheses) and the part of plant of biological interest are reported. some labdane diterpenoids, isolated from plant matrix, include the following: andrographolide ( figure ) (from andrographis paniculata (burm.f.) nees) [ ] , labda- ( ), -diene- , -dial (from curcuma amada roxb) [ ] , podoimbricatin c (a , -cyclo-labdane diterpenoid from dacrycarpus imbricatus (blume) de laub) [ ] chapecoderins a-c (from echinodorus macrophyllus (kunth) micheli), [ ] , ( r, s, s, r)- -des-ethyl- -oxolabda- ( ), e-dien- -oic acid (from juniperus oblonga m. bieb) [ ] , leoheteronin d and leojaponin a (from leonurus japonicus houtt) [ ] , marrubasch a-f and marrubenol (from marrubium aschersonii p.magnus), marrulibanoside (from marrubium globosum boiss. and balansa) [ ] , vitexlimolides a-c (from vitex limonifolia wall. ex c.b. clarke) [ ] . labdane diterpenes have been found in the various matrix of vegetal origin (leaves, rizomes, fruits, etc.) of different plants. in table , some of them (where diterpenes have been found), their botanical family (in parentheses) and the part of plant of biological interest are reported. some labdane diterpenoids, isolated from plant matrix, include the following: andrographolide ( figure ) (from andrographis paniculata (burm.f.) nees) [ ] , labda- ( ), -diene- , -dial (from curcuma amada roxb) [ ] , podoimbricatin c (a , -cyclo-labdane diterpenoid from dacrycarpus imbricatus (blume) de laub) [ ] chapecoderins a-c (from echinodorus macrophyllus (kunth) micheli), [ ] , ( r, s, s, r)- -des-ethyl- -oxolabda- ( ), e-dien- -oic acid (from juniperus oblonga m. bieb) [ ] , leoheteronin d and leojaponin a (from leonurus japonicus houtt) [ ] , marrubasch a-f and marrubenol (from marrubium aschersonii p.magnus), marrulibanoside (from marrubium globosum boiss. and balansa) [ ] , vitexlimolides a-c (from vitex limonifolia wall. ex c.b. clarke) [ ] . imbricatus (blume) de laub) [ ] chapecoderins a-c (from echinodorus macrophyllus (kunth) micheli), [ ] , ( r, s, s, r)- -des-ethyl- -oxolabda- ( ), e-dien- -oic acid (from juniperus oblonga m. bieb) [ ] , leoheteronin d and leojaponin a (from leonurus japonicus houtt) [ ] , marrubasch a-f and marrubenol (from marrubium aschersonii p.magnus), marrulibanoside (from marrubium globosum boiss. and balansa) [ ] , vitexlimolides a-c (from vitex limonifolia wall. ex c.b. clarke) [ ] . figure . chemical structure of andrographolide. this has been obtained using chemspider ® chemical structure database. the body's defense responses can be improved through various properties induced by plants. some of them, referring to the plants in table , are shown below. for each plants and plant-derived nutraceutical, only properties potentially attributable to the labdane skeleton and useful to improve the immune system are reported. as both inflammation (biological response of body tissues to harmful stimuli) and oxidative stress (imbalance between reactive oxygen species and a biological system's ability to detoxify/repair the resulting damage of the reactive intermediates) are the main self-defend methods to eliminate pathogens and protect living bodies, plants with antinflammatory and/or radical scavenger properties are considered too [ ] . indeed, labdane diterpenoids have recently gained greater attention from the scientific point of view, due to a wide range of biological activities, including the anti-inflammatory modulation of immune cell functions [ ] . a. paniculata exhibited, in vitro and in vivo, various pharmacological activities, including antihyperglycemic, antiplatelet aggregation, anti-microbial, anti-inflammatory, anti-hiv, anti-cancer, anti-nociceptive activity, etc. it has also been used for autoimmune encephalomyelitis and, in indian and chinese medicine, for respiratory tract infections [ , ] . more recently, a. paniculata has been used to stimulate the immune system and treat myocardial ischemia [ ] . a. paniculata inhibited interleukin (il)- , tnf-α mrna, lps-induced expression, and suppressed levels of tnf-α, il- β, jnk, c-reactive protein, and nf-κb [ ] . many labdane diterpenoids compounds have been found to act on the latter. the activation of the nf-κb pathway leads to several physiological responses, including inflammatory or innate immune response [ ] . in vitro, andrographolide (the main phytoconstituent of a. paniculata) can inhibit inflammation, by regulating protein expression (cytokines, chemokines) and by reducing immune cell infiltration. andrographolide was shown to inhibit also oxidative stress by binding to adenosine a a receptor, by inducing nuclear factor (erthroid-derived )-like (nrf ) translocation, and by increasing the expression of superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase- [ ] . these effects can contribute to the immunoregulatory activity of this plant-derived nutraceutical, as it can modulate the innate and adaptive immune responses by regulating macrophage phenotypic polarization and antibody productions [ ] . moreover, it was found to exert cytotoxic/anticancer effects on almost all types of tumour cell lines (human leukemia, renal tubular epithelial cells, breast cancer cells, etc.), mainly by cell cycle arrest, autophagy, cell death, anti-inflammatory and immune system mediated effects [ ] . other preclinical studies have highlighted pharmacological properties of labdane diterpenoidscontaining plants. data are limited, and consequently also their preclinical evidence. some examples are reported below. c. amada, also known as mango ginger, and its labdane-diterpenoids have shown antiflammatory, antibacterial, insecticidal, antifungal, antipyretic, antioxidant, anticancer, and antitubercolar properties in preclinical trials [ ] . d. imbricatus displayed cytotoxic and anti-neuroinflammatory activities, but it had no cytotoxic activity against human tumour cell lines [ ] . e. macrophyllus, brazilian plant, also known as "leather hat", is used as a methanolic (which contains mainly labane diterpenoids, steroids, alcaloids, etc.) or aqueous extract (rich in flavonoids) of the aerial parts, leaves in particular. in folk medicine, e. macrophyllus is used for various illnesses (respiratoy and urinary diseases, rheumatoid arthritis, atherosclerosis, etc.), as it has been shown to possess tissues protective activity and immunosuppressive effects (impaired secretion and function of b and/or t cells), on humoral or cellular immune responses and on autoimmune rheumatic diseases [ ] . in in vitro/vivo studies, the aqueous extract of e. macrophyllus exhibited strong antinflammatory activities by decreasing rats paw edema, inflammatory exudates, infiltrate tissues, no production, ltb release, and neutrophil migration [ ] . in preclinical studies, the methanolic extract of e. macrophyllus was not cytotoxic, genotoxic, mutagenic, and no acute toxicity (up to the maximum dose of mg/kg b.w.) has been observed in tested animals [ ] . however, the extrapolation of animal experiments to clinical practice must be done with caution [ ] . compounds from j. oblonga have shown anti-tumor effects, through moderate cytotoxicity against human tumor cell lines obtained from various human tissues, including: hepatocellular carcinoma (hepg ), breast cancer (mcf- ), and cervical carcinoma cancer (hela) [ ] . the berries from j. oblonga also have antimicrobial activity and anti-inflammatory effects. labdane diterpenoid (e.g., leonurine), extracted from l. japonicus, exhibited cytotoxicity and cell cicle arrest against cancer cell lines and presented immunomodulatory and antinflammatory activities (suppresses tnf-α, nf-κb, and down-regulated expression of inos, cox , and conseguently peg and no levels) [ ] . marrubium spp. (aschersonii, globosum, etc.) have multiple actions, including antimicrobial and anti-inflammatory activities. marrubasch a-f and marrubenol, isolated from the ethanolic extract of m. aschersonii, exhibited weak reduction in inos activity and, consequentely, no production [ ] . marrulibanoside, obtained from the aerial parts of m. globosum, inhibited catalytic activity of inos and cox- enzymes, and consequentely, the peg and no production. v. limonifolia, in preclinical trial, have shown a strong antiviral activity against coxsackievirus b , human rhinovirus b, and enterovirus (ev ). all of them could be responsible for various illnesses, ranging from common cold, hand, foot, and mouth diseases, to acute flaccid paralysis [ ] . the clinical efficacy of the medicinal plants, and plant-derived nutraceuticals discussed above are almost totally lacking. only for a. paniculata there are some evidence in humans. andrographis extract (various and not standardized), andrographolide, and its derivatives have been studied in the treatment of various disease (multiple sclerosis, infection disease, gastro-intestinal upsets, respiratory ailments, pain), and in the maintenance of immune function. in this last context, it seemed to improve the response to cough and sore throat, shortening the sick leave/time to resolution [ ] . the most interesting activity is the increase of cd + lymphocyte levels, in hiv-positive patients [ ] . the increase in these lymphocytes testifies an improvement in the state of the immune system. moreover, a chinese product containing andrographolide improved the efficacy of glucocorticoids and immunoglobulin in patients with severe hand, food, and mouth disease. however, andrographolide is considered a hazard, as it is irritating, and its injectable use is limited because it could induce allergic reactions (erythema, pruritus, etc.), which are sometimes life-threatening [ ] . preclinical data suggested that andrographolide could be responsible of pharmacokinetics interactions, as it induced cyp a [ ] . the european medicines agency (ema) reports a possibility of causing reproductive toxicity of andrographis extracts (decreases in sperm motility and counts) [ ] . on the other hand, no major adverse effects have been reported for a. paniculata; only minor side effects, mainly gastrointestinal, are known [ ] . notably, even if a. paniculata presents numerous pharmacological properties, andrographolide possess poor solubility (principally in dmso), which severely limits the possibility of achieving a therapeutic effect (if not properly formulated). its better absorption could be achieved by nano-formulations (e.g., nano-emulsion, nano-capsules). immunomodulation by plant-based nutraceuticals represents an interesting tool to be exploited for the treatment and preventing purposes of immune system disorders, due to their multiple bioactivities, well tolerability and good patient compliance. however, as often reported for several herbal medicinal products, some points require being underlined to improve the research in the field and provide solid evidence to support their rational use. according to previous stated critical issues [ , ] , herbal products under study must be characterized for the phytochemical composition, using validated analytical methodologies, and for the extraction procedures; moreover, the starting material should be fully defined in terms of origin (country and region), cultivation conditions, botanical identity and plant part. the content of specific compounds, used as analytical or active markers, should be determined too. these requirements are needed to ensure reproducible pharmacological/clinical activity and to compare different studies. indeed, using nonstandardized phytocomplexes increases variability of the biological response, thus limiting the reliability and validity of the studies. furthermore, to assess the pharmacological activity of specific compounds, purity (at least %) and identity should be characterized. indeed, when assessed as mixtures, the subtle interactions which can be established among phytochemicals make it difficult to understand whether the observed benefits are attributable to a specific class or to the whole phytocomplex. for instance, both fatty acids and polyphenols can be involved in the immunomodulating effects of pufa-enriched plant oils. moreover, as found for both polysaccharides and fatty acids, among the same class, different subclasses can co-occur, thus contributing to the whole effects. regarding preclinical studies, detailed methodologies, including information about specific extraction process, the choice of the tested concentrations and experimental procedures, vehicle effects, and comparison with standard effective compounds (positive controls) should be reported. in order to validate the "goodness" of the treatment, promising results in preclinical studies should be confirmed by clinical evidence of efficacy and lack of toxicological concerns for both the isolated compounds and the whole phytocomplex. at last, possible interactions with diet constituents or possible pharmacological treatments, as reported for andrographolide, which is a cyp a inducer, should be considered. as highlighted for a number of natural products, clinical evidence is a major challenge for plant-based immunomodulating nutraceuticals too, due to limited specific studies. moreover, methodological quality of the available trials was overall poor, the studies often being not blinded, protocol unavailable and lacking the standardization of tested products, thus making the claimed effect difficult to be reproduced. at last, standardized methodologies for systematic reviews and meta-analyses, such as the prisma guidelines [ ] , would allow a rational interpretation of the results and suggestions for future research. medicinal plants are rich sources of bioactive phytochemicals, characterized by multiple and often pleiotropic activities, which can be exploited both therapeutically and as nutraceutical strategies for preventive purposes. among plant-based nutraceuticals, immunomodulators have been highlighted to be of interest as boosters of the immune system, to counteract infectious or exogenous injuries, immunosuppressor, to control the abnormal immune response occurring during autoimmune diseases, or as adjuvants, which contribute by modulating nonimmune targets. in this review, we highlighted the scientific evidence about the immunomodulating properties of three emerging classes of nutraceuticals, including polysaccharides, fatty acids and labdane diterpenes. some of them, especially polysaccharides and labdane diterpenes, act as immune system booster, while fatty acids (mufa and pufa) mainly act as immunosuppressor, although punicic acid (a conjugate pufa) exhibited immunostimulant properties. 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reviews and meta-analyses: the prisma statement this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license a.d.s. and s.d.g. fellowships were funded by grants from sapienza university (ateneo ) and regione lazio. the authors thank "enrico and enrica sovena" foundation (italy) for supporting the study. the authors declare no conflict of interest. key: cord- -b zev zb authors: sobocińska, justyna; roszczenko-jasińska, paula; ciesielska, anna; kwiatkowska, katarzyna title: protein palmitoylation and its role in bacterial and viral infections date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: b zev zb s-palmitoylation is a reversible, enzymatic posttranslational modification of proteins in which palmitoyl chain is attached to a cysteine residue via a thioester linkage. s-palmitoylation determines the functioning of proteins by affecting their association with membranes, compartmentalization in membrane domains, trafficking, and stability. in this review, we focus on s-palmitoylation of proteins, which are crucial for the interactions of pathogenic bacteria and viruses with the host. we discuss the role of palmitoylated proteins in the invasion of host cells by bacteria and viruses, and those involved in the host responses to the infection. we highlight recent data on protein s-palmitoylation in pathogens and their hosts obtained owing to the development of methods based on click chemistry and acyl-biotin exchange allowing proteomic analysis of protein lipidation. the role of the palmitoyl moiety present in bacterial lipopolysaccharide and lipoproteins, contributing to infectivity and affecting recognition of bacteria by innate immune receptors, is also discussed. geranylgeranyl cysteine thioether h-and n-ras ( ) rab proteins ( ) attachment of glycosylphosphatidylinositol anchor or phosphatidylethanolamine ( ) to the c-terminus of proteins is also a form of lipidation but is not shown here. a n-myristoylation is in most cases co-translational, but during apoptosis caspases can cleave some proteins, such as bid, exposing their n-terminal glycine residue, which is then modified by attachment of myristate ( ) . b hedgehog proteins are additionally modified by covalent attachment of cholesterol to their c-terminus ( ) . c o-acylation of wnt proteins is reversed by notum of the α/β hydrolase superfamily ( ) . exceeds the consumption of other saturated fatty acids and, in the usa it accounts for about % of the total intake of saturated fatty acids ( ) . a growing body of experimental and clinical evidence points to a link between a westernized diet, including a high intake of saturated fatty acids, and chronic inflammatory diseases ( ) ( ) ( ) . as dietary saturated and unsaturated fatty acids apparently modulate activity of immune cells, their influence on the immune responses triggered upon infection is also beginning to be investigated ( ) . these facts drive the interest in palmitic acid with an aim of elucidating the molecular mechanisms of its immunomodulatory properties. in this review, we focus on s-palmitoylation of proteins crucial for the interactions of pathogenic bacteria and viruses with the host. we emphasize novel data on the role of s-palmitoylated proteins in the invasion of host cells by pathogens and those involved in the host innate immune responses to the infection, which have been obtained thanks to the application of new technical approaches. recently, substantial progress in the understanding of protein palmitoylation was made possible by the development of methods allowing high-throughput analysis of cellular/tissue palmitoyl proteomes. we begin, however, by showing how unique protein s-palmitoylation is among other protein lipidations. s-palmitoylation is a posttranslational modification of proteins consisting in a potentially reversible covalent attachment of palmitoyl chain to a cysteine residue(s) of proteins through a thioester bond ( table ) . thus, s-palmitoylation resembles other reversible regulatory posttranslational protein modifications, including phosphorylation or acetylation, well-established factors affecting protein structure and functions. in particular, s-palmitoylation modifies cellular localization of proteins and their stability. the most dramatic changes of localization concern cytosolic proteins which upon s-palmitoylation acquire a hydrophobic anchor facilitating their docking into membranes (figure ) . however, several integral membrane proteins also undergo s-palmitoylation. it often occurs on cysteine residue(s) located in the proximity of the junction of the transmembrane and cytoplasmic domains of the protein. s-palmitoylated transmembrane proteins occupy various cellular compartments, such as endoplasmic reticulum, golgi apparatus, and the plasma membrane. in accordance, for some proteins, such as transmembrane adaptor proteins in leukocytes, s-palmitoylation was found secondary to the length and hydrophobicity of the transmembrane domain as a determinant of plasma membrane destination ( ) . s-palmitoylation also contributes to the compartmentalization of proteins to distinct domains of membranes-rafts and tetraspanin-rich microdomains. in fact, the interest in s-palmi toylation was boosted when it was found to be required for the targeting of some signaling proteins to rafts. rafts are nanodomains of the plasma membrane and some intracellular membranes, mainly of the trans-golgi apparatus, rich in sphingolipids, glycerophospholipids with saturated fatty acid chains, and cholesterol ( ) . the plasma membrane nanodomains are sites of signal transduction by distinct receptors of immune cells involved in both acquired immune reactions, such as t cell receptor (tcr), fcε receptor i, fcγ receptor ii, and in innate immune responses, such as toll-like receptor (tlr ) ( , ) . rafts are also sites of virion assembly and budding, as established, e.g., for influenza a virus and human immunodeficiency virus- (hiv- ) ( , ) . peripheral membrane proteins acylated with saturated fatty acids are likely to anchor preferentially between the ordered saturated lipids of rafts rather than between the disordered lipids of the surrounding membrane. it has been shown that, owing to their raft localization, s-palmitoylated kinases of the src family interact with raft-associating plasma membrane immunoreceptors and initiate signaling cascades fundamental to acquired immunity ( , , ) . it is worth noting that also the acyl chains attached to proteins can affect the membrane structure. studies on model membranes have revealed that palmitic and myristic acids facilitate formation of ordered lamellar membrane regions ( , ) . in accordance, s-palmitoylation of erythrocyte peripheral membrane protein called membrane-palmitoylated protein (mpp ) was found to be required for the proper lateral organization and fluidity of erythrocyte membrane. in the absence of mpp s-palmitoylation, raft assembly was disturbed and erythrocyte functioning compromised leading to hemolytic anemia in patients deficient in the enzyme catalyzing this reaction ( , ) . preferential raft association is a feature of some s-palmitoylated transmembrane proteins, e.g., adaptor proteins pag, lat, and ntal, which collaborate with the abovementioned immunoreceptors. in fact, palmitoylation is required for the raft association of most integral raft proteins ( , , ) . on the other hand, s-palmitoylation does not obligatorily confer raft localization on transmembrane proteins. certain s-palmitoylated proteins, such as transferrin receptor, glycoprotein g of vesicular stomatitis virus (vsv), and anthrax toxin receptor, tumor endothelial marker (tem ), are actually excluded from rafts. apparently, a combination of s-palmitoylation and the properties of the transmembrane domain of the protein contribute to its destination to the raft or non-raft environment ( , ) . it has also been proposed that the attachment of a fatty acyl chain at the juxtamembrane cysteine(s) of a protein can induce tilting of its transmembrane fragment, determining in which part of the membrane it will accommodate to avoid a hydrophobic mismatch potentially caused by the thickness of the bilayer ( ) . that not all s-palmitoylated proteins associate with rafts has been shown convincingly for macrophage-like raw cells, where only about half of those proteins were found in the triton x- -resistant membrane fraction enriched in rafts ( , ) . in accordance, proteomic data on the distribution of s-palmitoylated proteins in prostate cancer cells have revealed that several such proteins are recovered in the non-raft (triton x- -soluble) fraction and are likely localized to microdomains enriched in scaffold proteins called tetraspanins ( ) . the tetraspanins are small integral membrane proteins found in the plasma membrane and other cellular membranes, having four transmembrane helices and undergoing s-palmitoylation at several conserved cysteine residues. the tetraspanins interact with each other and with various transmembrane and cytosolic partners, often also s-palmitoylated, forming microdomains ("tetraspanin web") ( ) . it has been suggested that the amino acid composition of the s-palmitoylation site in some transmembrane proteins, such as the adaptor proteins involved in acquired immune responses, determines the association of those s-palmitoylated proteins with rafts or with the tetraspanin-enriched microdomains ( ) . an intriguing and still poorly addressed question concerns the relation between rafts and the tetraspanin-enriched microdomains, apparently of functional significance, e.g., during virus budding from host cells ( ) . this uncertainty stems partially from the fact that s-palmitoylation of tetraspanins governs their interactions with cholesterol and gangliosides leading at certain conditions to the recovery of tetraspanins in detergent-resistant membrane fractions enriched in rafts ( , ) . besides its involvement in targeting proteins to rafts or tetraspanin-enriched microdomain, s-palmitoylation has been found to govern accumulation of the transmembrane chaperone protein calnexin in the perinuclear domain of endoplasmic reticulum ( ) . s-palmitoylation also affects protein stability through its interplay with ubiquitination or phosphorylation, as found for the anthrax toxin receptor tem , antiviral interferon-induced transmembrane protein ifitm , calnexin, and zdhhc , one of palmitoyl acyltransferases described below ( ) ( ) ( ) ( ) . possibly the most intriguing is the reversible character of s-palmitoylation. enzymes catalyzing palmitoylation and depalmitoylation of proteins have been characterized ( , ) . palmitate is transferred onto the thiol group of cysteine from cytosolic palmitoyl-coa by palmitoyl acyltransferases, enzymes containing the zinc finger dhhc domain named after the highly conserved asp-his-his-cys peptide (figure ). this is a twostep reaction comprising transient autoacylation of zdhhc enzymes and transfer of the fatty acyl chain from this intermediate to a protein substrate ( ) . in mammals, the zdhhc enzyme family consists of proteins, and zdhhc proteins are also found in other eukaryotes but not in bacteria nor are they encoded by viral genomes. mammalian zdhhc enzymes, each having at least four transmembrane helices, are located in the plasma membrane, endoplasmic reticulum, and golgi apparatus ( ) . they display some specificity toward their protein substrates and also selectivity toward fatty acyl moieties other than palmitate, which contributes to the heterogeneity of lipids attached to proteins, such as viral glycoproteins described below ( ) . in the opposite process, the thioester bond is cleaved by acyl-protein thioesterases (apts) (apt and apt ) and palmitoyl protein thioesterases (ppts) (ppt and ppt ), which are localized in the cytosol and in lysosomes, respectively. apt and apt likely govern the dynamic functional changes of s-acylation of proteins ( ) while ppt and ppt depalmitoylate proteins during their degradation ( , ) . recently, serine hydrolases of the abhd family have also been identified as depalmitoylating enzymes, and their specific substrate proteins determined ( , ) . of note, the zdhhcs, apt /apt , and abhd proteins are s-palmitoylated themselves, and palmitoylation of zdhhcs and depalmitoylation of apt / can occur in a cascade manner ( , ) . the dynamic cycles of palmitoylation/depalmitoylation detected for several peripheral membrane proteins are often synchronized with intracellular trafficking of those proteins. they circulate between the plasma membrane and the golgi apparatus or endosomes, as exemplified by n-and h-ras, r -regulator of g protein and apts. in fact, it is proposed that palmitoylationdependent anchoring of apt in the plasma membrane allows it to depalmitoylate h-ras at this location, while subsequent autodepalmitoylation releases apt guiding it, alongside h-ras, for another round of palmitoylation at the golgi apparatus ( , ( ) ( ) ( ) . cycles of palmitoylation/depalmitoylation are crucial for signaling by distinct plasma membrane receptors and for their distribution ( ) ( ) ( ) . activation of tcr receptor or fas receptor in t cells was found to trigger quick and transient palmitoylation of lck kinase of the src family ( , ) , but the exact meaning of the dynamic protein s-palmitoylation for processes triggered during the host-pathogen interaction awaits elucidation. it is worth mentioning that although the zddhc enzymes catalyze bulk protein palmitoylation in eukaryotic cells ( ) , some proteins have a unique autopalmitoylation activity. these include bet , a component of a multisubunit transport protein particle complex involved in vesicular trafficking, tea domain transcription factors, and also bacterial evf protein ( ) ( ) ( ) ( ) . the palmitic acid residue is attached constitutively to a specific cysteine residue of those proteins, remains buried inside a hydrophobic pocked in their core thereby affecting the tertiary structure and, thus, interactions with other proteins ( , ) . an exhaustive discussion on the physiology of s-palmitoylated proteins in eukaryotic cells can be found in several recent reviews ( , , ) . it has been established that, in addition to palmitate, various other fatty acyl moieties, such as saturated stearate (c : ) or protein palmitoylation in host-pathogen interactions frontiers in immunology | www.frontiersin.org january | volume | article monounsaturated palmitoleate (c : ), and oleate (c : ) can be attached via the thioester linkage to proteins. the early reports on the heterogeneity of the fatty acyl moieties attached to cysteines obtained by analysis of selected immunoprecipitated proteins ( , , ( ) ( ) ( ) have recently been complemented by a comprehensive proteomic analysis of fatty-acylated proteins of macrophage-like raw cells ( ) . the latter study showed that an enrichment of culture medium of cells with monounsaturated fatty acids leads to their incorporation into a similar set of proteins as those normally modified with palmitate. among them, several proteins relevant to innate immune responses were found. all these data justify the use of a broader term s-acylation rather than s-palmitoylation ( table ). the physiological consequences of s-acylation of proteins with individual fatty acids are slowly being revealed. modification of fyn kinase with polyunsaturated fatty acid residue, such as arachidonate (c : ), disturbed its raft localization and, thereby, tcr signaling ( ) . a heterogeneity of s-acylation was also found in viral spike proteins, such as hemagglutinin (ha) of influenza a virus, and e and e of semliki forest virus, which are modified in host eukaryotic cells by attachment of both palmitate and stearate ( ) . in ha, stearate is attached at the transmembrane cysteine while palmitate is attached to two cysteine residues in a membrane-proximal region of the protein. the stearoyl chain seems to accommodate into a groove formed by amino acids of the transmembrane helix shaping the domain in a way that facilitates its fitting into rafts ( ) . s-stearoylation of human transferrin receptor at the juxtamembrane cysteine residues(s) is a key factor of the signaling cascade controlling mitochondrial morphology and functioning ( ) . of interest, the latter study also showed that dietary supplementation of stearic acid reversed the deleterious effects of a genetically determined mitochondria dysfunction in drosophila. taking into account that unsaturated fatty acids affect the profile of s-acylation of proteins in vitro ( , ) , it is of outmost interest whether a similar effect of unsaturated and saturated (palmitic) fatty acids could be achieved in vivo with respect to proteins of immune cells. beside s-acylation, less frequently palmitate can also be attached to the amine group of various amino acids (glycine, cysteine, and lysine) giving n-palmitoylation or to the hydroxyl group of serine or threonine in a process called o-palmitoylation ( table ) . as during s-palmitoylation, also other fatty acids can be utilized in these processes named then n-and o-acylation. thus, a type of protein n-acylation is n-myristoylation, a frequent modification contributing to membrane anchoring of peripheral proteins. the saturated myristate (c : ) is transferred to the protein from myristoyl-coa by n-myristoyl transferase (two isozymes in mammals). in a vast majority of cases, myristate is attached cotranslationally to the n-terminal glycine residue (after removal of the initiator methionine) via an amide linkage ( table ) . like most lipidations, this modification is irreversible. several viral proteins are n-myristoylated, such as gag of hiv- crucial for budding of newly formed virions from plasma membrane rafts of host cells, and proteins of parasitic protozoa plasmodium falciparum, trypanosoma brucei, and leishmania donovani (causing malaria, african sleeping sickness, and leishmaniosis, respectively). for this reason, n-myristoyl transferase is considered a potential drug target in the therapy of these diseases ( - , , ) . data on the n-and o-palmitoylation of proteins involved in the host-pathogen interactions are limited, but interesting conclusions can be drawn from the information concerning proteins taking part in other processes. n-palmitoylation of the n-terminal glycine of the α-subunit of a heterotrimeric g protein (gαs) has been described ( ) ( table ) besides the wellknown s-palmitoylation of this pivotal signaling protein. the n-palmitoylation of gαs is irreversible, and the enzyme responsible for this modification is unknown. it has been speculated that s-to n-palmitoyl migration can occur both in vivo and also in vitro during mass spectrometry analysis ( , ) . this suggests that caution is needed in interpreting results of this methodological approach, which is used with increasing frequency to study fatty acylation of proteins in immune cells (see next sections). probably the best-characterized is the n-palmitoylation of the n-terminal cysteine residue of hedgehog proteins (sonic, indian, and desert in mammals). it determines secretion of these proteins, which regulate embryonic patterning ( table ) . secreted wnt and ghrelin proteins are examples of o-acylation of serine residues with unusual fatty acid residues such as palmitoleate (c : ) and octaonoate (c : ) ( table ). the fatty acylation of hedgehog, wnt, and ghrelin is catalyzed by enzymes from the multipass membrane-bound o-acyl transferases family ( ) . besides these unusual fatty acid residues, attachment of palmitate to serine and threonine residues is found in secreted venom toxins of the spider plectreurys tristis, which selectively target neuronal ion channels ( ) . also histone h is o-palmitoylated at a serine residue in the nucleus by acyl-coa:lysophosphatidylcholine acyltransferase ( ) ( table ). the latter is of special interest in the context of innate immune responses since histone h o-palmitoylation regulates transcriptional activity, which is the final outcome of the pro-inflammatory signaling pathways triggered by receptors of the innate immune system. special attention should be devoted to ε-n-acylation consisting in the attachment of a fatty acid residue to the side chain of lysine by amide linkage ( table ) . ε-n-myristoylated are interleukin α (il- α) and tumor necrosis factor α (tnfα), the pro-inflammatory cytokines crucial in combating bacterial infections ( ) . the enzyme(s) catalyzing this reaction is unknown, but it has been established that sirtuins reverse this modification ( ) . the ε-n-acylation affects the release of tnfα by immune cells ( , ) . surprisingly, this rare modification is also found in toxins of so-called rtx (repeats-in-toxin) class released by some pathogenic gram-negative bacteria ( , ) . we describe these cases in more detail in the following sections. besides s-palmitoylation and n-myristoylation, s-prenylation is another common lipidation that endows proteins with a hydrophobic moiety and contributes to their association with membranes. this modification relies on the posttranslational and irreversible attachment of either farnesyl or geranylgeranyl chains to a cysteine residue in the c-terminal caax box (alternatively also cc and cxc motifs) via a thioether linkage. the process is catalyzed by protein prenyl transferases that use polyprenylpyrophosphate as the donor of the isoprenoid group ( table ) . in peripheral membrane proteins, the s-palmitoylation site is often located in proximity of n-myristoylation or s-prenylation sites or a polybasic motif, which all are likely to mediate initial weak binding of a protein to a membrane and thereby facilitate subsequent attachment of palmitate to the protein by the integral membrane zdhhc enzymes ( ) . in contrast to s-palmitoylation, data on the role of s-prenylation of proteins key to the host-pathogen interactions are scarce ( ) . however, since s-prenylation is typical for the ubiquitous small gtpases of ras superfamily, it is vital for proper functioning of b and t cells ( , ) . a glance at table indicates that palmitate can be covalently bound via oxyester, amide, and thioester linkages to respective amino acid residues creating an array of possible modifications. o-and n-palmitoylation of proteins seems to be stable, resembling in this regard the other common protein lipidations, n-myristoylation and s-prenylation. by contrast, there exist enzymes cleaving the thioester bond formed during s-palmitoylation. for a long time, our understanding of protein s-palmitoylation and its dynamics was poor in comparison with other reversible protein modifications due to technical difficulties. only recently have these difficulties been overcome with the introduction of methods allowing high-throughput identification of palmitoylated proteins, also those involved in the immune response to microbial pathogens, as discussed in the next sections. one of the basic problems hindering studies on protein s-palmitoylation lies in the fact that there is no identifiable consensus sequence for the palmitoylation site that could facilitate its prediction. from the technical point of view, the progress in a comprehensive survey of protein s-palmitoylation was also hampered by a lack of antibodies detecting this modification, with the sole exception of an antibody specific to palmitoylated psd- ( ) . a classical method used to demonstrate protein palmitoylation is based on metabolic labeling of living cells with [ h]-palmitic acid, subsequent immunoprecipitation of a selected protein and detection of the incorporated tritiated fatty acid by autoradiography ( ) . a major disadvantage of this method is its low sensitivity. only a minute fraction of the radioactive palmitate is bound to proteins, the majority being incorporated into lipids, which requires lengthy film exposure (counting in days). a methodological breakthrough in the identification of palmitoylated proteins came with the development of two nonradioactive methods based on so-called click chemistry ( ) ( ) ( ) and acyl-biotin exchange (abe) ( , ) . these techniques have paved the way for high-throughput mass spectrometry-based proteomic analysis of protein palmitoylation in various cells and tissues and facilitated identification of new palmitoylated proteins of both pathogens and host cells involved in the innate immune responses. in the click chemistry-based method, cells are metabolically labeled with a palmitic acid analog bearing an alkyne group at the ω carbon of the fatty acyl chain, such as -octadecynoic acid ( odya) or alk- (figure a) , and this step resembles the classic labeling of cells with [ h]-palmitic acid. however, in the click chemistry-based assay, the labeling and lysis of cells is followed by in vitro coupling of the function group of the palmitic acid analog to a reporter tag, which greatly enhances the sensitivity of detection of labeled proteins ( , ) . thus, after cell lysis, the labeled proteins are subjected to cu (i) -catalyzed cycloaddition known as "click" reaction with an azide-bearing detection tag. in this step, a triazol is formed between the alkyne group in the palmitic acid analog and the azide of the tag (figure a) . the azide-bearing tags can be either fluorescent, such as tetramethylrhodamine or dyes with infrared fluorescence, or carry a biotin moiety. depending on the tag used, subsequent sds-page separation of proteins allows global visualization of palmitoylated proteins by simple in-gel fluorescence or by blotting with a streptavidin-conjugated reporter ( , , ) . notably, proteins biotinylated via the click reaction can also be enriched on streptavidin-coated beads and then subjected to on-bead tryptic digestion (or in-gel digestion if eluted from the beads) followed by identification by mass spectrometry. such comprehensive click chemistry-based proteomic analysis has brought about identification of an array of palmitoylated proteins in dendritic cells ( , ) , macrophage-like raw cells ( ) , and t cells ( , , ) . some of the s-palmitoylated proteins newly identified in those studies, such as ifitm and tlr , are involved in the host-pathogen interactions regulating innate immune responses ( , ) , while many others are known to contribute to adaptive immunity ( , ) , as described below. recently, global profiling of toxoplasma gondii (the causative agent of toxoplasmosis) has been performed revealing that many components of the parasite's motility complex are palmitoylated ( ) . similar studies on cryptococcus neoformans (the fungus causing cryptococcal meningitis) have revealed a contribution of specific zdhhc palmitoyl acyltransferase, called pfa , to its virulence ( ) . moreover, application of analogs of various saturated and unsaturated fatty acids confirmed the heterogeneous nature of the fatty acylation of proteins in raw cells and suggested that dietary unsaturated fatty acids, after incorporation to proteins, can change their properties and thereby affect the functioning of immune cells ( ) . the major advantage of the click chemistry-based method is that it can reveal the time course of protein s-palmitoylation. by using click chemistry-based labeling in the pulse-chase mode, one can follow the dynamics of protein palmitoylation. with such an approach, it was found that the palmitate turnover on lck, an src-family tyrosine kinase, is accelerated by t cell activation ( ) . additional introduction of stable isotope labeling by amino acids in cells (silac) has provided quantitative proteomic data , and after cell lysis, the click reaction is conducted with azido-tagged biotin or fluorescent probes allowing enrichment and detection of labeled proteins in various ways. biotinylated proteins can be bound on a streptavidin resin and then released using, e.g., high concentrations of urea and sds ( ) . when a cleavable derivative of biotin, azido-azo-biotin, is used the labeled proteins are eluted from streptavidin beads with sodium dithionite, which cleaves the diazobenzene moiety in the linker arm of azido-azo-biotin, and analyzed by mass spectrometry or immunoblotting ( ) . (b) abe method. cells or tissues are lysed, free thiol groups of proteins are blocked by alkylation, and palmitoyl moieties are released with hydroxylamine. the newly exposed protein thiol groups are subjected to labeling with biotin-hpdp allowing selective binding, elution, and analysis of the originally s-palmitoylated proteins. the proteins can also be captured without biotinylation through a direct interaction of their thiol residues with a thiol-reactive resin (acyl-rac technique). on the dynamics of protein palmitoylation in the cell ( , ) . this approach revealed, rather unexpectedly, that in unstimulated t cell hybridoma, the palmitoylation of most protein species does not undergo turnover ( ) . another advantage of the click chemistry-based assay is its high specificity, because the alkyne group introduced in the analog of palmitic acid is not normally found in cells ( , ) . the click chemistry-based methods can also be used to follow the cellular localization of palmitoylated proteins by immunofluorescence when combined with the proximity ligation technique ( , ) . palmitoylation of individual proteins can also be studied after their immunoprecipitation ( , , , ). despite its unquestionable success, the click chemistry-based methods have limitations. they will detect only those proteins that undergo palmitoylation during the period of the metabolic labeling of cells. one should also bear in mind that the palmitic acid analog can be incorporated at s-, n-, and o-palmitoylation sites alike ( , ) . in addition, although odya (alk- ) is preferentially used to mimic palmitoylation of proteins, it can also be incorporated with low efficiency at n-myristoylation sites of proteins ( , ) . another group of proteins that will be labeled with the palmitic acid analog but are not s-palmitoylated are those bearing the glycosylphosphatidylinositol (gpi) anchor ( , ) . most of these limitations can be overcome using various fatty acid reporters, inhibitors, and by exploiting the sensitivity of the thioester bond to hydroxylamine treatment. given the large variety of chemical reporters preferentially mimicking distinct fatty acids, recent years have witnessed a plethora of chemistry-based proteomic studies not only on palmitoylated but also myristoylated proteins and proteins bearing the gpi anchor, including those of pathogens and immune cells ( , , , , ) the abe method reveals protein the abe method can be used as a complement to the click chemistry-based approach in cell studies but unlike the latter it is uniquely suitable for studying whole tissues. abe does not require metabolic labeling of proteins in living cells, thus some of the abovementioned limitations and difficulties do not apply. the abe method relies on in vitro exchange of thioester-linked palmitate to a derivative of biotin which allows subsequent affinity purification of the resulting biotin-labeled proteins on streptavidin-coated beads ( figure b) . the first step of the abe involves lysis of cells or tissues followed by irreversible blockage of free thiol groups in the solubilized proteins by alkylation, most often with n-ethylmaleimide. subsequently, the thioester bonds existing in s-palmitoylated proteins are broken with hydroxylamine, releasing palmitoyl moieties. the newly exposed thiol groups can now be tagged with sulfhydryl-reactive derivatives, such as biotin-hpdp, forming disulfide bonds with thiols. the biotinylated proteins are subsequently captured on streptavidin-coated beads and eluted with agents that reduce the disulfide bond between the protein and biotin-hpdp, such as β-mercapthoethanol, dtt, or tcep ( , , , ) . as an alternative to biotinylation, in the so-called acyl-rac technique, the newly exposed protein thiol groups in hydroxylamine-treated cell lysates are captured on a resin containing sulfhydryl-reactive groups ( ) . in both abe and acyl-rac, the eluted proteins can be separated by sds-page and visualized by gel staining or immunoblotting, or identified by mass spectrometry. furthermore, when the hydroxylaminereleased palmitoyl moieties are exchanged for a polyethylene glycol-maleimide derivative of a distinct molecular weight, a shift in-gel migration of tagged proteins is observed reflecting the number of fatty acyl residues originally s-bound to the protein ( , ) . the abe method has so far been used successfully for proteomic profiling of s-acylated proteins in immune cells, such as raw cells ( ), several types of blood cells, such as platelets, primary t cells, and immortalized b cells ( ) ( ) ( ) , pathogenic microorganisms such as t. brucei and t. gondii ( , ) , and tissues ( , ) . to quantify the aberrations in protein palmitoylation in a mouse model of huntington's disease, whole animal stable isotope labeling of mammals (silam) was applied followed by tissue isolation and abe procedure ( ) . in another approach, for quantitative analysis of the t-cell palmitoylome, abe was combined with labeling of proteins with various oxygen isotopes during their digestion with trypsin before mass spectrometry analysis ( ) . in addition, preselection of tryptic peptides obtained by abe on streptavidin-coated or sulfhydrylreactive resins greatly facilitates the identification of s-acylation sites by mass spectrometry ( , , ) . some aspects of the abe method deserve a comment. since the assay relies on the sensitivity of thioester bonds to hydroxylamine, abe detects all s-acylation without distinguishing between s-palmitoylation and the other cases. furthermore, there is a possibility of false-positive detection of proteins bearing a thioester linkage with compounds other than fatty acyl residues, such as ubiquitin in the e ubiquitin conjugase ubc ( ) . another source of false-positives is proteins in which free thiol groups were not completely alkylated before biotinylation. on the other hand, insufficient deacylation of bonafide fatty-acylated proteins with hydroxylamine results in their absence in the final sample ( ) . in summary, the click chemistry-based method relies on metabolic labeling of cells with a palmitic acid analog which incorporates into proteins and next tagging it with reporter molecules greatly enhancing the sensitivity of detection. it only reveals proteins undergoing s-palmitoylation during metabolic labeling of cells and allows revealing turnover of this modification. by contrast, the abe method is based on direct binding of sulfhydryl-reactive derivatives to thiol groups of cysteines unraveled by hydroxylamine treatment after lysis of cells or tissues. it allows the investigation of the whole but static palmitoylome. a comparative proteomic study of protein palmitoylation in p. falciparum found that the sets of proteins identified using these two approaches overlapped in . % ( ) , indicating that they provide complementary data on the cellular palmitoyl proteome. thanks to the application of the click chemistry-and abe-based methods numerous new palmitoylated proteins have been identified. in , a swisspalm database was launched, ( ) which provides an excellent, manually curated resource of information on palmitoylated proteins, palmitoylation sites, etc., available at http://swisspalm.epfl.ch/. all these efforts have greatly furthered our knowledge on molecular mechanisms regulating diverse aspects of cell functioning, including host-pathogen interactions and progress of infectious diseases, as highlighted below. bacteria lack protein palmitoyl acyltransferases of the zdhhc family and, therefore, are essentially devoid of s-palmitoylated proteins. yet, they have developed unique mechanisms utilizing fatty acids, such as palmitic acid, to modify their glycolipids and proteins. these modifications augment infectivity and help bacteria evade recognition by the host innate immune system. for example, the vast majority of gram-negative bacteria produce lipopolysaccharide (lps) as a part of their outer membrane. lps is composed of the variable polysaccharide o-antigen and more-conserved lipid a containing two glucosamine residues hexa-acylated with hydroxymyristic, myristic, and lauric acid. lipid a is recognized by cd protein and tlr receptor complexed with md protein on the plasma membrane of the host immune and some non-immune cells. activation of tlr triggers strong pro-inflammatory reactions aiming at eradication of the bacteria, but when exaggerated, eventually leading to sepsis ( ) . incorporation of an additional palmitoyl chain into lipid a markedly diminishes its ability to activate tlr and to induce the host pro-inflammatory responses, which is correlated with an increased survival of bacteria forming a biofilm ( , ) . this strategy is utilized among others by salmonella typhimurium, a causative agent of gastroenteritis, by bordetella bronchiseptica, a respiratory pathogen of human and other mammals, and by protein palmitoylation in host-pathogen interactions frontiers in immunology | www.frontiersin.org january | volume | article yersinia pestis causing plague ( , ) . the formation of the extra-acylated lps relies on the transfer of palmitate from phospholipids onto the hydroxymyristate chain at position of glucosamine of lipid a. the reaction is catalyzed by lipid a palmitoyltransferases (pagp in salmonella and its homologs in other bacteria) localized in the outer membrane of these pathogens ( , ) . in addition to causing steric hindrance preventing the binding to the tlr /md complex, the hepta-acylation of lps also protects bacteria from the lytic activity of cationic antimicrobial peptides, most likely by reducing the fluidity of the bacterial outer membrane ( , ) . apart from being incorporated into lps in diverse bacteria, palmitate has also been found to modify a virulence factor of gram-negative erwinia carotovora, the evf protein. the palmitoyl chain is linked via a thioester bond to the cys residue at the center of evf, plausibly by a self-palmitoylating activity of the protein. e. carotovora is a phytopatogen using insects such as drosophila as vectors for dissemination between plants. the palmitoylation of evf is required for infectivity of e. carotovora and its persistence in the insect gut, however, its mode of action of unknown. it has been speculated to be linked with an ability of evf to associate with lipid bilayers, but the lack of similarities between evf and any other bacterial protein of known function makes prediction on this subject difficult ( ) . a number of bacterial toxins of so-called rtx class released during infection of mammals by pathogenic gram-negative bacteria undergo ε-n-acylation of the side chain of internal lysines. these toxins include adenylate cyclase of bordetella pertussis, acylated with palmitic acid, and α-hemolysin of extraintestinal (uropathogenic) escherichia coli, acylated with myristic acid and also -and -carbon fatty acids. the acylation is catalyzed by an endogenous bacterial acyltransferase which, unlike its eukaryotic counterparts, transfers the acyl chain not from acyl-coa but from acyl-carrier protein. the acylated toxins secreted by the bacteria bind to the plasma membrane of the host cells, oligomerize and form pores causing cell lysis. in the case of the toxin of b. pertussis, essential is also the delivery of the adenylate cyclase moiety to the cell interior. acylation is required for virulence possibly being involved in oligomerization of the toxins ( , , ) . although lacking s-palmitoylated proteins (with the single known exception of evf), bacteria express a wide range of membrane-bound proteins modified by a complex lipidation at the n-terminus, with palmitate frequently being a component of the lipid moiety ( , ) . the bacterial lipoproteins are synthesized in a multistep process catalyzed by a unique set of lipoprotein processing enzymes, lgt, lspa, and lnt, absent in eukaryotic cells. the formation of these lipoproteins begins with the attachment of a diacylglycerol via a thioester bond to a cysteine residue located in the so-called lipobox motif of the signal sequence of the transmembrane lipoprotein precursor. the signal sequence is then cleaved next to the lipid-modified cysteine leaving it at the n-terminus of the mature protein ( ) . in gramnegative and less frequently also gram-positive bacteria, a third fatty acid residue is additionally attached via an amide linkage to the amino group of the cysteine in a reaction analogous to the n-acylation of hedgehog proteins (see table ). this di-and tri-lipidation ensures membrane anchoring of the lipoproteins. all such lipoproteins of gram-positive bacteria are exposed to the milieu while in gram-negative bacteria some face the periplasm. the lipoproteins of gram-positive bacteria, e.g., streptococcus pneumoniae (causing pneumonia), mycobacterium tuberculosis (tuberculosis), and gram-negative bacteria, such as neisseria meningitidis (meningitis), y. pestis (plague), the spirochaete borrelia burgdorferi (lyme disease) and treponema pallidum (syphilis) are crucial for their virulence. they control several aspects of the host-pathogen interactions, like adhesion and entry to host cells, protection against proteolysis and oxidative stress in the host cell, and regulation of expression of genes encoding cytokines both during initiation and progress of the disease ( ) ( ) ( ) . the surface exposure of the lipoproteins allows their involvement in the host cell invasion while on the other hand forming the so-called pattern signal recognized by the tlr receptor, which triggers the pro-inflammatory responses helping to combat the bacteria ( ) . of interest, tlr is s-palmitoylated, as discussed below. the involvement of lipoproteins in pathogenesis fuels studies on their properties. one such recent work employing click chemistry to profile the lipoproteins of e. coli identified lipoproteins with high/medium confidence, % of them predicted before by bioinformatics analysis ( ) . notably, in that study a -carbon alkynyl fatty acid analog alk- rather than alk- was preferentially incorporated into the lipoproteins, contradicting earlier studies using gas chromatography and tlc, which found that palmitate was predominantly used for bacterial protein modification ( ) . further studies are required to establish whether the fatty acid found in lipoproteins varies depending on culture conditions or is species specific. for example, odya labeling for click reaction confirmed incorporation of palmitate into pallilysin (tp ), a lipoprotein of t. pallidum. pallilysin is a metalloprotease that degrades human fibrinogen and laminin. it is suggested that its exposure on the bacteria surface enables degradation of host structural proteins to facilitate rapid dissemination of this highly invasive pathogen ( ) . bacteria occasionally high-jack the palmitoylation machinery of host cells to modify the environment so as to favor their internalization, survival, and replication inside the cells. bacillus anthracis (the causative agent of anthrax) is an example of such bacteria that modify s-palmitoylation of host proteins to their ends. the anthrax toxin produced by this pathogen binds to the tem and cmg (capillary morphogenesis protein- ) proteins which, under physiological conditions, are involved in cell-cell and cell-extracellular matrix interactions. they are s-palmitoylated at multiple (two to four) cysteines ( ) . the s-palmitoylation of tem was found to inhibit its association with plasma membrane rafts preventing its ubiquitination by the raft-associated e ubiquitin ligase cbl. the binding of anthrax toxin drives association of the receptor-toxin complexes with rafts possibly correlated with depalmitoylation of the receptor. this allows subsequent ubiquitination of the receptor, an uptake of the receptor/toxin complexes in a clathrin-dependent manner and eventual delivery of the toxin to endosomes. these events are facilitated by s-palmitoylation of partner(s) of the receptors, most likely including kinases of the src family ( , , ) . while b. anthracis utilizes palmitoylated host proteins to induce its internalization, a growing body of data suggests that protein palmitoylation in host-pathogen interactions frontiers in immunology | www.frontiersin.org january | volume | article also bacterial proteins can undergo s-palmitoylation inside the host cells. this type of modification concerns so-called effectors, bacterial proteins that are injected into the host cell cytoplasm either across the plasma membrane or the membrane of vesicles enclosing internalized pathogens, with the help of their secretion systems. these are secretion systems type iii and type iv, homologs of which have been described for pathogens and symbionts of mammals, insects, and plants ( , ) . the bacterial effectors can be s-palmitoylated to reach host cell membranes and thereby accumulate at a location most suitable for their activity. application of the click chemistry-based method utilizing an analog of palmitic acid (alk- ) for cell labeling has revealed s-palmitoylation of two effector proteins of salmonella enterica, such as ssph and ssei ( ) . s. enterica invades gut endothelial cells and is a leading cause of gastroenteritis and typhoid fever. ssph carries an e ubiquitin ligase domain while ssei shows sequence homology to bacterial proteins that have a deamidase activity, and inhibits migration of salmonella-infected cells. the latter activity requires s-palmitoylation of ssei. both proteins are stably s-palmitoylated, most likely by zdhh and zdhh of the host and bind to the plasma membrane in a palmitoylationdependent manner ( ) . also two effector proteins of the ipah family of shigella spp. were found to be s-palmitoylated in that study, suggesting that this modification can control the activity of effector proteins of other pathogens as well ( ) . indeed, gobx and lpda, effector proteins of legionella pneumophila, the causative agent of legionnaires' disease invading macrophages and lung endothelial cells, are s-palmitoylated as was found recently using click chemistry. lpda is a phospholipase hydrolyzing various phosphatidylinositols while gobx is an e ubiquitin ligase. gobx is targeted in a palmitoylation-dependent manner to the golgi apparatus, and lpda to the plasma membrane and a subset of intracellular vesicles ( , ) . thus, the diversified subcellular localization of bacterial effector proteins reflects that of eukaryotic proteins. it is worth noting that global profiling of acylated proteins with the application of click chemistry and an alkyne-functionalized analog of myristic acid, alk- , for cell labeling was effective in reveling the mechanism of action of shigella flexneri effector protein ipaj of type iii secretion system. this is a unique protease that cleaves off the n-terminal myristoylated glycine. this proteolytic demyristoylation activity of ipaj is specific toward golgi-associated arf/arl family of gtpases regulating cargo transport through the golgi apparatus, inhibition of which is apparently pivotal for virulence of the bacteria causing diarrhea in humans ( ) . in addition to the s-palmitoylation of the effectors of pathogenic bacteria of mammals mentioned earlier, double acylation, n-myristoylation and s-palmitoylation, has been reported of the so-called avirulence (avr) proteins (effectors of type iii secretion system) of pseudomonas syringae, a causative agent of diverse plant diseases. among them, avrrpm and arvb are n-myristoylated and s-palmitoylated by host acyltransferases at neighboring glycine and cysteine residues localized at the n-terminus of the proteins (similarly to eukaryotic kinases of the src family), while in avrpphb and two avrpphb-like effectors-orf and nopt, the double acylation motif is exposed after auto-cleavage of the proteins (similarly to some eukaryotic proteins cleaved by caspases). the acylation of the avr proteins ensures their anchoring in the host plasma membrane, which is required for their functioning. in disease-susceptible plants avr proteins contribute to successful infection; however, in plants expressing host resistance (r) genes they trigger plant defense signals, in both cases engaging plasma membrane-associated host proteins ( , ) . the importance of palmitoylation of bacterial effector proteins for their infectivity is only beginning to be uncovered, in no small part owing to the development of the click chemistry-based method for detection of this protein modification. however, the strategy of high-jacking the host palmitoylation machinery to modify own proteins seems to be much more commonly employed by viruses. viruses do not encode palmitoyl acyltransferases but exploit extensively the host palmitoylation machinery to modify their proteins essential for infection of host cells and own replication. in fact, s-palmitoylation of proteins was discovered in as a modification of envelope glycoproteins of sindbis virus and vsv. in those studies [ h]-palmitic acid was used for metabolic labeling of virus-infected cells and labeled proteins were identified by autoradiography ( , ) . subsequently, a number of other viral proteins have been found to be palmitoylated using this approach. the most-studied group of viral palmitoylated proteins is those found in enveloped viruses, i.e., viruses covered by a lipid bilayer obtained during their replication from a membrane of the host cell, such as the plasma membrane or endoplasmic reticulum. influenza virus, hiv- , hepatitis c virus (hcv), and herpes simplex virus (hsv) are the best known enveloped viruses. the envelope is rich in transmembrane, often s-palmitoylated, glycoproteins called spikes, which can bind to cognate receptors on the host cell plasma membrane triggering endocytosis of the virion, mediate subsequent fusion of the viral and cellular membranes allowing entry of the viral genome to the cytoplasm, and are also involved in the budding of newly formed virus particles from the cell. an example of such multifunctional palmitoylated transmembrane glycoproteins is ha present in the envelope of influenza virus together with another palmitoylated transmembrane protein, the matrix protein m , which forms a proton channel earning the protein the name viroporin. as mentioned earlier, ha of influenza a virus is s-stearoylated and s-palmitoylated, respectively, at one cysteine residue located in the transmembrane domain of ha and two cysteines found in the cytoplasmic (intraviral) tail in close proximity to the membrane ( ) . on the other hand, m is s-palmitoylated on the amphiphilic helix located in the cytoplasmic part of the protein. due to the s-palmitoylation and the presence of a cholesterol-binding motif the helix bends toward and associates with membranes ( , ) . during infection, ha binds to sialic acid residues of glycans localized on the surface of airway and alveolar epithelial cells. the bound virions are endocytosed and next the viral and endosome membranes fuse. the membrane fusion is driven by ha, which undergoes conformational changes induced by low ph of endosomes. acidification of endosomes activates also the m proton channel activity, protons entering viral core facilitate dissociation of the viral genome which then moves to the nucleus where rna replication occurs. the s-palmitoylation of ha is required for the fusion of the viral and endosome membranes at least in some subtypes of the virus while the ion channel activity of m is not dependent on its s-palmitoylation ( ) . newly synthesized viral proteins and rna are assembled into virions in the plasma membrane rafts which merge into lager platforms crucial for the virion assembly and budding off. the triple fatty acylation of ha is required for its targeting to plasma membrane rafts ( , ) . besides s-palmitoylation, also the amino acid sequence of the transmembrane domain of ha determines its association with rafts ( ) . on the other hand, among the amino acids of the cytoplasmic tail of ha no other than the two s-palmitoylated cysteines are required for viral assembly and replication, although it is still not clear whether raft targeting (in cooperation with the transmembrane fragment) is the only mechanism of their participation. it is proposed that they affect conformation of the ha tail controlling its interaction with structural matrix protein m lying beneath the viral envelope ( , ) . the budding off of the virion is facilitated by m which localizes at the edges of rafts as a result of a combination of its s-palmitoylation, cholesterol binding, and properties of the transmembrane fragment. m protein can create a "wedge" altering membrane curvature thereby facilitating membrane scission and release of the virion ( , ) . the influenza virus s-palmitoylated proteins are the archetype for many other viral proteins. thus, s-palmitoylated spike glycoproteins include s-protein of coronaviruses (e.g., severe acute respiratory syndrome virus), the fusion (f) protein of paramyxoviruses (e.g., measles virus), env of retroviruses [e.g., hiv- , feline immunodeficiency virus (fiv)], and filoviruses (e.g., ebola). other viral proteins modified with palmitate are viroporins, such as e protein of coronaviruses, and also peripheral membrane proteins or nucleocapsid proteins absent in influenza virus. it has been found that s-palmitoylation of f l, a peripheral protein of the envelope of vaccinia virus, controls the association of the protein with intracellular membranes, thereby the formation of the envelope ( ) . the core protein of the nucleocapsid of hcv resides on the surface of lipid droplets and binds in a palmitoylation-dependent manner to membranes of the droplet-associated endoplasmic reticulum. subsequently, it recruits viral proteins and newly synthesized rna for viral particle formation ( ) . besides the interest in the role of viral protein s-palmitoylation for infectivity and possible use of host zdhhc enzymes as targets of anti-influenza drugs ( ) , viral proteins often serve as a model to study the consequences of fatty acylation for protein functioning and localization in distinct membrane domains (see s-palmitoylation of proteins and its influence on protein localization, trafficking, and stability of this review). readers are referred to recent exhaustive reviews that consider these topics ( , , ) while we will focus here on the recent advances in the field of viral protein palmitoylation brought about mainly by proteomic studies. the click chemistry-based approach has led to the identification of s-palmitoylation in the cytoplasmic domain of the transmembrane spike protein env of fiv, considered to be the cat equivalent of hiv- . env comprises three transmembrane gp glycoproteins and three associated gp which bind to cd receptor and coreceptors on the surface of t lymphocytes allowing fusion of the viral envelope and the plasma membrane and entry of viral capsid. four cysteines in fiv env are s-palmitoylated vis-a-vis two found in the env of hiv- . the two most membrane-proximal cysteines, and , are required for the fiv membrane-fusion activity and incorporation of env into virions ( ) , in agreement with the importance of env s-palmitoylation for virion assembly of some hiv- strains ( ) ( ) ( ) . the assembly of hiv- virions takes place in plasma membrane rafts and is driven by n-myristoylated gag protein which anchors and oligomerizes preferentially in these plasma membrane domains due to the presence of the fatty acyl chain ( ) . the development of click chemistry-based methods allowed for the first time global profiling of acylated proteins in virusinfected cells. in addition to identifying acylated viral proteins this approach has also revealed how the viral infection modulates the acylation pattern of the host cell proteins. thus far, click chemistry has been used to study protein myristoylation and palmitoylation in cells infected with hiv- and with hsv. in the latter case, the standard metabolic labeling with alkynefunctionalized myristic and palmitic acid analogs followed by click chemistry and mass spectrometry was combined with silac to discern between the changes in the extent of protein acylation and those in their abundance following viral infection. this approach allowed an elaborate quantitative analysis of host protein acylation and has revealed an overall downregulation of the level of both host protein modifications in infected cells. while the decreased content of myristoylated proteins resulted mainly from suppression of host protein synthesis, the drop in several s-palmitoylated proteins ensued from the inhibition of their palmitoylation in infected cells. the affected proteins were localized mainly to the plasma membrane and the golgi apparatus and were involved in vesicle-mediated transport and ion transport. in addition, the study has expanded the list of hsv-encoded acylated (mostly palmitoylated) proteins that play different functions in the viral cycle, such as ge, gi, gk, us , and us ( ) . similar results pointing to global changes of host protein acylation were obtained upon analysis of protein myristoylation and palmitoylation in cells infected with hiv- . in that study, the cells were labeled with analogs of palmitic or myristic acid tagged with an azide moiety for click chemistry reaction; however, the following mass spectrometry analysis did not address the relation between changes of protein acylation vs. alteration of protein level. the study identified palmitoylated and myristoylated proteins significantly differing in abundance between hiv- infected and uninfected cells. several of the proteins affected by the infection were of host origin. the abundance of myristoylated proteins was in general increased while that of the palmitoylated ones-decreased in infected cells ( ) . in other words, the two studies have revealed that hsv and hiv- not only encode proteins that are acylated in the host cell but protein palmitoylation in host-pathogen interactions frontiers in immunology | www.frontiersin.org january | volume | article also alter the palmitoylation of host proteins, likely to adapt the cellular environment to favor their replication and budding. the majority of the acylated proteins affected by hiv- or hsv infection had not been described earlier in this context; therefore, further studies on these proteins could be crucial for better understanding of viral infection. thus, the click chemistry-based approach has been highly effective in revealing changes of the host protein palmitoylation and opening new possibilities for the identification of novel antiviral drug targets. the innate immune responses are the first line of active defense against microbial infections. the application of click chemistrybased and abe methods and their use for large-scale analysis of protein palmitoylation in murine dendritic cd . cells ( , ) , and murine macrophage-like raw cells ( , ) complemented by proteomic analysis of the raft fraction of those cells ( ) have contributed significantly to the understanding of the role of palmitoylation of host receptors and signaling proteins involved in innate immune responses. thus, the palmitoyl proteome analysis of murine dendritic cells unraveled s-palmitoylation of tlr , a receptor expressed in cells of myeloidal lineage, which heterodimerizes with tlr or tlr to bind bacterial tri-or diacylated lipoproteins, respectively, and also other microbial components, such as glycolipids (e.g., lipoarabinomannan) of mycobacterium and yeast zymosan ( ) . besides tlr , two other human tlrs out of ectopically expressed in hek cell, flagellin receptor tlr , and tlr , a unique tlr negatively regulating the pro-inflammatory activity of tlr , were also found to be palmitoylated. the s-palmitoylation site of human tlr was mapped to cys adjacent to its transmembrane domain. the modification was present in unstimulated cells and was linked with up-regulation of the cell surface localization of tlr . mutation of cys abolished the ability of the receptor to induce pro-inflammatory signaling in response to microbial ligands of tlr ( ) . further studies are needed to reveal whether s-palmitoylation of tlr controls its association with rafts as sites of tlr activation ( ) and/or affects endocytosis of the receptor, as found for the anthrax toxin receptor ( ) . one of the most extensively studied tlrs, tlr activated by bacterial lps, is not palmitoylated. yet, saturated fatty acids have been indicated to trigger pro-inflammatory signaling of tlr . thus, the tlr /md receptor complex is involved in the pro-inflammatory outcome of a diet rich in palmitic acid, as was found when analyzing markers of inflammation in the heart and adipose tissue of high fat diet-fed mice ( , ) . the molecular mechanisms underlying the pro-inflammatory properties of palmitic acid can involve its influence on the plasma membrane lipid order, hence raft organization, in a way that facilitates translocation of tlr (and tlr ) toward rafts ( , ) . palmitic acid also directly binds to the tlr -associated md protein ( , ) . an influence of palmitic acid on sphingomyelin/ceramide metabolism, which enhances the lps-induced responses, has also been considered ( ) . recent proteomic studies based on odya labeling of raw macrophage-like cells followed by click chemistry have revealed that stimulation of cells with lps induces profound changes of the abundance of palmitoylated proteins ( ) . the data are in agreement with earlier findings showing that lps induces accumulation of s-palmitoylated lyn kinase in the raft-enriched fraction of cells, allowing it to downregulate tlr signaling ( ) . one of the upregulated s-palmitoylated proteins was type ii phosphatidylinositol -kinase iiβ, which phosphorylates phosphatidylinositol to phosphatidylinositol -monophosphate. it was shown that palmitoylation determines the involvement of the kinase in lps-induced signaling ( ) . these data suggest that s-palmitoylated proteins, including enzymes catalyzing phosphatidylinositol synthesis and turnover, are important factors affecting the pro-inflammatory responses triggered by lps. notably lps induces production of tnfα, a pro-inflammatory cytokine that is s-palmitoylated itself. tnfα is synthesized as a transmembrane -kda precursor (tmtnfα) transported from the endoplasmic reticulum to the plasma membrane through the golgi apparatus and recycling endosomes ( ) . human tmtnfα is s-palmitoylated at cys located at the boundary between its transmembrane and cytosolic fragments, as was found independently by radiolabeling and by labeling with odya followed by click chemistry ( , ) . poggi et al. ( ) arrived at a complex model explaining how the s-palmitoylation of tnfα affects its activity ( figure a) . the modification was shown to favor the association of tmtnfα with rafts. upon cell activation, the extracellular domain of tmtnf is cleaved by adam metalloproteinase whereupon the soluble tnfα (stnfα) is released to the extracellular milieu and activates tnf receptor (tnfr) and tnfr . as adam localizes to both non-raft and raft regions of the plasma membrane, the s-palmitoylation of tmtnfα does not affect its cleavage and production of the soluble cytokine. however, s-palmitoylated tmtnfα interacts with tnfr in rafts thereby reducing the binding of stnfα and consequently reducing the sensitivity of the cell to this cytokine. in addition, the fragment of tmtnfα which remains after the release of stnfα in rafts if further processed by intramembrane sppl a and b proteases giving rise to icd (intracellular domain) of an own biological activity. by contrast, the non-raft fragment of the adam -cleaved tmtnfα is rapidly degraded ( ) . the transport and maturation of tnfα are also regulated by another posttranslational acylation, ε-n-myristoylation ( ) . as shown in figure b , myristic acid residues are attached to two lysines (lys and ) of human tmtnfα. this modification is reversed by sirtuin catalyzing the demyristoylation. depletion of sirtuin decreases the release of stnfα since the ε-n-acylated tnfα precursor is redirected to and accumulates in lysosomes ( , ) . it is worth noting that exogenous palmitic acid stimulates the ε-n-myristoylation of tmtnfα, thereby reducing the release of stnfα in favor of accumulation of tmtnfα in lysosomes ( , ) . this somehow surprising anti-inflammatory effect of palmitic acid can be explained by competitive binding between long-chain fatty acids (in this case, palmitic) and myristoylated substrates of sirtuin found in vitro- ( ) and adds a new dimension to the potential effects of palmitic acid. (a) non-palmitoylated tmtnfα is localized outside rafts while that s-palmitoylated on cys -in rafts of the plasma membrane. tmtnfα is cleaved by adam protease in both these plasma membrane environments giving rise to stnfα, which subsequently activates tnf receptor (tnfr) receptor leading to activation of nfκb and erk / . however, only the raft-residing tmtnfα is further processed by sppl b protease to yield icd, which activates the promoter of interleukin (il)- β and expression of il- . on the other hand, a pool of s-palmitoylated tmtnfα interacts in rafts with tnfr preventing its activation by stnfα. (b) tmtnfα is transported from the endoplasmic reticulum via golgi apparatus and recycling endosomes [ , ] to the plasma membrane [ ] . in the plasma membrane, tnfα is cleaved by adam giving rise to stnfα [ ] or is internalized [ ] and either returns from the endosomes to the plasma membrane [ , ] or is directed to lysosomes for degradation [ ] . ε-n-myristoylation of tmtnfα at lys and lys facilitates its degradation [ , ] at the expense of processing to stnfα [ ] . oligomerization of tmtnfα and tnfr is not shown. s-palmitoylation of host proteins is also vital in antiviral defense. viral nucleic acids, which are recognized by several tlrs and also cytoplasmic pattern-recognition receptors, induce robust production of type i interferons (ifns), mainly infα and ifnβ. the ifnα and ifnβ released from cells which first encounter viruses, e.g., dendritic cells, induce an antiviral reaction in an autocrine and paracrine manner upon binding to plasma membrane ifnα/β receptor (ifnar) consisting of subunits and . both human ifnar subunits are s-palmitoylated, as has been found by classical radiolabeling. the s-palmitoylation of ifnar on cys , localized near the cytoplasmic end of the transmembrane domain, is required for downstream activation of stat and stat and the following transcription of ifnα-activated genes ( ) . among the ifn-induced proteins, some have been shown to be palmitoylated, using click chemistry and abe. they include the immunity-related gtpase irgm , bst also known as tetherin, and ifitm and ( , ) . ifitms are potent restriction factors against a wide range of enveloped viruses, e.g., influenza, west nile, dengue, and zika viruses ( , ) . ifitms localize primarily to endolysosomal membranes where they inhibit viral replication by blocking their fusion with these membranes and also facilitate virus degradation ( ) . the exact mechanism of this antiviral activity is not clear, but it seems to rely on a perturbation of the organization of endolysosomal membranes. this can be linked with the intramembrane topology of ifitms and their s-palmitoylation. ifitm and likely possess two loops embedded in but not spanning the membrane with both the n-and c-termini facing the cytoplasm ( , ) . s-palmitoylation of conserved cysteine residues adjacent to these loops, cys , , and in murine ifitm , contributes to the membrane binding, similarly as found earlier for caveolins ( , ) . the s-palmitoylation also facilitates clustering of ifitm in the membranes, which is of potential significance for its antiviral activity ( ) . in support of the latter, the antiviral capacity was markedly reduced for non-palmitoylated mutant forms of ifitm ( , ) . however, s-palmitoylation did not affect the endolysosomal localization or stability of ifitm . subsequent studies have revealed that the localization and degradation of murine ifitm , both shaping its antiviral capacity, are orchestrated by numerous posttranslational modifications comprising polyubiquitination, tyrosine phosphorylation by the src-family kinase fyn, and methylation ( , ) . by contrast, s-palmitoylation alone of the closely related murine ifitm endowed it with an antiviral activity and enhanced stability by preventing proteasomal degradation ( ) , which indicates diverse effects of this modification on individual ifitm isoforms. the presented data are only beginning to fill the gap which existed in our understanding of the role of protein palmitoylation in innate immune responses. for a long time, it was lagging behind that on acquired immune responses, in which a plethora of s-palmitoylated proteins have long been known to be involved. they include receptors (cd and cd ), tyrosine kinases of the src family, transmembrane adaptor proteins (e.g., lat, ntal, and pag/cbp), and α subunits of heterotrimeric g proteins. their s-palmitoylation in most cases targets them to rafts and is a prerequisite for their involvement in the signaling pathways triggered by immunoreceptors [tcr, b cell receptor (bcr), and fcγ and fcε receptors] crucial for the acquired immune responses. an association of some components of these signaling pathways with tetraspanin-enriched domains has also been considered. these topics are discussed in several earlier reviews ( , , , ) . it is worth noting that large-scale proteomic analyses of fatty-acylated proteins of t cells ( , , , ) and b cells ( ) , identifying numerous new palmitoylated proteins, have been published recently. further studies will shed light on the possible engagement of those proteins in acquired immune responses and/or in the cross talk between the innate and the acquired immune system, in which phagocytic cells, such as macrophages and dendritic cells, are essential ( ) . protein s-palmitoylation affects their localization, trafficking, and stability. it has long been known as an important factor controlling signal transduction by the bcr and tcr receptors involved in acquired immune responses. it is now becoming evident that palmitic acid is also a key lipid affecting the diverse processes at the host-pathogen encounter. palmitate is a component of bacterial lps and lipoproteins; s-palmitoylation of viral, some bacterial, and numerous host proteins is recognized as a crucial factor affecting both the virulence of pathogens and the innate immune reactions of the host. our understanding of the latter has benefited greatly from the development of novel methods of detection of this protein modification. their application has led to the identification of numerous proteins involved in the host-pathogen interaction. the methods have also allowed highthroughput proteomic analysis of palmitoylation of proteins in infected cells, showing widespread changes of the host cell palmitoylome. future studies will tell whether complex feedback loops comprising palmitoyl acyltransferases and acylthioesterases, similar to those of kinases and phosphatases carrying out protein phosphorylation/dephosphorylation, are involved in controlling protein s-palmitoylation in infected cells. revealing how the s-palmitoylation of particular proteins is regulated during the host-pathogen interactions should allow its modulation to favor the host defense. all authors contributed to writing and critically revised the paper. the authors thank prof. andrzej sobota from the laboratory of molecular membrane biology of the nencki institute (warsaw, poland) and dr. jan fronk from the faculty of biology, university of warsaw for helpful comments and critical discussion. the work was supported by the national science centre, poland, grant number dec- / /a/nz / to kk. mechanisms of nutritional and hormonal regulation of lipogenesis atherothrombosis and coronary artery disease microbial induction of immunity, inflammation, and cancer nutritional modulation of metabolic inflammation the impact of western diet and nutrients on the microbiota and immune response at mucosal interfaces 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mediated by clathrin, actin and unconventional adaptors anthrax toxin triggers the activation of src-like kinases to mediate its own uptake type iii secretion systems and disease exploitation of eukaryotic subcellular targeting mechanisms by bacterial effectors subcellular targeting of salmonella virulence proteins by host-mediated s-palmitoylation host cell-catalyzed s-palmitoylation mediates golgi targeting of the legionella ubiquitin ligase gobx legionella pneumophila effector lpda is a palmitoylated phospholipase d virulence factor myristoylome profiling reveals a concerted mechanism of arf gtpase deacylation by the bacterial protease ipaj eukaryotic fatty acylation drives plasma membrane targeting and enhances function of several type iii effector proteins from pseudomonas syringae a family of bacterial cysteine protease type iii effectors utilizes acylation-dependent and -independent strategies to localize to plasma membranes evidence for covalent attachment of fatty acids to 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saturated fatty acids and inhibition by docosahexaenoic acid palmitic acid is a toll-like receptor ligand that induces human dendritic cell secretion of il- β acid sphingomyelinase plays a key role in palmitic acid-amplified inflammatory signaling triggered by lipopolysaccharide at low concentrations in macrophages lps upregulates palmitoylated enzymes of the phosphatidylinositol cycle. an insight from proteomic studies cytokine secretion in macrophages and other cells: pathways and mediators transmembrane tnf (pro-tnf) is palmitoylated palmitoylation of tnf alpha is involved in the regulation of tnf receptor signalling palmitoylation of interferon-α (ifn-α) receptor subunit ifnar is required for the activation of stat and stat by ifn-α ifitm-family proteins: the cell's first line of antiviral defense the ifitms inhibit zika virus replication s-palmitoylation and ubiquitination differentially regulate interferon-induced transmembrane protein (ifitm )-mediated resistance to influenza virus phosphorylation of the antiviral protein interferon-inducible transmembrane protein (ifitm ) dually regulates its endocytosis and ubiquitination greasing their way: lipid modifications determine protein association with membrane rafts protein acylation and localization in t cell signaling (review) patterns, receptors, and signals: regulation of phagosome maturation key: cord- - sukdb authors: quade, bianca n.; parker, mark d.; occhipinti, rossana title: the therapeutic importance of acid-base balance date: - - journal: biochem pharmacol doi: . /j.bcp. . sha: doc_id: cord_uid: sukdb baking soda and vinegar have been used as home remedies for generations and today we are only a mouse-click away from claims that baking soda, lemon juice, and apple cider vinegar are miracles cures for everything from cancer to covid- . despite these specious claims, the therapeutic value of controlling acid-base balance is indisputable and is the basis of food and drug administration-approved treatments for constipation, epilepsy, metabolic acidosis, and peptic ulcers. in this narrative review, we present evidence in support of the current and potential therapeutic value of countering local and systemic acid-base imbalances, several of which do in fact involve the administration of baking soda (sodium bicarbonate). furthermore, we discuss the side effects of pharmaceuticals on acid-base balance as well as the influence of acid-base status on pharmacokinetic properties of drugs. our review considers all major organs systems as well as information relevant to several clinical specialties such as anesthesiology, infectious disease, oncology, dentistry, and surgery. the normal function of nearly all physiological processes in the body depends on maintenance of appropriate acid-base balance. the value of intracellular ph and interstitial ph strongly depends on the value of arterial blood ph, which ranges between . and . under normal physiological conditions. when ph deviates from its normal range, ph-dependent enzymes and membrane transport proteins may not work properly and metabolic pathways can be negatively affected. acidemia, which is defined as arterial ph lower than . , can cause a variety of disturbances including arterial vasodilation, insulin resistance, compromised immune function, and reduced neuronal excitability. alkalemia, which is defined as arterial ph greater than . , can also cause many disturbances including reduced myocardial blood flow and seizures. thus, it is imperative that the value of blood ph is tightly controlled. therapies for acid-base disturbances are not new. infusion of sodium carbonate (na co ) into cholera patients to compensate for loss of serum alkali in diarrhea was recorded in the s [ ] and the commercial production of sodium bicarbonate (nahco ) for use as an antacid (brioschi®) apparently dates back to the s. since then, decades of research advances have led to a broad appreciation of the importance of acid-base balance in health and disease. this research is now coming to fruition in the form of inspired and effective medical advances. at the same time, some in the alternative medical community have seized on anecdotes and the results of limited trials to generate ubiquitous clickbait headlines about the miraculous properties of household acids and bases such as baking soda and, in some cases, propagate conspiracy theories about suppression of this information. in the first major section of our review ( acid-base homeostasis) we discuss how the body controls the abundance and distribution of acids and bases in order to achieve acid base homeostasis. we describe the importance of the powerful co /hco buffer system, the vital functions of the lungs and kidneys in excreting excess acids and bases, the role of membrane transport proteins and carbonic anhydrases in the local redistribution of acids and bases, and the drugs that can be harnessed to control these processes. in the second major section of our review ( systemic acid-base disturbances) we discuss the causes and consequences of generic acidbase imbalance caused by disturbances in co and hco levels and how our knowledge of their etiology has informed therapeutic strategies. our third and fourth major sections ( applications by organ system and other applications by clinical specialty) bring together a wealth of information from in vitro, in vivo, and clinical studies that demonstrate the current and potential utility of acid-base-balance correcting therapies. for each organ system or clinical specialization, as appropriate, we provide the fundamental physiological aspects of normal acid-base balance, the pathological consequences of systemic and local acid-base disturbances, as well as considerations of corrective therapies based on restoring (or harnessing the agents of) acid-base balance. in our fifth and final major section ( ph-dependent aspects of pharmaceutical therapy) we discuss how acid-base chemistry can influence drug pharmacokinetic properties and how this phenomenon can be advantageous for optimizing therapeutic interventions. our review highlights an emerging and dynamic field of research that is in the process of translating numerous basic scientific findings into clinical therapies. these findings appear to touch on nearly all aspects of health. we note a wide array of therapeutic paradigms developed around the control of acid-base balance including numerous reports of the successful application of nahco , the so-called 'enemy of the pharmaceutical industry,' to the amelioration of disease signs in animal models and in limited clinical trials. although studies of the role of acid-base balance in health and disease have resulted in the generation of several fda-approved pharmaceuticals such as contraceptive gels and gastric-acid suppressors, systematic reviews of random trials of the clinical effectiveness of nahco itself tend to be circumspect in their conclusions. a note to the reader: we, the authors, are basic scientists and do not intend this review to serve as a diagnostic or therapeutic guide. in many cases, a lack of consistency among study methods and subject demographics makes it difficult to draw firm conclusions regarding outcomes. for these reasons it is also impossible to extrapolate findings of therapeutic effectiveness in animal models and limited trials into an assessment of general clinical utility. but, in as much as we are reporting potential, we have not discounted any positive outcomes. finally, we note that the scope of this narrative review is extremely broad and the literature is extensive. for this reason, we have often cited reviews instead of primary literature in order to simplify the document and provide a cue to further reading. we apologize in advance to any authors whose work we have omitted. the maintenance of blood ph in the face of a ~ - meq h + /day acid load imposed by diet and metabolism (net endogenous acid production: neap [ , ] ), requires robust homeostatic mechanisms. regulation of blood ph and, by extension, the entire extracellular fluid compartment depends on the interplay between (i) the urinary system, which controls the blood bicarbonate concentration ([hco − ]), and (ii) the neuro-respiratory systems, which control the partial pressure of co (pco , see figure ). the kidneys perform the tasks of generating hco -, depositing it into circulation, and recycling hco from filtered plasma back into circulation (see figure ). the lungs exhale co , with respiratory drive being controlled by chemosensitive neural circuitry [ ] . a third mechanism of defense, which does not exactly regulate ph but only tends to minimize its changes, is provided by the multitude of buffer systems present in the extracellular fluid compartment. among these, the most powerful is the co /hco − buffer system, the efficacy of which is conferred by the body behaving as an open system, with respect to co , from which co can escape [ ] . a feature of the co /hco − buffer system is that the first of the two-step reactions that describe the interconversion between co and hco − (co +h o ⇌ h co ⇌ hco − + h + ) is very slow unless catalyzed by a carbonic anhydrase (ca) enzyme. thus, efficient buffering requires the presence of a ca. the henderson-hasselbalch equation [ ] describes how [hco − ] and pco determine ph:   co [hco ] ph = p + log . pco k s four of the members of the monocarboxylate transporter family (mcts, slc a - [ ] ) are h + -coupled lactate transporters (mct - ). mct and mct , being expressed in tumors, are of main therapeutic interest. the directionality of their transport process is determined by kinetic, thermodynamic, and situational considerations; for example the widely expressed isoform mct typically mediates h + /lacimport, while mct , which is abundantly expressed in glycolytic (i.e., lactate-producing) cells such as cancer cells and astrocytes, mediates h + /lacexport [ ] . members of several other solute carrier families also cotransport h + with their substrates such as the h + -coupled oligopeptide transporters of the slc family (e.g., peptt [ ]) or the h + -coupled neurotransmitter transporters of the slc family (e.g., the excitatory amino acid transporter eaat [ ]). the mct inhibitor azd [ ] is currently in phase i clinical trial for its effectiveness in treating cancer (clinicaltrials.gov identifier: nct ). there are several membrane proteins that conduct h + but share no obvious commonality in protein sequence. one group act as h + -selective channels and include the voltage-gated h +channel hv (hvcn [ ] ) and the voltage-independent h + conductors slc a [ ] and otopetrin (otop [ ]). all permit the movement of h + down their transmembrane electrochemical gradient, but each differ in their regulation of gating. hv only opens when the gradient favors h + efflux, slc a favors h + influx (particularly at elevated phi [ ] , perhaps to defend phi), and otop also favors h + influx (particularly at low phe, perhaps consistent with its sensory role). in acidotic conditions, the transient receptor potential cation channel subfamily v member (trpv ) can also mediate a significant, but non-canonical, acid-loading h + conductance [ ] . we are unaware of any fda-approvals for inhibition of this class of proteins, with the exception of trpv agonists whose influence on h + conductance is untested. although there is no description of a hco --specific ion channel, several anion channels have significant hco permeability. the electrochemical gradient for hco typically favors hco -efflux. these channels include the cystic fibrosis transmembrane regulator (cftr [ ]), the ca +activated cl --channel anoctamin (ano [ ]), as well gaba-and glycine-activated cl -channels (gabr and glr families [ ] ). drugs such as ivacaftor (kalydeco®, increases cftr channel open probability), or cocktails that include ivacaftor and one or more of the cftr folding chaperone drugs elexacaftor/lumacaftor/tezacaftor (e.g., orkambi®, symdeko®, trikafta®) are indicated for the treatment of cystic fibrosis by the restoration of certain defective cftr channels. both ivacaftor and tezacaftor rescue the hco permeability of the Δ -cftr mutant. in fact the rescued mutant has a greater hco -:clpermeability ratio than wild-type cftr, which may be therapeutically valuable. the importance of cftr-mediated hco secretion is discussed in sections . the respiratory system, . the lower digestive system, and . the urinary system). five of the ten members of the slc family of proteins mediate some form of na + -coupled hco --transport [ ]. nbce and nbce (slc a and slc a ) are electrogenic na + / hco cotransporters that may either act as acid-extruders or acid-loaders, depending on the electrochemical gradient. for example, nbce mediates hco efflux in renal proximal tubule epithelia, but hco influx in pancreatic duct epithelia. the remaining three are all acid extruders. nbcn (slc a ) is an electroneutral na + -hco cotransporter, while ndcbe (slc a ) is an electroneutral na + -driven cl -/hco exchanger. nbcn /ncbe (slc a ) has been described as being capable of both actions. we are unaware of any fda-approvals for inhibition of this class of proteins, nor of any drug that is specific for this class of protein. we note however that the nonsteroidal anti-inflammatory drug tenidap, which failed clinical trials for the treatment of rheumatoid arthritis due to renal and hepatic toxicity, is an effective blocker of nbce and nbce [ , ]. equation ( ) and figure show that, provided that pco remains constant, (i) a fall in extracellular [hco − ] causes ph to decrease whereas (ii) a rise in extracellular [hco − ] causes blood ph to increase. these two cases describe states of metabolic acidosis (mac) and metabolic alkalosis (malk), respectively. equation ( ) also indicates that ph can return towards its normal physiological value by a decrease in extracellular pco (in case 'i') or an increase in extracellular pco (in case 'ii'). this compensatory normalization of the [hco -]:pco ratio to restore ph, describes the physiological response of the neuro-respiratory system to mac and malk. moreover, equation ( ) and figure show that, provided that [hco -] remains constant, (iii) a rise in extracellular pco causes blood ph to decrease whereas (iv) a decrease in extracellular pco causes blood ph to rise. these two cases describe states of respiratory acidosis (rac) and respiratory alkalosis (ralk), respectively. again, equation ( ) indicates that ph can return towards its normal value by an increase (case 'iii') or decrease (case 'iv') in extracellular [hco − ], describing the compensatory physiological response of the urinary system to rac and ralk. mac, malk, rac, and ralk are usually referred to as the four classic/simple acid-base disturbances. respiratory compensations usually occur quite rapidly, within an hour of the appearance of the metabolic disorders and are fully resolved within to hours. in contrast, metabolic responses to respiratory disorders occur more slowly and may take up to several days to fully resolve as they require remodeling of acid-base handling mechanisms in the urinary system. the most rapid response-and the first line of defense of our body-to an acid-base disorder is given by chemical buffering which usually occurs within minutes. in the following four sub-sections we will review the causes, consequences, and therapeutic paradigms for each of the four systemic acid-base disturbances. several of these considerations are also relevant to the resolution of local acid-base disturbances and will be revisited in sections and . for a more complete and clinical perspective on these disturbances in isolation, and in combination, we refer the reader to reference [ ] . metabolic acidosis is defined as acidemia due to a primary pathological deficit in [hco -] rather than a physiological, compensatory lowering of [hco -] in response to respiratory alkalosis [ ] . the body can counter acid shifts in plasma ph in the short term by increasing respiratory drive to lower co and, in the longer term, by increasing renal h + excretion/hco generation. however, if these compensatory systems are defective or overwhelmed, mac will result. for example: mac can result from diet, chronic kidney disease, and diabetes (diabetic ketoacidosis) or can follow acute myocardial infarction (lactic acidosis), mutations in renal acid-base transporters (renal tubular acidosis, see section . ), intoxication with compounds (e.g., aspirin), and diarrhea (loss of hco --rich secretions) [ ] [ ] [ ] [ ] [ ] . clinical manifestations vary depending on underlying cause, but generally include weakness, nausea, and flushed skin [ ] . as we shall see, chronic mac has severe consequences for long-term health. in cases where mac is secondary to another disturbance such as in diabetes or diarrhea, treatment of the underlying disorder is the ultimate goal. however, for short term remediation of mac, or for situations in which the primary defect is with acid-base homeostatic mechanisms, the typical course of action is 'alkali therapy' to address mac by normalizing plasma ph. this can be achieved via two mechanisms. the following paragraph describes therapies that increase base load while the final paragraph describes therapies that lower acid load. increasing base load. the simplest paradigm is administration of hco salts. a direct rise in plasma [hco -] can be achieved either intravenously or by peritoneal dialysis. an indirect rise in plasma [hco -] can be achieved by oral dosing; as the parietal cells of the stomach replace neutralized stomach acid, they also generate new hco -, which is absorbed into circulation [ ] . there are however a number of caveats associated with hco administration [ ] . one caveat is that the counter anion (usually na + or k + ) may contribute to fluid retention or k + imbalance. a second caveat is that the treatment has the potential to rapidly generate co . with oral administration this can manifest as bloating or even gastric rupture, whereas with intravenous administration, the co , if not effectively eliminated by the lungs, can enter cells causing a paradoxical intracellular acidification. a third caveat is that ph overshoot (i.e., overcompensation that creates its own ph disturbance) is possible if the dose is not well titrated. however, in practice, manifestation of the side effects associated with nahco administration is not a foregone conclusion [ , ] . alternative vehicles for intravenous alkali delivery such as na co and caco produce less co per neutralized h + and impose less of an osmotic stress [ ] . carbicarb is a mixture of nahco and na co that does not cause intracellular acidification [ , ] . citrate salts provide a gentler, indirect mean of raising hco as citrate is converted into hco in the liver. alternative buffers such as tham (tris-hydroxymethyl aminomethane aka tris-base aka tromethamine aka trometamol) bind h + without generating co and the protonated product is readily cleared by the kidneys [ ] . furthermore, because a certain proportion of tham is uncharged at physiological ph, it is cell permeable and can counter intracellular acidosis. other hco --replacing bases include lactate and acetate [ , ] . finally, potential side effects can be ameliorated by administering buffers at a lower dose as part of an intravenous cocktail of buffers. for example, tribonat is a mixture that includes nahco , na hpo , and sodium acetate [ , ] . an added bonus of that mixture is that the inclusion of phosphate counters the hypophosphatemia associated with mac. dietary acid load is associated with lower serum hco - [ , , ] and thus there is scope for dietary correction of mac by, for example, adherence to a very low protein [ ] or otherwise "alkaline" diet [ ] . ph imbalance in mac can also be redressed by increasing h + excretion. the thiazide diuretic hydrochlorothiazide increases h + secretion by the renal collecting duct and has been used as an adjunct therapy with nahco for mac [ ] . its role as a diuretic ought also to assist with excretion of the na + load associated with nahco treatment. veverimer is an orally dosed h + binding polymer that is in phase iii clinical trials at the time of writing for the treatment of mac in the context of chronic kidney disease (clinicaltrials.gov identifier: nct ). it binds h + in the stomach for eventual excretion in the feces [ , , ] . moreover, the raising of gastric ph by veverimer prompts parietal cells to deposit hco into circulation, mimicking the alkaline tide associated with feeding. another approach to counter mac, is to increase cellular h + consumption (and/or decrease lactic acid production) by metabolic means either by pyruvate administration or by stimulating pyruvate dehydrogenase using dichloroacetate (dca) [ , ] . malk is defined as alkalemia caused by a primary excess of hco -. malk may follow volume depletion or hyperaldosteronism (promotes renal h + secretion), vomiting (eliminate gastric acid, stimulating an alkaline tide), or the use of certain pharmaceuticals that mimic those responses (loop diuretics, antacids). clinical manifestations can include confusion and tetany [ ] . malk can also have a genetic cause. for example, liddle syndrome is associated with hyperactivity of the epithelial na + channel enac, the action of which promotes renal h + secretion [ ] . besides treatment of the underlying conditions, correction of malk has been achieved using the ca inhibitor acz, which by itself results in mac [ ] , by intravenous infusion of hcl [ ] , or (if malk follows loss of gastric acid) the use of h -receptor agonists to prevent alkaline tide [ ] (see section . . ). rac is defined as acidemia with a plasma pco > mmhg at rest and at sea level [ ] . it usually occurs when there is a disruption in the ventilatory system that causes a mismatch between the rate of co removal and the rate of co production, with consequent accumulation of co into the blood (i.e., co retention). this disruption can be caused by (i) inability of the lungs to remove the metabolically produced co (i.e., reduced ventilation), (ii) defects in co transport from tissue to lungs and (iii) overproduction of co . reduced ventilation can result from a depression of the respiratory center (e.g., due to sedative overdose or brain injury), airway obstruction (e.g., due to vomit aspiration or laryngospasm), neuromuscular disorders (e.g., due to guillain-barré syndrome) or restrictive defects of the chest (e.g., due to impaired functioning of the diaphragm) [ ] . defects of co transport that lead to hypercapnia are less common and usually the result of reduced pulmonary perfusion in response, for example, to cardiac arrest or pulmonary embolism. overproduction of co is rarely the sole cause of rac. in fact, under normal circumstances the body responds to increases in co production by appropriately increasing ventilation in order to remove the excess co and prevent hypercapnia. situations in which the lungs are unable to match the increased co production can occur in patients undergoing mechanical ventilation or with reduced respiratory reserve [ ] . in fact, for therapeutic reasons, individuals on mechanical ventilation are often deliberately maintained in a state of ''permissive hypercapnia'' (see section . ). as for metabolic disturbances, rac can be either acute or chronic. acute rac occurs when pco rises very rapidly and the kidneys are unable to adequately increase hco production to compensate in such a short amount of time. thus, only a very modest renal compensation occurs. on the contrary, during the longer timespan of chronic rac (such as with chronic obstructive pulmonary disease, copd), the kidneys are able to restore the acid-base balance by increasing acid excretion and hco production [ ] . treatment is usually directed towards reversing the underlying cause and also at restoring adequate alveolar ventilation, which can be accomplished by endotracheal intubation with mechanical ventilation or positive pressure ventilation [ ] . because the sum of pco and po must be constant in the alveolar gas of patients breathing room air, hypercapnia leads to hypoxemia, a condition that can have consequences far more dangerous than those caused by hypercapnia [ ] . consequently, management of acute respiratory acidosis is often also directed towards ensuring adequate oxygenation. administration of o must be performed carefully because it may lead to increased co retention, especially in patients with copd [ ] . correction of hypercapnia in chronic rac usually occurs slowly because rapid reduction of pco can lead to overshoot alkalosis due to the renal compensation that increases [hco -]. in the central nervous system (cns), rapid alkalinization of the cerebrospinal fluid (csf) can cause seizures and even coma [ ] . the use of alkali therapy in rac is controversial and indicated only in patients with acute hypercapnia and concurrent metabolic acidosis [ ] . administration of nahco is contraindicated because it may increase co production, reduce alveolar ventilation as well as cause a paradoxical acidosis in the cns. as noted above for mac, alterative alkali therapies such as carbicarb that do not generate as much co as nahco alone (see section . . ) may be preferable to correct ph in rac. in patients with copd, the ca inhibitor acz is sometimes used to stimulate respiration in order to improve oxygenation, reduce co retention and possibly remove the need for mechanical ventilation [ ] . however, because ca is ubiquitous, the inhibitory effect of acz may impact a variety of tissues and have potential negative consequences on patients with pulmonary diseases. for this reason the role of acz as a respiratory stimulant is controversial, especially in patients with severe copd with or without hypercapnia [ ] . finally, co can be de-gassed from blood using an extracorporeal co removal (ecco r) device or [ ] lowered by dialysis using a dialysate that has a low [hco -] [ ] . ralk refers to alkalemia with a plasma pco < mmhg at rest and sea level [ ] . it occurs when the ventilatory system does not work properly causing an increase in alveolar ventilation and/or reduced co production with consequent co depletion in the blood. hyperventilation can result from stimulation of the respiratory centers (e.g., due to drugs or disorders of the cns), hypoxemia or tissue hypoxia (e.g., due to high altitude), lung diseases (e.g., pneumonia). reduced co production can result from a decrease in the basal metabolic rate (e.g., due to hypothermia) or in physical activity (e.g., due to muscle paralysis). clinical manifestations can include rapid breathing and dizziness [ ] . although usually considered not life-threatening, severe ralk can have serious consequences on the brain, lungs and the heart. treatment is usually directed towards correcting the underlying disorders. hormone replacement therapy caused ralk in a study of postmenopausal women [ ] . abrupt correction of severe ralk should be avoided because of the risks of cerebral and pulmonary reperfusion injury. acz is used in the prevention and treatment of ralk associated with hyperventilation at high altitude (acute mountain sickness: ams) in part because it enhances hco excretion in the urine, providing a compensatory lower of ph [ ] . neuronal activity presents a substantial challenge to local acid-base balance. neurotransmitterfilled vesicles release h + into the synaptic cleft [ , ] (h + themselves may be considered to be neurotransmitters [ ] ) and are removed from the synaptic cleft by h + -coupled neurotransmitter transporters such as the excitatory amino acid transporter eaat . gaba-activated anion channels in neurons and astrocytes release hco - [ , ] , and the ca + /h + exchange activity of the plasma membrane ca + -atpase (pmca) in neurons causes a rise in extracellular ph as it restores intracellular ca + following an action potential [ ] . the acid load that results from intensive neuronal firing can result in a drop in phi that dampens neuronal activity: a mechanism that prevents excessive firing via effects upon ph-sensitive channels such as asic a and nmda receptors [ ] [ ] [ ] . conversely, alkalosis is associated with an increase in neuronal activity and seizures [ ] . neurons and astrocytes express numerous abts and cas to maintain ph homeostasis and their importance is highlighted by the effects of their disruption [ ] . for example, genetic disruptions in ae or nbcn are associated with epilepsy [ , ] , although the mechanism is not simply related to effects of neuronal phi on excitability as ae is an acidloader while nbcn is an acid extruder and may depend on whether the neurons in question are excitatory or inhibitory. several abts and cas are expressed in the choroid plexus epithelia where their action supports the secretion of cerebrospinal fluid (csf). genetic ablation of these transporters (e.g., nbcn , nbce ) in rodents is linked to reductions in ventricle fluid volume [ ] while pharmacological inhibition of cas results in reduction of intracranial pressure [ ] . however, it is unclear whether these changes are accompanied by a fall in ph of the csf. besides its role in determining ph, hco plays an important role in neuronal plasticity because the transmembrane gradients of cland hco determine the reversal potential of gabaactivated channels and consequently whether gabaergic signals are depolarizing and excitatory or hyperpolarizing and inhibitory [ ] . changes in these gradients are important in two ways. firstly, developmental changes in the gradient during central nervous system maturation promote the switch to inhibitory gaba signaling [ ] . secondly, activity-dependent changes in the gradient contribute to the pathophysiology of epilepsy by promoting a pathological switch to excitatory gaba signaling [ ] . neuronal cl − -hco − exchangers such as ae and ndcbe are likely to contribute to the status of these gradients [ ] . finally, mutations in endosomal nhe cause intellectual disability and are associated with defective synaptic remodeling [ ] . acidosis has a number of other consequences. for example in stroke, lactic acidosis is linked to ischemic damage [ ] . in protein-aggregating neurodegenerative diseases, acidic ph promotes the aggregation of alzheimer's amyloid proteins [ ] . a major genetic risk factor for alzheimer's disease is incidence of the apolipoprotein e allelic variant apoe , which causes the epigenetic downregulation of nhe [ ] . loss of nhe from endosomes causes aberrant acidification and defective clearance of amyloid deposits [ ] . brain acid-base status also has consequences for mental health (see mental health). the role of ph in the retina is considered in a later section (see the sensory systems). the link between ph and neuronal excitability is exploited in the anticonvulsant value of inhaled % co to induce hypercapnic acidosis [ ] . hypercapnia also has a neuroprotective role in stroke, by inhibiting caspase and other cytotoxic activities [ ] , and during reperfusion [ ] . ca inhibitors are used as adjunct therapies for epilepsy [ ] and have potential application for treatment of neuropathic pain [ ] , alzheimer's disease [ ] , and cognitive disorders [ ] . however, mac is a side effect of systemic ca inhibition [ ] . lowered seizure thresholds in some strains of abt-null mice suggest that abts may be potential targets for anticonvulsant therapy. however, the need for caution is shown by the observation that, at least in the case of nbcn -null mice, a reduced seizure-threshold does not mean reduced neuronal excitability [ ] . the role of abts and cas in csf secretion hints at the potential for targeting of these proteins to lower intracranial pressure in idiopathic intracranial hypertension (iih). the use of ca inhibitors in patients with iih produces some symptom relief, but the mechanism of action is uncertain [ ] . regarding therapies for neurodegenerative diseases, histone deacetylase inhibitors have shown potential to release nhe from its epigenetic restraints to restore amyloid protein processing in apoe mice [ ] . another strategy that has been proposed to have potential to reverse amyloid deposition in alzheimer's disease is the raising of brain ph [ ] . therapies that target the peripheral nervous system are discussed in the following section. sight. most ocular tissues express one or more abt or ca for the purpose of maintaining fluid and ph balance. perhaps the most therapeutically tractable tissue is the ciliary body that employs caii and a range of abts to secrete hco --containing aqueous humor into the anterior chamber [ , ] ( figure ). this fluid leaks into the corneal stroma to flush out metabolic wastes and is returned to the anterior chamber by corneal endothelial cells which express a similar array of abts including nbce , mct , and the h + channel slc a [ , ] . finally, the fluid is drained from the anterior chamber via the trabecular meshwork. individuals with mutations in nbce have band keratopathy, glaucoma, cataracts, and corneal edema linked to fluid/ph imbalance in the cornea, lens, and elsewhere [ ] . abts and hco are also important for retinal function [ ] [ ] [ ] , as suggested by the link between nbcn mutation and progressive rod-cone dystrophy [ ] , or retinal degeneration in mice with defective expression of nbce and mcts [ , ] . hearing loss is a symptom of several systemic diseases linked to defects in abts, including pendrin (pendred syndrome [ ] ), the h + /k + -atpase (distal renal tubular acidosis [ ] ), and slc a (harboyan syndrome [ ] ). all of these abts are expressed in the inner ear where they help to maintain inner ear fluid ph and endocochlear potential [ ] . although a human correlate has not yet been reported, progressive hearing loss is also a feature of nbcn null mice [ ] . disruption of the h + -channel otop and the anion exchanger pendrin in mice is associated with malformation of the caco crystals (otoconia) that are essential for maintenance of balance [ , ] . taste. in addition to its role in the inner ear, otop is required for sour taste sensation [ ]. pain sensation. it is generally recommended to keep the ph of injected formulation close to physiological ph to avoid injection-site pain, with the added note that the inclusion of certain buffers may increase pain (hence new citrate-free formulations of adalimubab aka humira®) [ ] . low phe exacerbates sensation of pain due to its effects on trpv channel activation in nociceptive neurons. furthermore, activation of these channels under acidotic conditions is associated with a drop in neuronal phi that is mediated in part by a trpv -mediated h + sight. ca inhibitors applied as eye drops have long been used to treat glaucoma by virtue of their ability to reduce the production of aqueous humor, although even their localized ophthalmic use has been documented to lead to the side-effect of systemic mac in some prone individuals [ , ] . corneal edema that results from the expression of mutant misfolded slc a may be amenable to correction by small molecule folding chaperones [ ] . nhe blockers are cytoprotective in a rat model of diabetic cataract formation and retinopathy [ ] . hypercapnia is protective against ischemia-reperfusion injury in the retina [ ] , as it is elsewhere in the central nervous system (see section . ). nahco solution is useful for softening and dispersing hardened ear wax [ ] . however, we are unaware of any therapies specifically targeted to restoring the acid-base chemistry necessary for correct generation of endolymph or ostoconia. on a related topic there is one side effect of ear drops that pertains to acid-base balance. the acetic acid in some ear drops used to treat outer ear infection can be ototoxic because acetic acid can move across the round window into the inner ear, resulting in a drop in endocochlear potential (perhaps by acid inhibition of the na + /k + -atpase) and endolymph and perilymph ph [ ] . we are unaware of any demonstrations of the usefulness of otop modulation in this area, but inhibitors of proteins that mediate bitter taste sensation have been used to mask bitter tastes, suggesting potential utility of otop block for masking sour tastes and increasing the palatability of sour-tasting medications [ , ] . pain sensation. adjuvant nahco raises the ph of an injectable lidocaine solution and lowers perception of pain associated with lidocaine injection in one study, but the mechanism of the effect is uncertain [ ] . see also sections . (anesthesiology). besides the increased respiratory drive to exhale co in response to rac [ ] and the bohr effect (see section . the circulatory system) the highest profile link between ph and respiration relates to the role of cftr. defects in cftr are devastating because the cland hco secretion that this channel normally mediates is a fundamental part of the mechanisms that drive fluid secretion in our bodies [ ] ( figure ). the majority of deaths associated with cf are caused by respiratory failure [ ] . in the lungs, secretions are required to provide a moist surface for gas exchange, to liquefy mucus, and to flush inhaled particles and pathogens out towards the throat (mucociliary clearance). besides the general importance of anion secretion, cftr-mediated hco secretion plays a further role in ph homeostasis in the airway surface liquid (asl); hco helps to unfold and hydrate mucus [ ] and, by defending airway ph, has been hypothesized to promote a healthy local immune response to airway bacteria [ , ] . hco secretion is modulated by epithelial h + secretion mediated by a host of acid-extruding transporters [ ] ( figure ). airway acidification is a feature of individuals with cystic fibrosis, as well as those with asthma and tuberculosis [ ] and is exacerbated by lactic acid production by airway pathogens and airway epithelia [ ] . the new personalized cf therapies have focused on stimulation of defective cftr to restore fluid secretion [ ] , but are targeted to individuals with specific cf genotypes and thus alternative general therapies are still required. strategies specifically focused on correcting asl ph include inhalation of nebulized bases such as nahco [ , ] and tham [ ] as well as block of airway h + secretion using h + /k + -atpase inhibitors [ ] . all these strategies result in improvements in asl ph and some also improve mucus viscosity and/or pathogen clearance. an in vitro study suggests that mct blockade could also be protective of asl ph in individuals with cf [ ] by reducing epithelial h + secretion. a newly described paracellular pathway for hco secretion by cf airway epithelia might also be amenable to therapeutic modulation [ ] . the new personalized cf therapies have focused on stimulation of defective cftr to restore fluid secretion [ ] (e.g. lumafactor/ivacaftor, see section . . ), but are targeted. heart. mac is associated with reduced cardiac contractility. this phenomenon is explained by diverse mechanistic elements such as the ph-dependence of the channels and transporters that regulate ca + handling in myocytes as well as the dampening effect of acidosis on the responsiveness of the contractile apparatus to ca + [ ] . whether the heart rate is lowered by acidosis is harder to predict because of the complex effects of acidosis upon the sympathoadrenal system [ ] . intracellular acidosis in myocardial infarction after a period of ischemia, is countered, during reperfusion, by the action of acid-extruders such as ncbts and nhes [ ] . however, the accompanying na + load can be sufficient to reverse the action of the na + / ca + exchanger, raising [ca + ]i and increasing susceptibility to ventricular arrhythmias [ , ] . paradoxically, the loss of nbce function can also result in ca + overload because compensatory acid-extrusion mediated by nbcn and nhe imposes double the na + load per hco equivalent; a mechanism proposed to promote hypertrophy of cardiomyocytes in spontaneously hypertensive rats [ ] . ca activity is also pro-hypertrophic [ ] . on the other hand, the action of the acidloading anion exchange ae is considered to be protective against hypertrophy [ ] . finally, nhe action in the mitochondria is proposed to contribute to mitochondrial damage in the diseased heart [ ] . vasculature. typically, acidosis causes arterial vessels to dilate resulting in a fall in peripheral resistance, while veins may constrict [ ] . it is perhaps then no surprise that numerous blood pressure traits are linked to polymorphisms in abt genes [ ] . at least at the level of the vascular response of arteries, nbcs and nhes are required for normal vascular smooth muscle contractility and sensitivity to vasodilators [ ] . however, blood pressure is a complex trait that is not determined by vascular response alone, so explanation of these linkages is not simple. another important aspect is that mac inhibits progression of vascular calcification [ ] . the bohr effect describes the influence of ph and pco upon the oxygen carrying capacity of hemoglobin. in systemic capillaries, metabolically produced co enters the red blood cells (rbcs) where it is hydrolyzed into hco and h + by the action of caii. the newly produced hco is then extruded by ae , causing a fall in rbc phi which, by the bohr effect, reduces the hb-o binding affinity, promoting o release from hb to tissue ( figure b ). the reverse process occurs in the pulmonary capillaries. here, as co leaves rbc phi rises thereby favoring o binding to hb ( figure b ). thus acidosis enhances o delivery into tissues, but diminishes o loading in the lungs [ ] . this relationship between ph and gas exchange is partly sensitized by the content of the hemoglobin-regulating molecule , -dpg (diphosphoglyceric acid) in rbcs, a parameter which itself is ph-dependent; , -dpg levels increase with chronic acidosis promoting o release [ ] . heart. exogenous expression of skeletal muscle caiii in mouse cardiomyocytes enhances defense of phi and preserves cardiac function during mac [ ] . nahco is used to counter lactic acidosis in cardiac arrest and during prolonged cardiopulmonary resuscitation, but aside from its value at normalizing pre-existing mac or hyperkalemia (acidosis promotes cellular k + release), compelling data that this treatment improves outcomes are lacking [ ] [ ] [ ] . nhe blockers have shown promise as cardioprotective agents in reperfusion injury [ ] and likely act by targeting both plasma membrane and mitochondrial nhe [ , ] . although the nhe blocker cariporide caused serious side-effects in clinical trials (see section . . ), alternative approaches are available. for example, a microrna that lowers nhe expression protects cardiomyocytes from apoptosis during prolonged endoplasmic reticulum stress [ ] . in addition, antibodies and drugs that block nbcs have also demonstrated cardioprotective properties in animal models of ischemia reperfusion injury [ , ] . just as blockade of acid-extruders is cardioprotective, so too is the stimulation of the acid loader ae . this has been achieved in cell models using the glycoside sasanqua saponin [ ] , an extract from a herb used in traditional chinese medicine. we are unaware of any reports of acid-base based therapies for blood pressure that directly target the vasculature, but a discussion of diuretics for lowering blood pressure in congestive heart failure is provided in the urinary system section. some alkali-containing therapies may enhance progression of vascular calcification [ ] while use of the ca blocker acz has therapeutic value in calcifying disease [ , ] , perhaps by lowering ph. one study has cautioned the use of nahco in congestive heart failure because, in the face of adaptively elevated , -dpg levels, a sudden rise in ph could result in a maladaptive increase in hb-o affinity and risk of myocardial ischemia [ ] . in skeletal muscles, the build-up of lactic acid during intense exercise correlates with muscle weakness and self-limiting fatigue. however, the contribution of lactic acidosis to those symptoms may not be as direct or major as once thought [ , ] . generalized acidosis may contribute to weakness via alterations in neuromuscular drive [ ] and/or a decreased driving force for lactate efflux [ ] . regardless, acidosis promotes degradation of muscle protein [ ] . a high estimated dietary acid load has been associated with frailty in elderly japanese women [ ] recovery from lactic acidosis is mediated by mcts, nbcs, and nhes [ ] , while caiii specifically has been shown to plays a role in defense from muscle fatigue [ ] . many studies suggest the utility of nahco for improving exercise performance. for example, induction of malk by ingestion of oral nahco solutions has been shown to improve exercise endurance [ ] and reduce perception of effort [ ] in limited trials. however, taking a broader view of the field, the results of trials that link ph and exercise performance are deemed inconclusive due to inconsistent methodology and subgroup effects [ , ] . it has also been suggested that any competitive benefits that could be gained from nahco administration, from an athletic viewpoint, may be outweighed by gastrointestinal side effects such as bloating [ ] . away from the arena, hco administration or a reduced dietary acid load could have value in maintaining muscle mass in older adults [ , ] . mineralized material is eroded by acids as is evident in the case of tooth enamel, which is subject to demineralization by dietary acids (see oral health). however acidosis also inhibits bone growth by inhibiting osteoblasts, stimulating the activity of bone-resorbing osteoclasts [ ] , and influencing hormonal axes [ , ] (see also section . about the effects of ph on the endocrine system). accordingly, serum [hco -] positively correlates with bone mineral density (bmd) [ ] and negatively correlates with levels of serum parathyroid hormone (which promotes bone resorption) [ ] . at a local level, the process of bone remodeling, as well as the hormonal mobilization of ca + and pi from bone, requires that osteoclasts secrete h + onto the bone surface. these cells express intracellular caii to generate h + and hco -, an apical v-type h + -atpase to secrete h + onto the bone surface, and basolateral ae to export hco and defend osteoclast phi from alkalosis during h + secretion ( figure ). mutations in the v-type atpase and caii disable bone resorption by osteoclasts and are associated with increased bone density and osteopetrosis in humans [ , ] . the acid-base regulating proteins of osteoclasts are amenable to pharmaceutical modulation and their blockade ought to be protective of osteoporosis. for example, ae may be a useful target for increasing bmd because bmd is elevated in ae -null mice and cattle (reviewed in ref. [ ] ). regarding caii, one study showed a fortuitous bone-sparing effect in post-menopausal women were chronic users of ca-inhibitors for glaucoma treatment, [ ] . another study showed a paradoxical, but therapeutically valuable, bmd-lowering effect of ca inhibition in three children with sclerosing bone dysplasias. in these children, osteoclasts are already defective so the predominant effect of ca-inhibition is induction of chronic mac which promotes bone resorption [ ] . the therapeutic utility of proton pump inhibitors (ppis) to treat osteoporosis is negated by their negative influence on intestinal ca + absorption [ ] . in fact, several studies link ppi use with fracture susceptibility and low bmd (reviewed in refs. [ , ] ). because of these side effects, ppis are used with caution in some groups who may take them as antacid therapy [ , ] (and see next section). the three major health-related aspects of acid-base in this system are the roles of salivary hco in defense of enamel (which are discussed in section . oral health), gastric acid secretion, and peptic ulceration with helicobacter pylori. gastric acid is required to activate digestive enzymes, stimulate downstream secretory processes, and to kill ingested pathogens. it is secreted by parietal cells using similar transport mechanisms employed by osteoclasts (described in the previous section). thus, the secretion of acid across the apical membrane is mediated by a h + /k + -atpase and is balanced by the extrusion of hco into the plasma via ae (the alkaline tide associated with feeding [ , ] , see figure ). stomach epithelia are protected from acid injury by a mucus lining. the pathogenic bacterium h.pylori is able to survive in gastric acid because it can take up urea from its environment, via a h + -gated urea channel, and convert it into nh to neutralize acid in its immediate environment [ ] . in the vicinity of the mucus layer, this action causes h.pylori to raise mucus ph, lowering its viscoelasticity, promoting bacterial infiltration, and ultimately resulting in inflammation, ulceration [ ] , and risk of gastric cancer [ ] . another condition, gastroesophageal reflux disease, is caused by reflux of gastric acid into the esophagus and can cause heartburn and, in severe cases, can lead to esophageal damage. salivary hco plays an important role in esophageal acid defense [ ] [ ] [ ] by neutralizing gastric acid [ ] . conversely, the action of nhe in esophageal epithelia may exacerbate the damage, perhaps by indirectly stimulating pro-apoptotic pathways [ ] . acid reflux symptoms can be relieved by neutralizing gastric acid with antacids, which at their simplest are just bicarbonate or carbonate salts (e.g., tums® is calcium carbonate). however, an early antacid regimen for peptic ulcers, based on administration of milk and caco and still observed in the modern age in self-medicating individuals, results in adverse outcomes: the so-called 'milk-alkali syndrome' characterized by malk and hypercalcemia [ , ] . an alternative approach to lowering gastric ph is to use ppis or h -receptor agonists which dampen the signaling pathways that stimulate h + secretion. h agonists, in addition, therapeutically lower the activity of esophageal nhe [ ] . some over-the-counter formulations combine these drugs with an antacid to lower the dose of each and minimize side effects of each such as bloating (from gastric co generation) and osteoporosis (from chronic inhibition of intestinal ca + reabsorption, see section . ). orally-dosed acid-chelators such as veverimer, also raise gastric acid ph [ ] but have not been tested as a therapy for heartburn. the achlorhydric phenotype of ae -null mice suggests that ae blockage may have potential as a therapeutic target [ ] . ppis in combination with antibiotics are used to treat h.pylori infections: it has been proposed that raising stomach ph permits faster bacterial growth, potentiating the effects of antibiotics that act on dividing bacteria [ ] . inhibitors of the urease and h + -gated urea transporter of h.pylori are potential therapeutic modalities that remain in development [ ] . the exocrine pancreas secretes a hco --rich fluid that is vital for neutralizing gastric juices passing into the duodenum. the alkaline ph of pancreatic juice holds digestive enzymes such as amylase and lipase in an inactive state until the secreted fluid is neutralized in the duodenal lumen by chyme, preventing damage to the pancreatic ducts. in cf, duodenal hyperacidity also holds pancreatic enzymes in an inactive state, but without the neutralization of chyme, they are not even active in the duodenum leading to malabsorption of nutrients such as lipids [ ] . all along the intestine, hco --containing fluid secretions are required to promote gastric motility. the loss of this fluid in feces represents a substantial acid load. consequently, cftr mutations result in intestinal blockage [ ] and secretory diarrhea can result in mac [ ] . balancing secretory processes, nhe and slc a promote fluid reabsorption ( figure ). accordingly, downregulation of intestinal nhe by the enterotoxigenic bacteria (e.coli and c.difficile) and inactivating genetic defects in nhe and slc a are all associated with hypersecretion and diarrhea [ ] [ ] [ ] [ ] . on the subject of gut microbiota, intestinal ph can both influence and be influenced by the composition of gut microbiome [ ] . in individuals with insufficient intestine to absorb nutrients (short bowel syndrome), unabsorbed carbohydrates promote the growth of lactic acid-producing bacteria, which can lead to d-lactic acidosis [ ] . finally, the absorption of many nutrients depends on the action of h + -coupled abts (e.g. the h + -coupled oligopeptide transporters of the slc family: [ ] ), which in turn require the presence of acid extruders such as nbce to maintain epithelial ph during nutrient absorption. indeed, nbce -null mice exhibit defective nutrient absorption, which contributes to their general failure to thrive [ ] . the ability of small molecule inhibitors of nhe (tenapanor) and slc a (the , dimethylcoumarin drug "drainh-a ") to reduce intestinal fluid absorption makes them valuable therapies for irritable bowel syndrome with constipation and for relief of constipation in cf [ , ] . the cftr corrector ivacaftor improves intestinal hco secretion and nutrient absorption in individuals with cf [ ] . the mode of action of the anti-constipation drug linaclotide (linzess®: a guanylate cyclase c receptor agonist) encompasses both paradigms by the cgmpmediated reduction of nhe [ ] and activation of cftr activities [ ] . nephron function. the kidneys are vital to whole body ph balance (see acid-base homeostasis). it is the kidneys that generate hco -, reabsorb hco from the glomerular filtrate to prevent its loss in urine, and excrete h + in the form of nh + or titratable acids such as phosphate [ ] ( figure ). thus, it is no surprise that defects in renal transport mechanisms result in mac. these acidifying diseases can be acquired or genetic. fanconi syndrome is a degeneration of the proximal tubule, while renal tubular acidosis (rta [ ] ) can result from mutations in acid-base transporters such as nbce (type ii proximal rta: prta), h + -atpase or ae (type i distal rta: drta), caii (type iii rta), or disruption of h + secretion due to hypoaldosteronism (type iv drta). chronic kidney disease (ckd) is also associated with mac [ ] , and mac itself promotes progression of ckd (see below). malk is a common finding in cf patients. cf-model mice are less capable of defending against hco loads than their wild-type counterparts, due to downregulation of the renal hco -secreting anion exchanger pendrin [ ] and presumably loss of direct cftr-mediated hco secretion. concurrently, acid-base status has profound influence on kidney function. the very mechanisms that allow the kidneys to increase acid excretion in response to acute increases in acid load (e.g., ammoniagenesis and the renal endocrine response to acidosis) can be maladaptive in chronic mac, leading to inflammation and fibrosis [ ] . it is perhaps then not coincidental that low serum [hco -] is linked to a higher risk of chronic kidney disease in both adults and children [ , ] . an additional set of renal pathologies follows the integration of acid/base and salt/water handling by the nephron. for example, states and conditions of increased sodium reabsorption by the proximal tubule (e.g., volume contraction) or collecting duct (e.g., hyperactivity of the epithelial na + channel enac in liddle's syndrome) result in malk (see metabolic alkalosis) while hyperkalemia can cause mac [ ] . stone formation. urinary ph can influence stone formation which can lead to inflammation and obstructive kidney injury. a high urinary ph can cause the formation of calcium oxalate or calcium phosphate crystals, while a low urinary ph promotes uric acid crystallization [ ] . urinary ph can be modified by uropathogenic bacteria. urease-expressing bacteria generate nh , which can substantially raise urinary ph, promoting deposition of struvite and apatite crystals [ ] . besides the consequences of stone formation in the urinary tract, these deposits can cause the encrustation and blockage of indwelling catheters [ ] . it is interesting to recall that the pathogenic action of another bacterium, h.pylori, in the stomach also depends on urease action (see section . ). nephron function. many studies point to the value of correcting mac for preserving the function of the failing kidney and slowing ckd progression [ ] [ ] [ ] . we outlined corrective strategies based around alkali therapy in section . . ), but there is an additional prophylactic value in emergency settings. nahco infusion is protective against the kidney damage that can result from traumatic rhabdomyolysis (due to a crush injury), preventing development of mac and tempering the renal toxicity of myoglobin [ , ] . another consideration related to therapies is that a number of drugs cause metabolic acid-base disturbances because they are nephrotoxic [ ] or incidentally interfere with the kidneys ability to excrete acid [ ] . for example, ca inhibitors such as acz that are used as diuretics, due to their ability to interfere with fluid reabsorption, also cause mac [ ] . on the other hand, loop diuretics use can cause malk [ ] . another example are penicillin antibiotics which, acting as significant non-reabsorbed anions in the collecting duct lumen, promote hypersecretion of k + and h + , resulting in hypokalemia and malk [ , ] . finally in this section, the ability of cf kidneys to secrete excess hco is restored by treatment with the cftr restoring drug cocktail lumacaftor/ivacaftor (orkambi®) [ ] . stone formation. both citrate and low ph discourage the formation of calcium precipitates [ ] . thus, ingesting lemon juice, which raises urinary citrate while lowering urinary ph, decreases the propensity to form kidney stones and catheter-blocking deposits [ ] . dietary supplementation with citrate salts is also effective for this purpose because, despite resulting in a rise in urinary ph, the accompanying rise in urinary citrate increases the ph of crystal nucleation to an even higher value [ , ] . urinary ph can also exert a meaningful influence on drug excretion as discussed later (see section . ). at this point in our review we have presented ample evidence that abts are necessary to sustain life, and now we will see that they are also necessary to create new life. in the male reproductive tract, h + secretion by clear cells in the tail of the epididymis is required to maintain an acidic luminal ph for storage of sperm [ ] . hco secretion along the length of epididymis is necessary to functionally activate sperm before ejaculation [ ] and prevent their inactivation by the acidic vaginal environment (discussed later). indeed low levels of hco are associated with lowered sperm motility [ ] . in the female reproductive tract, endometrial epithelial cells further secrete a hco --rich fluid that is necessary for sperm capacitation and fertilization [ ] . furthermore, the secretory phase of the uterine cycle is associated with a dramatic rise in the ph of the oviduct lumen, corresponding with a level of hco that is sufficient to promote thinning of mucus during ovulation to promote sperm mobility [ ] and to promote dispersal of the egg-surrounding corona cells to allow the sperm access for fertilization [ ] . ultimately hco is even a prerequisite for the acrosome reaction [ ] , by virtue of its ability to stimulate soluble adenylyl cyclase to produce camp and initiate requisite signaling cascades [ ] . finally, once fertilization has occurred, acidification of uterine fluid is a necessary prerequisite for embryo implantation [ ] . numerous abts are involved in these processes; for example, loss of ae , slc a , cftr, or nhe are all associated with infertility or reduced fertility in male mice [ , [ ] [ ] [ ] . the acidic ph of the vagina noted earlier is caused by the metabolic activity of lactobacilli and serves to defend against sexually transmitted disease pathogens. loss of the acidity in bacterial vaginosis is associated with increased susceptibility to std infection [ ] . with regard to ultimate reproductive success, maternal-fetal acid-base balance is an important determinant of perinatal outcomes [ ] . for example, obstructed labor has poor outcomes due to intermittent hypoxia and lactic acidosis [ ] . because vaginal acidity tends to dampen sperm motility, vaginal douching with nahco improves fertility [ , ] . conversely, vaginal acidification is contraceptive and prophylactic. the spermicidal properties of lemon juice have long been appreciated [ ] . phexxi™ (formerly known as acidform: [ ] ), is the most recent of a series of acidic contraceptive gels. phexxi™ is a vaginally-applied gel of lactic acid, citric acid, and potassium bitartrate that is indicated by the fda to prevent pregnancy [ ] . an alternate approach 'buffergel' utilizes an acidic polymer for the same purpose [ ] . by lowering vaginal ph these products also confer microbicidal benefits [ ] . finally, some abt-targeted drugs interfere with fertility: ca inhibitors, for example, prevent dispersal of corona cells [ ] and ppis inhibit sperm motility [ ] . finally, with regard to childbirth, peri-operative nahco infusion has been proposed as a possible measure to improve outcomes in obstructed labor [ ] . many metabolic reactions that consume or generate acids and bases can, in disease, result in mac. the liver makes important contributions to acid-base balance by consuming lactate (countering lactic acidosis), generating albumin (a weak acid: hypoalbuminemia is associated with malk) and producing keto acids (contributing to diabetic ketoacidosis) [ ] . furthermore, acidbase status impacts the activity of the enzymes that constitute several key metabolic pathways. for example, acidosis inhibits glycolysis [ ] and lipolysis [ ] but stimulates gluconeogenesis [ , ] . the ability of disturbed acid-base balance to interfere with glycemic control is further evidenced by the following observations: (i) acidosis and alkalosis both lower glucose-stimulated insulin release from pancreatic islets [ ] ; (ii) the hco content of plasma correlates with insulin solubility [ ] , (iii) acidosis is associated with decreased insulin sensitivity [ , ] . this latter observation is due in part to the ph-sensitivity of the interaction between insulin and its receptor: a bell-shaped ph-dependence that exhibits strongest binding at ph ~ . [ ] . in fact, insulin can also influence abt action. for example, insulin promotes renal nbce activity. in type diabetes the resulting pathological increase in renal fluid absorption caused by nbce upregulation is thought to contribute to hypertension [ ] . other hormones that influence acid-base balance include secretin (increases pancreatic hco secretion in response to duodenal acidity [ ] ), angiotensin ii and aldosterone (increases renal acid excretion in acidosis [ , ] ), and parathyroid hormone (increases renal acid excretion and excretion of urinary-buffer phosphate in acidosis: [ ] ). interestingly, licorice can cause malk because one of its constituent compounds (glycyrrhizic acid) indirectly causes overstimulation of the aldosterone receptor [ ] . hormones whose levels are pathologically altered in acidosis include aldosterone and endothelin (increased: [ , ] ) cortisone (increased: [ ] ), and igf- (decreased: [ ] ). metabolic reprogramming of cancer cells in the hypoxic and acidotic tumor environment is discussed further in a later section (see . oncology.) the influence and therapeutic relevance of ph upon endocrine and metabolic aspects of heart function, muscle mass, and bone growth have been discussed in earlier sections ( . , . , and . ). the link between dietary acid load and development of insulin sensitivity has made dietary control an appealing target for lowering the incidence of type diabetes, although overwhelming evidence of efficacy is currently lacking [ ] . because of the ph-dependence of insulin solubility, the dissolution time of administered insulin is slower in plasma from diabetics with dka than in otherwise normal plasma, suggesting at face value that combined insulin-bicarbonate therapy might be valuable [ ] . however, such treatment in practice may be of limited value and has been linked to development of cerebral edema in children [ ] . the influence of ph on drug solubility is discussed in more detail in section . . ph plays a role in all aspects of the immune response. first we will enumerate the subcellular effects: (i) the cytosolic alkalinization that promotes cytoskeletal rearrangement during neutrophil spreading (the morphological change that is important for capillary adhesion and extravasation) requires nhe action [ ] . (ii) inflammatory sites are usually acidic due to the metabolic activities of invading bacteria and neutrophils, an environment that promotes the production of proinflammatory cytokines [ ] (see also section . ). (iii) during bacterial killing, h + efflux into the phagosome is necessary for charge compensation during the respiratory burst that produces cytotoxic superoxide anions. this action is mediated by the voltage-gated h + channel hv [ ] . (iv) some of the generated superoxide is converted into cytotoxic hypochlorous acid (hocl) which is able to diffuse back into the neutrophil cytoplasm. thus the action of phagosomal hv , together with the action of nhe in the plasma membrane, defends the neutrophil cytoplasm from acidosis which would otherwise dampen nadph oxidase activity [ , , ] . secondly we can consider the effect of acid-base disturbance on the whole cell. although the ph sensitivity of individual immune cell types are well characterized in vitro (e.g., acidosis decreases leukocyte and neutrophil mobility [ ] , promotes complement activation [ ] , modulates expression of inflammatory mediators [ ] ), there are many subtleties to these effects. for example the type of acidosis, type of cell, activation state of the cell, and effects on phagocytic activity versus migration may all influence the effect of acid-base disturbance on the immune response mediated by a given cell type [ ] . thus, for example, acidosis enhances bacterial killing by neutrophils [ ] and leukocytes [ ] but not by macrophages [ ] . although it is complicated to tease out a set of concerted mechanisms, in general, systemic acidosis is associated with compromised immune function [ , ] . [ ] . the link between ph and disturbed immune response is demonstrated by the following example. single nucleotide polymorphisms in the acid-loading protein ae are linked to progression of primary biliary cholangitis. a mechanism is suggested by studies of ae -null mice in which loss of ae from cytotoxic t-cells causes a phi increase that promotes their proliferation, activation and survival, amplifying the autoimmune response against damaged liver cells. furthermore, type iv drta is linked to systemic lupus erythematosus, although the causal relationship is unclear [ , ] . several paradigms have been proposed to suppress a pathological immune response. oral nahco dosing in rats stimulates an anti-inflammatory response; this effect could be harnessed to prevent tissue damage in autoimmune diseases such as rheumatoid arthritis [ ] . one preliminary study in mice even suggests that oral nahco dosing could be useful to suppress peanut allergy [ ] . regarding the pharmacological aspect, mct inhibitors act as immunosuppressors by interfering with the disposal of lactate during t-cell activation [ ] . finally, the nhe blocker cariporide suppresses the systemic immune response to burn injury in rats, although the mechanism is unclear [ ] . on the other hand, knowledge of acid-base balance can be exploited to promote an immune response. it has been suggested that blocking ae to promote a stronger cytotoxic t-cell response could be useful for treatment of chronic infections [ ] . considerations about the role of ph in cancer immunotherapy are included in section . . neuronal activity is ph dependent (see section . ) and there is emerging evidence that agents of acid-base balance can influence behavior and progression of neuropsychiatric disorders. inhalation of co (despite its general dampening action on neuronal excitability, see section . . ) invokes anxiety and panic, and the influence of co is exacerbated in individuals with panic disorders [ ] . studies in mice indicate that lowering brain ph triggers the action of the acidsensing ion channel asic a in the regions of the brain responsible for stress, fear, and social behavior [ , ] . the acquisition of fear-related freezing behavior in mice is enhanced by asic a overexpression [ ] and dampened by asic a disruption [ ] . in humans, decreased brain ph is also associated with schizophrenia, bipolar disorder, and autism spectrum disorder [ ] . one study even suggests that a high dietary acid load correlates with incidence of emotional problems and hyperactivity in young children, but causality could not be established as could not be discounted that behavior influences dietary habits [ ] . in terms of the linkage between abts and neuropsychiatric disorders, slc a is a biomarker (both in terms of incidence of a specific single nucleotide polymorphism and in terms of reduced expression determined by microarray) of suicide ideation and completion, especially with bipolar disorder [ ] . although the mechanistic details of the linkage are unknown, the role of nbce in control of brain ph is likely to be relevant. perhaps also of tangential relevance, due to its impact upon systemic acid-base balance [ ] , is that estimated glomerular filtration rate in ckd, which typically correlates with ability to excrete acid, is inversely correlated to depressive symptoms and suicide ideation [ ] . the anxiety response to co inhalation is a useful clinical test to follow the effectiveness of treatments for panic disorders [ ] . the response itself can be quelled by acz [ ] but, as this is just a model of panic disorder, the use of ca blockers to treat actual panic disorders is unclear. the above-mentioned studies of asic a-null mice suggest that asic a inhibition could be therapeutic for panic disorder. regarding the link between slc a and suicide, it is unclear how modulating nbce activity might influence depression, although detection of the biomarker could help to identify high risk individuals and thereby inform therapeutic strategies [ ] . finally, it has been suggested that antipsychotic medications could contribute to lactic acidosis and be partly responsible for decreased ph in the brains of individuals with schizophrenia [ ] . as mentioned previously, plasma ph depends on adequate ventilation to exhaust co thus mechanical ventilation can induce respiratory acid-base disturbances. another important aspect for consideration in this section is that the bioavailability of anesthetic agents can be influenced by acid-base status. when using mechanical ventilation, two important considerations, related to acid-base balance, must be raised. the first consideration relates to low-flow anesthesia or closed-circuit rebreathing systems that return exhaled anesthetic gas mixtures. in these systems co must be removed from the recirculated air. for this purpose, co scrubbers are used to adsorb co . the archetypal scrubber is soda lime (a mixture of naoh and ca(oh) , which reacts with carbonic acid to form an insoluble caco precipitate), although a number of other technologies are available [ , ] (see also section . . ). the choice of technology can be important as many co scrubbers can have undesirable reactions with anesthetic gases, producing toxins such as carbon monoxide [ ] . the second consideration is for individuals in which low-tidal-volume ventilation is indicated, such as those with acute respiratory distress syndrome or copd. these individuals may be deliberately under-ventilated to prevent mechanical stress on the lungs. thus, patients are maintained in a state of compensated rac called "permissive hypercapnia". under some circumstances, such as in critically ill patients, this hypercapnic state may be protective due to its anti-inflammatory influence [ ] (see also the immune system). acid-base balance concerns are not limited to inhaled anesthetics. prolonged infusion with the intravenously administered anesthetic propofol can cause severe lactic acidosis [ ] . the influence of ph on the pharmacokinetics of drugs in general is discussed in section . perioperative interventions have the potential to disturb acid-base homeostasis with negative consequences for outcome. post-operative metabolic acidosis is a well described but complex phenomenon related to issues including lactate accumulation in poorly perfused tissues and hyperchloremic acidosis due to dilution/displacement of hco --containing plasma by infused saline [ ] [ ] [ ] [ ] . pre-operative acidosis has also been described and has been linked to stress and fasting [ ] . post-operative malk has also been described following general surgery and has been linked to the infusion of citrate-buffered plasma [ , ] . peri-operative malk is linked to the removal of stomach acid by nasogastric suction. poor outcomes have been associated with both post-operative mac [ ] and malk [ ] , although this is not a universal finding [ ] . on a related theme, the usefulness of stored blood for transfusion can be compromised by numerous storage lesions including low ph that follows anaerobic metabolism by stored cells [ , ] . finally, an acidic tissue environment appears to favor natural wound healing, yet alkaline ph favors the success of skin grafts [ ] . altering the chemistry of infused fluids is the obvious strategy to counter post-operative acid-base disturbances. with specific regard to post-operative mac, dichloroacetate treatment tempered the pathological rise in lactate following liver transplantation, although no effect on outcome was observed [ ] . other treatments for mac are discussed in section . . . treatment for malk are discussed in section . . . finally, a study in mice suggests that raising the ph of stored blood enhances red blood survival after transfusion [ ] . the ph-dependence of wound healing suggests that therapeutic maintenance of the ph of the wound or graft could aid healing [ ] . hco is a major buffer in stimulated saliva [ ] , defending oral ph against acidic foods and drinks and against those acids produced from sugars by acidophilic bacteria in the oral cavity. acid defense protects enamel from erosion and oral ph is also an important determinant of a healthy oral microbiome; low salivary flow and ph generally encourage the presence of pathogenic and cariogenic bacteria (e.g., [ , ] ). as a consequence, low salivary ph, [hco -], and/or buffer capacity are predictors of cavity formation [ ] [ ] [ ] . salivary ph is lowered in many groups of individuals such as patients undergoing chemotherapy for head/neck cancer [ ] , cocaine users [ ] , or tobacco smokers [ ] . low salivary ph is also described in individuals with diseases such as cf [ ] , sjögren's syndrome [ ] , and juvenile idiopathic arthritis [ ] . not all studies report a major impact on dental health in these cases as compensating factors may be in play [ ] . abts and cas play important roles in salivary secretion and well as in enamel formation [ , ] . for example, defective dentition is a feature of some individuals with nbce mutations [ ] and appears not to be a secondary consequence of acidemia [ ] . salivary ph is a useful biomarker for oral health [ ] and the usefulness of baking soda in oral hygiene was first suggested as long ago as [ ] . nahco delivery either as a mouth wash [ ] , mucoadhesive spray [ ] or sugar-free gum [ ] raises salivary ph and in some cases may lower colonization of acidophilic bacteria. in individuals undergoing chemotherapy for leukemia, use of a nahco mouthwash lowered susceptibility to mouth ulcers [ ] . in smokers the similar treatment reduced levels of the inflammatory biomarker il- β [ ] . nahco containing dentifrices have been shown to be effective at neutralizing plaque ph [ ] , enhancing plaque removal [ ] , and inhibiting formation of caries [ ] . ph-stabilizing resins used in restorative dentistry have also been suggested to be useful cariostatic by releasing oh - [ ] . finally, reducing dietary intake of sugars is also beneficial to oral ph because it limits the acidification that can be caused by acidophilic bacteria. hence sugar-free gum is effective at raising plaque ph [ ] . we have dealt with various aspects of acid-base-related medical microbiology in earlier sections (see sections on the respiratory system, the upper digestive system, the lower digestive system, the reproductive system, the immune system and oral health). in this section we will confine our considerations to viruses and parasites, using influenza and malaria as examples. . both of these mechanisms are necessary to support viral replication. in defending phi against the increased acid load, infected cells extrude h + , creating a concomitant acidification of phe at the cell surface [ ] . there are multiple acid-base-related aspects to the malarial lifecycle and its pathological impact. first of all, mosquitos are attracted by co and co sensitizes them to human odors [ ] . secondly, the erythrocytic phase of malaria infection requires that plasmodium invades red blood cells. one of the surface antigens that plasmodium exploits for host-cell recognition and invasion is the ae -glycophorin a complex. consequently, ae -null mice are immune to infection [ ] as are individuals with an ae defect that causes the abnormal red cell morphology (south east asian ovalocytosis, sao) [ ] . finally, malarial infection causes numerous metabolic disturbances, such as mac that, in part, follows the tissue hypoxia and hyperlactemia caused by blocked microvasculature. mac is a strong prognosticator of fatal outcome in infected individuals [ , ] . influenza. m h + -channel blockers have potential to target influenza strains that are resistant to currently available antiviral treatments [ ] . on the other hand, the use of ppis may paradoxically increase susceptibility to viral infection in the gastrointestinal tract by neutralizing the stomach acidity that typically destroys viral particles [ ] . lactic acidosis in malaria can be ameliorated by dichloroacetate treatment [ ] . malarial resistance in individuals with sao suggests that transfusions with sao blood may be a useful therapy for individuals infected with drug-resistant plasmodium [ ] . finally, the parasite's own abts may be a target for antimalarial action [ ] . the rapid proliferation of cancerous cells is associated with the warburg effect (also known as 'aerobic glycolysis'): a shift from aerobic to anaerobic metabolism even in the presence of oxygen [ , ] . in this state, cells increase their atp production by increasing their glucose uptake with the consequence of increased lactate and h + production. because of their high metabolic rates, such cells would tend to have a much lower phi than normal cells. however, cancer cells are able to maintain near-normal phi by upregulating acid-extruding abts such as nbce , nbcn , nhe , h + -atpase, and mct to facilitate the removal of h + , as well as extracellular cas (caix, caxii) to facilitate the removal of co [ ] [ ] [ ] [ ] [ ] . aquaporins (aqps)-which can serve as a conduit for transmembrane movements of co [ ] -also promote tumor growth and survival, but it is not clear that promotion of co removal is a major part of their pathological importance [ , , ] . as shown in cancer cell-lines [ ] , acidosis can also increase the drive on the tca cycle (promoting atp production for h + extrusion) and the pentose phosphate pathway (promoting nadph production to counter ros, promoting cell survival) [ ] . the combined action of these processes results in a drastic reduction in local phe. these changes allow cancer cells both to outcompete neighboring non-cancer cells and to mobilize and spread to other parts of the body. metastasis and tumor survival are further enhanced by acid-dependent remodeling of the extracellular matrix [ ] and suppression of anti-tumor immune responses [ ] (see also the immune system). it is noteworthy that a high level of net endogenous acid production was associated with higher mortality in breast-cancer recurrence in a cohort of early stage breast cancer survivors [ ] . moreover, acid-treatment of melanoma cells selects for more invasive phenotypes [ ] and the extent of upregulation of various abts and cas can be an adverse prognostic factor (e.g., [ ] [ ] [ ] ). the importance of acid-base balance in cancer has suggested that targeting tumor ph could be a valuable adjuvant therapy. the three main therapeutic modalities are (i) interfering with the ability of cancer cells to defend their phi, (ii) interfering with the ability of cancer cells to create an acidic extracellular environment, and (iii) bolstering the immune response in an acidic tumor microenvironment. interfering with phi defense. blocking the defense of phi by cancer cells can be achieved by inhibiting acid-extruding abts and/or cas [ , ] . the value of these approaches is demonstrated in diverse studies that report anything from delayed tumor-growth in nbcn -null mice [ ] to positive clinical outcomes with adjuvant use of proton pump inhibitors [ ] . however, reports from clinical trials are currently sparse. given the potential for side effects due to the physiological importance of these proteins in all organ systems, much attention has focused on inhibiting those proteins, such as caix, which are specifically upregulated in cancer cells (in response to hypoxia) and not highly expressed elsewhere [ ] . another approach, the combinatorial use of blockers, allows for a lower dose of each and thus fewer undesired effects on non-tumor cells. the use of a combination of five compounds, each of which targets a different abt/ca, revealed effectiveness at reducing intracellular brain tumor acidification in mice and the consequent activation of the pro-apoptotic marker caspase- in tumor cells, with little negative effect on non-tumor cells [ ] . interestingly, the potency of such approaches is enhanced by glucose loading, which increases the acid-load in these glycolytic tumor cells [ ] . finally, as different abts/cas are upregulated in different tumor types, generic approaches are also valuable to consider. for example, a number of anticancer drugs, such as salinomycin, that are not known to specifically target abts or cas, also promote cellular acidification [ ] . interfering with extracellular acidification. anecdotal evidence suggests that interfering with extracellular acidification might be achieved by dietary means [ ] . indeed, oral nahco supplementation can raise tumor phe and inhibit metastasis in mice [ ] , but the overall consequence is complex, with one recent study suggesting that such therapy may confoundingly promote tumor proliferation [ ] . however, the addition of adjuvant nahco to a chemotherapeutic agent that was fed directly into a hepatic tumor via catherization of tumorfeeding arteries (tila-tace: targeting intra-tumoral lactic acidosis with trans-arterial chemoembolization) was associated with markedly improved outcomes [ ] . in short, there is currently insufficient data to show that lowering dietary acid load improves outcomes, except by virtue of the association of such diets with general well-being [ ] and the role of alkalinization in enhancing the safety of certain chemotherapeutic drugs ( [ ] ). the acidic tumor microenvironment can also be exploited to promote local drug delivery as discussed in section . . bolstering antitumor immune response. the acidic tumor microenvironment promotes an antiinflammatory t-cell response (see section . . ), but the deletion of ae promotes a proinflammatory t-cell response (see section . . ). therefore inhibition of t-cell ae could be a valuable paradigm for enhancing a cytotoxic immune response [ ] . ph-dependent aspects of pharmaceutical therapy in this section, we will focus on the influence of ph upon pharmacokinetic properties of drugs. we will also consider how all of these ph-related properties can be harnessed to therapeutic advantage, particularly in diseases associated with acid-base imbalance. oral drug delivery is the most common and convenient method used to administer drugs whereby they can either directly access their targets or be absorbed into circulation to reach their intended targets. thus, oral drug delivery will serve as our paradigm for discussion. however, these concepts are also relevant to other, parenteral, methods of drug delivery such as inhalation of nebulized substances and injection into subcutaneous, intramuscular, or intravenous compartments. gastrointestinal (gi) ph is one of the major determinants of oral drug bioavailability as it affects various properties including solubility and dissolution rate, stability, and absorbability. ph varies drastically along the gi tract [ ] [ ] [ ] . starting from a value of ~ . in the stomach, it rises to ~ . in the duodenum, reaches ~ . in the terminal ileum, and falls again to ~ . in the colon [ ] . an additional consideration is that these values can vary with age, presence of food, diseases, and also by the co-administration of other drugs [ ] [ ] [ ] [ ] . finally, we note that the influence of ph on bioavailability could, in ph-disturbed states, result in underdose or overdose. most drugs are weak acids and bases. the relationship between the drug ionization constant (pka) and the ph of the environment to which the drug is exposed (hereafter referred to as the environmental ph) is a critical determinant of drug solubility and dissolution rate in aqueous compartments [ ] . this relationship determines the ratio of the concentration of the unionized (i.e., uncharged) form (u) to the concentration of the ionized (i.e., charged) form (i) of the drug and is described by the henderson-hasselbalch equation. now, according to equation ( ), . if ph<pka (i.e., ph−pka < ), the ionized form i (i.e., the acidic conjugate weak base bh + ) prevails; . if ph>pka (i.e., pka−ph > ) , the unionized form u (i.e., the basic neutral for b) prevails. thus, because the ionized form i is more water-soluble than the unionized form (due to better solvation between the ionized form i and the dipole of the water molecule), weakly acidic drugs have higher solubility at high ph whereas weakly basic drugs have higher solubility at low ph. this means that weakly acidic drugs tend to dissolve more easily in the intestine whereas weakly basic drugs tend to dissolve more easily in the stomach. because drug solubility is ph-dependent, the dissolution profile of the drug (i.e., the process by which a solid drug dissolves into solution) may also be ph-dependent [ , ] . drug solubility and dissolution may be enhanced by several techniques including (i) chemical derivatization to alter drug pka, (ii) administration of the drug in ionic form (as a salt) rather than as a free acid or base, or (iii) alteration of environmental ph by the adjuvant use of acids or bases to better match the drug's pka [ ] [ ] [ ] . in some cases, it may be desirable to lower the solubility and dissolution of parenterally-administered drugs in order to prolong their half-life. as we discuss in the next section, the influence of drug pka and environmental ph on drug ionization has important ramifications for drug absorption and distribution. besides solubility in aqueous solvent, the ionization state of a drug can influence its solubility in the lipid phase (e.g., drug pka can be modified to increase drug polarity and therefore reduce drug lipophilicity) and therefore affect its ability to permeate cell membranes [ , ] . assuming that passive diffusion (i.e., transmembrane concentration gradient is the driving force) is the mechanism by which a drug moves across a membrane and that only uncharged ions can freely diffuse across the membrane, we can turn again to the henderson-hasselbalch equation to describe the importance of drug pka and environmental ph to drug absorption and distribution between body compartments. we note that, according to point ) above, the henderson-hasselbalch equation predicts that weak acids tend to be absorbed in an acidic environment (e.g., in the stomach). similarly, according to point ) above, the henderson-hasselbalch equation predicts that weak bases tend to be absorbed in a basic environment (e.g., in the small intestine). in , shore and coworkers proposed the ph partition hypothesis (first described by jacobs in [ ] ) to describe the influence of pka and ph upon the gastric secretion of a variety of intravenously-administered weakly acidic and basic drugs [ ] . the results of their experiments could be explained with their theoretical model of drug absorption across a lipoid barrier (i.e., the gastric mucosa) that separates two compartments (i.e., the stomach lumen and the blood) with different ph and permeable only to the unionized form, which was assumed at equilibrium. they found that the extent to which a drug moves between compartments (the gi tract and blood) depends on the value of the drug pka and the environmental ph values of the two compartments such that (using equations ( ) and ( ) that is to say that weakly acidic drugs such as aspirin (pka= . ) can be effectively absorbed from the stomach. in the case of a weakly basic drug. in practice, the model has several limitations because the ph partition hypothesis ignores other substantial influences upon drug absorption. for example, the assumption of equilibrium is unrealistic in such a dynamic system. furthermore, even weakly acidic drugs can be substantially absorbed in the small intestine because of the large luminal surface area that it presents [ ] . finally, the ph-partition hypothesis does not consider the mechanism by which drugs can move across epithelial layers. today we know that both the transcellular and paracellular pathways play important roles in the absorption and elimination of both charged and uncharged drug forms [ ] . low-specificity membrane transporter proteins that contribute to transcellular drug transport around the body include organic anion transporters (oats), organic cation transporters (octs), some mcts, and members of the abc transporter superfamily, such as the p-glycoprotein transporters (p-gp) [ ] [ ] [ ] [ ] [ ] . in some cases, the transporters themselves may be ph-sensitive or coupled to the transport of acids and bases. the importance of such considerations is exemplified by lowered absorption of weakly basic drugs in individuals with an unusually high stomach ph such as those taking ppis or individuals with achlorhydria [ ] . for example, the dissolution and absorption rate of the weakly basic antifungal agent ketoconazole, which is soluble only at ph lower than , can be enhanced by coadministration of an acidic, carbonated beverage [ ] . a related example, albeit related to absorption of drugs by bacteria, is that the adjuvant use of nahco enhances the in vitro potency of antibiotics by interfering with the proton motive force that drives antibiotic efflux from bacteria [ ] . as we will see in section . , considerations of drug solubility and transepithelial movement also influence drug elimination in urine by the kidneys. when developing drugs for oral delivery it is important to account for the adverse acidic environment of the stomach, which can cause instability and rapid degradation of drugs before they reach the small intestine for absorption. this could happen because the polymers used for the tablet coating may be susceptible to ph. for this reason, carriers for oral drug delivery are tested for ph-sensitivity and endurance in acidic environment. acid-resistant polymers that only dissolve above certain ph values are sometimes used as enteric or gastro-resistant coatings [ ] [ ] [ ] [ ] . this is the case, for example, for oral delivery of insulin [ , ] . the ph partition hypothesis provides the theoretical framework for understanding how urinary ph influences renal drug excretion; acidification of urine favors elimination of weakly basic drugs (because their absorption is reduced) whereas alkalinization of urine favors elimination of weakly acidic drugs. for example, urine acidification via administration of ammonium chloride increases elimination of the weakly basic drug amphetamine [ ] , whereas urine alkalinization via intravenous administration of nahco enhances elimination of acidic drugs like salicylic acid (i.e., aspirin) and can be helpful in the management of drug poisoning like aspirin intoxication [ ] [ ] [ ] . as we considered earlier, partitioning is just one aspect of drug distribution. many oats and octs [ ] are expressed in nephron epithelia where their action is vital for delivering drugs from the peritubular capillaries into the nephron lumen for excretion in urine and for delivering diuretics (e.g., furosemide) to their therapeutic targets in the nephron lumen. the direct secretion of these drugs into the nephron lumen is necessary because many such drugs are substantially bound to albumin in circulation and are not effectively filtered into the nephron lumen at the glomerulus. . exploiting ph for targeted drug delivery acidity can be harnessed to target drug release to acidic environments such as the stomach or pathological acidotic microenvironments such as tumors. one example under development is a gastro-floating matrix tablet that contains adjuvant nahco with the drug, produces co upon reaction with gastric acid causing the dosage form to remain buoyant in the stomach for prolonged release [ ] . another example is the use of ph-sensitive vehicles such as micelles that could release cytotoxic chemotherapeutic agents only in the acidic tumor environment [ ] . similarly, as suggested by the disparity between the usefulness of an anticancer drug that was identified in an in vitro screening performed at neutral ph and its value in vivo in the acidic tumor microenvironment [ ] , it is possible that some drugs may be inactive in circulation and may not be activated until they reach an acidic environment. abts and cas play major roles in a variety of pathologies and provide an array of potential therapeutic targets, but many are currently lacking specific or safe drugs. the application of epigenetic modulators and other genetic tools to alter their expression is an underexplored area of research. it is interesting to note that, among the many treatment paradigms for diverse acid disturbances, nahco administration is a common thread. its low cost and ready availability have prompted its nickname "enemy of the pharmaceutical industry." however, despite the promise of numerous limited trials, robust evidence in favor of its broad effectiveness in many fields is currently lacking. this is perhaps due to a lack of appreciation of subgroup effects or a lack of standardization among trails. nonetheless, research into the therapeutic importance of balancing ph remains robust and promises the delivery of many more effective treatments in the coming years. figure . targeting carbonic anhydrase to treat glaucoma. glaucoma is retinal degeneration caused by increased intralocular pressure. eye drops containing ca-inhibitors such as acetazolamide (acz) target cas in the ciliary body and reduce the production of aqueous humor, lowering intraocular pressure. the ciliary body is a complex epithelial tissue comprised of two cell layers joined by gap junctions. a variety of abts and other transporters are required to move nacl, which is followed by water, from the interstitial fluid into the anterior chamber of the eye. figure . enhancing fluid secretion in the lungs. cftr promotes the movement of hco -containing fluid onto the airway surface to promote mucociliary clearance and lung health. drugs such as lumacaftor/ivacaftor rescue this function in some individuals with cf by helping misfolded cftr molecules to function normally. alternative ph-based strategies have been suggested as adjunct cf therapy, such as blockade of the many h + secreting abts. figure . the bohr effect. the action of ae and cas promote o release in systemic capillaries (panel a) and co release in pulmonary capillaries because of the ph-dependence of the affinity of hemoglobin for o (the bohr effect). figure . the role of abts and cas in bone remodeling. osteoclasts secrete acid onto the bone surface to resorb minerals during bone growth/remodeling and in response to hormonal requirements for release of mineralized ca + and phosphate. one report suggested an off-target bone-sparing effect of ca inhibitors, used to treat glaucoma, in a group of post-menopausal women. figure . targeting the stomach h + /k + -atpase to treat acid-reflux disease. proton pump inhibitors are widely used to reduce gastric acid secretion, as an alternative or adjunct strategy to neutralizing stomach acid with an antacid such as caco . figure . targeting intestinal abts to treat constipation. intestinal fluid absorption is promoted by the combined action of nhe and slc a , which perform the net uptake of nacl, and therefore water. inhibition of either, to reduce fluid absorption from the intestinal lumen is a useful therapy for irritable bowel syndrome with constipation. ivacaftor is similarly useful in cf by restoring intestinal fluid secretion. figure . the role of abts and cas in cancer. numerous acid-base handling proteins are upregulated in rapidly proliferating tumor calls to help them dispose of metabolic acids and create an acidic microenvironment that disadvantages non-tumor cells in their vicinity. as most of these abts and cas are gainfully expressed elsewhere in the body, therapies in development are focused on blocking those rarer targets that are preferentially expressed in the hypoxic tumor environment such as mct and caix. other approached include exploiting the acidic milieu of the tumor for the targeted delivery of chemotherapeutic drugs (see section . ). figure . the influence of ph on drug distribution. theoretical distribution of a hypothetical weakly acidic drug (pka = . , panel 'a') and a hypothetical weakly basic drug (pka = . , panel 'b') between two aqueous compartments with different ph (gi tract at ph = . and blood at ph = . ). assuming that only the unionized form u (ha in panel 'a' and b in panel 'b') can cross the membrane and that u is equilibrated across the plasma membrane, panel 'a' shows that a weakly acidic drug is more concentrated in the alkaline compartment. this result suggests that weakly acidic drugs tend to be absorbed from the more acidic compartment to the more basic compartment (blue arrows). panel 'b' shows that a weakly basic drug is more concentrated in the acidic compartment, indicating that weakly basic drugs are poorly absorbed in an acidic compartment (blue arrows). weakly basic drugs are in fact poorly absorbed from the stomach. 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neutrophils: role of fcgamma receptors and tyrosine kinases science review: extracellular acidosis and the immune response: clinical and physiologic implications the effect of ph and nucleophiles on complement activation by human proximal tubular epithelial cells unravelling the interplay between extracellular acidosis and immune cells acute asphyxia affects neutrophil number and function in the rat ph changes observed in the inflamed gingival crevice modulate human polymorphonuclear leukocyte activation in vitro acidosis differently modulates the inflammatory program in monocytes and macrophages the effects of extracellular ph on immune function anion exchanger is critical for cd (+) t cells to maintain phi homeostasis and modulate immune responses systemic lupus erythematosus associated with type renal tubular acidosis: a case report and review of the literature systemic lupus erythematosus with distal renal tubular acidosis presenting as hypokalemic paralysis with respiratory failure oral nahco activates a splenic anti-inflammatory pathway: evidence that cholinergic signals are transmitted via mesothelial cells sodium bicarbonate facilitates low-dose oral tolerance to peanut in mice monocarboxylate transporter mct is a target for immunosuppression inhibition of na+/h+ exchanger by cariporide alleviates burn-induced multiple organ injury a. van den hout, co vulnerability in panic disorder overshadowed by the amygdala: the bed nucleus of the stria terminalis emerges as key to psychiatric disorders acidsensing ion channel is localized in brain regions with high synaptic density and contributes to fear conditioning overexpression of acid-sensing ion channel a in transgenic mice increases acquired fear-related behavior decreased brain ph as a shared endophenotype of psychiatric disorders dietary acid load and mental health outcomes in children and adolescents: results from the giniplus and lisa birth cohort studies understanding and predicting suicidality using a combined genomic and clinical risk assessment approach metabolic acidosis of ckd: an update prevalence of depression and suicidal ideation increases proportionally with renal function decline, beginning from early stages of chronic kidney disease carbon dioxide test as an additional clinical measure of treatment response in panic disorder responses to hypercarbia induced by acetazolamide in panic disorder patients increased lactate levels and reduced ph in postmortem brains of schizophrenics: medication confounds possible alternatives to soda lime absorption of carbon dioxide interaction of inhalational anaesthetics with co absorbents hypercapnia and acidosis in sepsis: a double-edged sword? propofol infusion associated metabolic acidosis in patients undergoing neurosurgical anesthesia: a retrospective study perioperative metabolic acidosis: the bradford anaesthetic department acidosis study chasing the base deficit: hyperchloraemic acidosis following . % saline fluid resuscitation factors related to postoperative metabolic acidosis following major abdominal surgery cause of metabolic acidosis in prolonged surgery postoperative metabolic alkalosis following general surgery: its incidence and possible etiology citrate metabolism and its complications in non-massive blood transfusions: association with decompensated metabolic alkalosis+respiratory acidosis and serum electrolyte levels outcome of surgical patients who present acidosis postoperatively biochemical changes in stored donor units: implications on the efficacy of blood transfusion clinical impact of blood storage lesions influence of ph on wound-healing: a new perspective for wound-therapy? dichloroacetate stabilizes the intraoperative acid-base balance during liver transplantation ph modulation ameliorates the red blood cell storage lesion in a murine model of transfusion an analysis of the buffer systems in saliva change of saliva composition with radiotherapy effect of decreased salivation and ph on the adherence of klebsiella species to human buccal epithelial cells evaluation of non-microbial salivary caries activity parameters and salivary biochemical indicators in predicting dental caries salivary parameters and oral health status amongst adolescents in mexico salivary flow rate, ph, buffering capacity, total protein, oxidative stress and antioxidant capacity in children with and without dental caries salivary buffer capacity, ph, and stimulated flow rate of crack cocaine users effect of sodium bicarbonate mouth wash on salivary ph and interleukin- β levels among smokers salivary biomarkers and oral microbial load in relation to the dental status of adults with cystic fibrosis salivary changes and dental caries as potential oral markers of autoimmune salivary gland dysfunction in primary sjögren's syndrome unstimulated salivary flow, ph, proteins and oral health in patients with juvenile idiopathic arthritis new paradigms on the transport functions of maturation-stage ameloblasts a novel mutant na + /hco -cotransporter nbce in a case of compound-heterozygous inheritance of proximal renal tubular acidosis extrarenal signs of proximal renal tubular acidosis persist in nonacidemic nbce b/c-null mice salivary ph: a diagnostic biomarker baking soda dentifrices and oral health the effect of sodium bicarbonate oral rinse on salivary ph and oral microflora: a prospective cohort study salivary ph after a glucose rinse: effect of a new mucoadhesive spray (cariex) based on sodium bicarbonate and xylitol effect of chewing bicarbonate-containing sugar-free gum on the salivary ph: an in vivo study sodium bicarbonate solution versus chlorhexidine mouthwash in oral care of acute leukemia patients undergoing induction chemotherapy: a randomized controlled trial the effect of bicarbonate/fluoride dentifrices on human plaque ph enhancement of plaque removal efficacy by tooth brushing with baking soda dentifrices: results of five clinical studies bicarbonate-based powder and paste dentifrice effects on caries ph stabilizing properties of a posterior light cured resin composite: an in vivo study six months of daily high-dose xylitol in high-risk schoolchildren: a randomized clinical trial on plaque ph and salivary mutans streptococci the influence of virus infection on the extracellular ph of the host carbon dioxide instantly sensitizes female yellow fever mosquitoes to human skin odours merozoite surface protein recognition of host glycophorin a mediates malaria parasite invasion of red blood cells reduced risk of plasmodium vivax malaria in papua new guinean children with southeast asian ovalocytosis in two cohorts and a case-control study the pathophysiologic and prognostic significance of acidosis in severe adult malaria unidentified acids of strong prognostic significance in severe malaria put a cork in it: plugging the m viral ion channel to sink influenza proton pump inhibitors are risk factors for viral infections: even for covid- ? pharmacokinetics and pharmacodynamics of dichloroacetate in children with lactic acidosis due to severe malaria can exchange transfusions using red blood cells from donors with southeast asian ovalocytosis prevent or ameliorate cerebral malaria in patients with multi-drug resistant plasmodium falciparum? na(+) regulation in the malaria parasite plasmodium falciparum involves the cation atpase pfatp and is a target of the spiroindolone antimalarials the warburg effect: how does it benefit cancer cells? we need to talk about the warburg effect ph control mechanisms of tumor survival and growth regulation and roles of bicarbonate transporters in cancer role of ph regulatory proteins and dysregulation of ph in prostate cancer disrupting hypoxia-induced bicarbonate transport acidifies tumor cells and suppresses tumor growth carbonic anhydrases: role in ph control and cancer sharpey-schafer lecture: gas channels rapid co permeation across biological membranes: implications for co venting from tissue water transport proteins-aquaporins (aqps) in cancer biology acidosis induces reprogramming of cellular metabolism to mitigate oxidative stress the acidic tumor microenvironment as a driver of cancer tumor immunoevasion via acidosis-dependent induction of regulatory tumor-associated macrophages increased acid-producing diet and past smoking intensity are associated with worse prognoses among breast cancer survivors: a prospective cohort study acid treatment of melanoma cells selects for invasive phenotypes the hypoxic response expression as a survival biomarkers in treatment-naive advanced breast cancer, asian pac increased expression of na+/h+ exchanger isoform predicts tumor aggressiveness and unfavorable prognosis in epithelial ovarian cancer monocarboxylate transporters in breast cancer and adipose tissue are novel biomarkers and potential therapeutic targets cellular acidification as a new approach to cancer treatment and to the understanding and therapeutics of neurodegenerative diseases disrupting na + , hco₃ − -cotransporter nbcn (slc a ) delays murine breast cancer development intermittent high dose proton pump inhibitor enhances the antitumor effects of chemotherapy in metastatic breast cancer development of a small molecule tubulysin b conjugate for treatment of carbonic anhydrase ix receptor expressing cancers brain tumor acidification using drugs simultaneously targeting multiple ph regulatory mechanisms glucose-dependent growth arrest of leukemia cells by mct inhibition: feeding warburg's sweet tooth and blocking acid export as an anticancer strategy manipulating ph in cancer treatment: alkalizing drugs and alkaline diet bicarbonate increases tumor ph and inhibits spontaneous metastases targeting the acidic tumor microenvironment: unexpected pro-neoplastic effects of oral nahco therapy in murine breast tissue a nonrandomized cohort and a randomized study of local control of large hepatocarcinoma by targeting intratumoral lactic acidosis pros and cons of dietary strategies popular among cancer patients irinotecan-induced neutropenia is reduced by oral alkalization drugs: analysis using retrospective chart reviews and the spontaneous reporting database martínez-climent, targeting the anion exchanger with specific peptides as a new therapeutic approach in b lymphoid neoplasms intestinal luminal ph in inflammatory bowel disease: possible determinants and implications for therapy with aminosalicylates and other drugs measurement of gastrointestinal ph profiles in normal ambulant human subjects advances in oral drug delivery for regional targeting in the gastrointestinal tract -influence of physiological, pathophysiological and pharmaceutical factors impact of gastrointestinal disease states on oral drug absorption -implications for formulation design -a pearrl review food, gastrointestinal ph, and models of oral drug absorption literature review of gastrointestinal physiology in the elderly prediction of ph-dependent drug-drug interactions for basic drugs using physiologically based biopharmaceutics modeling: industry case studies study of ph-dependent drugs solubility in water acidic and basic drugs in medicinal chemistry: a perspective drug solubility: importance and enhancement techniques, isrn pharm physicochemical properties, formulation, and drug delivery a quantitative assessment of herg liability as a function of lipophilicity some aspects of cell permeability to weak electrolytes the gastric secretion of drugs: a ph partition hypothesis surface area of the digestive tract -revisited tight junction modulation and its relationship to drug delivery polyspecific organic cation transporters and their impact on drug intracellular levels and pharmacodynamics the human organic cation transporter oct mediates high affinity uptake of the anticancer drug daunorubicin the organic anion transporter (oat) family: a systems biology perspective the p-glycoprotein transport system and cardiovascular drugs drugs as p-glycoprotein substrates, inhibitors, and inducers impaired drug absorption due to high stomach ph: a review of strategies for mitigation of such effect to enable pharmaceutical product development bicarbonate alters bacterial susceptibility to antibiotics by targeting the proton motive force ionotropically cross-linked ph-sensitive ipn hydrogel matrices as potential carriers for intestine-specific oral delivery of protein drugs peppas, ph-responsive and enzymatically-responsive hydrogel microparticles for the oral delivery of therapeutic proteins: effects of protein size, crosslinking density, and hydrogel degradation on protein delivery nanotechnology for protein delivery: overview and perspectives eudragit: a technology evaluation preparation of layer-by-layer thin films containing insulin and its ph-sensitive decomposition oral delivery of insulin using ph-responsive complexation gels fundaments of toxicology-approach to the poisoned patient the role of sodium bicarbonate in the management of some toxic ingestions acid-alkaline balance: role in chronic disease and detoxification., alternative therapies in health and medicine relationship between the urinary excretion mechanisms of drugs and their physicochemical properties gastro-floating matrices designed for simultaneous improvement of floating and drug release capabilities: an in vitro case study of matrices loaded with ciprofloxacin hydrochloride environmental ph-sensitive polymeric micelles for cancer diagnosis and targeted therapy a drug screening assay on cancer cells chronically adapted to acidosis key: cord- -rtg fs authors: lim, t. k. title: punica granatum date: - - journal: edible medicinal and non-medicinal plants doi: . / - - - - _ sha: doc_id: cord_uid: rtg fs nan the pomegranate tree is native from the middle east to the himalayas in northern india. it has been cultivated and naturalised since ancient times throughout the mediterranean region of asia, caucasus, northern africa and europe. the fruit has manifold uses as it is today and was featured in egyptian mythology and art, in the old testament of the bible and in the babylonian talmud. from its native range, it was introduced to central and southern india and southeast asia. it was reported growing in indonesia in . it was introduced into latin america and california by the spanish in , it is now grown in california and arizona. it has been widely cultivated throughout india and drier parts of southeast asia and tropical africa. the most important growing regions are egypt, china, afghanistan, turkey, syria, pakistan, bangladesh, iran, iraq, india, myanmar and saudi arabia. there are some commercial orchards in israel on the coastal plain and in the jordan valley. pomegranate is primarily mild-temperate to subtropical and naturally adapted to regions with cool winters and hot summers, but can also be grown in warm tropical areas, such as in southern india, southeast asia and various islands in the caribbean. areas with mean annual temperature of - °c is ideal. it suffers severe, irrecoverable injuries at temperatures below - . °c. the plant thrives in a semi-arid condition with mean annual rainfall of to , mm and is extremely drought-tolerant. it does not fl ower and fruit well in very humid and wet climates. it is cultivated up to altitudes of , m as occur throughout the western range (baluchistan, n. & s. waziristan, nwfp, kurram, dir, chitral) in pakistan. the species is adaptable to a wide range of soil types including soils on which other fruit species will not grow. it thrives on calcareous soil, alkaline soil, gravelly soil and on deep, acidic loams. for commercial cultivation well-drained, heavy, light and medium soils are preferred although it can withstand seasonal waterlogging. irrigation is required to sustain high yields in drier areas. the fruit is relished fresh, out of hand by quartering the fruit and lifting out the rind to exposed the juice-laden arils around the seeds, both of which are eaten. the fruit is also consumed as juice which is the basis for lemonades or a beverage similar to wine. in the middle east, caucasus and india, pomegranate juice is a very popular beverage. for beverage purposes, the juice is usually sweetened. pomegranate juice is widely made into grenadine syrup for use in mixed drinks, cocktails and often processed into wine. in saudi arabia, the juice sacs may be frozen intact or the extracted juice may be concentrated and frozen, for future use. the juice can be processed into jellies by the addition of pectin and sugar. pomegranate is also used in food and as a spice condiment. fresh pomegranate arils are used in preparation of curd rice dadhojanam (telugu) in andhra pradesh in india. in northern india, the reduced juice is used for desserts and for marinating and tenderising meat due to its proteolytic enzymes. dried pomegranate arils are used in various cuisines such as trail mix, granola bars, or as toppings for ice-cream, yogurt and salads. dried whole arils are commonly sold in ethnic indian subcontinent markets. they impart a subtle, sweet-sour and tart fl avour popular in punjab and gujarat. dried pomegranate seeds, ' anardana ', has culinary importance as spice for vegetable and legume dishes in northern india and in pakistani cuisine. these dried seeds are used as an acidic condiment for chutney and curry preparation. the dried seeds can also be ground and used, which results in a deeper fl avoring in dishes and prevents the seeds from getting stuck in teeth. seeds of the wild pomegranate variety known as daru from the himalayas are renown as quality sources for this spice. in turkey, pomegranate seeds are also used in salads and sometimes as garnish for desserts such as güllaç . in greece and cyprus, pomegranate is used to make kolliva , a mixture of pomegranate seeds and sugar. pomegranate juice can also be processed into a concentrate, syrup and sauces for juice in food dishes and desserts. in iran, a traditional recipe fesenjan is made from a thick pomegranate sauce and ground walnuts used for duck and poultry or in a popular pomegranate soup ash-e nar . in azerbaijan, pomegranate sauce narsharab , made from pomegranate juice, is served with fi sh or tika kabab (grilled, roasted or stewed meat). in turkey, pomegranate sauce called nar ekşisi is used as a salad dressing, to marinate meat, or simply to drink straight. pomegranate syrup used in muhammara , a roasted red pepper, walnut, and garlic spread popular in syria and turkey. pomegranate is also popular in greek cuisine such as kollivozoum i, a creamy broth made from boiled wheat, pomegranates and raisins, legume salad with wheat and pomegranate, traditional middle eastern lamb kebabs with pomegranate glaze, pomegranate eggplant relish, and avocadopomegranate dip. pomegranate is also processed into a liqueur and fruit confectionery used as icecream toppings or mixed with yogurt and jam on toast. a deciduous, much-branched, small tree or shrub . - m high with a smooth, dark grey bark (plate ). branches are terete, opposite and branchlets usually ending in spines. leaves are opposite, glabrous, coriaceous, glossy green, entire, simple, oblong-lanceolate (plates , and ) to obovate or elliptic, - (− ) × - (− ) mm, subpetiolate, apex sub-actue to obtuse. flowers are large, showy, scarlet red or white, bisexual, to cm across, solitary or clustered at the shoot apex (plates and ). calyx campanulate, reddish or purplish with six triangular, persistent lobes, petals , broadly obovate, wrinkled, alternating with the sepal lobes, stamens numerous, multiseriate, persistent, inserted on fl ower tube, ovary subglobose, inferior with three cells in two-series, style one thick, reddish, stigma simple slightly bilobed. fruit globose to subglobose, - cm in diameter, pale red to scarlet to purple or brownish,; the rind thick and coriaceous (plates , , and ). internally, the fruit is partitioned by thin leathery yellow septa into compartments fi lled with transparent sacs (arils) fi lled with tart, fl avourful, fl eshy, juicy, red, pink or whitish pulp (plates and ). in each sac, there is one white, pink or red, angular, soft or hard seed - mm long. food value of raw, pomegranate fruit (refuse % skin and membrane) per g edible portion based on the california wonderful variety is as follows (usda ) : water . g, energy kcal ( kj), protein . g, total lipid (fat) . g, ash . g, carbohydrate . g; fi bre (total dietary) g, total sugars . g, minerals -calcium mg, iron . mg, magnesium mg, phosphorus those of seeds, except potassium which was . ppm in the juice. the seed lipids had a refractive index of . , melting point . °c, iodine value . , acid number . , unsaponi fi able matter . %, saponi fi cation value . , ester value . and glycerol content . %. the lipids contained fatty acids, with caprylic ( . %), the predominant acid, followed by stearic acid ( . %); linoleic acid ( . %) and oleic acid ( . %). the saturated fatty acids of the seed lipids constituted . % of the total fatty acids content. vitamin c content in different turkish cultivars of pomegranate had a range of - . mg/ g, oil content a range of . - . %, sterol content a range of . - . %, anthocyanin content a range of , - , mg/l, potassium a range of - , ppm, calcium a range of - ppm, magnesium a range of - ppm, iron a range of - ppm, sodium a range of - ppm, and phosphorus a range of - ppm (dumlu and gürkan ) . elfalleh et al. ( ) found total sugars of pomegranate juice comprised about g/ ml fructose and about g/ ml glucose, soluble proteins about g/l, . mg/ ml of phosphorus, and . mg/ ml of potassium. the peel contained . and . mg/ g of phosphorus and potassium respectively. the sodium contents were nearly mg/ ml in both peel and juice. nutrient composition of the juice for most components was comparable to the whole fruit. protein and fat values were higher in the whole fruit compared to the juice due to the seeds, which are % of the aril (juice sac) weight (thomas and gebhardt ) . pomegranate aril juice was reported to provide about % of an adult's daily vitamin c requirement per ml serving, and to be a good source of vitamin b (pantothenic acid), potassium and antioxidant polyphenols. the tocopherol ( a -tocopherol, g -tocopherol, and d -tocopherol) contents were, respectively, . , . , and . mg/ g from dry pomegranate seed (elfalleh et al. a, b ) . a total of compounds were found in pomegranate aroma pro fi les, including monoterpenes, aldehydes, alcohols, monoterpenoids and linear hydrocarbons . the most abundant compounds were trans - -hexenal, -carene, a -terpinene and a -terpineol. the total concentration of volatiles ranged from . to . g/kg. overall consumer preference of pomegranate juices was associated with the presence of monoterpenes such as a -pinene, b -pinene, b -myrcene, limonene and g -terpinene. the presence of aldehydes such as hexanol, hexanal and cis - -hexenol was correlated with poor overall consumer liking. high overall consumer liking was associated with intense and acceptable fresh pomegranate odour and fl avour (high scores of satisfaction degree), medium intensity of red colour and low sourness. pomegranate is a fruit rich in polyphenols that include fl avonoids, tannins and hydrolyzable tannins (gil et al. ; seeram et al. a, b ) . pomegranate contain a complex mixture of gallotannins, ellagitannins, ellagic acid and anthocyanins (madrigal-carballo et al. ) . pomegranate juice was found to be rich in tannins, anthocyanins, ellagic acid derivatives, and hydrolyzable tannins (gil et al. ) . the predominant organic acid in pomegranate was citric acid followed by malic acid (pande and akoh ) . the peel fraction had the highest total hydrolyzable tannins content ( , . - , . mg/ g of fw). a total of dimers of fl avanol-anthocyanin adducts were detected, consisting of mono-and disubstituted hexoside derivatives of the adducts between the fl avan- -ols (epi)gallocatechin, (epi) catechin and (epi)afzelechin and the anthocyanidins delphinidin, cyanidin and pelargonidin in pomegranate juice (sentandreu et al. ) . anthocyanidins found in pomegranate fruit included: delphinidin, cyanidin, and pelargonidin (noda et al. ) . pomegranate fruit was reported to contain ellagic acid, gallagic acid, punicalins and punicalagins (reddy et al. ) ; ellagic acid, caffeic acid, luteolin and punicic acid (lansky et al. a, b ) ; pelargonidin- -galactose, cyanidin- -glucose, gallic acid, quercetin, and myricetin (naz et al. ) ; gallic acid, methyl gallate, ellagic acid, (+) catechin, isoquerecitrin, d-mannitol, ursolic acid, oleanolic acid, b -sitosterol and daucosterol (rena et al. ) . pomegranate fruit was found to have a low level of indolamines ( - m g/g serotonin, - m g/g tryptamine, and - ng/ g melatonin) (badria ) . gözlekçi et al. ( ) found that in all the turkish pomegranate cultivars the highest levels of total phenolic content were obtained from the peel extracts. the total phenolic content ranged from , . to , . mg gallic acid equivalent (gae)/l among the cultivars. however, the total phenolic content of pomegranate juice and seed extract ranged from . to , . mg gae/l and . - . mg gae/l, respectively. four phenolic compounds were identi fi ed and quanti fi ed in pomegranate peel and pulp: hydroxybenzoic acids (gallic and ellagic acids) and hydroxycinnamic acids (caffeic and p -coumaric acids) (elfalleh et al. a ) . ellagitannins isolated from pomegranate pericarp included: inhibitors punicalin, punicalagin, granatin b, gallagyldilactone, casuarinin, pedunculagin, tellimagrandin i, gallic acid, granatin a, corilagin and ellagic acid (satomi et al. ) . pomegranate fruit peel had been reported to be a rich source of hydrolyzable tannins called ellagitannins (ets); pomegranate ets were found to show potent antioxidant, antiatherosclerotic and anticancer activities (seeram et al. b ) . the major fruit peel ets were punicalagin ( - % w/w) and ellagic acid (ea; . % w/w) and unquanti fi ed amounts of punicalin and ellagitannin-glycosides (hexoside, rhamnoside and pentoside). pomegrante fruit peel is currently an underutilized food by-product with potential to develop phytoceuticals with potential health bene fi ts or to develop products for use in the cosmetic and food biopreservative industries (seeram et al. b ) . prodelphinidins and gallocatechins including gallocatechin, gallocatechin-( - )-catechin, gallocatechin-( - )-gallocatechin and catechin-( - )-gallocatechin were identi fi ed from pomegranate peels (plumb et al. ) . luteolin, luteolin -o -glucoside, kaempferol, kaempferol- -o -glucoside, kaempferol- -orhamnoglycoside and quercetin were found in the fruit peel (van elswijk et al. ) . of the ellagi-tannins isolated from pomegranate pericarp, seven namely punicalin, punicalagin, granatin b, gallagyldilactone, casuarinin, pedunculagin and tellimagrandin i were found to be active carbonic anhydrase inhibitors and four namely gallic acid, granatin a, corilagin and ellagic acid to be weakly active inhibitors. the type of inhibition by three and seven punicalagin and gallagyldilactone was found to be noncompetitive. a new antifungal peptide designated as pomegranin with a molecular mass of kda, was isolated from fresh pomegranate peels (guo et al. ) . epicatechin, epigallocatechin -gallate, fl avan- -ol, catechin were found in the fruit peel and juice (de pascual-teresa et al. ) . pomegranate fruit and juice were found to contain the following lignans: isolariciresinol, medioresinol, matairesinol, pinoresinol, secoisolariciresinol and syringaresinol (bonzanini et al. ). total lignin contents in the seeds was determined as . m g/g, in wood knots . m g/g, in fruit pulp . m g/g and in the endocarp. . m g/g. syringaresinol was most abundant in the seed ( . m g/g), pinoresinol in knots ( . m g/g), pulp ( . m g/g) endocarp ( . m g/g) and juice ( . m g/g). lignans were also found in two concentrated juices and three pomegranate beverages at levels of . - . m g/g. in addition to the peel, mesocarp, and twigs, lignans were detected in two juices obtained from entire pomegranate fruits, four commercial juices, and three encapsulated pomegranate extracts (fischer et al. ) . isolariciresinol was the predominant lignan with contents of . , . , and . mg/kg dry matter in processed pomegranate mesocarp, peel, and twigs, respectively. six anthocyanin pigments identi fi ed as delphinidin -glucoside, delphinidin , -diglucoside, cyanidin -glucoside, cyanidin , -diglucoside, pelargonidin -glucoside and pelargonidin , -diglucoside were found to be responsible for the red colour of pomegranate juice (cv 'mollar') (gil et al. ) . the fruit skin contained only the cyanidin and pelargonidin derivatives. generally, there was an increase in juice pigmentation with fruit ripening. the concentration of pigments in juice obtained from mature pomegranates ranged between and m g of anthocyanin per gram fresh weight of arils. six anthocyanin pigments delphinidin -glucoside and , -diglucoside, cyanidin -glucoside and , -diglucoside and pelargonidin -glucoside and , -diglucoside were found to be responsible for the red color of pomegranate juice (hernández et al. ) . generally, juice pigmentation increased as the fruit ripened. in the early fruit-ripening stages, delphinidin , -diglucoside was the major pigment, followed by cyanidin , -diglucoside, while in the later stages, the monoglucoside derivatives cyanidin -glucoside and delphinidin -glucoside increased considerably. the pelargonidin derivatives were always present in small amounts. rp-hplc analysis of pomegranate arils' anthocyanins revealed mono-and diglucosylated delphinidins and cyanidins as the major anthocyanins and pelargonidins as minor components (borochov-neori et al. ) . anthocyanin accumulation changed inversely to the season's temperatures. cyanidins were generally more abundant but delphinidin accumulation was enhanced in cooler season. monoglucosylated anthocyanins prevailed at cooler temperatures and subsided during seasonal warming with a concomitant rise in diglucoside proportion. the major anthocyanins detected in the iranian pomegranate varieties were as follows: delphinidin -glucoside ( . - . mg/l), delphinidin , -diglucoside ( . - . mg/l), pelargonidin -glucoside ( . - . mg/l), pelargonidin , -diglucoside ( . - . mg/l), cyanidin -glucoside ( . - . mg/l), and cyanidin , -diglucoside ( . - . mg/l) (alighourchi et al. ) . the major anthocyanins in the juice of iranian pomegranate cultivars were delphinidin , -diglucoside ( - , mg/l), cyanidin , -diglucoside ( - , mg/l), delphinidin -glucoside ( - , mg/l) and pelargonidin , -diglucoside ( - mg/l) (mousavinejad et al. ) . the cultivar, saveh black leather had the highest level of ellagic acid ( mg/l). pomegranate juices obtained from six iranian pomegranate cultivars were found to have . - . total soluble solids content (brix), ph values of . - . , titrable acidity concentration from . to . g/ g, total sugars content from . to . g/ g (faroogh), total antho-cyanins . - . mg/ g, ascorbic acid . - . mg/ , total phenolics content . - . mg tannic acid/ g, the total tannins level . - . mg tannic acid/ g, condensed tannins from . mg to . catechin/ g, antioxidant activity from . to . % (zarei et al. ) . phenolics, fl avonoids, anthocyanins, and tannins of pomegranate juices, obtained from nine tunisian ecotypes were quanti fi ed by el kar et al. ( ) . phenolics ranged from , to , mg gallic acid equivalents/l and fl avonoids from to mg quercetin equivalent/l of juice. highest anthocyanin content was mg cyanidin- -glucoside equivalent/l and highest tannin content was , mg catechin equivalent/l of juice. tartaric and quinic acids were con fi rmed in pomegranate juice at concentrations of - and ~ mg/l, respectively (ehling and cole ) . twenty-one volatile compounds were found in fresh pomegranate juices from nine spanish cultivars, including aldehydes, monoterpenes, and alcohols . the most abundant compounds were hexanal, limonene, trans - -hexenal, and cis - -hexenol. the presence of monoterpenes ( a -terpineol) was correlated with overall consumer preference of pomegranate juice while high aldehydes ( trans - -hexenal) concentrations were correlated with poor overall consumer liking. -hydroxymethyl furfural was determined to be at a signi fi cant level in traditional sour concentrate of pomegranate juice (orak ) . pomegranate was known to contain estrogens (estradiol, estrone, and estriol) (mori-okamoto et al. ) . polysaccharide (psp ) was isolated from pomegranate rind (joseph et al. ) . pomegranate seed oil was found to have % saturated fatty acids, % monounsaturated, % diunsaturated and approximately % conjugated acid, most probably punicic acid (el-shaarawy and nahpetian ) . pomegranate seed was found to have high contents of a -tocopherol ( . - . mg/ g) and g -tocopherol ( . - . mg/ g). the seeds of punica granatum also contained ursolic acid and b -sitosterol along with a long straightchain hydrocarbon -nonacosene (ahmed et al. ) . presence of estrogens and glycosides were also detected. estrone, an estrogen, was identi fi ed in pomegranate seeds (heftmann et al. ) . cold pressed pomegranate seed oil was found to contain punicic acid ( . %), palmitic acid ( . %), stearic acid ( . %), oleic acid ( . %), linoleic acid ( . %) and three unidenti fi ed peaks from which two ( . %) were probably isomers of punicic acid (schubert et al. ) . pomegranate seed had an average lipid content of . % with punicic acid as the predominant fatty acid (pande and akoh ) . pomegranate seed oil was found to be rich in -o -trans , cis , trans - , , -octadecatrienoyl glycerol and also to have small amounts of -o -isopentyl- -o -octadec- -enoyl glycerol and the known cis - -octadecenoic, octadecanoic and eicosanoic acids (fatope et al. ) . pomegranate seed oil (pgo) was reported to be rich in % cis (c) , trans (t) ,c - : as conjugated linolenic acids (cla) (kohno et al. ) . a triglyceride, di-o -punicyl-o -octadeca- z , z , e -trienylglycerol, was isolated and characterized from the seeds of punica granatum from india and iran (yusuph and mann ) . four compound were isolated from pomegranate seeds namely . pomegranate seed oil from pomegranate cultivars was found to have mainly unsaturated fatty acids (about %) (el kar et al. ) . the predominant fatty acid was linolenic acid ( . - . %), followed by linoleic acid ( . - . %), oleic acid ( . - . %), palmitic acid ( . - . %), stearic acid ( . - . %), gadoleic acid ( . - . %), lignoceric acid (< . %), arachidic acid (< . %) and myristic acid (< . %). pomegranate seed linolenic acid isomers, punicic acid and α-eleostearic acid were found in pomegranate seeds (tran et al. ) . a high yield ( . - . %) of unsaponi fi able matter containing tocopherol, aliphatic alcohol (including policosanol), squalene, phytosterols and triterpene was obtained from pomegranate seed oil (caligiani et al. ) . the levels of squalene (up to mg/kg), policosanol ( - mg/kg), b -sitosterol (up to , mg/kg) and cycloartenol ( , - , mg/kg) were found while b -and d -tocopherol were the most abundant vitamin e forms. the seed oil of p. granatum may be an interesting alimentary source of substances of nutraceutical value involved in the modulation of cholesterol metabolism. linolenic acid isomers like punicic acid and αeleostearic acid were reported from pomegranate seeds (tran et al. ) . qualitatively, the pomegranate fatty acid composition of pomegranate cultivars ( tunisian and chinese) seed oil was identical comprising mainly unsaturated about % (elfalleh et al. b ). the predominant fatty acid was linolenic acid ( . - . %), followed by linoleic acid ( . - . %), oleic acid ( . - . %), palmitic acid ( . - . %), stearic acid ( . - . %), gadoleic acid ( . - . %), lignoceric acid ( < . %), arachidic acid ( < . %) and myristic acid ( < . %). ) isolated the following bioactive compounds from pomegranate seeds: coniferyl methy lellagic acid; , ¢ , ¢ -tri-o -methylellagic acid; phenethyl rutinoside; icariside d and daucosterol. a new class iii chitinase (pomegranate seed chitinase) with a molecular weight of approximately kda was isolated and puri fi ed from pomegranate seeds (yang et al. ) . this chitinase was found to naturally bind calcium ions with high capacity and low af fi nity, suggesting it to be a calcium storage protein. this enzyme was found to be widely distributed in the stroma of amyloplasts of the embryonic cells, suggesting that amyloplasts in seeds could serve as an alternative plastid for calcium storage. two new b -sitosterol esters elucidated as stigmast- en- b -ol- b -dodecanoate ( b -sitosterol laurate) and stigmast- -en- b -ol- b -tetradecanoate ( b -sitosterol myristate) along with the known com pounds n-tricosane, n-heptacosanyl n-hexanoate olean- , -dien- b -ol- -oic acid and olean- -en- bol- -oic acid were isolated from pomegranate fl owers (bagri et al. b ) . a new polyphenol compound named pomegranatate, together with, ellagic acid, , ¢ , ¢ -tri-o -methylellagic acid, ethyl brevifolincarboxylate, urolic and maslinic acids, and daucosterol were isolated from the ethanolic extract of the fl owers of punica granatum . maslinic acid exhibited antioxidant activity as evaluated by measurement of ldl susceptibility to oxidation. a taraxastane-type triterpene, punicanolic acid; two galloyl glucoses, , , -tri-o -galloyl b -d-glucopyranoside, , -di-o -galloyl- , -o -(s)-hexahydroxydiphenoyl b -d-glucopyranoside; fl avones, luteolin; triterpnenes oleanolic acid, maslinic acid; and b -sitosterol were isolated from pomegranate fl owers (xie et al. ) . an alkaloid -( -propenyl)-d -piperideine was isolated from pomegranate leaves (roberts et al. ) . pomegranate leaves were found to contain tannins granatin a, granatin b, corilagin, strictinin, , , , -tetra-o -galloyl-b -d-glucose and , , , , -penta-o -galloyl-b -d-glucose and an ellagitannin, punicafolin elucidated as , , -tri-o -galloyl- , -(r)-hexahydroxydiphenoyl-b -dglucose (tanaka et al. (tanaka et al. , . pomegranate leaves were found to be rich in polyphenols: brevifolin carboxylic acid, brevifolin, corilagin, ellagic acid, , , , , -pentahydroxydibenzo[ b,d ] pyran- -one, granatin-b and punicafolin (nawwar et al. b ) ; n-( ¢ , ¢ -dihydroxyphenyl) pyridinium chloride, as well as the known fl avone glycosides, apigenin (nawwar et al. a ) ; ellagitannin, punicafolin, tannins, granatins a and b, corilagin, strictinin, , , , -tetra-o -galloyl-b -d-glucose and , , , , -penta-o -galloyl-b -d-glucose (tanaka et al. ) ; gallotannins, , , -tri-o -galloylb -glucopyranose and , , -tri-o -galloyl-b -glucopyranose together with the hitherto unknown ellagitannins, , -di-o -galloyl- , -( r )-hexahydroxydiphenyl-b -glucopyranose and brevifolin carboxylic acid -monopotassium sulphate (hussein et al. ) . a hydroquinone pyridinium alkaloid in the form a mixture of a con jugated and a cross-conjugated heterocyclic mesomeric betaine was isolated from the leaves of punica granatum (schmidt et al. ) . balwani et al. ( ) isolated a novel compound, -methyl-pyran- -one- -o -b -d-glucopyranoside from pomegranate leaves. these alkaloid isopelletierine, methylisopelletierine, pelletierine, pseudopelletierine were isolated from pomegranate bark (chilton and partridge ; wibaut et al. ) and roots (chilton and partridge ) ; isopelletierine, methylisopelletierine and y pelleterine from bark (wibaut and hollstein ) ; and n-acetyl-sedridine from bark and root (neuhöfer ) . the bark is rich in punicotannic acid (about %) and also contains gallic acid, mannite and four alkaloids isopelletierine, methylisopelletierine, pelletierine, pseudopelletierine (grieve ) . the following alkaloids were isolated from pomegranate bark and roots: pelletierine, methylisopelletierine, pseudopelletierine and from roots norpseudopelletierine, sedridine, -( ¢ -hydroxypropyl) d -piperidine; -( ¢ -propenyl) d -piperidine, hygrine and norhygrine (neuhöfer et al. ) . tannins and related compound were isolated from pomegranate bark and included punicalin and punicalagin elucidated as to , -(s, s)-gallagyl-d-glucose ( ) and , -(s)hexahydroxy-diphenoyl- , -(s, s)-gallagyl-dglucose ( ), respectively and a hydrolyzable tannin, -o -galloyl- , -(s, s)-gallagyl-d-glucose (tanaka et al. a ) ; ellagitannins, punicacorteins a, b, c and d, punigluconin, casuariin and casuarinin (tanaka et al. b ) . punicacor teins a, b, c and d were established as novel c-glycosideic ellagitannins, the former two possessing a unique tetraphenyl (gallagyl) ester group, and the latter two containing a galloyl group in place of the gallagyl group, while punigluconin was elucidated as , -di-o -galloyl- , -(s)-hexahydroxydiphenoyl gluconic acid. a fl avonoid diglycoside, quercetin- , ¢ -dimethyl ether- -o -a -l-arabinofuranosyl ( → )-b -dglucopyranoside, quercetin, pelargonidine- , diglucoside and ellagic acid were isolated from pomegranate bark (chauhan and chauhan ) . the heartwood of punica grantum was found to contain ellagitannins: diellagic acid rhamnosyl toumy and rauwald ) ; ¢ -o -methyl- , methylenedioxyellagic acid, as well as eight known ellagitannins and gallotannins (el-toumy et al. ) . a new dimeric gallic acid glycoside named humarain was isolated from stem bark of punica granatum (tantray et al. ) . punica granatum is a unique medicinal plant with a long and extensive ethnomedicinal uses since ancient times. various parts of the plant viz. seed, aril, fruit juice, peel, leaf, fl ower, bark, and roots have been reported to contain bioactive phytochemicals with interesting medicinal values and pharmacological activities. the phytochemistry and pharmacological properties of pomegranate plant parts suggest a wide range of clinical applications for the treatment and prevention of ailments such as cancer as well as other diseases where chronic in fl ammation is believed to play an essential etiologic role . the synergistic action of the pomegranate constituents appears to be superior to that of single constituents. in the past two decade, numerous invitro, in-vivo and preclinical studies on the antioxidant, anticarcinogenic, and anti-in fl ammatory properties of pomegranate constituents have been published, focusing on treatment and prevention of cancer, cardiovascular disease, diabetes, dental conditions, erectile dysfunction, bacterial infections and antibiotic resistance, and ultraviolet radiation-induced skin damage (jurenka ) . other potential applications include infant brain ischemia, male infertility, alzheimer's disease, arthritis, and obesity. aqueous and ethyl acetate extracts of pomegranate arils, juice and peels exhibited good antioxidant activity (ricci et al. ) . pomegranate juice, peel, and seed oil antioxidants were con fi rmed by ferric reducing antioxidant power (frap) and oxygen radical absorbance capacity (orac) methods (elfalleh et al. ) . the highest values were recorded in peels with . mmol trolox equivalent/ g and . mmol te/ g for frap and orac assay, respectively. the tocopherol ( a -tocopherol, g -tocopherol, and d -tocopherol) contents were, respectively, . , . , and . mg/ g from dry pomegranate seed. four phenolic compounds were identi fi ed and quanti fi ed in pomegranate peel and pulp: hydroxybenzoic acids (gallic and ellagic acids) and hydroxycinnamic acids (caffeic and p -coumaric acids). results showed that the antioxidant potency of pomegranate extracts was correlated with their phenolic compound content. in particular, the highest correlation was reported in peels. high correlations were also found between peel hydroxybenzoic acids and frap orac antioxidant capacities. identi fi ed tocopherols appeared to contribute in major part to the antioxidant activity of pomegranate seed oil. gil et al. ( ) found that the antioxidant activity of commercial pomegranate juices ( − teac) was three times higher than those of red wine and green tea ( − teac). commercial juices extracted from whole pomegranates showed higher antioxidant activity than in experimental juices obtained from the arils only ( − teac). further, they found that commercial juices contained the pomegranate abundant tannin punicalagin ( , − , mg/l) while only traces were detected in the experimental juice obtained from arils showing that pomegranate industrial processing extracts some of the hydrolyzable tannins present in the fruit rind. also, anthocyanins, ellagic acid derivatives, and hydrolyzable tannins were found in the pomegranate juices. the results of studies by tzulker et al. ( ) showed that the antioxidant activity in pomegranate aril juice correlated signi fi cantly to the total polyphenol and anthocyanin contents. however, the homogenates prepared from the whole fruit exhibited an approximately -fold higher antioxidant activity than the level found in the aril juice. unlike the arils, the antioxidant level in the homogenates correlated signi fi cantly to the content of the four hydrolyzable tannins in which punicalagin was predominant, while no correlation was found to the level of anthocyanins. pomegranate juice was found to be a potent inhibitor of superoxide anion-mediated disappearance of nitric oxide . it was much more potent than concord grape juice, blueberry juice, red wine, ascorbic acid, and dl-α-tocopherol. as little as three μl of a six-fold dilution of pomegranate juice, in a reaction volume of , μ l, produced a marked antioxidant effect, whereas μ l of undiluted blueberry juice or nearly , μ l of undiluted concord grape juice were required to produce similar effects. pomegranate juice and other antioxidant-containing products were found to augment the anti-proliferative action of nitric oxide (deta/no) on vascular smooth muscle cell (rat aorta) proliferation. and other antioxidant-containing products were found to augment the antiproliferative action of no on vascular smooth muscle cell (rat aorta) proliferation. pomegranate juice was much more effective than the other products tested and elicited no effects when tested alone in the absence of added no. pomegranate juice elicited no effects on enos protein expression or catalytic activity and did not enhance promoter activity in the enos gene. the observations indicated that pomegranate juice possessed potent antioxidant activity that resulted in marked protection of nitric oxide against oxidative destruction pande and akoh ( ) in their study found the highest antioxidant capacity to be in pomegranate leaves followed by peel, pulp, and seed. the tannin rich mixtures from pomegranate by-product exhibited ic values against reactive oxygen species (ros) generation at . - m g/ ml. the antioxidant capacity (orac) of pomegranate juice was , m mol te/ g pomegranate juice which was comparable to blueberry and grape juice (thomas and gebhardt ) . oral administration of fl avonoid rich fractions from pomegranate fruits to rats at a dose of mg/kg/day exhibited potential antiperoxidative activity (sudeesh and vijayalakshmi ) . malondialdehyde, hydroperoxides and conjugated dienes levels in the liver were signi fi cantly decreased antioxidative enzymes catalase, superoxide dismutase (sod), glutathione peroxidase and glutathione reductase were signi fi cantly elevated. glutathione content in the tissues were also increased. pomegranate fermented juice and cold pressed seed oil exhibited potent antioxidant activity almost equivalent to butylated hydroxyanisole (bha) and green tea ( thea sinensis ), but signi fi cantly higher than that of red wine ( vitis vitifera ) (schubert et al. ) . flavonoids extracted from cold pressed pomegranate seed oil exhibited - % inhibition of sheep cyclooxygenase and - % inhibition of soybean lipoxygenase. flavonoids extracted from pomegranate fermented juice displayed - % inhibition of soybean lipoxygenase but showed no signi fi cant inhibition of sheep cyclooxygenase. total polyphenols in cold pressed pomegranate seed oil showed a concentration by weight of approximately . %. fatty acid composition in cold pressed pomegranate seed oil showed punicic acid ( . %) along with palmitic acid ( . %), stearic acid ( . %), oleic acid ( . %), linoleic acid ( . %) and three unidenti fi ed peaks from which two ( . %) are probably isomers of punicic acid. acetone extract ( %) of pomegranate fruit displayed scavenging activity against hydroxyl (·oh) and superoxide (o ·-) radicals (noda et al. ) . its three major anthocyanindins, delphinidin, cyanidin, and pelargonidin, scavenged o ·in a dose-dependent fashion with id values of . , , and m m, respectively but did not effectively scavenge nitric oxide. the anthocyanidins inhibited a fenton reagent ·oh generating system. further, the anthocyanidins inhibited hydrogen peroxide-induced lipid peroxidation in the rat brain homogenates with id values . , . , and m m, respectively for delphinidin, cyanidin, and pelargonidin (noda et al. ) . in another study, pomegranate elagitanninsellagic acid, gallagic acid, punicalins and punicalagins from pomegranate fruit showed ic values of . , . , . and . m m, respectively, against reactive oxygen species (ros) generation and no toxicity up to . m g/ml against hl- cells (reddy et al. ) . the good antioxidant action of punicalagin a high molecular weight polyphenol isolated from pomegranate fruit pith and carpellary membrane was expressed not only through its scavenging reactions but also by its ability to form metal chelates (kulkarni et al. ) . binding of punicalagin with bovine serum albumin and metal ions such as iron and copper revealed different binding af fi nities, whereas its binding with dna was very weak and non-speci fi c. in-vitro cytotoxic studies against three cell lines, namely, vero (normal african green monkey kidney cell line), hep- (human larynx epithelial cancer cell line), and a- (human small cell lung carcinoma cell line) showed that punicalagin, was toxic only at higher concentration. studies found that pomegranate peel had the highest antioxidant activity among the peel, pulp and seed fractions of kinds of fruits commonly consumed in china as determined by frap (ferric reducing antioxidant power) assay (guo et al. ) . in a subsequent study pomegranate peel extract was shown to have markedly higher antioxidant capacity than the pulp extract in scavenging or preventive capacity against superoxide anion, hydroxyl and peroxyl radicals as well as inhibiting cuso -induced ldl oxidation. the contents of total phenolics, fl avonoids and proathocyanidins were also higher in peel extract than in pulp extract. the large amount of phenolics contained in peel extract may cause its strong antioxidant ability. the authors concluded that pomegranate peel extract appeared to have more potential as a health supplement rich in natural antioxidants than the pulp extract. separate studies showed pomegranate peel extracts to have both antioxidant and antimutagenic properties and may be exploited as biopreservatives in food applications and neutraceuticals (negi et al. ) . all the pomegranate peel extracts (ethyl acetate, acetone, methanol and water) exhibited marked antioxidant capacity, but the water extract was the lowest. the order of antioxidant capacity varied because of differential responses at four concentrations ( , , and m g/ml) in each solvent (negi et al. ) . studies in male rats showed that pomegranate fruit peel extract decreased lipid peroxidation in hepatic, cardiac, and renal tissues and serum glucose concentration (parmar and kar ) . pomegranate peels were found to contain potent antioxidant contents, as evidenced by free radical dpph scavenging value of . m g/ml and abts scavenging value of . mm trolox equivalent antioxidant capacity/ g dry weight (elfalleh et al. ) . aqueous and alcoholic extracts of pomegranate rind showed good antioxidant effect with ic ranging from . to . /ml for aqueous and . - . m g/ml for alcoholic extracts (rajan et al. ) . phenolic compounds, tannins and fl avonoids were the major phytochemicals present in both the extracts. the aqueous and alcoholic extract yielded . and mg/g gallic acid equivalent phenolic content, . snf . mg/g quercetin equivalent fl avonoid and . and . mg/g tannic acid equivalent tannins respectively. plumb et al. ( ) found that the prodelphinidin dimers from pomegranate peels were potent antioxidants in the aqueous phase, being much more effective than the gallocatechin monomer in scavenging of the radical cation of , -azinobis ( -ethyl-benzothiazoline- -sulphonate, abts) relative to the water-soluble vitamin e analogue trolox c (expressed as trolox c equivalent antioxidant capacity, teac). in the lipid phase, only one of the dimers (gallocatechin-( - )-catechin) was signi fi cantly more effective than the monomer in the inhibition of lipid peroxidation of phosphatidylcholine vesicles. the water, methanol, acetone and ethyl acetate (etoac) extracts of pomegranate peel phenolics showed enhanced inhibitory effect on lard peroxidation as the phenolic concentrations increased . acetone extract exhibited the highest antiliperoxidant activity followed by water, methanol and etoac extracts. acetone extract at . % (w/w) and water extract at . % (w/w) exhibited an antiliperoxidant effect close to that of tea polyphenols ( . %, w/w) and higher than that of bht (butylated hydroxytoluene) ( . %, w/w). at . % (w/w), acetone extract exerted a higher inhibitory activity on lard oxidation than that of tea polyphenols and bht. studies by guo et al. ( ) showed that showed that red pomegranate peel extract had the best effect on the scavenging ability of superoxide anion with lowest ic value ( . m g/ml) among all pomegranate extracts (peel, juice, and seed of three varieties). the peel extract of white pomegranate had the best scavenging ability on hydrogen peroxide with the lowest ic value ( . m g/ml) of the nine extracts. the seed extract of white pomegranate could scavenge hydroxide radical most effectively of the nine extracts (the ic value . m g/ ml). the seed extract of white pomegranate (the ic value was . m g/ml) was the most powerful on the dna damage-preventing effect of the extracts. the results of studies by xu et al. ( ) indicated that pomegranate peel extracts exerted protective effects on oxidative stress in mice loaded with restraint stress which may be attributed to its free radical scavenging activity and lipid peroxidation inhibitory effect. the extract decreased alanine aminotransferase and malondialdehyde levels and increased antioxidant capacity in the liver and glutathione levels in plasma as compared with restraint stress control mice. the methanol fraction of pomegranate peel showed highest antioxidant activity by all the four in vitro assays viz. dpph free radical scavenging, phosphomolybdenum, frap (fe( +) reducing power) and cuprac (cupric ions (cu( +)) reducing ability) comparable to ascorbic acid and butylated hydroxy toluene (bht) followed by activity in ethanol, acetone, and ethyl acetate fractions (zahin et al. a ) . in cell free-systems, preparations from various parts of pomegranate displayed displayed good antioxidant capacity as assayed by , -diphenyl- -picrylhydrazyl (dpph), chemiluminescence luminol/xanthine/xanthine oxidase and lipoxygenase assays, with relative potency sequence of rind extract > pomegranate juice > aril juice (sestili et al. ) . however, only the rind extract was capable of preventing the deleterious effects -cytotoxicity, dna damage and depletion of non-protein sulphydrils (npsh) pool, caused by treatment of cells with hydroxide peroxide, tert-butylhydroperoxide or oxidized lipoproteins (ox-ldl) via a mechanism which was postulated to involve both direct scavenging of radical species and iron chelation. the results suggested that the aril juice the major and tasty part of pomegranate fruit, did not contain ellagic acid and punicalagin (i.e. the polyphenols highly represented in the rind which appeared to be responsible for the antioxidant capacity) in amounts suf fi cient to exert cytoprotection in oxidatively injured, living cells. based on these results, the authors advocated that development and evaluation of rinds-only based derivatives of pomegranate for antiatherogenic preventive purposes in humans should be encouraged. the antioxidant activity (percentage of inhibition of on peroxidation in linoleic acid system) of cpj (traditional sour concentrate of pomegranate juice) was determined to be higher ( . %) than that of pj (pomegranate juice) ( . %) (orak ). during the concentration process, the reducing sugars, glucose and fructose level of cpj showed an increase to . , . , and . %, respectively. in cpj the amounts of sodium, iron, zinc, copper and lead were found lower than those of pj. in contrast, potassium and magnesium mineral contents increased during concentration. the total phenolics were also found to be , and , m g/ml in pj and cpj, respectively. the total anthocyanin content of pj was found to be . mg/l but it was not determined in cpj. -hydroxymethyl furfural was determined to be at a signi fi cant level in cpj as a result of the heat process. sezer et al. ( ) found that pomegranate and red wines decreased low-density lipoprotein (ldl) diene levels following a -min incubation period compared with controls. however, pure pomegranate wine demonstrated a greater antioxidant effect on diene level ( m mol/mg of ldl protein) than pure red wine ( m mol/mg of ldl protein). the phenol levels of pomegranate and red wines ( , mg/l gallic acid equivalents and mg/l gallic acid equivalents, respectively) were in accordance with their total antioxidant activity ( . and . %, respectively). four compound from pomegranate seeds namely coniferyl ( ) displayed antioxidant activity, which was evaluated by measurement of low-density lipoprotein (ldl) susceptibility to oxidation and by in-vitro determination of malondialdehyde (mda) levels in the rat's brain . ethanolic extract of pomegranate fl owers was found to contain a large amount of polyphenols and to exhibit potent reducing ability, both indicative of potent antioxidant ability (kaur et al. ) . the extract showed . % antioxidant activity in dpph model system. the fl ower extract was found to signi fi cantly scavenge superoxide (o − ) (by up to . %), hydrogen peroxide (h o ) (by up to %), hydroxyl radicals ( − oh) (by up to %) and nitric oxide (no) (by up to . %). the extract also inhibited ( − oh) induced oxidation of lipids and proteins in vitro. these results indicated pomegranate fl ower extract to exert a signi fi cant antioxidant activity in-vitro. daily consumption of pomegranate juices was found to be potentially better than apple juice in improving antioxidant function in the elderly (guo et al. ) . as the plasma ascorbic acid, vitamin e, and reduced glutathione contents did not differ signi fi cantly between the apple and pomegranate groups in the study, the phenolics may be the functional components contained in pomegranate juice that accounted for the observations. recent in-vitro studies and preclinical animal studies have shown that pomegranate extracts selectively inhibit the growth of breast, prostate, colon and lung cancer cells (adhami et al. ) . an initial phase ii clinical trial of pomegranate juice in patients with prostate cancer reported signi fi cant prolongation of prostate speci fi c antigen doubling time. some of these researches are further elaborated herein. various parts of the pomegranate fruit e.g. seed oil, juice, fermented juice and peel extract, had been shown to exert suppressive effects on human breast cancer cells in-vitro and in this context, three estrogenic compounds, i.e. luteolin, quercetin and kaempferol, were detected in the fruit peel extract (van elswijk et al. ) . studies showed pomegranate fruit possessed chemopreventive and adjuvant therapeutic potential for human breast cancer (kim et al. ) . polyphenols from fermented pomegranate juice, pericarp, and oil inhibited aromatase activity by - % indicating its ability to effect a blockade of endogenous active estrogen biosynthesis. fermented juice and pericarp polyphenols, and whole seed oil, inhibited -b -hydroxysteroid dehydrogenase type from to %, at concentrations ranging from to , m g/ml in the sequence seed oil > > fermented juice polyphenols > pericarp polyphenols. lyophilized fresh pomegranate juice elicited a % inhibition of the estrogenic activity of -b -estradiol; whereas the lyophilized juice by itself displayed only minimal estrogenic action. inhibition of cell lines by fermented juice and pericarp polyphenols was according to estrogen-dependent (mcf- ) > > estrogen-independent (mb-mda- ) > normal human breast epithelial cells (mcf- a). in both mcf- and mb-mda- cells, fermented pomegranate juice polyphenols consistently displayed about twice the anti-proliferative effect as fresh pomegranate juice polyphenols. pomegranate seed oil elicited % inhibition of proliferation of mcf- at m g/ml medium, % inhibition of invasion of mcf- across a matrigel membrane at m g/ml, and % apoptosis in mda-mb- estrogen receptor negative metastatic human breast cancer cells at m g/ml. in a murine mammary gland organ culture, fermented juice polyphenols effected % inhibition of cancerous lesion formation induced by the carcinogen , -dimethylbenz[a] anthracene (dmba). pomegranate seed oil and fermented pomegranate juice polyphenols were found to have anti-angiogenic activity (toi et al. ) . in-vitro studies showed that these pomegranate fractions strongly suppressed vascular endothelial growth factor in normal human breast epithelial cells (mcf- a) and in estrogen sensitive (mcf- ) human breast cancer cells, but upregulated migration inhibitory factor in estrogen resistant (mda-mb- ) human breast cancer cells. an anti-proliferative effect on angiogenic cells was shown in human umbilical vein endothelial cell (huvec) and in myometrial and amniotic fl uid fi broblasts, and inhibition of huvec tubule formation was also demonstrated in an invitro model employing glass carrier beads. additionally, they showed a signi fi cant reduction in new blood vessel formation using the chicken chorioallantoic membrane (cam) model in-vivo. in another study, pretreatment of mouse mammary organ culture with pomegranate fermented juice polyphenols (w), a high-performance liquid chromatographic (hplc) peak separated from w (peak b), or pomegranate seed oil prior to exposure to the to the carcinogen , -dimethylbenz[a] anthracene (dmba) resulted in a % reduction in the number of lesions for w compared with control, peak b and pomegranate seed oil each effected an % reduction (mehta and lansky ) . both pomegranate extracts and genistein inhibit the growth of mcf- breast cancer cells through induction of apoptosis, with combination treatment being more ef fi cacious than single treatments (jeune et al. ) . more recent studies demonstrated that pomegranate fruit extract dose-dependently inhibited nf-kb-dependent reporter gene expression associated with proliferation, invasion, and motility in aggressive breast cancer phenotypes while suppressing rhoc and rhoa protein expression ) . the bioactive components of the fruit extract comprised mainly ellagitannins and phenolic acids in the aqueous fruit extract and conjugated octadecatrienoic acids in the lipid fruit extract derived from seeds. the results suggested a role of pomegranate fruit extract in lowering the metastatic potential of aggressive breast cancer species. pomegranate extract inhibited the proliferation and viability of mmtv-wnt- mouse mammary cancer stem cells in-vitro in a timeand concentration-dependent manner (dai et al. ) . its constituents ellagic ursolic acid and luteolin also caused a time-and concentrationdependent reduction of cell proliferation and viability, suggesting that they contribute to the inhibitory effect of the extract, while caffeic acid had no effect. the methanolic pomegranate fruit peel extract was found to reduce cell proliferation and induce apoptosis on mcf- breast cancer cells (dikmen et al. ) . in addition, expression of the pro-apoptotic gene bax was increased, and that of the anti-apoptotic gene bcl- was decreased after pomegranate extract treatment. the extract exhibited high antioxidant activity and yielded total phenolic content of . mg of gallic acid equivalents/g of extract with ellagic acid as the most abundant constituent. among the ten pomegranate ellagitanninderived compounds (namely ellagic acid, gallagic acid, urolithins a and b and their acetylated, methylated, and sulfated analogues), urolithin b (ub) was shown to most effectively inhibit aromatase activity in a live breast cancer cell assay (adams et al. ) . ub signi fi cantly inhibited testosterone-induced mcf- aro cell proliferation. the remaining test compounds also exhibited antiproliferative activity, but to a lesser degree than ub. the results suggested pomegranate et-derived compounds to have potential for the prevention of estrogen-responsive breast cancers. pomegranate seed linolenic acid isomers, punicic acid and α-eleostearic acid were found to be selective estrogen receptor modulators (serms) in-vitro (tran et al. ) . punicic acid inhibited (ic ) estrogen receptor (er) α at . m m, estrogen receptor β at . m m. α-eleostearic acid (aea) inhibited erα/erβ at . / . m m. punicic acid agonized erα/erβ (ec ) at . / m m, antagonizing at / m m. α-eleostearic acid antagonized erα/erβ at / m m. both isomers induced erα and erβ mrna expression in mcf- breast cancer cells, but not in mda-mb- breast cancer cells. punicic acid, an omega- fatty acid in pomegranate seed oil, was found capable of inhibiting breast cancer proliferation (grossmann et al. ) . proliferation was inhibited and % for mda-mb- and mda-erα cells, respectively. further punicic acid induced apoptosis in the mda-mb- and mda-erα cells by and %, respectively compared to untreated control cells and disrupted cellular mitochondrial membrane potential. the results suggested the breast cancer inhibitor properties of punicic acid were dependent on lipid peroxidation and the protein kinase c signalling pathway. treatment of human lung carcinoma a cells with pomegranate fruit extract resulted in a decrease in the viability of a cells and dosedependent arrest of cells in g -g phase of the cell cycle (khan et al. a, b ) . treatment of cells with pomegranate fruit extract inhibited (i) phosphorylation of mapk proteins, (ii) pi k, (iii) phosphorylation of akt at thr , (iv) nf-kappab and ikkα, (v) degradation and phosphorylation of ikappabα, and (vi) ki- and pcna. oral administration of pomegranate fruit extract ( . and . %, wt/vol) to athymic nude mice implanted with a cells resulted in a signi fi cant inhibition in tumour growth. treatment of mice with pomegranate juice prior to exposure to carcinogens benzo(a)pyrene (b(a) p) and n-nitroso-tris-chloroethylurea (ntcu), resulted in statistically signi fi cant lower lung tumour multiplicities than mice treated with carcinogens only (khan et al. a ) . treatment of cells with pomegranate fruit extract caused inhibition of (a) activation of nuclear factor-kappab and ikappabα kinase, (b) degradation and phosphorylation of ikappabα, (c) phosphorylation of mitogen-activated protein kinases (extracellular signal-regulated kinase / , c-jun nh( )-terminal kinase / , and p ), (d) phosphatidylinositol -kinase (p and p ), (e) phosphorylation of akt at thr( ), (f) activation of mammalian target of rapamycin signaling, (g) phosphorylation of c-met, and (h) markers of cell proliferation (ki- and proliferating cell nuclear antigen) and angiogenesis (inducible nitric oxide synthase, cd , and vascular endothelial growth factor) in lungs of b(a)p-and ntcu-treated mice. overall, the results suggested that pomegranate fruit extract could be a useful chemopreventive/chemotherapeutic agent against human lung cancer. flavonoid-rich polyphenol fractions from the pomegranate fruit had been reported to exert antiproliferative, anti-invasive, anti-eicosanoid, and pro-apoptotic actions in breast and prostate cancer cells and anti-angiogenic activities in-vitro and in-vivo (kawaii and lansky ) . they found that various fruit extracts had proportional inhibitory effects on human hl- promyelocytic leukemia cell proliferation. fermented pomegranate juice and aqueous extract of pomegranate pericarps were found to be strong promoters of differentiation in all settings, while fresh juice extract showed only a relatively mild differentiation-promoting effect. li et al. ( ) found that pomegranate ellagitannins bound with gelatin to form self-assembled nanoparticles. ellagitannins encapsulated in nanoparticles were less effective in inducing the early stage of apoptosis on human promyelocytic leukemia cells hl- . but they had similar effects in inducing late stage of apoptosis and necrosis. differentiation refers to the ability of cancer cells to revert to their normal counterparts, and its induction represents an important noncytotoxic therapy for leukemia, and also breast, prostate, and other solid malignancies (kawaii and lansky ) . pomegranate emulsion treatment ( or g/ kg) to rats, started weeks prior to the dietary carcinogen diethylnitrosamine (dena) challenge and continued for weeks thereafter, showed striking chemopreventive activity demonstrated by reduced incidence, number, multiplicity, size and volume of hepatic nodules, precursors of hepatocellular carcinoma (bishayee et al. ) . both doses of the emulsion signi fi cantly attenuated the number and area of g -glutamyl transpeptidase-positive hepatic foci compared with the dena control. the emulsion also attenuated dena-induced hepatic lipid peroxidation and protein oxidation and elevated protein and messenger rna expression of the hepatic nuclear factor e -related factor (nrf ). the methanolic extract of punica granatum fl owers was exhibited inhibitory effect on tumour necrosis factor-α (tnf-α, ng/ml)induced cytotoxicity in l (murine fi broblast) cells (xie et al. ) . a new taraxastane-type triterpene, punicanolic acid ( ), was isolated from the active fraction (ethyl acetate-soluble fraction) together with four triterpenes ( - ), two galloyl glucoses ( , ), two fl avones ( , ) , and b -sitosterol. among the constituents, , oleanolic acid ( ), maslinic acid ( ), , , -tri ( ), and luteolin ( ) signi fi cantly inhibited tnf-α-induced cytotoxicity in l cells at m m. four pure chemicals, ellagic acid (e), caffeic acid (c), luteolin (l) and punicic acid (p), all important components of the aqueous compartments or oily compartment of pomegranate fruit exhibited anticancerous activities by inhibiting human pc- prostate cancer cell invasion of matrigel arti fi cial membranes (lansky et al. a ) . all compounds signi fi cantly inhibited invasion when employed individually. when c, p, and l were equally combined at the same gross dosage ( m g/ml) as when the compounds were tested individually, a supra-additive inhibition of invasion was observed. pomegranate cold-pressed seed oil, fermented juice polyphenols (w), and pericarp polyphenols (p) each acutely inhibited in-vitro proliferation of human prostate cancer, lncap, pc- , and du human cancer cell lines (albrecht et al. ) . these effects were mediated by changes in both cell cycle distribution and induction of apoptosis. for example, the androgen-independent cell line du showed a signi fi cant increase from to % in g( )/m cells by treatment with pomegranate oil ( m g/ ml) with a modest induction of apoptosis. in other cell lines/treatments, the apoptotic response predominated, for example, in pc- cells treated with pomegranate pericarp polyphenols, at least partially through a caspase -mediated pathway. all agents potently suppressed pc- invasion through matrigel, and furthermore pomegranate pericarp polyphenols and seed oil demonstrated potent inhibition of pc- xenograft growth in athymic mice. overall, the study demonstrated signi fi cant antitumour activity of pomegranatederived materials against human prostate cancer. in another study, combinations of the anatomically discrete pomegranate fractions: fermented pomegranate juice polyphenols (w), pomegranate pericarp (peel) polyphenols (p) or pomegranate seed oil (oil) exhibited synergistic prostate cancer suppression (lansky et al. b ) . supraadditive, complementary and synergistic effects were proven in all models. proliferation effects were additionally evaluated with compusyn software median effect analysis and showed a concentration index ci < , con fi rming synergy. pomegranate fruit extract (pfe) exhibited antiproliferative and proapoptotic activities against human prostate cancer cells (malik et al. ; malik and mukhtar ) . pfe ( - m g/ ml; h) treatment of highly aggressive human prostate cancer pc cells resulted in a dosedependent inhibition of cell growth/cell viability and induction of apoptosis. immunoblot analysis revealed that pfe treatment of pc cells resulted in (i) induction of bax and bak (proapoptotic); (ii) down-regulation of bcl-x(l) and bcl- (antiapoptotic); (iii) induction of waf /p and kip /p ; (iv) a decrease in cyclins d , d , and e; and (v) a decrease in cyclin-dependent kinase (cdk) , cdk , and cdk expression. findings established the involvement of the cyclin kinase inhibitor-cyclin-cdk network during the antiproliferative effects of pfe. oral administration of pfe ( . and . %, wt/vol) to athymic nude mice implanted with androgen-sensitive cwr rnu cells resulted in a signi fi cant inhibition in tumour growth concomitant with a signi fi cant decrease in serum prostate-speci fi c antigen levels. the results suggested that pomegranate juice may have cancer-chemopreventive as well as cancerchemotherapeutic effects against prostate cancer in humans. in a phase ii, simon two-stage clinical trial for men with a rising prostate-speci fi c antigen (psa), daily consumption of pomegranate juice was found to have a positive effect following surgery or radiation for prostate cancer (pantuck et al. ) . there were no serious adverse events reported and the treatment was well tolerated. mean psa doubling time signi fi cantly increased with treatment from a mean of months at baseline to months posttreatment. in-vitro assays comparing pretreatment and posttreatment patient serum on the growth of human prostate cancer lncap showed a % decrease in cell proliferation and a % increase in apoptosis, a % increase in serum nitric oxide, and signi fi cant reductions in oxidative state and sensitivity to oxidation of serum lipids after versus before pomegranate juice consumption. in further studies, a standardized ellagitannins (ets)-enriched pomegranate extract (pe), signi fi cantly inhibited lapc- xenograft growth in severe combined immunode fi cient (scid) mice as compared to vehicle control seeram et al. ) . ellagic acid and several synthesized urolithins were shown to inhibit the growth of human prostate cancer cap cells in-vitro. the chemopreventive potential of pomegranate ets and localization of their bioactive metabolites in mouse prostate tissue suggested that pomegranate may play a role in cap treatment and chemoprevention. the results of studies demonstrated that an ellagitannin-rich pomegranate extract could inhibit tumour-associated angiogenesis as one of several potential mechanisms for slowing the growth of prostate cancer in chemopreventive applications (sartippour et al. ) . a pomegranate extract standardized to ellagitannin content (pomx) inhibited the proliferation of lncap and huvec cells signi fi cantly under both normoxic and hypoxic conditions. hif- α (hypoxia-inducible factor- α) and vegf (vascular endothelial growth factor) protein levels were also reduced by pomx under hypoxic conditions. pomx decreased prostate cancer xenograft size, tumour vessel density, vascular endothelial growth factor (vegf) peptide levels and hif- α expression after weeks of treatment in severe combined immunode fi cient (scid) mice. studies showed that pomegranate extract inhibited androgen-independent prostate cancer growth through a nuclear factor-kappabdependent mechanism . pomegranate extract (pe) inhibited nf-kappab and cell viability of prostate cancer cell lines in a dose-dependent fashion in vitro. maximal pe-induced apoptosis was dependent on pe-mediated nf-kappab blockade. in the lapc xenograft model, pe delayed the emergence of lapc androgen-independent xenografts in castrated mice through an inhibition of proliferation and induction of apoptosis. the scientist also showed that pomegranate polyphenols inhibited gene expression and androgen receptor (ar) most consistently in the human prostate cancer lncap-ar cell line (hong et al. ) . therefore, inhibition by pomegranate polyphenols of gene expression involved in androgen-synthesizing enzymes and the ar may be of particular importance in androgen-independent prostate cancer cells and the subset of human prostate cancers where ar is up-regulated. koyama et al. ( ) demonstrated that pomegranate extract derived from rind and arils (minus seeds) inhibited cell proliferation and induced apoptosis in human lapc prostate cancer cells by modulation of the igf-igfbp (insulin growth factor -insulin growth factor binding proteins) axis. pomegranate extract treatment also decreased igf- mrna expression in a dose-dependent manner indicating that its actions also involved tumour-speci fi c suppression of igf- . pomegranate peel extracts increased the levels of oxygen radical absorbance capacity (orac) in plasma and the density of lecithin and the levels of zn in prostatitic rats (kuang et al. ) . it decreased the levels of malondialdehyde of prostate and the activity of acid phosphatase and the number of white blood cell and adjusted the levels of no in plasma compared with the prostatitis model group. the results indicated that pomegranate peel extracts could markedly improve the protective function of oxidation resistance. pomegranate ellagitannins/microbial metabolites were found to have cyp b (a target in prostate cancer chemoprevention) inhibitory activity in prostate cancer cells (kasimsetty et al. ) . urolithin a, a microbial metabolite, was the most potent uncompetitive inhibitor of cyp b -mediated ethoxyresoru fi n-o -deethylase (erod) activity, exhibiting two-fold selectivity over cyp a , while urolithin b was a noncompetitive inhibitor with three-fold selectivity. the punicalins and punicalagins exhibited potent cyp a inhibition with - -fold selectivity over cyp b . cellular uptake experiments demonstrated a fi ve-fold increase in urolithin uptake by rv cells. western blots of the cyp b protein indicated that the urolithins interfered with the expression of cyp b protein. thus, urolithins were found to display a dual mode mechanism by decreasing cyp b activity and expression. wang et al. ( ) showed that in addition to causing cell death of hormonerefractory prostate cancer cells, pomegranate juice also increased cell adhesion and decreased cell migration of the unkilled cells. pomegranate juice was found to upregulate genes involved in cell adhesion such as e-cadherin, intercellular adhesion molecule (icam- ) and down-regulated genes involved in cell migration such as hyaluranan-mediated motility receptor (hmmr) and type i collagen. in addition, pomegranate juice signi fi cantly decreased the level of secreted pro-in fl ammatory cytokines/chemokines such as il- , il- p , il- b and rantes, thereby having the potential to decrease in fl ammation and its impact. pomegrante juice also inhibited the ability of the chemokine sdf a to chemoattract these cancer cells. faria et al. ( ) found that pomegranate juice consumption decreased total hepatic cytochrome p (cyp) content as well as the expression of cyp a and cyp a in male mice. prevention of procarcinogen activation through cyp activity/expression inhibition may be involved in pomegranate juice's effect on tumour initiation, promotion, and progression pomegranate juice showed greatest antiproliferative activity against all cell lines namely human oral (kb, cal ), colon (ht- , hct , sw , sw ) and prostate (rwpe- , rv ) tumour cells by inhibiting proliferation from to % (seeram et al. a ) . at m g/ml, pomegranate juice, ellagic acid, punicalagin and a standardized total pomegranate tannin (tpt) extract induced apoptosis in ht- colon cells. however, in the hct colon cells, ellagic acid, punicalagin and tpt but not pomegranate juice induced apoptosis. the trend in antioxidant activity was pomegranate juice > tpt > punicalagin > ellagic acid. their data indicated the superior bioactivity of pomegranate juice compared to its puri fi ed individual polyphenolic active ingredients illustrating the multifactorial effects and chemical synergy of the action of multiple compounds. in further studies, they (adams et al. ) found that pomegranate juice signi fi cantly suppressed tnf-α-induced cox- protein expression by %, total pomegranate tannin extract (tpt) %, and punicalagin % in ht- colcon cells. in addition, pomegranate juice reduced phosphorylation of the p subunit and binding to the nfkappab response element . -fold, tpt suppressed nfkappab binding ten-fold, punicalagin . fold, whereas ellagic acid was ineffective. pomegranate juice also abolished tnfαinduced akt activation, needed for nfkappab activity. pomegranate fruit rich in ellagitannins may have bene fi cial effects against colon cancer. in the stomach and gut, ellagitannins were reported to be hydrolyzed to release ellagic acid (ea) and were converted by gut microbiota to urolithin a ( , -dihydroxy- h-dibenzopyran- one) type metabolites (sharma et al. ) . they reported that pomegranate ellagitannin extract, ellagic acid, and their colonic metabolite, urolithin a inhibited wnt signaling, which plays a pivotal role in human colon carcinogenesis, suggesting that et-rich foods may have potential against colon carcinogenesis and that urolithins were relevant bioactive constituents in the colon. studies by gonzález-sarrías et al. ( ) showed that elagic acid and its colonic metabolites, urolithin-a ( , -dihydroxy- h-dibenzo [b,d] pyran- -one) and urolithin-b ( -hydroxy- hdibenzo[b,d]pyran- -one), modulated phase i and phase ii detoxifying enzymes in colon cancer caco- cells. ellagic acid and urolithins may exert some blocking chemopreventive effects in the colon but this effect may be critically affected by interfering factors, such as the food matrix nature. saruwatari et al. ( ) found that pomegranate juice potently inhibited the sulfoconjugation of -naphthol in caco- human colon carcinoma cells. the inhibition was both doseand culture time-dependent, with a % inhibitory concentration (ic ) value of . % (vol/vol). punicalagin, the most abundant antioxidant polyphenol in pomegranate juice, was also found to strongly inhibit sulfoconjugation in caco- cells with an ic of m m. additionally pomegranate juice and punicalagin both inhibited phenol sulfotransferase activity in caco- cells. the data also suggested that constituents of pomegranate juice, most probably punicalagin, impaired the enteric functions of sulfoconjugation and that this may have effects upon the bioavailability of drugs and other compounds and may be related to the anticarcinogenic properties of pomegranate juice. pomegranate seed oil (pgo) rich in % cis (c) , trans (t) ,c - : as conjugated linolenic acids (cla) could suppress by azoxymethane -induced colon carcinogenesis, and the inhibition was associated in part with the increased content of cla in the colon and liver and/or increased expression of peroxisome proliferator-activated receptor (ppar) γ protein in the colon mucosa (kohno et al. ) . pomegranate extract was found to induce cell cycle arrest and alter cellular phenotype of human pancreatic cancer cells panc- and aspc- (nair et al. ) studies by weisburg et al. ( ) showed that pomegranate extract exerted greater antiproliferative effects towards cancer (such as hsc- carcinoma), than to normal, cells, isolated from the human oral cavity. the antiproliferative mechanism of pomegranate extract was, in part, by induction of oxidative stress. the mode of cell death was by apoptosis, as activation of caspase- , and cleavage of parp. reduction of caspase- activation and of parp cleavage in cells co-treated with pomegranate extract and either cobalt or pyruvate, respectively, as compared to pomegranate extract alone, indicated that apoptosis was through the prooxidant nature of pomegranate extract. pomegranate seed oil ( %) signi fi cantly decreased mice skin tumour incidence, multiplicity, and -o -tetradecanoylphorbol -acetate (tpa)-induced ornithine decarboxylase activity, an important event in skin cancer promotion (hora et al. ) . the results suggested the potential of pomegranate seed oil as a safe and effective chemopreventive agent against skin cancer. afaq et al. ( a, b ) demonstrated that topical application of pomegranate fruit extract (pfe) prior to -o -tetradecanoylphorbol- -acetate (tpa) application on mouse skin afforded signi fi cant time-dependent inhibition, against tpa-mediated increase in skin edema and hyperplasia, epidermal ornithine decarboxylase (odc) activity and protein expression of odc and cyclooxygenase- . also, application of pfe resulted in inhibition of tpa-induced phosphorylation of erk / , p and jnk / , as well as activation of nf-kappab and ikkα and phosphorylation and degradation of ikappabα. pretreatment of pfe on tpa-induced skin tumour promotion in , -dimethylbenz(a)anthracene-initiated cd- mouse substantially reduced tumour incidence and lower tumour body burden when assessed as total number of tumours per group, percent of mice with tumours and number of tumours per animal as compared to animals that did not receive pfe. skin application of pfe prior to tpa application also resulted in a signi fi cant delay in latency period from to weeks and afforded protection when tumour data were considered in terms of tumour incidence and tumour multiplicity. studies by george et al. ( ) suggested that pomegranate fruit extract (pfe) and diallyl sul fi de (das) in combination afforded better suppressive activity of mouse skin tumours than either of these agents alone. pfe and das alone delayed onset and tumour incidence by ~ and ~ %, respectively, while their combination at low doses synergistically decreased tumour incidence more potentially (~ %,). further, regression in tumour volume was seen with continuous combinatorial treatment. the inhibition was associated with decreased expression of phosphorylated erk / , jnk and activated nf-k b/p , ikk a , i k b a phosphorylation and degradation in skin tissue/ tumour. polysaccharide (psp ) isolated from pomegranate rind was found to have antioxidant, antitumour and immunomodulatory properties (joseph et al. ) . psp exhibited a dosedependent enhancement in antioxidant activity using concentrations from to , m g/ml when evaluated using various assays such as, ferric reducing antioxidant power assay, linoleic acid emulsion thiocyanate assay, and superoxide, hydroxyl and nitric oxide radical scavenging assays except for the dpph assay for which the highest activity was obtained at m g/ml. psp exhibited anticancer activity with ic values of . and . m g/-ml following h incubation for mcf- (breast cancer), and k (leukemia) cells, respectively. all the pomegranate peel extracts (ethyl acetate (etoac), acetone, methanol and water) decreased sodium azide mutagènicity in salmonella typhimurium strains (ta and ta ), either weakly or strongly (negi et al. ) . at , m g/ plate all the extracts showed strong antimutagenicity. the antimutagenicity of the water extract was followed by acetone, etoac and methanol extracts. the methanol pomegranate peel fraction with promising antioxidant activity showed antimutagenic activity against sodium azide and methyl methane sulphonate with percent inhibition of mutagenicity ranging from . to . % in a concentration-dependent manner using the ames salmonella/microsome assay (zahin et al. a ) . similar trend of inhibition of mutagenicity ( . - . %) against indirect mutagens ( -amino fl uorene and benzo(a)pyrene) was also recorded. phytochemical analysis by hplc, lc-ms of total phenolic content revealed high content of ellagitannins which might be responsible for promising antioxidant and antimutagenic activities of p. granatum peel extract. methanol extract of punica granatum fl owers ( mg/plate) showed the highest antimutagenic activity in salmonella typhimurium ta and ta , respectively (wongwattanasathien et al. ) . the protective effects of these fl ower extracts might be due to the presence of antimutagenic components that were supposed to be fl avonoids. studies demonstrated that tannin from the pericarp of punica granatum was an effective agent against genital herpes simplex virus (hsv- ) (zhang et al. ) . the tannin not only inhibited hsv- replication, but also showed stronger effects of killing virus and blocking its absorption to cells. punica granatum extract showed anti-human herpes simplex virus type (hsv- ) activity, which was possibly contributed by the polyphenolic compounds in the herbal extract (li et al. ) . studies by neurath et al. ( ) indicated that hiv- entry inhibitors from pomegranate juice adsorbed onto corn starch and the resulting complex blocked virus binding to cd and cxcr /ccr and inhibited infection by primary virus clades a to g and group o. their results suggested the possibility of producing an anti-hiv- microbicide from inexpensive, widely available sources. pomegranate juice containing polyphenols, β-sitosterol, sugars and ellagic acid) was reported to inactivate hiv and further shown to inactivate in fl uenza, herpes viruses and poxviruses (kotwal ) . a formulation consisting of fulvic acid, a complex mixture of compounds was previously reported to render vaccinia virus, hiv and sars virus non-infectious. recently, both fulvic acid and pomegranate juice were shown to inactivate genetically diverse strains of in fl uenza including h n , further con fi rming the broad spectrum nature of these agents. sundararajan et al. ( ) found that the acidity of pomegranate juice and concentrated liquid extract contributed to rapid anti-in fl uenza activity, but this was not a factor with pomegranate polyphenols powder ( %) extract. studies using pomegranate powder extract showed that min treatment at room temperature with m g/ml pomegranate polyphenols resulted in at least a log reduction in the titers of in fl uenza viruses pr (h n ), x (h n ), and a reassortant h n virus derived from a human isolate. however, the antiviral activity was less against a coronavirus and reassortant h n in fl uenza viruses derived from avian isolates. electron microscopic analysis indicated that viral inactivation by pomegranate polyphenols was primarily a consequence of virion structural damage. pomegranate polyphenol extract was shown to have anti-in fl uenza virus properties (haidari et al. ) . of four major polyphenols in pomegranate polyphenol extract (ppe) (ellagic acid, caffeic acid, luteolin, and punicalagin) punicalagin was the effective, anti-in fl uenza component. punicalagin blocked replication of the virus rna, inhibited agglutination of chicken rbc's by the virus and had virucidal effects. further, the combination of ppe and oseltamivir synergistically increased the anti-in fl uenza effect of oseltamivir. the data showed ppe inhibited the replication of human in fl uenza a/ hong kong (h n ) virus in-vitro. exposure of foodborne virus surrogates feline calicivirus (fcv-f ), murine norovirus (mnv- ), and ms (ssrna) bacteriophage to pomegranate juice and pomegranate polyphenols resulted in titer reductions after one hour at room temperature, suggesting promise for use in hurdle technologies and/or for therapeutic or preventive use (su et al. ) . ethanolic extracts of garcinia mangostana , punica granatum and quercus infectoria were found to have good antimicrobial activity of nine thai medicinal plants with mics for methicillin-resistant staphylococcus aureus (mrsa) isolates of . - . , . - . and . - . mg/ml, respectively, and for s. aureus atcc of . , . and . mg/ml, respectively (voravuthikunchai and d kitpipit ) . mbcs for mrsa isolates were . - . , . - . and . - . mg/ml, and for s. aureus atcc were . , . and . mg/ml, respectively. punica granatum was found to have anti-quorum-sensing activity and may be useful in combating pathogenic bacteria and reduce the development of antibiotic resistance (koh and tham ; zahin et al. b ) . in another study the ethanolic extract of p. granatum exhibited bacteriostatic and bactericidal activities against two enterohemorrhagic escherichia coli strains (voravuthikunchai and limsuwan (pai et al. ) . ethanol extract of p. granatum exhibited strong antibacterial activity against escherichia coli (sharma et al. ) . studies showed that punica granatum (pomegranate) methanolic extract (pgme) dramatically enhanced the activity of all antibiotics tested (braga et al. a ) . synergic activity was detected between pgme and the fi ve antibiotics tested, chloramphenicol, gentamicin, ampicillin, tetracycline, and oxacillin, ranging from to %. using pgme ( . × mic) in combination with ampicillin ( . × mic), cell viability was reduced by . and . % in methicillin-sensitive staphylococcus aureus (mssa) and methicillin-resistant staphylococcus aureus (mrsa) populations, respectively. pgme increased the post-antibiotic effect (pae) of ampicillin from to h. pomegranate extract inhibited staphylococcus aureus growth and subsequent enterotoxin production (braga et al. b ) . of several thai medicinal plants, the ethanol extract of p. granatum fruit rind displayed the most outstanding in-vitro antibacterial activity with mic of . and . mg/ml and mbc of . and . mg/ml against staphylococcus aureus and escherichia coli respectively (chansakaow et al. ) . the extract was found to contain both hydrolysable and condensed tannins. the methanol pomegranate pericarp extract exhibited maximum antibacterial activity against salmonella typhimurium , salmonella typhi and shigella dysenteriae serotype (pradeep et al. ) . studies showed that the antibacterial activity of pomegranate rind can be enhanced by the addition of metal salts and vitamin c (mccarrell et al. ) . pomegranate rind extracts (pre) exhibited activity against the gram positive organisms at h were inactive against gram negative bacteria. addition of cu + salts to pre solutions extended the activities resulting in no detectable growth being observed for the pre/cu + combination against escherichia coli , pseudomonas aeruginosa and proteus mirabilis . minimal antimicrobial activity was observed following incubation with fe + , mn + or zn + salts alone or in combination with pre against any of the organisms in the test panel. the addition of vitamin c markedly enhanced the activities of both pre/fe + and pre/cu + combinations against staphylococcus aureus . pelargonidin- -galactose, cyanidin- -glucose, gallic acid, quercetin, and myricetin isolated from the methanolic extract of pomegranate fruit exhibited appreciable activity against species of corynebacteria , staphylococcus , streptococcus , shigella , salmonella , bacillus subtilis , vibrio cholera , and escherichia coli (naz et al. ) . however, all these compounds were more inhibitory against gram-positive species. gallic acid exerted the highest inhibitory activity against all the tested bacteria. various tannin-rich fractions from pomegranate byproduct and the ellagitannins, ellagic acid ( ), gallagic acid ( ), punicalins ( ), and punicalagins ( ) displayed antimicrobial activity when assayed against escherichia coli , pseudomonas aeruginosa , candida albicans , cryptococcus neoformans , methicillin-resistant staphylococcus aureus (mrsa), aspergillus fumigatus and mycobacterium intracellulare (reddy et al. ) . compounds and showed activity against p. aeruginosa , c. neoformans , and mrsa. a new antifungal peptide designated as pomegranin, isolated from fresh pomegranate peels, was found to inhibit mycelial growth of the fungi botrytis cinerea and fusarium oxysporum with an ic of and . m m, respectively (guo et al. ) . lyophilized pomegranate juice (lpj) exhibited antilisterial activity in-vitro and in ground beef (lucas and were ) . against fi ve listeria monocytogenes strains, lpj had a mic of . - . % (wt/vol). the lpj ( , , , and min of heating) signi fi cantly inhibited growth of all fi ve l. monocytogenes strains in refrigerated ground cooked beef by . - . log cfu/g at day . heating did not negatively impact lpj antilisterial activity. ethanol peel extract of pomegranate exhibited in-vitro and in-vivo antimicrobial activity against salmonella typhimurium (choi et al. ) . the minimal inhibitory concentrations of their extract were in the range of . - , m g/ml. in a s. typhimurium infection mouse model. the extract was found to have signi fi cant effects on mortality and the numbers of viable s. typhimurium recovered from faeces. although clinical signs and histological damage were rarely observed in the treated mice, the untreated controls showed signs of lethargy and histological damage in the liver and spleen. the results of this study indicated that the peel extract had the potential to provide an effective treatment for salmonellosis. studies on patients with denture stomatitis showed that gel extract of p. granatum may be used as a topical antifungal agent for the treatment of candidosis associated with denture stomatitis (vasconcelos et al. ) . in subsequent studies, punica granatum phytotherapeutic gel and miconazole (daktarin oral gel) exhibited antimicrobial effect against three standard streptococci strains ( streptococcus mutans , streptococcus sanguis and streptococcus mitis ), s. mutans clinically isolated and candida albicans either alone or in association (vasconcelos et al. ) . the minimum inhibitory concentrations of adherence of punica granatum gel against the test organisms were: : for s. mutans (atcc), s. mutans (ci) and s. sanguis ; : for s. mitis and : for c. albicans . the minimum inhibitory concentrations of adherence of miconazole against the same organisms were: : , : , : , : and : , respectively. in experiments with three and four associated microorganisms, the punica granatum gel had greater ef fi ciency in inhibiting microbial adherence than the miconazole. the results of this study suggest that this phytotherapeutic agent might be used in the control of adherence of different microorganisms in the oral cavity. studies showed that the hydroalcoholic extract from punica granatu m fruits was very effective against dental plaque microorganisms, decreasing the colony forming units per milliliter (cfu/ml) by % (menezes et al. ) . while similar values were observed with chlorhexidine, used as standard and positive control ( % inhibition). however, another study found that the gel containing % punica granatum extract was not ef fi cient in preventing supragingival dental plaque formation and gingivitis (salgado et al. ) . methanolic extract of pomegranate peel exhibited antibacterial activity against oral pathogens: staphylococcus aureus and s. epidermidis (abdollahzadeh et al. ) . only at concentration of mg/ml and mg/ml the extract was effective against lactobacillus acidophilus , streptococcus mutans and streptococcus salivarius . the extract did not inhibit actinomyces viscosus and candida albicans . pomegranate rind extract (pre) singularly showed limited ef fi cacy against methicillin-sensitive and -resistant staphylococcus aureus ( mssa, mrsa) respectively but in combination with cu(ii) ions (cupric sulphate), it exhibited moderate antimicrobial effects against clinical isolates of mssa, mrsa and panton-valentine leukocidin positive community acquired mssa (pvl positive ca-mssa) isolates. (gould et al. ) . sastravaha et al. ( ) showed that adjunctive local delivery of extracts from centella asiatica in combination with p. granatum signi fi cantly improved clinical signs of chronic periodontitis such probing pocket depth, attachment level, gingival index at and months and of bleeding index at months in the test group as compared to control. no signi fi cant differences in plaque index were found between the two treatment modalities. the test group also showed statistically greater reduction of interleukin il- β at both and months and lower il- concentration. a study of young adults showed that weeks of thrice daily mouth rinsing with the extract improved salivary measures relevant to oral health including gingivitis (disilvestro et al. ) . salivary changes observed included a reduction in total protein (associated with plaque forming bacteria readings), activities of aspartate aminotransferase (an indicator of cell injury) and α-glucosidase activity (a sucrose degrading enzyme). the changes also included increased activities of the antioxidant enzyme ceruloplasmin and radical scavenging capacity. pomegranate mouth-rinse was found to have an antiplaque effect (bhadbhade et al. ) . aggregatibacter actinomycetemcomitans , porphyromonas gingivalis , and prevotella intermedia strains in-vitro. pomegranate mouth-rinse could be explored as a long-term antiplaque rinse with prophylactic bene fi ts. probiotication improved the antioxidant activity of sweet pomegranate aril juice from . to . %, and sour pomegranate juice from . to . % (fazeli et al. ) . based on the ferric reducing antioxidant power (frap) value, the reducing power of the probioticated pomegranate juices was also much stronger than the nonprobioticated juices. the frap values for sweet and sour probioticated pomegranate juices were . and . mmol/l, respectively, which were notably higher than . and . mmol/l for sweet and sour nonprobioticated juices. total counts of lactobacillus casei gg increased by about three log in sweet and two log in sour juices after h incubation. both fermentated and nonfermentated juices exhibited a potent and widespectrum antibacterial effect, with the highest activity against pseudomonas aeruginosa with the sweet juice showing wider zones of growth inhibition. the results showed that probiotication of sweet and sour pomegranate juices could add to their bene fi cial antioxidant activities. pomegranate byproducts and punicalagins inhibited the growth of pathogenic clostridia and staphylococcus aureus (bialonska et al. b ) . the growth of probiotic lactobacilli and bi fi dobacteria were generally not affected by ellagitannins. the effect of pomegranate ellagitannins on bi fi dobacteria was species-and tannin-dependent. the growth of bi fi dobacterium animalis ssp. lactis was slightly inhibited by punicalagins, punicalins, and ellagic acid. pomegranate ellagitannin-enriched polyphenol extract (pomx) supplementation signi fi cantly enhanced the growth of bi fi dobacterium breve and bi fi dobacterium infantis. bialonska et al. ( a ) found that products of the intestinal microbial transformation of pomegranate ellagitannins may account for systemic antioxidant effects. while moving through the intestines, pomegranate ellagitannins namely ellagic acid and punicalagins are metabolized by gut bacteria into urolithins that readily enter systemic circulation. their study found that the antioxidant activity of urolithins was correlated with the number of hydroxy groups as well as the lipophilicity of the molecule. the most potent antioxidants were urolithins c and d with ic values of . and . m m, respectively, when compared to ic values of . and . m m of the parent ellagic acid and punicalagins, respectively. the dihydroxylated urolithin a showed weaker antioxidant activity, with an ic value . m m, however, the potency was within the range of urolithin a plasma concentrations. numerous laboratory research, animal and human pilot studies had reported on the effectiveness of pomegranate fruit, pomegranate juice and pomegranate fruit polypehnols in reducing heart disease risk factors ldl oxidation, blood pressure, serum angiotensin converting enzyme (ace) activity, cholesterol esteri fi cation, macrophage oxidative status, and macrophage foam cell formation, all of which are steps in atherosclerosis and cardiovascular disease (aviram et al. (aviram et al. , (aviram et al. , aviram and dornfeld ; kaplan et al. ; esmaillzadeh et al. ; fuhrman et al. ; de nigris et al. ; rosenblat et al. a, b ; fuhrman and aviram ; bagri et al. ) . in healthy humans, pomegranate juice consumption decreased ldl susceptibility to aggregation and retention and increased the activity of serum paraoxonase by % (aviram et al. ) . paraoxanase an hdlassociated esterase, could protect against lipid peroxidation. in apolipoprotein e-de fi cient e o mice, oxidation of ldl by peritoneal macrophages was reduced by up to % after pomegranate juice consumption and this effect was associated with reduced cellular lipid peroxidation and superoxide release. the uptake of oxidized ldl and native ldl by mouse peritoneal macrophages obtained after pomegranate juice administration was reduced by %. further, pomegranate juice supplementation of e o mice reduced the size of their atherosclerotic lesions by % and also the number of foam cells compared with control e o mice supplemented with water. the potent antiatherogenic effects in healthy humans and in atherosclerotic mice may be attributable to its antioxidative properties. anti-atherosclerotic properties was attributed to pomegranate potent anti-oxidative characteristics. after consumption of pomegranate juice, a % reduction in serum angiotensin converting enzyme (ace) activity and a % reduction in systolic blood pressure were noted in hypertensive patients . similar dose-dependent inhibitory effect ( %) of pomegranate juice on serum ace activity was observed also in-vitro. additional studies showed that pomegranate juice supplementation to atherosclerotic mice reduced macrophage lipid peroxidation, cellular cholesterol accumulation and development of atherosclerosis (kaplan et al. ) . pomegranate juice supplementation reduced each of the proatherogenic variables. it signi fi cantly induced serum paraoxonase activity and reduced mouse peritoneal macrophage (mpm) lipid peroxide content compared with placebo-treated mice and control mice. pomegranate juice administration to apolipoprotein e-de fi cient e o mice signi fi cantly reduced the oxidized (ox)-ldl mpm uptake by % and mpm cholesterol esteri fi cation and increased macrophage cholesterol ef fl ux by % compared with age-matched, placebo-treated mice. pomegranate juice consumption reduced macrophage ox-ldl uptake and cholesterol esteri fi cation to levels lower than those in -month-old, unsupplemented controls. pomegranate juice supplementation to e o mice with advanced atherosclerosis reduced the lesion size by % compared with place botreated mice. in a separate study, supple mentation of young ( -month-old) e o mice for months with a tannin fraction isolated from pomegranate juice reduced their atherosclerotic lesion size, paralleled by reduced plasma lipid peroxidation and decreased ox-ldl mpm uptake. studies indicated that the proatherogenic effects induced by perturbed shear stress in cultured human coronary artery endothelial cells could be reversed by chronic administration of pomegranate juice (de nigris et al. (de nigris et al. , . pomegranate juice concentrate and pomegranate fruit extract rich in punicalagin reduced the activation of redox-sensitive genes elk- , p-jun, p-creb, and increased enos expression (which was decreased by perturbed shear stress) in cultured endothelial cells and in atherosclerosis-prone areas of hypercholesterolemic mice. furthermore, oral administration of pomegranate juice to hypercholesterolemic mice at various stages of disease reduced signi fi cantly the progression of atherosclerosis and isoprostane levels and increased nitrates. de found that pomegranate juice reverted the potent downregulation of the expression of endothelial nitricoxide synthase (nosiii) induced by oxidized low-density lipoprotein (oxldl) in human coronary endothelial cells. their data suggested that pomegranate juice could exert bene fi cial effects on the evolution of clinical vascular complications, coronary heart disease, and atherogenesis in humans by enhancing the nitric-oxide synthase bioactivity. aviram et al. ( ) reported that pomegranate polyphenols protected low-density lipoprotein (ldl) against cell-mediated oxidation via two pathways, including either direct interaction of the polyphenols with the lipoprotein and/or an indirect effect through accumulation of polyphenols in arterial macrophages (aviram et al. ) . pomegranate polyphenols were shown to reduce the capacity of macrophages to oxidatively modify ldl, due to their interaction with ldl to inhibit its oxidation by scavenging reactive oxygen species and reactive nitrogen species and also due to accumulation of polyphenols in arterial macrophages; hence, the inhibition of macrophage lipid peroxidation and the formation of lipid peroxide-rich macrophages. additionally, pomegranate polyphenols increased serum paraoxonase activity, resulting in the hydrolysis of lipid peroxides in oxidized lipoproteins and in atherosclerotic lesions. these antioxidative and antiatherogenic effects of pomegranate polyphenols were demonstrated in-vitro, as well as in-vivo in humans and in atherosclerotic apolipoprotein e de fi cient mice. dietary supplementation of polyphenol-rich pomegranate juice to atherosclerotic mice signi fi cantly inhibited the development of atherosclerotic lesions and this may be attributed to the protection of ldl against oxidation. subsequent studies indicated that that pomegranate juice consumption by patients with carotid artery stenosis cas decreased carotid carotid intima-media thickness (imt) and systolic blood pressure and these effects could be related to the potent antioxidant characteristics of pomegranate juice polyphenols (aviram et al. ) . for all studied parameters, the maximal effects were observed after year of pomegranate juice consumption. further consumption of pomegranate juice, for up to years, had no additional bene fi cial effects on imt and serum paraoxonase (pon ) activity, whereas serum lipid peroxidation was further reduced by up to % after years of pomegranate juice consumption. the antiatherogenic properties of pomegranate juice (pj) were attributed to its antioxidant potency and to its capacity to decrease macrophage oxidative stress, the hallmark of early atherogeneis (rosenberg et al. ). pomegranate juice polyphenols and sugar-containing polyphenolic anthocyanins were shown to confer pj its antioxidant capacity. their study showed that pj sugar consumption by diabetic mice for days resulted in a small but signi fi cant decrement in their peritoneal macrophage total peroxide levels and an increment in cellular glutathione content, compared to mouse peritoneal macrophages harvested from control diabetic mice administrated with water. these antioxidant/antiatherogenic effects could be due to the presence of unique complex sugars and/or phenolic sugars in pj. they further showed the anti-oxidative characteristics of pj unique phenolics punicalagin and gallic acid could be related, at least in part, to their stimulatory effect on macrophage paraoxonase (pon ) expression, a phenomenon which was shown to be associated with activation of the transcription factors papr γ and ap- (shiner et al. ) . similar results were obtained by pomegranate byproduct (which includes the whole pomegranate fruit left after juice preparation) ( or . m g of gallic acid equiv/kg/day) administration to apolipoprotein e-de fi cient mice that resulted in attenuation of atherosclerosis development as a result of decreased macrophage oxidative stress and reduced cellular uptake of oxidized low-density lipoprotein (rosenblat et al. ) . in-vitro studies showed that preincubation of j .a macrophages with pomegranate juice resulted in a signi fi cant reduction in ox-ldl degradation by % (fuhrman et al. ) . macrophage cholesterol biosynthesis was inhibited by % after cell incubation with pomegranate juice. this inhibition, however, was not mediated at the -hydroxy- methylglutaryl coenzyme a reductase level along the biosynthetic pathway. it was concluded that pomegranate juice-mediated suppression of ox-ldl degradation and of cholesterol biosynthesis in macrophages could lead to reduced cellular cholesterol accumulation and foam cell formation. studies in iran reported that consumption of concentrated pomegranate juice may modify heart disease risk factors in hyperlipidemic ( cholesterol ³ . mmol/l or triacylglycerol ³ . mmol/l) patients (esmaillzadeh et al. (esmaillzadeh et al. , . after consumption of concentrated pomegranate juice, signi fi cant reductions were seen in total cholesterol, low-density lipoprotein (ldl)-cholesterol, ldl-cholesterol/high-density lipoprotein (hdl)-cholesterol, and total cholesterol/hdl-cholesterol. but, there were no signi fi cant changes in serum triacylglycerol and hdl-cholesterol concentrations. anthropometric indices, physical activity, kind and doses of oral hypoglycemic agents, and the intakes of nutrients and fl avonoid-rich foods showed no change during the concentrated pomegranate juice consumption period. rosenblat et al. ( ) reported that pomegranate juice consumption by diabetic patients did not affect serum glucose, cholesterol and triglyceride levels, but it resulted in a signi fi cant reduction in serum lipid peroxides and tbars (thiobarbituric acid reactive substance) levels by and %, whereas serum sh (sulfhydryl) groups and paraoxonase (pon ) activity signi fi cantly increased by and %, respectively. in the patients versus controls monocytes-derived macrophages (hmdm), they observed increased level of cellular peroxides (by %), and decreased glutathione content (by %). pomegranate juice consumption signi fi cantly reduced cellular peroxides (by %), and increased glutathione levels (by %) in the patients' hmdm. the patients' versus control hmdm took up oxidized ldl (ox-ldl) at enhanced rate (by %) and pomegranate juice consumption signi fi cantly decreased the extent of ox-ldl cellular uptake (by %). they thus concluded that pomegranate juice consumption by diabetic patients did not worsen the diabetic parameters, but rather resulted in anti-oxidative effects on serum and macrophages, which could contribute to attenuation of atherosclerosis development in these patients. pomegranate juice was found to have potent antiatherogenic activity . in-vitro studies demonstrated a pomegranate juice dose-dependent antioxidant capability against lipid peroxidation in plasma (by %), in ldl (by %), and in hdl (by %). pomegranate juice consumption by hypertensive patients reduced their systolic blood pressure (by %), along with inhibition (by %) of angiotensin converting enzyme (ace). pomegranate juice supplementation to atherosclerotic apolipoprotein e-de fi cient (e°) mice reduced their atherosclerotic lesion size by % and the number of foam cells in their lesion. consumption of pomegranate juice by ten patients with carotid artery stenosis (cas) for year reduced the patients' carotid intima-media thickness (imt) by %. these effects were associated with exvivo reduced lipid peroxidation in plasma and in isolated lipoproteins in humans and mice. furthermore, pomegranate juice consumption by humans increased the activity of their serum paraoxonase (pon ), an hdl-associated esterase that protects against lipid peroxidation. macrophage atherogenicity was studied in mouse peritoneal macrophages (mpm) harvested from e° mice. following pomegranate juice consumption, uptake of oxidized ldl and cell-mediated oxidation of ldl by macrophages was reduced by and by %, respectively, in association with reduced cellular lipid peroxidation, reduced superoxide anion release due to decreased nadph-oxidase activation, and elevated glutathione content. in-vitro studies demonstrated that pomegranate juice reduced macrophage ox-ldl degradation by %, and macrophage cholesterol biosynthesis by %. overall, the results of the above studies demonstrated that pomegranate juice consumption had very potent antiatherogenic properties, which could be associated mainly with pomegranate juice hydrolysable tannin antioxidative properties. in a recent study ) pomegranate juice (pj), fruit peels (pomxl, pomxp), arils (poma), seeds (pomo), and fl owers (pomf), extracts all were found to possess antioxidative properties in-vitro. after consumption of pomegranate juice, fruit peel, aril and fl ower extracts the atherosclerotic lesion area in atherosclerotic apolipoprotein e-de fi cient (e ) mice was signi fi cantly decreased by , , , , or %, respectively, as compared to placebo-treated group, while pomegranate seed oil had no effect. pomegrante fl ower consumption reduced serum lipids, and glucose levels by - %. consumption of the extracts except for the seed oil resulted in a signi fi cant decrement, by , , , , or %, respectively, in mpm (mouse peritoneal macrophage) total peroxides content, and increased cellular paraoxonase (pon ) activity, as compared to placebo-treated mice. the uptake rates of oxidized-ldl by e ( )-mpm were signi fi cantly reduced by approximately % after consumption of juice and the two fruit peel extracts. similar results were obtained on using j a. macrophage cell line. finally, pomegranate phenolics (punicalagin, punicalin, gallic acid, and ellagic acid), as well as pomegranate unique complexed sugars, could mimic the antiatherogenic effects of the pomegranate extracts. rock et al. ( ) reported that after weeks of pomegranate juice consumption by male patients, basal serum oxidative stress was signi fi cantly decreased by %, whereas serum concentrations of thiol groups signi fi cantly increased by %. moreover, hdlassociated paraoxonase (pon ), arylesterase, paraoxonase, and lactonase activities increased signi fi cantly after pomegranate juice consumption by - %, as compared to the baseline levels. pon protein binding to hdl was signi fi cantly increased by % following pomegranate juice consumption, and the enzyme became more stable. in male patients that consumed pomegranate polyphenol extract and in female patients that consumed pomegranate juice, a similar pattern was observed, although to a lesser extent. these bene fi cial effects of pomegranate consumption on serum pon stability and activity could lead to retardation of atherosclerosis development in diabetic patients. results of a randomized, double-blind, parallel trial involving men ( - years old) and women ( - years old) with moderate coronary heart disease risk suggested that in subjects at moderate coronary heart disease risk, pomegranate juice consumption had no signi fi cant effect on overall carotid intima-media thickness progression rate but may have slowed carotid intima-media thickness progression in subjects with increased oxidative stress and disturbances in the triglycerides-rich lipoprotein/high-density lipoprotein axis (davidson et al. ) . lei et al. ( ) reported that the pomegranate leaf extract could inhibit the development of obesity and hyperlipidemia in high-fat diet induced obese mice. mice treated with the extract presented a signi fi cant decrease in body weight, energy intake and various adipose pad weight percents and serum, serum total cholesterol (tc), triglyceride (tg), glucose levels and tc/highdensity lipoprotein cholesterol (hdl-c) ratio after weeks treatment. further, the extract signi fi cantly attenuated the raising of the serum tg level and inhibited the intestinal fat absorption in mice given a fat emulsion orally. the effects were postulated to be partly mediated by inhibiting the pancreatic lipase activity and suppressing energy intake. yamasaki et al. ( ) found that mice fed dietary pomegranate seed oil (pso) high in levels of punicic acid showed signi fi cant increases in serum triacylglycerol and phospholipid levels but not in total cholesterol. punicic acid could be detected in serum, liver, and adipose tissues in mice fed the . or . % pso diet. oral administration of streptozotocin-induced diabetic wistar rats with of and mg/kg of aqueous pomegranate fl ower extract for days resulted in a signi fi cant fall in fasting blood glucose, total cholesterol, triglycerides, low-density lipoprotein cholesterol , very low density lipoprotein, lipid peroxidation level (bagri et al. ) . pomegrante extract elevated levels of high density lipoprotein cholesterol (hdl-c), reduced glutathione (gsh) and the antioxidative enzymes, glutathione peroxidase (gpx), glutathione reductase (gr), glutathione-s-transferase (gst), superoxide dismutase (sod) and catalase (cat). mcfarlin et al. ( ) found that weight gain in high fat diet mice was associated with an increase in biomarkers of cholesterol pro fi le, glucose sensitivity, adipose tissue accumulation and systemic low-grade in fl ammation. despite a similar level of energy intake, high-fate diet mice had a greater concentration of leptin and a lower concentration of adiponectin compared to high fat + pomegranate seed oil diet mice. pomegranate seed oil, a rich source of -cis , -trans conjugate linolenic acid, only altered body weight accumulation, fi nal body weight, leptin, adiponectin and insulin. pomegranate seed oil intake was associated with an improvement in insulin sensitivity, suggesting that risk of developing type two diabetes may have been reduced; however, cvd risk did not change. lan et al. ( ) demonstrated that ellagic acid in pomegranate leaf tannins could be transported into hepg cells and this correlated with total cholesterol alteration in the cells. vroegrijk et al. ( ) found that pomegranate seed oil, a rich source of punicic acid, ameliorated high-fat diet induced obesity and insulin resistance in mice, independent of changes in food intake or energy expenditure. compared to high fat diet mice, its intake resulted in a lower body weight and improved peripheral insulin sensitivity but did not affect liver insulin sensitivity. in a randomized, double-blind, placebo-controlled clinical trial of obese adult volunteer, pomegranate juice administration for month did not modify insulin secretion and sensitivity in the obese patients, however, the natural evolution to increased weight and adiposity was halted (gonzález-ortiz et al. ) . rosenblat and aviram ( ) found that the inhibitory effect of pomegranate juice on triglyceride biosynthesis could be attributed to a direct effect of pomegranate juice on the activity of diacylglycerol acyltransferase (dgat ) the rate-limiting enzyme in triglyceride biosynthesis. pomegranate juice and its constituent punicalagin signi fi cantly and dose-dependently decreased the triglyceride content and triglyceride biosynthesis rate in j a. macrophages or in c bl/ mouse peritoneal macrophages. both pomegranate juice and punicalagin increased ( . -fold) mouse peritoneal macrophages paraoxonase (pon ) mrna expression, and pon was previously shown to inhibit dgat activity. however, the addition of pj or punicalagin ( m m) to microsomes from pon -de fi cient mouse peritoneal macrophages still resulted in a signi fi cant reduction ( - %) in dgat activity. in a randomised block design study of student volunteers, supplementation of pomegranate juice caused a fall in diastolic blood pressure and this could be related to ros scavenging activity rather than to angiotensin-converting enzyme inhibitors (wright and pipkin ) oral administration of pomegranate juice extract ( and mg/kg) to angiotensin-ii treated rats for weeks signi fi cantly reduced the mean arterial blood pressure and vascular reactivity changes to various catecholamines (waghulde et al. ) . pomegranate juice administration signi fi cantly decreased the serum levels of ace (angiotensin converting enzyme) and the levels of thiobarbituric acid reactive substances (tbars); while enzyme activity of superoxide dismutase (sod), catalase (cat), glutathione reductase (gsh) in kidney tissue showed a signi fi cant elevation in pomegranate juice treated angiotensin-ii induced hypertensive rats. the results suggested that pomegranate juice extract could prevent the development of high blood pressure induced by angiotensin-ii probably by combating the oxidative stress and antagonizing the physiological actions of angiotensin-ii. chronic administration of pomegranate fruit juice (pj) extract ( and mg/kg; p.o. for weeks) reduced the mean arterial blood pressure and vascular reactivity changes to various catecholamines and also reversed the biochemical changes induced by diabetes and angiotensin ii (ang ii) (mohan et al. b ) . acute subcutaneous administration of angiotensin ii causes a rise in blood pressure in streptozotocin-induced diabetic wistar rats. pj treatment also caused a signi fi cant decrease in levels of thiobarbituric acid reactive substances (tbars) in the kidney and pancreas while activities of enzymes superoxide dismutase (sod), catalase (cat), and glutathione reductase (gsh) showed signi fi cant elevation. pj treatment prevented the tubular degenerative changes induced by diabetes. the results suggested that the pj extract could prevent the development of high blood pressure induced by ang ii in diabetic rats probably by combating the oxidative stress induced by diabetes and ang ii and by inhibiting ace activity. pomegranate in particular its fl owers, seeds, and juice have been employed for the treatment of various diseases in traditional unani and ayurvedic systems of medicine in india but only the fl ower has been prescribed for the treatment of diabetic disorders katz et al. ) . the mechanisms for it hypoglycaemic effects are largely unknown, though recent research suggested pomegranate fl owers and juice may prevent diabetic sequelae via peroxisome proliferator-activated receptor (ppar) α/γ binding and nitric oxide production (katz et al. ; huang et al. a, b ; li et al. ; xu et al. ) . pomegranate compounds associated with such effects include oleanolic, ursolic, and gallic acids (katz et al. ) . another study suggested that punica granatum fl ower (pgf) extract inhibited increased cardiac fatty acid uptake and oxidation in the diabetic condition (huang et al. b ) . pgf extract and its component oleanolic acid enhanced peroxisome proliferator-activated receptor (ppar)-α luciferase reporter gene activity in human embryonic kidney cells. this effect was completely suppressed by a selective ppar-α antagonist mk- , consistent with the presence of ppar-α activator activity in the extract and this component. the fi ndings suggested that pgf extract improved abnormal cardiac lipid metabolism in zucker diabetic fatty rats by activating ppar-α and thereby lowering circulating lipid and inhibiting its cardiac uptake. excess triglyceride (tg) accumulation and increased fatty acid (fa) oxidation in the diabetic heart contribute to cardiac dysfunction. in subsequent in-vitro studies, the scientists (huang et al. a ) demonstrated that -week oral administration of methanol extract from pgf ( mg/kg, daily) inhibited glucose loading-induced increase of plasma glucose levels in zucker diabetic fatty rats (zdf), a genetic animal model for type two diabetes, whereas it did not inhibit the increase in zucker lean rats (zl). the treatment did not lower the plasma glucose levels in fasted zdf and zl rats. further, rt-pcr results demonstrated that the pgf extract treatment in zdf rats enhanced cardiac ppar-γ mrna expression and restored the down-regulated cardiac glucose transporter (glut)- (the insulin-dependent isoform of gluts) mrna. these results suggest that the anti-diabetic activity of pgf extract may result from improved sensitivity of the insulin receptor. from the in-vitro studies, it was demonstrated that the pgf extract enhanced ppar-γ mrna and protein expression and increased ppar-γ-dependent mrna expression and activity of lipoprotein lipase in human thp- -differentiated macrophage cells. phytochemical investigation demonstrated that gallic acid in pgf extract was mostly responsible for this activity. further in-vitro studies showed that punica granatum fl ower extract and its components oleanolic acid, ursolic acid, and gallic acid inhibited lipopolysaccharide-induced nf-kappab activation in macrophages. the fi ndings indicated that punica granatum fl ower extract reduced cardiac fi brosis in zucker diabetic fatty rats, at least in part, by modulating cardiac et- and nf-kappab signalling. recent studies suggested that pomegranate fl ower (pgf) medicine ameliorated diabetes and obesity-associated fatty liver, at least in part, by activating hepatic expression of genes responsible for fatty acid oxidation ) . pgf-treated zdf rats showed reduced ratio of liver weight to tibia length, hepatic triglyceride contents and lipid droplets. these effects were accompanied by enhanced hepatic gene expression of peroxisome proliferator-activated receptor (ppar)-α, carnitine palmitoyltransferase- and acyl-coa oxidase (aco), and reduced stearoyl-coa desaturase- . in contrast, pgf showed minimal effects on expression of genes responsible for synthesis, hydrolysis or uptake of fatty acid and triglycerides. pgf treatment also increased ppar-α and aco mrna levels in hepg cells. li et al. ( ) reviewed the dual ppar-α/-γ activator properties of pomegranate fl ower and its potential treatment of diabetes and its associated complications. ppars are nuclear transcription factors and are the major regulators of lipid and glucose metabolism. ppar-α is involved in the regulation of fatty acid (fa) uptake and oxidation, in fl ammation and vascular function, while ppar-γ participates in fa uptake and storage, glucose homeostasis and in fl am mation. synthetic ppar-α or ppar-γ agonists have been widely used in the treatment of dyslipidaemia, hyperglycaemia and their complications. however, they are associated with an incidence of adverse events. given the favourable metabolic effects of both ppar-α and ppar-γ activators, as well as their potential to modulate vascular disease, com bined ppar-α/-γ activation has recently emerged as a promising concept, leading to the development of mixed ppar-α/-γ activators. hontecillas et al. ( ) demonstrated that punicic acid (pua), a conjugated linolenic acid isomer found in pomegranate, caused a dosedependent increase ppar α and γ reporter activity in t -l pre-adipocyte cells and bound although weakly to the ligand-binding domain of human ppar γ. dietary pua decreased fasting plasma glucose concentrations, improved the glucose-normalizing ability, suppressed nf-kappab activation, tnf-α expression and upregulated ppar αand γ-responsive genes in skeletal muscle and adipose tissue. pua improved glucose homeostasis and suppress obesity-related in fl ammation studies in india showed that pomegranate seed extract ( , and mg/kg) administered orally to streptozotocin (stz)-induced diabetic rats caused a signi fi cant reduction of blood glucose levels by and %, respectively, at the end of h (das et al. ) . kim et al. ( ) found that administration of pomegranate extract to streptozotocin (stz)-induced diabetic mice for weeks improved postprandial glucose regulation. further elevated na(+)-dependent glucose uptake by brush border membrane vesicles isolated from stz mice was normalized by pomegranate treatment. the results suggested that pomegranate extract could play a role in controlling the dietary glucose absorption at the intestinal tract by decreasing sodium-coupled glucose transporter sglt expression, and may contribute to blood glucose homeostasis in the diabetic condition. oral administration of pomegranate fl ower (pgf) extract markedly lowered plasma glucose levels in non-fasted zucker diabetic fatty rats (a genetic model of obesity and type two diabetes), whereas it had little effect in the fasted animals, suggesting it affected postprandial hyperglycemia in type two diabetes . the extract was found to markedly inhibit the increase of plasma glucose levels after sucrose loading, but not after glucose loading in mice, and it had no effect on glucose levels in normal mice. in-vitro, pgf extract demonstrated a potent inhibitory effect on α-glucosidase activity (ic : . m g/ml). these fi ndings strongly suggested that pgf extract improved postprandial hyperglycemia in type two diabetes and obesity, at least in part, by inhibiting intestinal α-glucosidase activity. postprandial hyperglycemia plays an important role in the development of type two diabetes and has been proposed as an independent risk factor for cardiovascular diseases. in a recent paper, bagri et al. ( a ) reported that oral administration of pomegranate aqueous extract at doses of and mg/kg for days to stzinduced diabetic rats resulted in a signi fi cant reduction in fasting blood glucose, total cholesterol (tc), triglycerides (tg), low-density lipoprotein cholesterol (ldl-c), very low density lipoprotein (vldl), and tissue lipid peroxidation levels coupled with elevation of high density lipoprotein cholesterol (hdl-c), glutathione (gsh) content and antioxidant enzymes in comparison with diabetic control group. the results suggested that pg could be used, as a dietary supplement, in the treatment of chronic diseases characterized by atherogenous lipoprotein pro fi le, aggravated antioxidant status and impaired glucose metabolism and also in their prevention. in-vitro studies showed that pomegranate juice polyphenols increased recombinant paraoxonase- binding to high-density lipoprotein (hdl) beyond their antioxidative effect (fuhrman et al. ) . further recombinant paraoxonase- was found to be associated more ef fi ciently with hdls isolated from diabetic patients after pomegranate juice consumption versus hdls isolated before pomegranate juice consumption. studies by ahmed et al. ( ) showed that pomegranate fruit extract or compounds derived from it may inhibit cartilage degradation in osteoarthritis and may also be a useful nutritive supplement for maintaining joint integrity and function. the extract inhibited interleukin (il)- β induced expression of matrix metalloproteinases by suppressing the activation of mitogen-activated protein kinases and nuclear factor-kappab in human chondrocytes in-vitro. pomegranate methanol extract was found to dose-dependently inhibit tumour necrosis factor α (tnf-α) production in lipopolysaccharide (lps) stimulated cells (jung et al. ) . the data suggested that the extract may suppress lps-stimulated tnf production through inhibition of nfkappab in bv microglia cells. shukla et al. ( b ) reported that consumption of hydrolyzable tannins-rich pomegranate extract potently delayed the onset and reduced the incidence and severity of collagen-induced arthritis in mice. pomegranate extract -fed mice had reduced joint in fi ltration by the in fl ammatory cells, and the destruction of bone and cartilage were alleviated. levels of interleukin il- were signi fi cantly decreased in the joints of pomegranate-fed mice with collagen-induced arthritis. in mouse macrophages, pomegranate extract abolished multiple signal transduction pathways and downstream mediators implicated in the pathogenesis of rheumatoid arthritis. in another study, rabbit plasma samples collected after oral ingestion of polyphenol rich pomegranate fruit extract were found to inhibit the il- β-induced pge and no production in chondrocytes (shukla et al. a ) . these same plasma samples also inhibited both cox- and cox- enzyme activity ex-vivo but the effect was more pronounced on the enzyme activity of cox- enzyme. the studies suggested that pomegranate fruit extract-derived bioavailable compounds may exert an anti -in fl ammatory effect by inhibiting the in fl ammatory cytokineinduced production of pge and no in-vivo. pomegranate extract rich in polyphenols was found to inhibit the interleukin- b -induced activation of mkk- , p a -mapk and transcription factor runx- in human osteoarthritis chondrocytes (rasheed et al. ) . this pharmacological actions of pomegranate extract suggest that the extract or its derived compounds may be developed as mkk and p -mapk inhibitors for the treatment of osteoarthritis and other degenerative/ in fl ammatory diseases. in a pilot week openlabelled study, pomegranate consumption reduced the composite disease activity index (das ) and tender joint count in rheumatoid arthritis patients, and this effect could be related to the antioxidative property of pomegranates (balbir-gurman et al. ) . the results suggested dietary supplementation with pomegranates may be a useful complementary strategy to attenuate clinical symptoms in rheumatoid arthritis patients. supplementation of obese zucker rats with pomegranate fruit extract (pfe) or pomegranate juice (pj) signi fi cantly decreased the expression of vascular in fl ammation markers, thrombospondin (tsp), and cytokine tgfβ , whereas seed oil supplementation had a signi fi cant effect only on tsp- expression (de nigris et al. a ) . plasma nitrate and nitrite (no(x)) levels were signi fi cantly increased by pfe and pj. in addition, the effect of pfe in increasing endothelial no synthase (enos) expression was comparable to that of pj. the data suggested possible clinical applications of pfe in metabolic syndrome (clinical conditions such as obesity, hypertension, dislipidemia, and diabetes). in-vivo studies revealed that aqueous pomegranate peel extract inhibited neutrophil myeloperoxidase activity and attenuated lipopolysaccharide-induced lung in fl ammation in mice (bachoual et al. ) . inhibition of myeloperoxidase activity by pomegranate extract could explain its antiin fl ammatory action. balwani et al. ( ) demonstrated that -methyl-pyran- -one- -o -b -d-glucopyranoside (mpg) isolated from pomegranate leaves, inhibited tnf a -induced cell adhesion molecules expression by blocking nuclear transcription factor-k b (nf-k b) translocation and activation. the results suggested that mpg could be useful as a novel lead molecule for developing future antiin fl ammatory agents. oral pomegranate extract decreased reactive oxygen species concentration and acute in fl ammation in the tympanic membrane in rats after myringotomy (kahya et al. ) . the density of in fl ammatory cells was signi fi cantly less in rats treated with the extract and the lamina propria thickness and vessel density were also signi fi cantly reduced. pretreatment of wistar rats with a methanolic extract of pomegranate peel followed by carbon tetrachloride treatment retained catalase, peroxidase, and superoxide dismutase to values comparable with control values, whereas lipid peroxidation was reduced by % as compared to control (chidambara murthy et al. ) . histopathological studies of the liver supported the hepatoprotective effects exhibited by the extract by restoring the normal hepatic architecture. kaur et al. ( ) demonstrated that pre-treatment of mice with pomegranate fl ower extract at a dose regimen of - mg/ kg body weight for a week signi fi cantly and dose dependently protected against ferric nitrilotriacetate (fe-nta)-induced oxidative stress as well as hepatic injury. the extract conferred up to % protection against hepatic lipid peroxidation and preserved glutathione (gsh) levels and activities of antioxidant enzymes viz., catalase (cat), glutathione peroxidase (gpx) glutathione reductase (gr) and glutathione-s-transferase (gst) by up to , . , . , . and . % respectively. a protection against fe-nta induced liver injury was apparent as inhibition in the modulation of liver markers viz., aspartate aminotransferase (ast), alanine aminotransferase (alt), alkaline phosphatase (alp), bilirubin and albumin in serum. the histopathological changes produced by fe-nta, such as ballooning degeneration, fatty changes, necrosis were also alleviated by the extract. the fl ower extract was found to signi fi cantly scavenge superoxide radicals by up to . %, hydrogen peroxide by up to %, hydroxyl radicals by up to % and nitric oxide by up to . %. the extract also inhibited (.)oh induced oxidation of lipids and proteins in vitro. the potent antioxidant property of the fl ower extract was postulated to be responsible for its hepatoprotective effects. in another study, pomegranate fl ower infusion was found to exhibit hepatoprotective and antioxidant effect against trichloroacetic acid (tca)-exposed rats (celik et al. ) . the infusion signi fi cantly decreased levels of aspartate aminotransferase and alanine aminotransferase; increased signi fi cantly glutathione s-transferase activity in the liver, brain and spleen and maintained superoxide dismustase level in the liver. intake of pomegranate juice by mice for weeks was found to confer hepatic protection against protein and dna oxidation (faria et al. b ) . there was also a signi fi cant decrease in gsh (reduced glutathione) and gssg (oxidized glutathione), without change in the gsh/gssg ratio. all studied enzymatic activities (superoxide dismutase (sod), glutathione peroxidase (gpx), glutathione s-transferase (gst) and glutathione reductase (gr) and catalase) were found to be decreased by pomegranate juice treatment. also, glutathione s-transferase and glutathione synthetase transcription were also decreased in this group. chronic pomegranate peel extract administration to rats alleviated the bile duct ligation (bdl)-induced oxidative injury of the liver and improved the hepatic structure and function (toklu et al. ) . plasma antioxidant capacity and hepatic glutathione levels were signi fi cantly depressed by bdl but were increased back to control levels in the pomegranate extract-treated bdl group. increases in tissue malondialdehyde levels and myeloperoxidase activity due to bdl were reduced back to control levels by pomegranate extract treatment. similarly, increased hepatic collagen content in the bdl rats was reduced to the level of the control group with extract treatment. sumner et al. ( ) showed that daily consumption of pomegranate juice may improve stressinduced myocardial ischemia in patients who have coronary heart disease in a randomized, placebo-controlled, double-blind study. after months, the extent of stress-induced ischemia decreased in the pomegranate group (sds − . ± . ) but increased in the control group (sds . ± . ). this bene fi t was observed without changes in cardiac medications, blood sugar, hemoglobin a c, weight, or blood pressure in either group. mohan et al. ( a ) demonstrated that pre-treatment of male wistar rats with pomegranate juice ( and mg/kg, p.o.) and its butanolic extract( mg/kg., p.o.) for a period of days signi fi cantly inhibited the effects of isoproterenol-induced myocardial infarction such as heart rate, pressure rate index, ecg patterns, levels of lactate dehydrogenase, creatine kinase, superoxide dismutase and catalase in the serum and vascular reactivity changes. treatment with pj and b-pj ( mg/kg., p.o.) alone did not alter any of the parameters as compared to vehicletreated wistar rats. hassanpour et al. ( ) found that pomegranate fruit extract displayed cardioprotective doxorubicin (dox)-induced cardiotoxicity in rats. rats administered the extract showed decreased qt and increase in heart rate compared to the dox group signi fi cant decrease in creatine kinase-mb isoenzyme, lactate dehydrogenase and no such signi fi cant decrease in aspartate aminotransferase were observed as compared to the dox group. there was signi fi cant increase in the level of reduced glutathione, whereas inhibition of lipid peroxidation and increase in superoxide dismutase concentration was not signi fi cant in the extract treated group compared to the dox group. histopathological study of the extract -treated group showed slight protection against myocardial toxicity induced by dox. p. granatum fruit peel extract elicited % precipitation of ovine haemoglobin in-vitro and when orally administered to ethanol-induced gastric-damaged rats produced a signi fi cant decrease in gastric lesions (gharzouli et al. ) . the acid content of the stomach was signi fi cantly increased by pomegranate ( %) suggesting that monomeric and polymeric polyphenols could strengthen the gastric mucosal barrier. administration of % methanolic pomegranate rind extract inhibited aspirin-and ethanol-induced gastric ulceration (ajaikumar et al. ) . treated animals showed increased antioxidant levels of superoxide dismutase (sod), catalase, glutathione (gsh) and glutathione peroxidase (gpx) and decreased level of tissue lipid peroxidation. no erosion of gastric mucosa, sub-mucosal edema and neutrophil in fi ltration was observed in treated animals. pomegranate tannins ( , , mg/ kg) signi fi cantly inhibited ulcerative formation induced by both water immersion stress and pylorus ligation, and decreased the gastric mucosa damages induced by intragastric absolute ethanol, in dose-dependent manner in rats (lai et al. ) . its antiulcer effect was found to be due to increasing secretion of adherent mucus and free mucus from the stomach wall, which may inhibit generation of oxygen-derived free radicals, and decrease the consumption glutathione peroxidase (gsh-px) and superoxide dismutase (sod), decrease absolute alcohol-induced elevation of malondialdehyde and maintain content of no at normal level. punica granatum peel extract (ppe) supplementation of irradiated rats reduced oxidative damage in the ileal tissues and protected against ionizing radiation-induced enteritis and leukocyte apoptosis in rats, probably by a mechanism associated with the decreased production of reactive oxygen metabolites and enhancement of antioxidant mechanisms (toklu et al. ) . ppe treatment reversed all these biochemical indices induced by irradiations such as the decrease in glutathione and total antioxidant capacity associated with increases in malondialdehyde levels, myeloperoxidase activity, collagen content of the tissue with a concomitant increase -hydroxy- ¢deoxyguanosine (an index of oxidative dna damage) and increases in pro-in fl ammatory cytokines (tnf-α, il- β and il- ) and lactate dehydrogenase. histopathological alterations and the increase in leukocyte apoptosis and cell death induced by irradiation was also reversed by ppe. oral administration of aqueous methanolic extract of pomegranate ( and mg/kg bw) signi fi cantly reduced the ulcer lesion index produced by alcohol, indomethacin, and aspirin, at both doses in rats (alam et al. ) . in pylorusligated rats the extract signi fi cantly reduced the ulcer lesions, gastric volume, and total acidity and prevented the ulceration by increasing the ph and mucus secretion. oral administration of pomegranate extract and its ellagic acid rich fraction ( and mg/ kg) signi fi cantly attenuated dextran sulfate sodium -induced colonic in fl ammation in mice along with attenuation of histamine, myeloperoxidase and oxidative stress (singh et al. ) . the antiulcerative effect was comparable to sulphasalazine ( mg/kg, p.o.) and sodium cromoglycate ( mg/kg i.p). the authors stated that the antiulcerative effects may be attributed to mast cell stabilizing, antiin fl ammatory and antioxidant actions. pomegrante peel extracts exhibited remarkable in-vitro anti-helicobacter pylori activity against the clinical isolates of h. pylori (mean of inhibition zone diameter ranging from to mm/ m g disc). helicobacter pylori infection causes lifelong chronic gastritis, which can lead to peptic ulcer, mucosa-associated lymphoid tissue (malt) lymphoma and gastric cancer. pretreatment of rats with hydroalcoholic extract of pomegranate fl owers ( and mg/kg p.o. twice daily for days) signi fi cantly attenuated hypertonic glycerol-induced myoglobinuric renal dysfunction in a dose-dependent manner . the mechanism of renoprotective effects of punica granatum was found to involve activation of ppar-g and nitric oxide-dependent signalling pathway. pomegranate fruit rind powder at the dose of mg/kg orally as aqueous suspension was found to stimulate the cell-mediated and humoral components of the immune system in rabbits (gracious ross et al. ) . the pomegranate powder elicited an increase in antibody titer to typhoid-h antigen. it also enhanced the inhibition of leucocyte migration in leucocyte migration inhibition test and induration of skin in delayed hypersensitivity test with puri fi ed protein derivative (ppd) con fi rming its stimulatory effect on cell-mediated immune response. punicalagin isolated from pomegranate fruit was found to be a potent immune suppressant, based on its inhibitory action on the activation of the nuclear factor of activated t cells (nfat). punicalagin downregulated the mrna and soluble protein expression of interleukin- from anti-cd /anti-cd -stimulated murine splenic cd + t cells and suppressed mixed leukocytes reaction (mlr) without exhibiting cytotoxicity to the cells. in vivo, the punicalagin treatment inhibited phorbol -myristate -acetate (pma)induced chronic ear edema in mice and decreased cd + t cell in fi ltration of the in fl amed tissue. the results suggested that punicalagin could be a potential candidate for the therapeutics of various immune pathologies. yamasaki et al. ( ) found that dietary pomegranate seed oil (pso) high in levels of punicic acid ( c, t, c-octadecatrienoic acid), may enhance b-cell function in mice. splenocytes isolated from mice fed . or . % pso produced larger amounts of immunoglobulins g and m but not immunoglobulin a irrespective of stimulation with or without phorbol -myristate -acetate and the calcium ionophore a . dietary pso did not affect the percentages of b cells or cd -positive or cd -positive t cells in splenocytes. a polysaccharide (psp ) isolated from pomegranate rind was found to have immunomodulatory activity (joseph et al. ) . psp showed in-vitro growth stimulatory effect on isolated normal lymphocytes, and a proliferative index of . at a concentration of , m g/-ml was obtained, indicating immunomodulatory activity. wistar rats with excision wounds treated with % water-soluble gel formulated from the methanolic extract of dried pomegranate rind, showed good complete wound healing after days (chidambara murthy et al. ) . in comparison in rats treated with . % gel, healing was observed on day , and in the positive control animals receiving the blank gel took - days for complete healing. collagen content in terms of hydroxyproline level increased by two-fold in the group treated with . % gel. the gel extract was found to contain gallic acid and catechin as major components. aslam et al. ( ) found that pomegranate seed oil, but not aqueous extracts of fermented juice, peel or seed cake, stimulated human keratinocyte proliferation in monolayer culture. contrariwise, pomegranate peel aqueous extract (and to a lesser extent, both the fermented juice and seed cake extracts) stimulated type i procollagen synthesis and inhibited matrix metalloproteinase- (mmp- ; interstitial collagenase) production by dermal fi broblasts, but had no growth-supporting effect on keratinocytes. the results suggested that pomegranate peel aqueous extract could promote regeneration of dermis and pomegranate seed oil could promote regeneration of epidermis. pomegranate peel methanol extract-based ointment signi fi cantly enhanced wound contraction and the period of epithelialization as assessed by the mechanical (contraction rate, tensile strength), the biochemical (increasing of collagen, dna and proteins synthesis) and the histopathological characteristics in guinea pigs (hayouni et al. ) . the extract displayed antioxidant activity as potent as natural and synthetic compounds (trolox, butylated hydroxyanisole, quercetin). in addition, the extract exhibited signi fi cant antibacterial and antifungal activity against pseudomonas aeruginosa , staphylococcus aureus , escherichia coli , klebsiella pneumoniae , salmonella anatum , salmonella typhimurium , streptococcus pneumoniae , and fungi candida albicans , candida glabrata , trichopyton rubrum and aspergillus niger. the results suggested that the pomegranate formulated ointment may be used as skin repair agent without hazard to human health. the ethanol extract of p. granatum fl owers showed signi fi cant wound healing activity when topically administered in rats (pirbalouti et al. ) . the extract signi fi cantly increased the rate of wound contraction and collagen turnover. in-vitro studies using normal human epidermal keratinocytes, showed that pre-treatment with pomegranate fruit extract rich in anthocyannins and hydrolyzable tannins protected against the adverse effects of uv-b radiation by inhibiting uv-b-induced modulations of nuclear factor kappa b (nf-kappab) and mitogen-activated protein kinases (mapk) pathways (afaq et al. a, b ) . similarly, they reported pomegranate fruit extract to be an effective agent for ameliorating uva-mediated skin damages by modulating cellular pathways in-vitro (syed et al. ) . uva-mediated cellular damage occurs primarily through the release of reactive oxygen species and is responsible for immunosuppression, photodermatoses, photoaging and photocarcinogenesis. pretreatment of normal human epidermal keratinocytes with the extract ( - m g/ ml) for h before exposure to uva resulted in a dose-dependent inhibition of uva-mediated phosphorylation of signal transducers and activators of transcription (stat ), protein kinase b/ akt and mitogen activated protein kinases (mapks) viz. extracellular signal-regulated kinase (erk / ). the extract pretreatment also inhibited uva exposure-mediated increases in ki- antigen and pcna (proliferating cell nuclear antigen) and increased the cell-cycle arrest induced by uva in the g phase and the expression of bax and bad (proapoptotic proteins), while suppressing bcl-x(l) antiapoptotic protein expression. studies by zaid et al. ( ) showed that pretreatment of human immortalized hacat keratinocytes with polyphenol-rich pomegranate fruit extract inhibited uvb-mediated decrease in cell viability, decrease in intracellular glutathione content and increase in lipid peroxidation. immunoblot analysis showed that pretreatment of hacat cells with pomegranate fruit extract inhibited uvb-induced ( ) upregulation of mmp- , - , - and - , ( ) decrease in timp- , ( ) phosphorylation of mapks and (iv) phosphorylation of c-jun, whereas no effect was observed on uvb-induced c-fos protein levels. the results suggested that pomegranate fruit protected hacat cells against uvb-induced oxidative stress and markers of photoaging and could be a useful supplement in skin care products. pomegranate fruit extract ( - mg/l) was effective at protecting human skin fi broblasts from cell death following uv irradiation (pacheco-palencia et al. ) . this photoprotective effect was postulated to be related to a reduced activation of the pro-in fl ammatory transcription factor nf-kappab, suppression of proapoptotic caspase- , and an increased g /g phase, associated with dna repair. higher polyphenolic concentrations ( - , mg/l) were required to achieve a signi fi cant reduction in uv-induced reactive oxygen species levels and increased intracellular antioxidant capacity. pretreatment of reconstituted human skin "epiderm" with pomegranate-derived products pomx juice, pomx extract and pomegranate oil inhibited uvb-induced cyclobutane pyrimidine dimers (cpd), -dihydro- ¢deoxyguanosine ( -ohdg), protein oxidation and proliferating cell nuclear antigen (pcna) protein expression (afaq et al. ) . further, pretreatment of epiderm with pomegranate-derived products resulted in inhibition of uvb-induced collagenase (mmp- ), gelatinase (mmp- , mmp- ), stromelysin (mmp- ), marilysin (mmp- ), elastase (mmp- ), and tropoelastin. mmp- and mmp- activities were also inhibited. overall, the results suggested that all three pomegranate-derived products may be useful against uvb-induced damage to human skin. park et al. ( ) using cultured human skin fi broblasts, demonstrated that pomegranate fruit rind extract rich in polyphenols catechin, quercetin, kaempferol, and equol signi fi cantly protected against uvb-induced skin damage. the synthesis of collagen was increased and the expression of mmp- was decreased. oral feeding of pomegranate fruit extract to mice provided substantial protection from the adverse effects of uvb radiation via modulation in early biomarkers of photocarcinogenesis (afaq et al. ) . the extract inhibited uvb-induced: skin edema; hyperplasia; in fi ltration of leukocytes; lipid peroxidation; hydrogen peroxide generation; ornithine decarboxylase (odc) activity; and odc, cyclooxygenase- and proliferating cell nuclear antigen protein expression. the extract enhanced repair of uvbmediated formation of cyclobutane pyrimidine dimers (cpds) and -oxo- , -dihydro- ¢deoxyguanosine ( -oxodg). the extract inhibited uvb-mediated nuclear translocation of nf-k b; activation of ikk a ; and phosphorylation and degradation of i k b a . additionally, the extract further enhanced uvb-mediated increase in tumour suppressor p and cyclin kinase inhibitor p . in further studies, khan et al. ( ) reported that oral feeding of pomegranate fruit extract to skh- hairless mice inhibited uvbinduced epidermal hyperplasia, in fi ltration of leukocytes, protein oxidation and lipid peroxidation. immunoblot analysis demonstrated that oral feeding of pomegranate fruit extract to mice inhibited uvb-induced ( ) nuclear translocation and phosphorylation of nuclear factor kappa b/p , ( ) phosphorylation and degradation of i k b a , ( ) activation of ikk a / i k k b and ( ) phosphorylation of mitogen-activated protein kinase proteins and c-jun. pomegranate fruit extract consumption also inhibited uvb-induced protein expression of ( ) cox- and inos, ( ) pcna and cyclin d and ( ) matrix metalloproteinases- ,- and - in mouse skin. overall, the data showed that pomegranate fruit extract consumption afforded protection to mouse skin against the adverse effects of uvb radiation by modulating uvbinduced signalling pathways. in a double-blind, placebo-controlled trial involving female subjects age - s, kasai et al. ( ) found that oral administration of an ellagic acid-rich pomegranate extract had an inhibitory effect on a slight pigmentation (stains and freckles, brightness of face) in the human skin caused by uv irradiation. methanolic pomegranate extract showed . % invitro mushroom tyrosinase inhibitory activity (adhikari et al. ) . a pomegranate rind extract was found to have skin whitening activity (yoshimura et al. ) . the extract exhibited inhibitory activity against mushroom tyrosinase invitro comparable to that of the skin whitening agent, arbutin. when taken orally the extract inhibited uv-induced skin pigmentation on the back of brownish guinea pig. the results suggested the skinwhitening effect of the extract was probably due to inhibition of the proliferation of melanocytes and melanin synthesis by tyrosinase in melanocytes. a pomegranate polysaccharide fraction inhibited the formation of advanced glycation end-products (ages) by % and also inhibited the formation of fructosamine in the bsa/glucose system (rout and banerjee ) . the fraction inhibit , -diphenyl- -picrylhydrazyl (dpph) and , ¢ -azinobis[ ethylbenzothiazoline- -sulfonate] abts(+) radical activities by and %, respectively with m g/ ml concentration it also inhibited mushroom tyrosinase by % at m g/ml concentration suggesting its ef fi cacy as a potential skin whitener. pomegrante juice was shown to have a protective effect against ethylene glycol-induced nephrolithiasis in rats (tugcu et al. ) . ethylene glycol caused hyperoxaluria characterised by severe crystalization in renal tubules and granulovacuolar epithelial cell degeneration, marked elevation in malondialdehyde and nitric oxide levels and decrease of reduced glutathione (gsh) in rats there was no crystal formation in the rats treated with ethylene glycol and pomegranate juice. administration of pomegranate juice at medium and high dosage to rats was found to have inhibitory effects on renal tubular cell injury and oxidative stress caused by oxalate crystal deposition by reducing ros, inos, p -mapk, and nf-kb expression (ilbey et al. ) . rats treated with methanol pomegranate seed extract exhibited signi fi cant inhibitory activity against castor-oil induced diarrhoea and pge induced enteropooling (das et al. ) . the extract also displayed a signi fi cant reduction in gastrointestinal motility in charcoal meal test in rats. pomegranate juice and polyphenol-rich pomegranate fruit extract reduced platelet aggregation, calcium mobilization, thromboxane a( ) production, and hydrogen peroxide formation, induced by collagen and arachidonic acid (mattiello et al. ) . the polyphenol -rich fruit extract was more potent in reducing platelet activation. studies showed that pomegranate fruit components (mainly ellagic acid) modulated human thrombin amidolytic activity (cuccioloni et al. ) . pomegranate fruit ethanol extract was found to signi fi cantly increase the growth of osteoblastic mc t -e cells and caused a signi fi cant elevation of alkaline phosphatase (alp) activity and collagen content in the cells (kim and choi ) . treatment the extract decreased the tnfα-induced production of interleukin il- and nitric oxide in osteoblasts. promprom et al. ( ) found pomegranate seed extract to be a potent stimulator of phasic activity in rat uterus. pomegranate seed extract and b -sitosterol, the main constituent of the extract ( %), increased spontaneous contractions of the rat uterus in a concentration-dependent manner. their data suggested that the uterotonic effect was due to nonestrogenic effects of b -sitosterol acting to inhibit k channels and sarcoplasmic reticulum calcium atpase and thereby increasing contraction via calcium entry on l-type calcium channels and myosin light chain kinase. two b -secretase (bace ) inhibitors (anti-dementia agents) were isolated from pomegranate rind and identi fi ed as ellagic acid and punicalagin with ic values of . × − and . × − m (kwak et al. ) . ellagic acid and punicalagin were less inhibitory to α-secretase (tace) and other serine proteases such as chymotrypsin, trypsin, and elastase, thus indicating that they were relatively speci fi c inhibitors of bace . b -secretase is an aspartic-acid protease involved in the pathogenesis of alzheimer's disease studies showed that ethanolic extract of p. granatum seeds signi fi cantly exhibited the anxiolytic activity animal models of elevated plus maze test, barbiturate-induced sleeping time, tail suspension test, hot-plate and tail-fl ick tests (kumar et al. ) . the extract ( and mg/kg) signi fi cantly increased the sleeping latency and reduced the sleeping time. tail suspension test showed that the extract ( and mg/kg) was able to induce a signi fi cant decrease in the immobility time, similar to imipramine, a recognized antidepressant drug. tail-fl ick and hot-plate tests exhibited antinociceptive property of pomegranate extract, similar to morphine, a recognized antinociceptive agent. phytochemical screening and measurement of reducing power revealed the central nervous system (cns) activity of ethanol extract of pomegranate seeds may be due to its antioxidative pro fi le. supplementation of pomegranate fl owers led to improvements in learning and memory performances of streptozotocin-induced diabetic rats (cambay et al. ) . supplementation of pomegranate fl owers restored the elevated levels of lipid peroxidation and decreased level of glutathione towards their control values. daily pomegranate fl ower supplementation to diabetic rats reduced the increase in glial-fi brilar acidic protein (gfap) contents induced by diabetes in the hippocampus. the observations suggested that pomegranate fl ower supplementation decreased oxidative stress and amelioratedimpairment in learning and memory performances in diabetic rats and may be clinically useful in treating neuronal de fi cit in diabetic patients. loren et al. ( ) found that maternal dietary supplementation with pomegranate juice was neuroprotective in an animal model of neonatal hypoxic-ischemic brain injury. dietary supplementation with pomegranate juice resulted in markedly decreased brain tissue loss (> %) in all three brain regions assessed, with the highest pomegranate juice dose having greatest signi fi cance pomegranate juice also diminished caspase- activation by % in the hippocampus and % in the cortex. in further studies, the scientists showed that pomegranate polyphenols and resveratrol reduced caspase- activation following neonatal hypoxic-ischemic injury (west et al. ) . in separate study, transgenic mice (app(sw)/tg ) treated with pomegranate juice had signi fi cantly less (approximately %) accumulation of soluble aβ and amyloid deposition in the hippocampus as compared to control mice (hartman et al. ) . mice administered pomegranate juice learned water maze tasks more quickly and swam faster than controls. the results suggest that pomegranate juice may be useful in alzheimer's disease and warrant further studies. choi et al. ( b ) found that the ethanol pomegranate extract mitigated h o induced oxidative stress in pc cells. additionally, the extract inhibited neuronal cell death caused by a b -induced oxidative stress and a b -induced learning and memory de fi ciency in mice. studies showed that pomegranate fruit extract exhibited embryo protective effect against adriamycin-induced oxidative stress in chick embryos (kishore et al. ) . pre-administration of pomegranate fruit extract signi fi cantly ameliorated to normal, embryo gross morphological deformities and signi fi cant changes in the levels of biochemical parameters in amniotic fl uid observed in the adriamycin-treated group. pomegranate juice consumption by healthy male rats provided an increase in epididymal sperm concentration, sperm motility, spermatogenic cell density and diameter of seminiferous tubules and germinal cell layer thickness and antioxidant activity, and it decreased abnormal sperm rate when compared to the control group (türk et al. ) . a signi fi cant decrease in malondialdehyde level and marked increases in glutathione, glutathione peroxidase and catalase activities, and vitamin c level were observed in rats treated with different doses of pomegranate juice. studies showed that ethanolic extract of pomegranate could be useful for the treatment of the deleterious effect of lead acetate administration on sperm production in rats (leiva et al. ) . the extract exhibited antioxidant activity similar to that of ascorbic acid and prevented lead acetate -induced spermatogenic disruption in rats. its antioxidant activity could explain its capacity to reverse the damage produced by lead acetate on spermatogenesis. in a randomized, placebo-controlled, doubleblind, crossover study involving male patients with mild to moderate erectile dysfunction, of the subjects who demonstrated improvement in global assessment questionnaires (gaq) scores after beverage consumption, reported improvement in erectile function after drinking pomegranate juice (forest et al. ) . subjects were more likely to have improved scores when pomegranate juice was consumed. although overall statistical signi fi cance was not achieved, this pilot study suggested the possibility that larger cohorts and longer treatment periods may achieve statistical signi fi cance. studies in rabbits with atherosclerosis-induced erectile dysfunction showed that pomegranate extract signi fi cantly improved intracavernosal blood fl ow, erectile activity, smooth muscle relaxation and fi brosis of the atherosclerotic group in comparison with the atherosclerotic group receiving placebo, but did not normalize them to the agematched control levels . pomegranate extract appeared more effective in diminishing oxidative products, preventing superoxide dismutase and aldose reductase gene upregulation, and protecting mitochondrial, endothelial and caveolae structural integrity of the atherosclerotic group. the study showed that dietary antioxidants could improve arteriogenic erectile dysfunction. pomegranate known to contain estrogens (estradiol, estrone, and estriol) exhibited estrogenic activities in mice (mori-okamoto et al. ) . administration of pomegranate extract (juice and seed extract) for weeks to ovariectomized mice prevented the loss of uterus weight and shortened the immobility time compared with % glucose-dosed mice (control). further, ovariectomy-induced decrease of bone mineral density was normalized by administration of the pomegranate extract. the bone volume and the trabecular number were signi fi cantly increased and the trabecular separation was decreased in the pomegranate-dosed group compared with the control group. some histological bone formation/ resorption parameters were signi fi cantly increased by ovariectomy but were normalized by administration of the pomegranate extract. these changes suggested that the pomegranate extract inhibited ovariectomy-stimulated bone turnover. the authors concluded that pomegranate may be clinically effective on a depressive state and bone loss in menopausal syndrome in women. studies in human volunteers, found that in human liver microsomes, the mean % inhibitory concentrations (ic ) for pomegranate juice (pj) and grapefruit juice (gfj) versus cyp a (triazolam α-hydroxylation) were . and . %, (v/v) respectively without preincubation of inhibitor with microsomes (farkas et al. ) . after preincubation, the ic for pj increased to . % whereas the ic for gfj decreased to . % suggesting mechanism-based inhibition by gfj but not pj. administration of pj also did not affect c(max), total area under the curve (auc), or clearance of oral midazolam. however, gfj increased midazolam c(max) and auc by a factor of . and . , respectively, and reduced oral clearance to % of control values. the results suggested pj did not alter clearance of intravenous or oral midazolam, whereas gfj impaired clearance and elevated plasma levels of oral midazolam. jarvis et al. ( ) reported a potential interaction between pomegranate juice and warfarin as laboratory studies hade shown that pomegranate juice inhibited cytochrome p enzymes involved in warfarin metabolism. in a an open-label, randomized, single-center, two-period crossover study in healthy japanese volunteers, a single subtherapeutic doses of midazolam following weeks consumption of pomegranate juice did not signi fi cantly alter the pharmacokinetic pro fi le of midazolam compared with that of the control (misaka et al. ) . results of a -week randomized, double-blind, placebo-controlled study involving patients suggested that polyphenol-rich pomegranate juice (pj) supplementation added no bene fi t to the current standard therapy in patients with stable chronic obstructive pulmonary disease (cerdá et al. ) . the high teac (trolox equivalent antioxidant capacity) of pj could not be extrapolated in-vivo probably due to the metabolism of its polyphenols by colonic micro fl ora. the understanding of the different bioavailability of dietary polyphenols was thus critical before claiming any antioxidant-related health bene fi t. elbow fl exion strength was signi fi cantly higher during the -to -h period post-exercise with pomegranate juice compared with that of placebo (trombold et al. ) . elbow fl exor muscle soreness was also signi fi cantly reduced with pomegranate juice compared with that of placebo and at and h post-exercise. isometric strength and muscle soreness in the knee extensors were not signi fi cantly different with pomegranate juice compared with those using placebo. the results indicated a mild, acute ergogenic effect of pomegranate juice in the elbow fl exor muscles of resistance trained individuals after eccentric exercise. seven highly active ellagitannin inhibitors against carbonic anhydrase, punicalin ( ), punicalagin ( ), granatin b ( ), gallagyldilactone ( ), casuarinin ( ), pedunculagin ( ) and tellimagrandin i ( ), and four weakly active ellagitannin inhibitors, gallic acid ( ), granatin a ( ), corilagin ( ) and ellagic acid ( ), were isolated from pomegranate pericarps (satomi et al. ) . the type of inhibition by compounds ( ) and ( ) using p -nitrophenyl acetate as a substrate, was noncompetitive. carbonic anhydrase inhibitors are used as antiglaucoma drugs, and many potent carbonic anhydrase inhibitors have also been shown to inhibit the growth of several tumour cell lines in-vitro and in-vivo providing interesting leads for developing novel antitumour therapies (supuran et al. ) using the hot plate method in mice, pomegranate fl ower extracts showed signi fi cant analgesic activity at a dose of mg/kg body weight (chakraborthy ) . maximum analgesic activity was observed at min after drug administration, which was equivalent to the standard drug used morphine sulphate. two milliliters of aqueous extract of pomegranate roots exhibited higher activity on cultures from entamoeba histolytica than from entamoeba invadens strains, producing growth inhibitions of about and % respectively (segura et al. ) . alkaloid concentrations of mg/ml had no amoebicide activity, however tannins at concentrations of m g/ml for e. histolytica , and m g/ml for e. invadens were suf fi cient to produce an growth inhibition about %. tannic acid was also tested on the cultures of e. histolytica producing a high inhibitory activity on growth, this effect was produced at . mg/ ml similar to that observed with the tannin mixture. the methanolic extract of pomegranate was reported to in-vitro inhibit the growth of the malarial parasite, plasmodium berghei (dell'agli et al. ) . in another study, gallagic acid and punicalagin from pomegranate by-product exhibited antiplasmodial activity against plasmodium falciparum d and w clones with ic values of . , . , . and . m m, respectively (reddy et al. ) . pomegranate extract exhibited strong antimalarial activity against plasmodium falciparum (valdés et al. ) . p. grantum plant extract also exhibited in-vitro activity against the vaginal parasite, trichomonas vaginalis (el-sherbini et al. ) . hydroalcoholic pomegranate extract inhibited the growth of intracellular amastigotes of leishmania amazonensis with ic value of . m g/ml (garcía et al. ) . additionally, a low toxicity on macrophage from balb/c mice was observed. wibaut and hollstein ( ) found that the anthelminthic activity of pomegranate bark extract was mainly due to isopelletierine, methylisopelletierine while y pelleterine was less active the molluscicidal activity of p. granatum bark and canna indica root against the snail, lymnaea acuminata was found to be both time and dose dependent (tripathi and singh ) . the toxicity of p. granatum bark was more pronounced than that of c. indica . the h lc of the c. indica was . mg/l whereas that of the bark of p. granatum was . mg/l. the ethanol extract of p. granatum ( h lc : . mg/l) was more effective than the ethanol extract of c. indica ( h lc : . mg/l) in killing the test animals. p. granatum and c. indica may be used as potent molluscicides since the concentrations used to kill the snails were not toxic to the fi sh colisa fasciatus , sharing the same habitat with the snail. in a subsequent study, tripathi et al. ( ) reported that sub-lethal h exposure to active fraction of pomegranate bark separately or in combination with canna roots signi fi cantly inhibited the activity of acetylcholinesterase, acid/alkaline phosphatase, na(+)k(+)atpase and lactic dehydrogenase in the nervous tissue of lymnaea acuminata. lei et al. ( ) found that ellagic acid, the principal bioactive component of pomegranate leaf extract, had poor absorption and rapid elimination after oral administration pomegranate leaf extract, and part of it was absorbed from stomach. studies in rats showed that only - % of the ingested punicalagin was detected as such or as metabolites in urine and faeces (cerdá et al. b ) . only traces of punicalagin metabolites being detected in liver or kidney. the transformation of ellagic acid derivatives to h-dibenzo[b,d] pyran- -one derivatives in the rat was con fi rmed. studies of cerdá et al. ( ) found that the potential systemic biological effects of pomegranate juice ingestion should be attributed to the colonic micro fl ora metabolites rather than to the polyphenols present in the juice. neither the potent antioxidant punicalagin nor ellagic acid present in pomegranate juice were detected in both plasma and urine on ingestion of pomegranate juice. three microbial ellagitannin-derived metabolites were detected: , -dihydroxy- h-dibenzo[b,d]pyran- -one glucuronide, an unidenti fi ed aglycone (tentatively, trihydroxy- h-dibenzo[b,d]pyran- -one) and hydroxy- -hdibenzo [b,d] pyran- -one glucuronide in the plasma and urine. the metabolites did not show signi fi cant antioxidant activity compared to punicalagin from pomegranate juice. in separate studies, ellagic acid was detected in human plasma at a maximum concentration ( . ng/ml) after h post-ingestion of pomegranate juice but was rapidly eliminated by h (seeram et al. ) . six hours post-ingestion of pomegranate juice, ellagic acid (ea) was detected in plasma of all healthy human volunteers with a maximum concentration of . m mol/l, area under concentration time curve of . ( m mol × h) × l(- ), time of maximum concentration of . h, and elimination half-life of . h . ellagic acid metabolites, including dimethylellagic acid glucuronide (dmeag) and hydroxy- h-benzopyran- -one derivatives (urolithins), were also detected in plasma and urine in conjugated and free forms. dmeag was found in the urine obtained from of subjects on day , but was not detected on d − (day before) or + (day after), demonstrating its potential as a biomarker of intake. urolithin a-glucuronide was found in urine samples from subjects on d and in the urine from subjects on d + . urolithin b-glucuronide was found in the urine of three subjects on d and in the urine of fi ve subjects on d+ . the scientists asserted that urolithins, formed by intestinal bacteria, may contribute to the biological effects of pomegranate juice as they may persist in plasma and tissues and account for some of the health bene fi ts noted after chronic juice consumption. studies by seeram et al. ( ) found that pomegranate juice, pomegranate polyphenol liquid extract and pomegranate polyphenol powder extract provide similar levels of plasma and urinary ellagitannin metabolites such as urolithin a, in human subjects. there was a delay in time of maximum concentration of pomegranate powder extract compared to pomegranate juice and pomegranate polyphenol liquid. mertens-talcott et al. ( ) found ellagic acid from pomegranate extract to be bioavailable at h after consumption by healthy volunteers. its metabolites urolithin a, urolithin b, hydroxyl-urolithin a, urolithin a-glucuronide, and dimethyl ellagic acidglucuronide were found in the plasma. the antioxidant capacity measured with the oxygen radical absorbance capacity (orac) assay was increased with a maximum effect of % after . h, whereas the generation of reactive oxygen species (ros) was not affected. vidal et al. ( ) found that in chick embryo model doses of hydroalcoholic pomegranate fruit extract of less than . mg per embryo were not toxic. the ld of the extract, determined in of- mice of both sexes after intraperitoneal administration, was mg/kg. con fi dence limits were - mg/kg. at the doses of . and . mg/kg of extract, the repeated intranasal administration to wistar rats produced no toxic effects in terms of food intake, weight gain, behavioural or biochemical parameters, or results of histopathological studies. cerdá et al. ( a ) found that repeated oral administration of high doses of the pomegranate ellagitannin punicalagin to rats for days was not toxic. punicalagin and related metabolites were identi fi ed in plasma, liver, and kidney. five punicalagin-related metabolites were detected in liver and kidney, that is, two ellagic acid derivatives, gallagic acid, , -dihydroxy- h-dibenzo [b,d] pyran- -one glucuronide, and , , -trihydroxy- h-dibenzo [b,d] pyran- -one. feedstuff intake, food utility index, and growth rate were lower in punicalagin treated rats during the fi rst days without signi fi cant adverse effects, which could be due to the lower nutritional value of the punicalagin-enriched diet together with a decrease in its palatability (lower food intake). no signi fi cant differences were found in punicalagin treated rats in any blood parameter analyzed (including the antioxidant enzymes glutathione peroxidase and superoxide dismutase) with the exception of urea and triglycerides, which remained at low values throughout the study. clinical studies by heber et al. ( ) demonstrated the safety of a pomegranate ellagitannin-enriched polyphenol dietary supplement in overweight individuals with increased waist size and provided evidence of antioxidant activity in humans re fl ected by a signi fi cant reduction in thiobarbituric acid reactive substances (tbars) linked with cardiovascular disease risk. patel et al. ( ) found that the no observed-adverse-effect level (noael) for a standardized pomegranate fruit extract was determined as mg/kg body weight/day, the highest dose tested in rats. compared to the control group, administration of the extract did not result in any toxicologically signi fi cant treatmentrelated changes in clinical observations, ophthalmic examinations, body weights, body weight gains, feed consumption, clinical pathology evaluations and organ weights. toxicological evaluation of pomegranate seed oil (pso) showed that the no observable adverse effect level (noael) was , ppm pso (= . g pso/kg body weight/day) (meerts et al. ) . no mutagenicity of pso was observed in the absence and presence of metabolic activation up to precipitating concentrations of , m g/ plate (ames test) or m g/ml (chromosome aberration test). the acute oral toxicity study revealed no signi fi cant fi ndings at , mg pso/ kg body weight. results from reversion and gene-conversion test in microorganisms, sister chromatid exchanges, micronuclei and sperm-shape abnormality assays in mice, clearly showed that the hydroalcoholic extract of pomegranate whole fruit was genotoxic when tested both in-vitro and in-vivo (sánchez-lamar et al. ) . the bark of the roots, the fl owers, the rind of pomegranate fruit and the seeds, are of fi cial in many pharmacopoeias. various parts of the pomegranate plant have been extensively used for thousands of years in traditional medicine in the middle east, ancient greece and asia (burkill ; grieve ; stuart ) ; and in india such as in the ayurveda and unani systems of medicine (nadkarni and nadkarni. ; sharma et al. ; kapoor ; pradeep et al. ) . the fruit rind and stem bark have been used as a traditional remedy for diarrhoea, dysentery and intestinal parasites. pomegranate pericarp has been commonly employed as a crude drug in indian traditional medicine for the treatment of diarrhoea as well as for use as an astringent, antihelminthic, asphrodisacs, laxative, diuretic, stomachic, cardiotonic and refrigerant. the seeds and juice are considered as bitter and astringent and employed as a tonic for hear and throat ailments. the astringent qualities of the fl ower sap, fruit rind and tree bark are considered useful remedies for nose bleeds and gum bleeds, toning skin, (after mixing with mustard oil) fi rming-up sagging breasts and treating haemorrhoids. a syrup prepared from the fruit is useful in all bilious complaints. the juice of the fresh fruit is much esteemed in dyspepsia and as a cooling, thirst-quenching beverage in fever and sickness. the fruit juice is also found bene fi cial in leprosy. pomegranate fruit juice has been used as eyedrops to treat cataracts. dried, pulverized fl ower buds are employed as a remedy for bronchitis. pomegranate has been reported as a remedy for diabetes in the unani system of medicine practiced in the middle east and india. the ancient greeks and egyptians used the fruit rind, fl owers and root bark as astringents and the last as vermicide for treating tapeworms. in malaysia, the root bark is used as vermifuge and powdered root bark is administered to children for stomach pains. the root is also used for diarrhoea and tits sap used for treating sore-eyes. leaves are used in jamu preparations with a raft of other herbal ingredients for many medicinal complaints. pounded leaves are used in a complex bolus for stomach ache and the fruit juice is recommended for coughs. in singapore, the root bark has been used in as a component in a compound infusion or decoction taken by women for days after childbirth. other traditional uses of the fruit rind and root include as a treatment for snakebite (jain and puri ) , diabetes (singh ) , burns (siang ) , leprosy and assorted gynecological problems (singh et al. ) . in sri lanka, the fresh fruit has been used as a refrigerant to lower fever (arseculeratne et al. ) . in the philippines, a decoction of the tender leaves is used as a gargle for affections of the buccal cavity. the rind of the fruit is used internally in decoction as anthelmintic and taenifuge. in mexico, a decoction of the fl owers is gargled to relieve oral and throat in fl ammation. in korea, traditional uses of the fruit and rind include as an anthelminthic and for phlegm, cholethiasis, tineapedis and laryngitis. punica granatum is a drought tolerant tree suitable for arid and semi-arid zone afforestation. pomegranate has deep rooting system and is used for erosion control, planted along rivers to stabilize banks. an ideal suitable ornamental plant for gardens and amenity parks. pomegranate grows along well as intercrop with grapes in mediterranean countries. the tree is sometimes used for fencing and planted as boundary plants. pomegranate leaf litter decomposes slowly and is suitable for mulching. the leaves are foraged by domesticated stock. ink can be made by steeping the leaves in vinegar. both the fruit rind and the fl owers yield dyes for textiles. the light-coloured wood is hard and durable, mostly used in making farm implements, walking-sticks and in woodcrafts as it is only available in small dimension. tree branches are used as fi rewood. the bark is used in tanning and dyeing providing the yellow hue for moroccan leather. root bark yields a black ink rich in tannins. in japan, an insecticide is derived from the bark. studies revealed that pomegranate peel can prepared as an adsorbent in treating industrial ef fl uents containing phenols and safely disposed of by stabilizing into cement (bhatnagar and minocha ) . studies showed that that pomegranate peel waste can be used as adsorbent bene fi cially for nickel removal from aqueous solution (bhatnagar and minocha ) . pomegranate husk when converted into activated carbon exhibited its ability to remove hexavalent chromium from wastewater (nemr ) . studies showed that the nutritive value and the antioxidant capacity of pomegranate peel could be enhanced by ensiling into a favorable healthpromoting constituent of feedlot beef cattle diet (shabtay et al. ) . dietary supplementation with fresh peels promoted signi fi cant increases in feed intake and a-tocopherol concentration in the plasma, with positive tendency toward increased weight gain of bull calves. the pomegranate fruit is steeped in religious and cultural signi fi cance in judaism, christianity, islam, hinduism religions, persian, armenian, azerbaijani and chinese cultures (wikipedia ) . pomegrante germinates readily from seeds and are established from seedlings, rooted hardwood cuttings, from air layering and suckers. screening of nepalese crude drugs traditionally used to treat hyperpigmentation: in vitro tyrosinase inhibition pomegranate fruit extract modulates uv-bmediated phosphorylation of mitogen-activated protein kinases and activation of nuclear factor kappa b in normal human epidermal keratinocytes paragraph sign anthocyanin-and hydrolyzable tannin-rich pomegranate fruit extract modulates mapk and nf-kappab pathways and inhibits skin tumorigenesis in cd- mice protective effect of pomegranate-derived products on uvb-mediated damage in human reconstituted skin oral feeding of pomegranate fruit extract inhibits early biomarkers of uvb radiation-induced carcinogenesis in skh- hairless mouse epidermis short communication: studies on punica granatum -i isolation and identi fi cation of some constituents from the seeds of punica granatum punica granatum l. extract inhibits il- beta-induced expression of matrix metalloproteinases by inhibiting the activation of map kinases and nf-kappab in human chondrocytes in vitro the inhibition of gastric mucosal injury by punica granatum l. (pomegranate) methanolic extract protective effects of punica granatum in experimentally-induced gastric ulcers pomegranate extracts potently suppress proliferation, xenograft growth, and invasion of human prostate cancer cells anthocyanins characterization of iranian pomegranate ( punica granatum l.) varieties and their variation after cold storage and pasteurization antimicrobial activity of pomegranate ( punica granatum l.) fruit peels studies on medicinal plants of sri lanka. part pomegranate as a cosmeceutical source: pomegranate fractions promote proliferation and procollagen synthesis and inhibit matrix metalloproteinase- production in human skin cells pomegranate juice consumption inhibits serum angiotensin converting enzyme activity and reduces systolic blood pressure pomegranate juice consumption reduces oxidative stress, atherogenic modi fi cations to ldl, and platelet aggregation: studies in humans and in atherosclerotic apolipoprotein e-de fi cient mice pomegranate juice fl avonoids inhibit low-density lipoprotein oxidation and cardiovascular diseases: studies in atherosclerotic mice and in humans pomegranate juice consumption for years by patients with carotid artery stenosis reduces common carotid intima-media thickness, blood pressure and ldl oxidation pomegranate phenolics from the peels, arils, and fl owers are antiatherogenic: studies in vivo in atherosclerotic apolipoprotein e-de fi cient (e ) mice and in vitro in cultured macrophages and lipoproteins an aqueous pomegranate peel extract inhibits neutrophil myeloperoxidase in vitro and attenuates lung in fl ammation in mice flora of java (spermatophytes only), . noordhoff melatonin, serotonin, and tryptamine in some egyptian food and medicinal plants antidiabetic effect of punica granatum fl owers: effect on hyperlipidemia, pancreatic cells lipid peroxidation and antioxidant enzymes in experimental diabetes new sterol esters from the fl owers of punica granatum linn consumption of pomegranate decreases serum oxidative stress and reduces disease activity in patients with active rheumatoid arthritis: a pilot study -methyl-pyran- -one- -o -b -d-glucopyranoside isolated from leaves of punica granatum inhibits the tnf a -induced cell adhesion molecules expression by blocking nuclear transcription factor-k b (nf-k b) the antiplaque ef fi cacy of pomegranate mouthrinse adsorptive removal of , -dichlorophenol from water utilizing punica granatum peel waste and stabilization with cement biosorption optimization of nickel removal from water using punica granatum peel waste urolithins, intestinal microbial metabolites of pomegranate ellagitannins, exhibit potent antioxidant activity in a cell-based assay the effect of pomegranate ( punica granatum l.) byproducts and ellagitannins on the growth of human gut bacteria pomegranate-mediated chemoprevention of experimental hepatocarcinogenesis involves nrf -regulated antioxidant mechanisms climate effects on anthocyanin accumulation and composition in the pomegranate ( punica granatum l.) fruit arils synergic interaction between pomegranate extract and antibiotics against staphylococcus aureus pomegranate extract inhibits staphylococcus aureus growth and subsequent enterotoxin production a dictionary of the economic products of the malay peninsula, revised reprint, vols. ministry of agriculture and co-operatives characterization of a potential nutraceutical ingredient: pomegranate ( punica granatum l.) seed oil unsaponi fi able fraction volatile composition and sensory quality of spanish pomegranates ( punica granatum l.) pomegranate ( punica granatum l.) fl ower improves learning and memory performances impaired by diabetes mellitus in rats hepatoprotective role and antioxidant capacity of pomegranate ( punica granatum ) fl owers infusion against trichloroacetic acidexposed in rats repeated oral administration of high doses of the pomegranate ellagitannin punicalagin to rats for days is not toxic evaluation of the bioavailability and metabolism in the rat of punicalagin, an antioxidant polyphenol from pomegranate juice the potent in vitro antioxidant ellagitannins from pomegranate juice are metabolised into bioavailable but poor antioxidant hydroxy- h-dibenzopyran- -one derivatives by the colonic micro fl ora of healthy humans pomegranate juice supplementation in chronic obstructive pulmonary disease: a -week randomized, doubleblind, placebo-controlled trial analgesic activity of various extracts of punica granatum (linn) fl owers antibacterial activity of thai medicinal plant extracts on the skin infectious microorganisms flavonoid diglycoside from punica granatum studies on antioxidant activity of pomegranate ( punica granatum ) peel extract using in vivo models study on wound healing activity of punica granatum peel the partition chromatography of alkaloids: part iii.-the alkaloids of punica granatum in vitro and in vivo antibacterial activity of punica granatum peel ethanol extract against salmonella punica granatum protects against oxidative stress in pc cells and oxidative stress-induced alzheimer's symptoms in mice the wealth of india: a dictionary of indian raw materials and industrial products pomegranate extract inhibits the proliferation and viability of mmtv-wnt- mouse mammary cancer stem cells in vitro studies on antidiarrhoeal activity of punica granatum seed extract in rats studies on the hypoglycaemic activity of punica granatum seed in streptozotocin induced diabetic rats effects of consumption of pomegranate juice on carotid intima-media thickness in men and women at moderate risk for coronary heart disease bene fi cial effects of pomegranate juice on oxidation-sensitive genes and endothelial nitric oxide synthase activity at sites of perturbed shear stress pomegranate juice reduces oxidized low-density lipoprotein downregulation of endothelial nitric oxide synthase in human coronary endothelial cells the in fl uence of pomegranate fruit extract in comparison to regular pomegranate juice and seed oil on nitric oxide and arterial function in obese zucker rats effects of a pomegranate fruit extract rich in punicalagin on oxidation-sensitive genes and enos activity at sites of perturbed shear stress and atherogenesis quantitative analysis of fl avan- -ols in spanish foodstuffs and beverages antiplasmodial activity of punica granatum l. fruit rind the antioxidant potency of punica granatum l. fruit peel reduces cell proliferation and induces apoptosis on breast cancer pomegranate extract mouth rinsing effects on saliva measures relevant to gingivitis risk antimicrobial activity of six pomegranate ( punica granatum l.) varieties and their relation to some of their pomological and phytonutrient characteristics elemental and nutritional analysis of punica granatum from turkey analysis of organic acids in fruit juices by liquid chromatography-mass spectrometry: an enhanced tool for authenticity testing pomegranate ( punica granatum ) juices: chemical composition, micronutrient cations, and antioxidant capacity physico-chemical properties and dpph-abts scavenging activity of some local pomegranate ( punica granatum ) ecotypes antioxidant capacities of phenolic compounds and tocopherols from tunisian pomegranate ( punica granatum ) fruits fatty acids from tunisian and chinese pomegranate ( punica granatum l.) seeds chemical composition of juice and seeds of pomegranate fruit studies on pomegranate seed oil ef fi cacy of two plant extracts against vaginal trichomoniasis two ellagitannins from punica granatum heartwood two new ellagic acid rhamnosides from punica granatum heartwood ellagiand gallotannins from punica granatum heartwood concentrated pomegranate juice improves lipid pro fi les in diabetic patients with hyperlipidemia cholesterol-lowering effect of concentrated pomegranate juice consumption in type ii diabetic patients with hyperlipidemia pomegranate juice effects on cytochrome p s expression: in vivo studies effect of pomegranate ( punica granatum ) juice intake on hepatic oxidative stress pomegranate juice does not impair clearance of oral or intravenous midazolam, a probe for cytochrome p - a activity: comparison with grapefruit juice monoacylglycerol from punica granatum seed oil effect of probiotication on antioxidant and antibacterial activities of pomegranate juices from sour and sweet cultivars determination of lignans in edible and nonedible parts of pomegranate ( punica granatum l.) and products derived there from, particularly focusing on the quantitation of isolariciresinol using hplc-dad-esi/ms(n) ef fi cacy and safety of pomegranate juice on improvement of erectile dysfunction in male patients with mild to moderate erectile dysfunction: a randomized, placebo-controlled, double-blind, crossover study foundation for revitalisation of local health traditions pomegranate and cardiovascular diseases: pomegranate juice polyphenolic antioxidants protect against oxidative stress and atherosclerosis development pomegranate juice inhibits oxidized ldl uptake and cholesterol biosynthesis in macrophages pomegranate juice polyphenols increase recombinant paraoxonase- binding to high-density lipoprotein: studies in vitro and in diabetic patients screening of medicinal plants against leishmania amazonensis synergistic growth inhibition of mouse skin tumors by pomegranate fruit extract and diallyl sul fi de: evidence for inhibition of activated mapks/nf-k b and reduced cell proliferation effects of aqueous extracts from quercus ilex l. root bark, punica granatum l. fruit peel and artemisia herba-alba asso leaves on ethanolinduced gastric damage in rats arte's f, toma's-barbera'n f ( ) changes in pomegranate juice pigmentation during ripening antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing effect of pomegranate juice on insulin secretion and sensitivity in patients with obesity dissimilar in vitro and in vivo effects of ellagic acid and its microbiota-derived metabolites, urolithins, on the cytochrome p a anti-microbial activities of pomegranate rind extracts: enhancement by cupric sulphate against clinical isolates of s. aureus , mrsa and pvl positive ca-mssa total phenolic distribution of juice, peel, and seed extracts of four pomegranate cultivars immunomodulatory activity of punica granatum in rabbits -a preliminary study punicic acid is an omega- fatty acid capable of inhibiting breast cancer proliferation antioxidant activities of peel, pulp and seed fractions of common fruits as determined by frap assay evaluation of antioxidant activity and preventing dna damage effect of pomegranate extracts by chemiluminescence method pomegranate juice is potentially better than apple juice in improving antioxidant function in elderly subjects pomegranin, an antifungal peptide from pomegranate peels pomegranate ( punica granatum ) puri fi ed polyphenol extract inhibits in fl uenza virus and has a synergistic effect with oseltamivir in vitro antibacterial activity of some iranian medicinal plant extracts against helicobacter pylori pomegranate juice decreases amyloid load and improves behavior in a mouse model of alzheimer's disease cardioprotective effect of whole fruit extract of pomegranate on doxorubicin-induced toxicity in rat hydroalcoholic extract based-ointment from punica granatum l. peels with enhanced in vivo healing potential on dermal wounds safety and antioxidant activity of a pomegranate ellagitanninenriched polyphenol dietary supplement in overweight individuals with increased waist size identi fi cation of estrone in pomegranate seeds evolution of juice anthocyanins during ripening of new selected pomegranate ( punica granatum ) clones pomegranate polyphenols down-regulate expression of androgensynthesizing genes in human prostate cancer cells overexpressing the androgen receptor activation of ppar gamma and alpha by punicic acid ameliorates glucose tolerance and suppresses obesity-related in fl ammation chemopreventive effects of pomegranate seed oil on skin tumor development in cd mice anti-diabetic action of punica granatum fl ower extract: activation of ppargamma and identi fi cation of an active component pomegranate fl ower improves cardiac lipid metabolism in a diabetic rat model: role of lowering circulating lipids pomegranate fl ower extract diminishes cardiac fi brosis in zucker diabetic fatty rats: modulation of cardiac endothelin- and nuclear factor-kappab pathways tannins from the leaves of punica granatum pomegranate juice protects nitric oxide against oxidative destruction and enhances the biological actions of nitric oxide effects of pomegranate juice on hyperoxaluria-induced oxidative stress in the rat kidneys possible interaction between pomegranate juice and warfarin anticancer activities of pomegranate extracts and genistein in human breast cancer cells evaluation of antioxidant, antitumor and immunomodulatory properties of polysaccharide isolated from fruit rind of punica granatum suppressive effect of punica granatum on the production of tumor necrosis factor (tnf) in bv microglial cells therapeutic applications of pomegranate ( punica granatum l.): a review antioxidant effect of pomegranate extract in reducing acute in fl ammation due to myringotomy pomegranate juice supplementation to atherosclerotic mice reduces macrophage lipid peroxidation, cellular cholesterol accumulation and development of atherosclerosis crc handbook of ayurvedic medicinal plants effects of oral administration of ellagic acidrich pomegranate extract on ultraviolet-induced pigmentation in the human skin effects of pomegranate chemical constituents/intestinal microbial metabolites on cyp b in rv prostate cancer cells punica granatum : heuristic treatment for diabetes mellitus punica granatum (pomegranate) fl ower extract possesses potent antioxidant activity and abrogates fe-nta induced hepatotoxicity in mice differentiation-promoting activity of pomegranate ( punica granatum ) fruit extracts in hl- human promyelocytic leukemia cells oral consumption of pomegranate fruit extract inhibits growth and progression of primary lung tumors in mice pomegranate fruit extract inhibits prosurvival pathways in human a lung carcinoma cells and tumor growth in athymic nude mice pomegranate fruit extract impairs invasion and motility in human breast cancer pomegranate fruit extract inhibits uvb-induced in fl ammation and proliferation by modulating nf-k b and mapk signaling pathways in mouse skin stimulation of osteoblastic differentiation and inhibition of interleukin- and nitric oxide in mc t -e cells by pomegranate ethanol extract chemopreventive and adjuvant therapeutic potential of pomegranate ( punica granatum ) for human breast cancer inhibitory effect of pomegranate on intestinal sodium dependent glucose uptake embryo protective effect of pomegranate ( punica granatum l.) fruit extract in adriamycin-induced oxidative stress screening of traditional chinese medicinal plants for quorum-sensing inhibitors activity pomegranate seed oil rich in conjugated linolenic acid suppresses chemically induced colon carcinogenesis in rats genetic diversity-independent neutralization of pandemic viruses (e.g. hiv), potentially pandemic (e.g. h n strain of in fl uenza) and carcinogenic (e.g. hbv and hcv) viruses and possible agents of bioterrorism (variola) by enveloped virus neutralizing compounds (evncs) pomegranate extract induces apoptosis in human prostate cancer cells by modulation of the igf-igfbp axis effect of pomegranate peel extracts on experimental prostatitis rats in vitro studies on the binding, antioxidant, and cytotoxic actions of punicalagin central nervous system activity of acute administration of ethanol extract of punica granatum l. seeds in mice beta-secretase (bace ) inhibitors from pomegranate ( punica granatum ) husk effects of pomegranate tannins on experimental gastric damages transport behavior of ellagic acid of pomegranate leaf tannins and its correlation with total cholesterol alteration in hepg cells punica granatum (pomegranate) and its potential for prevention and treatment of in fl ammation and cancer pomegranate ( punica granatum ) pure chemicals show possible synergistic inhibition of human pc- prostate cancer cell invasion across matrigel possible synergistic prostate cancer suppression by anatomically discrete pomegranate fractions immune-suppressive activity of punicalagin via inhibition of nfat activation pharmacokinetic study of ellagic acid in rat after oral administration of pomegranate leaf extract evidence of anti-obesity effects of the pomegranate leaf extract in high-fat diet induced obese mice effect of punica granatum (pomegranate) on sperm production in male rats treated with lead acetate antiviral activities of medicinal herbs traditionally used in southern mainland china punica granatum fl ower extract, a potent alpha-glucosidase inhibitor, improves postprandial hyperglycemia in zucker diabetic fatty rats evaluation of antioxidant properties of pomegranate peel extract in comparison with pomegranate pulp extract pomegranate fl ower: a unique traditional antidiabetic medicine with dual ppar-alpha/-gamma activator properties fabrication of nanoparticles using partially puri fi ed pomegranate ellagitannins and gelatin and their apoptotic effects maternal dietary supplementation with pomegranate juice is neuroprotective in an animal model of neonatal hypoxic-ischemic brain injury anti-listeria monocytogenes activity of heat-treated lyophilized pomegranate juice in media and in ground top round beef pomegranate ( punica granatum ) supplements: authenticity, antioxidant and polyphenol composition prostate cancer prevention through pomegranate fruit pomegranate fruit juice for chemoprevention and chemotherapy of prostate cancer effects of pomegranate juice and extract polyphenols on platelet function antimicrobial activities of pomegranate rind extracts: enhancement by addition of metal salts and vitamin c pomegranate seed oil consumption during a period of high-fat feeding reduces weight gain and reduces type diabetes risk in cd- mice toxicological evaluation of pomegranate seed oil breast cancer chemopreventive properties of pomegranate ( punica granatum ) fruit extracts in a mouse mammary organ culture volatile composition of pomegranates from spanish cultivars using headspace solid phase microextraction punica granatum (pomegranate) extract is active against dental plaque absorption, metabolism, and antioxidant effects of pomegranate ( punica granatum l.) polyphenols after ingestion of a standardized extract in healthy human volunteers effect of weeks' consumption of pomegranate juice on the pharmacokinetics of a single dose of midazolam: an open-label, randomized, single-center, -period crossover study in healthy japanese volunteers cardioprotective potential of punica granatum extract in isoproterenol-induced myocardial infarction in wistar rats effect of pomegranate juice on angiotensin ii-induced hypertension in diabetic wistar rats pomegranate extract improves a depressive state and bone properties in menopausal syndrome model ovariectomized mice fruits of warm climates. julia f identi fi cation and quanti fi cation of phenolic compounds and their effects on antioxidant activity in pomegranate juices of eight iranian cultivars indian materia medica with ayurvedic pomegranate extract induces cell cycle arrest and alters cellular phenotype of human pancreatic cancer cells medicinal plants in the republic of korea nmr spectral analysis of polyphenols from punica granatum antibacterial activity directed isolation of compounds from punica granatum antioxidant and antimutagenic activities of pomegranate peel extracts potential of pomegranate husk carbon for cr(vi) removal from wastewater: kinetic and isotherm studies the existence of pelletierine derivatives in punica granatum alkaloids in the bark of punica granatum l. (pomegranate) from yugoslavia punica granatum (pomegranate) juice provides an hiv- entry inhibitor and candidate topical microbicide antioxidant activities of pomegranate fruit extract and its anthocyanidins: delphinidin, cyanidin, and pelargonidin evaluation of antioxidant activity, colour and some nutritional characteristics of pomegranate ( punica granatum l.) juice and its sour concentrate processed by conventional evaporation agroforestree database: a tree reference and selection guide version protective effects of standardized pomegranate ( punica granatum l.) polyphenolic extract in ultraviolet-irradiated human skin fi broblasts antifungal ef fi cacy of punica granatum , acacia nilotica , cuminum cyminum and foeniculum vulgare on candida albicans : an in vitro study antioxidant capacity and lipid characterization of six georgia-grown pomegranate cultivars phase ii study of pomegranate juice for men with rising prostate-speci fi c antigen following surgery or radiation for prostate cancer antimicrobial ellagitannin from pomegranate ( punica granatum ) fruits extract of punica granatum inhibits skin photoaging induced by uvb irradiation medicinal values of fruit peels from citrus sinensis , punica granatum , and musa paradisiaca with respect to alterations in tissue lipid peroxidation and serum concentration of glucose, insulin, and thyroid hormones safety assessment of pomegranate fruit extract: acute and subchronic toxicity studies the wound healing activity of fl ower extracts of punica granatum and achillea kellalensis in wistar rats antioxidant properties of gallocatechin and prodelphinidins from pomegranate peel searchable world wide web multilingual multiscript plant name database antibacterial activity of punica granatum l. against gastro intestinal tract infection causing organisms the effects of pomegranate seed extract and {beta}-sitosterol on rat uterine contractions antioxidant potentials of punica granatum fruit rind extracts pomegranate extract inhibits the interleukin- b -induced activation of mkk- , p a -mapk and transcription factor runx- in human osteoarthritis chondrocytes antioxidant, antimalarial and antimicrobial activities of tannin-rich fractions, ellagitannins and phenolic acids from punica granatum l studies on the chemical constituents from xinjiang punica granatum pomegranate extract inhibits androgen-independent prostate cancer growth through a nuclear factor-kappab-dependent mechanism antioxidant activity of punica granatum fruits the occurrence of -( -propenyl)-d -piperideine in the leaves of pomegranate consumption of wonderful variety pomegranate juice and extract by diabetic patients increases paraoxonase association with high-density lipoprotein and stimulates its catalytic activities pomegranate juice protects macrophages from triglyceride accumulation: inhibitory effect on dgat activity and on triglyceride biosynthesis anti-oxidative effects of pomegranate juice (pj) consumption by diabetic patients on serum and on macrophages pomegranate byproduct administration to apolipoprotein e-de fi cient mice attenuates atherosclerosis development as a result of decreased macrophage oxidative stress and reduced cellular uptake of oxidized lowdensity lipoprotein free radical scavenging, antiglycation and tyrosinase inhibition properties of a polysaccharide fraction isolated from the rind from punica granatum pomegranate juice sugar fraction reduces macrophage oxidative state, whereas white grape juice sugar fraction increases it antiplaque and antigingivitis effects of a gel containing punica granatum linn extract: a double-blind clinical study in humans assessment of the genotoxic risk of punica granatum l. (punicaceae) whole fruit extracts ellagitannin-rich pomegranate extract inhibits angiogenesis in prostate cancer in vitro and in vivo pomegranate juice inhibits sulfoconjugation in caco- human colon carcinoma cells carbonic anhydrase inhibitors from the pericarps of punica granatum l investigation of a betainic alkaloid from punica granatum antioxidant and eicosanoid enzyme inhibition properties of pomegranate seed oil and fermented juice fl avonoids bioavailability of ellagic acid in human plasma after consumption of ellagitannins from pomegranate ( punica granatum l.) juice in vitro antiproliferative, apoptotic and antioxidant activities of punicalagin, ellagic acid and a total pomegranate tannin extract are enhanced in combination with other polyphenols as found in pomegranate juice rapid large scale puri fi cation of ellagitannins from pomegranate husk, a by-product of the commercial juice industry pomegranate juice ellagitannin metabolites are present in human plasma and some persist in urine for up to hours pomegranate ellagitannin-derived metabolites inhibit prostate cancer growth and localize to the mouse prostate gland pomegranate juice and extracts provide similar levels of plasma and urinary ellagitannin metabolites in human subjects growth inhibition of entamoeba histolytica and e . invadens produced by pomegranate root ( punica granatum l.) lc-dad-esi/ms(n) determination of direct condensation fl avanol-anthocyanin adducts in pressure extracted pomegranate ( punica granatum l.) juice pomegranate wine has greater protection capacity than red wine on low-density lipoprotein oxidation nutritive and antioxidative potential of fresh and stored pomegranate industrial byproduct as a novel beef cattle feed central council for research in ayurveda & siddha ( ) database on medicinal plants used in ayurveda antibacterial activity of medicinal plants against pathogens causing complicated urinary tract infections effects of fruit ellagitannin extracts, ellagic acid, and their colonic metabolite, urolithin a, on wnt signaling macrophage paraoxonase (pon ) expression is up-regulated by pomegranate juice phenolic anti-oxidants via ppar gamma and ap- pathway activation bioavailable constituents/metabolites of pomegranate ( punica granatum l.) preferentially inhibit cox activity ex vivo and il- beta-induced pge production in human chondrocytes in vitro consumption of hydrolyzable tannins-rich pomegranate extract suppresses in fl ammation and joint damage in rheumatoid arthritis use of combined traditional chinese and western medicine in the management of burns traditional medicine in fiji: some herbal folk cures used by fiji indians medicinal plants from ujjain district madhya pradesh. part ii exploring the ameliorative potential of punica granatum in dextran sulfate sodium induced ulcerative colitis in mice pharmacological investigations of punica granatum in glycerol-induced acute renal failure in rats philippine alternative medicine. manual of some philippine medicinal plants in vitro effects of pomegranate juice and pomegranate polyphenols on foodborne viral surrogates flavonoids from punica granatum : potential antiperoxidative agents punica granaum l effects of pomegranate juice consumption on myocardial perfusion in patients with coronary heart disease in fl uenza virus variation in susceptibility to inactivation by pomegranate polyphenols is determined by envelope glycoproteins designing of novel carbonic anhydrase inhibitors and activators photochemopreventive effect of pomegranate fruit extract on uva-mediated activation of cellular pathways in normal human epidermal keratinocytes punicafolin, an ellagitannin from the leaves of punica granatum xl: revision of the structures of punicalin and punicalagin, and isolation and characterization of -o -galloylpunicalin from the bark of punica granatum l tannins and related compounds. xli: isolation and characterization of novel ellagitannins, punicacorteins a, b, c, and d, and punigluconin from the bark of punica granatum l tannins and related compounds. c. reaction of dehydrohexahydroxydiphenic acid esters with bases, and its application to the structure determination of pomegranate tannins, granatins a and b humarain: a new dimeric gallic acid glycoside from punica granatum l. bark nutritive value of pomegranate fruit and juice preliminary studies on the anti-angiogenic potential of pomegranate fractions in vitro and in vivo pomegranate peel extract prevents liver fi brosis in biliary-obstructed rats punica granatum peel extract protects against ionizing radiationinduced enteritis and leukocyte apoptosis in rats pomegranate ( punica granatum ) seed linolenic acid isomers: concentrationdependent modulation of estrogen receptor activity molluscicidal activity of punica granatum bark and canna indica root enzyme inhibition by the molluscicidal agent punica granatum linn. bark and canna indica linn. root the effect of pomegranate juice supplementation on strength and soreness after eccentric exercise protective effect of a potent antioxidant, pomegranate juice, in the kidney of rats with nephrolithiasis induced by ethylene glycol effects of pomegranate juice consumption on sperm quality, spermatogenic cell density, antioxidant activity and testosterone level in male rats antioxidant activity, polyphenol content, and related compounds in different fruit juices and homogenates prepared from different pomegranate accessions usda) ( ) usda national nutrient database for standard reference, release . nutrient data laboratory home page in vitro antimalarial activity and cytotoxicity of some selected cuban medicinal plants rapid dereplication of estrogenic compounds in pomegranate ( punica granatum ) using on-line biochemical detection coupled to mass spectrometry use of punica granatum as an antifungal agent against candidosis associated with denture stomatitis minimum inhibitory concentration of adherence of punica granatum linn (pomegranate) gel against s. mutans , s. mitis and c. albicans studies on the toxicity of punica granatum l. (punicaceae) whole fruit extracts activity of medicinal plant extracts against hospital isolates of methicillin-resistant staphylococcus aureus medicinal plant extracts as anti-escherichia coli o :h agents and their effects on bacterial cell aggregation pomegranate seed oil, a rich source of punicic acid, prevents diet-induced obesity and insulin resistance in mice punica granatum attenuates angiotensin-ii induced hypertension in wistar rats bioactive compounds from the seeds of punica granatum (pomegranate) constituents of the fl owers of punica granatum pomegranate: constituents, bioactivities and pharmacokinetics cellular and molecular mechanisms of pomegranate juice-induced anti-metastatic effect on prostate cancer cells pomegranate extract, a prooxidant with antiproliferative and proapoptotic activities preferentially towards carcinoma cells pomegranate polyphenols and resveratrol protect the neonatal brain against hypoxic-ischemic injury investigation of the alkaloids of punica granatum investigation of the alkaloids of punica granatum l. by partition chromatography antimutagenicity of some fl owers grown in thailand pomegranates ( punica granatum ), kiwifruit ( actinidia deliciosa ) and blood pressure: a pilot study medicinal fl owers. xxiii. new taraxastane-type triterpene, punicanolic acid, with tumor necrosis factor-alpha inhibitory activity from the fl owers of punica granatum effect of pomegranate peel extracts on oxidative stress in restrained mice pomegranate fl ower ameliorates fatty liver in an animal model of type diabetes and obesity dietary effect of pomegranate seed oil on immune function and lipid metabolism in mice chitinase iii in pomegranate seeds ( punica granatum linn.): a high-capacity calcium-binding protein in amyloplasts inhibitory effect of an ellagic acid-rich pomegranate extract on tyrosinase activity and ultravioletinduced pigmentation a triglyceride from punica granatum broad spectrum antimutagenic activity of antioxidant active fraction of punica granatum l. peel extracts screening of certain medicinal plants from india for their anti-quorum sensing activity inhibition of uvb-mediated oxidative stress and markers of photoaging in immortalized hacat keratinocytes by pomegranate polyphenol extract pomx studies on physicochemical properties and bioactive compounds of six pomegranate cultivars antiviral activity of tannin from the pericarp of punica granatum l. against genital herpes virus in vitro antiliperoxidant activity of pomegranate peel extracts on lard dietary antioxidants improve arteriogenic erectile dysfunction key: cord- - quigar authors: nan title: posters date: - - journal: j pept sci doi: . /psc. sha: doc_id: cord_uid: quigar no abstract is available for this article. laboratory of molecular biology and immunology, department of pharmacy, university of patras, patras, greece antimicrobial peptides (amps) are an important component of innate immune system of most living organisms. they have recently gained much attention as new anti-infective drugs with new modes of actions and few or no side effects. their antimicrobial spectrum covers gram-positive and -negative bacteria as well as fungi and certain viruses . fish have proven to be a rich source of antimicrobial peptides. three chrysophsin peptides (chrysophsin- , - , - ) have been identified in the gills of the red sea bream, chrysophrys major, which are all bactericidal to pathogenic bacteria at low concentrations . they are cationic α-helical peptides, rich in histidine residues and all end in an unusual rrrh motif. however, in addition to its high antimicrobial potency, chrysophsins have considerable hemolytic activity. the development of new analogues which would preserve high antimicrobial potency, but would lack the undesired hemolytic activity, could be a useful tool with possible commercial and clinical applications. in the present study, we synthesized a series of analogues of chrysophsin- with different ratios of lys and leu residues, utilizing the fmoc/but solid phase methodology . the synthesized analogues were purified and isolated by rp-hplc. the antimicrobial properties of the above peptide analogues are currently testing in gram positive (s. aureus, s. epidermidis, e. faecium) and gram negative (e. coli, p. aeruginosa) bacteria. the goal is to identify the minimum bacteriostatic and bactericidal concentrations of the analogues, under conditions that simulate the best possible that of the human organism. hemolytic or cytotoxic activity of the peptides will also be determined. the rise of antibiotic resistance demands the development of new antimicrobial agents. these should exhibit a novel mechanism of action so as to overcome the resistance and be invulnerable to 'not yet acquired resistance mechanisms'. such criteria are difficult to meet. however, cationic host defence peptides (hdps) have emerged as promising candidates. hdps target and disrupt bacterial membranes. in order to evade such a threat a bacterium would need to make substantial changes to its membrane composition disfavouring the development of resistance ( ) . however, exact role and mechanism of hpds in the regulation and monitoring of microbial invasions remain to be established. herein we will present new potential mechanisms of antimicrobial regulation by helical hdps using de novo ( ) and native systems ( ) . biophysical and microbiology aspects of the experimental designs will be discussed. the low number of the newly discovered antibiotics, emergence of multiple-drug resistance, and the alarming death rate due to the infection disease led to development the alternative means to combat the infections. the researchers accumulate information about antimicrobial drugs that could be result of the innate immunity mechanisms. armed only with the innate immunity, the insect has developed into the most widespread class in living kingdom. they produce several antimicrobial peptides with complementary and rapid mode of action. so far there are hundreds of antimicrobial peptides isolated from insect and lot of them are waiting to be discovered. the fleshly neobellieria bullata was chosen for isolation of these active compounds. its larvae in the third instar were squeezed to collect the haemolymph, which was gradually centrifuged and precipitated by acidified methanol. supernatant was subsequently separated by chromatographic methods (spe column, rp-hplc) to obtain fractions of short peptides. identification and characterization of these fractions were performed by tricine electrophoresis, mass spectrometry maldi-tof analysis and n-terminal sequencing. several fractions showed antimicrobial activity against institute of chemical kinetics and combustion, novosibirsk, russian federation in this work, we extracted d-structural information on newly synthesized, medium-length, double spin-labeled peptaibiotics using peldor spectroscopy. we investigated the magnetic dipole-dipole interactions between spin labels and the orientation selectivity effects. in particular, the medium-length peptaibiotics tylopeptin b , and heptaibin , double spin-labeled with the nitroxyl probe toac ( -amino- -oxyl- , , , -tetramethylpiperidine- -carboxylic acid), were studied by means of x-band peldor spectroscopy. this study was conducted on tylopeptin labeled at positions and (t ) and heptaibin labeled at positions and (h ) in frozen glassy methanol solutions at Κ. peldor data analysis was carried out using the theory developed for short interspin distances. the distance distribution functions between spin labels for Τ (maximum at . nm, halfwidth of . nm) and Η (maximum at . nm, half-width of . nm) were determined. the intramolecular distances observed between the labels allowed us to assign an essentially α-helical conformation to Τ and a largely prevailing -helical structure to Η under the aforementioned experimental conditions. are amidated at the c-terminus, as a result of a posttranslational enzymatic reaction. temporins are particularly active against gram-positive bacteria and are not toxic to eukaryotic cells. in this study we designed a series of analogues of tb with the aim to improve the peptide antimicrobial activity against both gram negative and gram positive strains and then to structurally elucidate the mechanism of interaction of active peptides with lps. the peptides have been synthesized substituting one or two amino acids with an alanine and lengthening the sequence with positively charged amino acids. among the designed peptides, one of the analogues, tb_kkg a, showed highly increased activity against gram negative bacteria and also a slightly increased activity against gram positive bacteria with a total lack of hemolytic activity. to develop ll- -derived short amps with prokaryotic selectivity and lipolysaccharide (lps)neutralizing activity, a series of amino acid-substituted analogs based on ig- (residues - of ll- ) were synthesized. analog a showed the highest prokaryotic selectivity, but much lower lps-neutralizing activity compared to ll- . the analogs, a , a , a and a with higher hydrophobicity displayed lps-neutralizing activity comparable to that of ll- , but much lesser prokaryotic selectivity. these results indicated that the proper hydrophobicity of the peptides is crucial to exert the amalgamated property of lps-neutralizing activity and prokaryotic selectivity. to increase lps-neutralizing activity of the analog a , we synthesized trp-substituted analogs (a -w and a -w ), in which phe or phe of a is replaced by trp. despite their same prokaryotic selectivity, a -w displayed much higher lps-neutralizing activity compared to a -w . this result suggested that the effective site for trp-substitution when designing novel amps with higher lps-neutralizing activity, without a remarkable reduction in prokaryotic selectivity, is the amphipathic interface between the end of the hydrophilic side and the start of the hydrophobic side rather than the central position of the hydrophobic side in their α-helical wheel projection. furthermore, d-enantiomeric peptides (a -w -e and a -w -e) of a -w and a -w possessed not only more improved prokaryotic selectivity and retained lpsneutralizing activity compared to a -w but also protease stability. taken together, a -w -e and a -w -e can serve as promising templates for the development of therapeutic agents for the treatment of endotoxic shock and bacterial infection. department of zoology, faculty of science, charles university, prague, czech republic antimicrobial peptides (amps) are among the most promising lead compounds for developing medicines in the fight against resistant pathogenic bacteria. we have already shown that the venom of wild bee is a rich source of pharmacologically interesting antimicrobial peptides [ ] [ ] [ ] [ ] . from the venom of solitary bee macropis fulvipes, we isolated and characterized the novel antimicrobial peptide named macropin (mac- ). by edman degradation and mass spectrometry, its primary sequence was established as gfgmalkllkkvl-nh . mac- possesses potent antimicrobial activity against both gram-positive andnegative bacteria and moderate hemolytic activity against human red blood cells. cd spectra confirmed that mac- can form an amphipathic α-helical secondary structure in the presence of membrane-mimicking substances as sodium dodecyl sulfate or organic solvents like trifluoroethanol. we prepared a series of mac- analogs to study the effect of incorporating d-amino acid residues into the sequence in various positions on antimicrobial and hemolytic activity, α-helicity and serum stability. the substitution of l-amino acid residues at n-terminal part of sequence by d-amino acid residues led to the improving hemolytic activity with maintaining or increasing antimicrobial activity. these modifications increased peptide stability in human serum. effect of the incorporation of d-amino acid residues into the mac- sequence on its α-helical structure will be discussed. the neutralization of endotoxins (lipopolysaccharide, lps) by suitable compounds has been shown to be a key step in the treatment of infectious diseases, in particular in the case of gram-negative bacteria. the active endotoxic center of lps is lipid a, its lipophilic part. an effective antimicrobial peptide against gram-negative bacteria is magainin , which was originally found in the skin of an african frog. here, we studied the interaction of hexa-acyl bisphosphoryl lipid a prepared from erwinia carotovora lps with magainin with some minor substitutions in the amino acid pattern. by using fourier-transform infrared spectroscopy, the gel to liquid crystalline phase transition of the acyl chains of lipid a, the conformation of their phosphate groups due to peptide binding, and the profile of the secondary structure of the peptides was investigated. the zeta potential of lipid a aggregates in the presence of the peptides was determined by measuring the electrophoretic mobility. small-angle x-ray scattering was performed for the elucidation of the aggregate structures in the absence and presence of the peptides, and isothermal titration calorimetry was applied for evaluating the thermodynamics of binding between peptides and lipid a. the data show that asp-or glusubstituted peptides improved the binding activity to lipid a correlated with characteristic changes in the physical parameters, which were stronger expressed for the aspsubstituted peptide. the new hydrogen bond connection between glu and asp by carboxylic acids apparently leads to a more pronounced -structure of the peptide. the conformation change of the peptide enhanced the activity of incorporation into the lipid a aggregates, along with changes in biochemical and biophysical parameters. royal college of surgeons, dublin, ireland cationic antimicrobial peptides (caps) have been reported to exhibit anticancer activity . one such peptide, p , has been shown to inhibit the growth of several cancer cell lines, with inhibiting concentration (ic ) in the range of to μm . however the concentration at which p and other caps act is too high to be clinically relevant. the enhancement of their activity can be achieved through the modification of their amino acid composition or the addition of other molecules. conjugation of naturally produced hydroxylated fatty acids to p showed a -fold improvement in its anticancer activity on a variety of human-derived cell lines. in addition to the enhancement of activity we wished to understand the mechanism of action of the peptide and conjugates. we investigated the uptake of conjugated and unconjugated peptides into hela (cervical) and miapaca (pancreatic) human cancer cells and the localisation of the peptide in the cell once taken up. we investigated the effect of altering the carbon number of the hydroxylated fatty acids ranging from hydroxyhexanoic acid (r ) to hydroxydodecanoic acid (r ) conjugated to p peptide and tested on hela and miapaca cell lines. circular dichroism studies were performed to investigate the effect on α-helical content due to amino acid composition alteration and hydroxyalkanoic acid conjugation. the effect of the position of the hydroxyl moiety on enhancement of activity was also investigated. in the current study p and its derivatives also lacked haemolytic activity with concentrations up to fold higher than ic values needed to observe any haemolysis. when current antibiotics become less efficient, there is a promise that some antibiotics can be replaced by other nature's substances, e.g. peptides. halictines are novel antimicrobial peptides isolated from the venom of the eusocial bee halictus sexcinctus. we obtained four analogues of the native peptide hal from iocb av cr. they already characterized structural properties of these peptides and their antimicrobial activity against selected bacteria . the analogues were prepared by point mutations of native peptide, which could increase antimicrobial activity and decrease undesirable hemolytic activity. our aim was to characterized membrane permeation activity of halictines through the use of a basic model of biological cells -large unilamellar phospholipid vesicles luvs. we prepared two basic types of leakage assays based on luvs with free dyes entrapped inside and one assay with laurdan content. we used classical steady state fluorescence spectroscopy and advanced fluorescence methods for study of dyes escape from luvs and we also used laurdan generalized polarization technique gp for better understanding peptide insertion into membrane. in this way we received complementary information and we can conclude that the most active peptides are the native hal and analogue hal / . however hal / requires presence of negatively charged phospholipids in membrane which may explain its higher selectivity against bacteria. furthermore, fcs results have shown that the leakage happens via pore formation. results from gp revealed that peptide insertion in the membrane do not lead directly to formation of pores. against a wide range of microorganisms, mainly by perturbing the permeability of bacterial membranes through the formation of pores. however, amps effects on membrane properties probably extend beyond poreformation. we performed a systematic spectroscopic analysis of the effects on membrane structure and dynamics of two very different amps: the cationic pmap- , which creates pores according to the "carpet" model , and alamethicin, which forms "barrel-stave" channels . by using fluorescence anisotropy measurements on liposomes comprising probes localized at different depths in the bilayer, we measured peptide effects on membrane fluidity and order. laurdan spectral shifts provided indications about water penetration in the bilayer. in the case of pmap- , it was possible to focus specifically on the lipids surrounding the peptide by following the membrane-probe fluorescence due to fret from the peptide trp residues. finally, peptide-induced perturbation of lateral mobility and domain formation were determined by several methods. all experiments were compared with liposome-leakage measurements: while for pmap- all membrane-perturbing effects are correlated with the vesicle leakage process, alamethicin does not significantly influence membrane dynamics at the concentrations in which it forms pores. surprisingly, in all cases the most significant peptide-induced effect is a reduction in membrane fluidity. we have reinvestigated -residue peptaibols named metanicins from an ascomycetous fungus originally described as metarhizium anisopliae strain cbs . (cbs = centraalbureau voor schimmelcultures, utrecht, the netherlands). however, due to unusually shaped conidia and based on rna-sequencing of its internal transcribed spacer (its) region, the identification of cbs . as metarhizium has been withdrawn and this particular strain is currently under taxonomic reinvestigation . sequencing of four isolated peptides by fab-ms, esi-ms and edman degradation of partial hydrolysates revealed structural relationship to -residue peptaibol antibiotics paracelsins from trichoderma reesei (=hypocrea jecorina). sequences determined are: ac-u-a-u-a-u-a(u)-q-u-v-u-g-l-u-p-v-u-u(j)-q-q-fol (exchange positions in parenthesis; ac, acetyl; u, aib, α-aminoisobutyric acid; j, d-isovaline; fol, l-upmc univ paris laboratoire des biomolécules; cnrs umr ; ens lbm; address: laboratoire des biomolécules, ens dpt de chimie, , rue lhomond f- , paris, france current data suggest that the cellular uptake of cellpenetrating peptides (cpps) occur by two processes: direct translocation across the plasma membrane and endocytosis . the large diversity of cpp sequences described in the literature (derived either from fragments of proteins, structurally constrained synthetic peptides, peptide libraries or dendrimers) has hampered the identification of general rules for their efficacy of internalisation. we have used a reductionist approach, restricting the cpp functional groups (amide and guanidinium) and tailoring cpp amphiphilic properties. two families of cpps have been designed: ) primary amphiphilic cpps corresponding to tetra-arginines functionalised with fatty acid chains of different lengths and ) secondary amphiphilic cpps containing arginine and alanine or tryptophan residues . these cpps were linked by a disulfide bridge to a peptide inhibitor of protein kinase c (pkci). the efficiencies of internalisation of the conjugates were quantified by a method based on maldi-tof mass spectrometry previously developed in our group . the mechanism of internalisation was studied by comparing the amounts of cell-surface bound and internalized pkci cargo on cho-k cells and glycosaminoglycan-deficient cho cells at o c and o c. conjugates were found to enter by both direct translocation and glycosaminoglycandependent endocytosis. in addition, the primary amphipathic cpps were found to be more efficient than the secondary amphipathic ones. furthermore, structural or mechanistic novelty does not guarantee immunity from resistance, with strains resistant to linezolid identified prior to fda approval. therefore, modifying existing antibiotics to overcome resistance mechanisms presents an opportunity to rationally develop effective new drugs more rapidly than screening for new structures. vancomycin is a glycopeptide commonly used as a front line treatment for infections caused by methicillinresistant staphylococcus aureus (mrsa). the emergence of vancomycin-resistant enterococci (vre), vancomycinintermediate s. aureus (visa) and vancomycin-resistant s. aureus (vrsa) has prompted the development of semisynthetic glycopeptides . we have generated a variety of glycopeptide derivatives that show superior antibacterial activity against mrsa and vre compared to vancomycin and second generation lipoglycopeptides. this was undertaken by employing a combination of solid phase and solution phase chemistry to attach a membraneassociative element that selectively binds to bacterial membranes in preference to eukaryotic membranes, thus increasing the local concentration at the lipid ii d-ala-d-ala peptidoglycan cell wall precursor target site. three novel antimicrobial peptides, named panurgines (png), were isolated from the venom of wild bee panurgus calcaratus. one of them is dodecapeptide with sequence lnwgailkhiik-nh (png- ). the next two peptides are almost identical. these are cyclic peptides containing amino acid residues and two intramolecular disulfide bridges ldvkkiicvackixpnpackkicpk-oh (x=k png-k and x=r png-r). all peptides exhibited antimicrobial activity against gram-positive bacteria and gram-negative bacteria, antifungal activity and low haemolytic activity against human erythrocytes. we prepared analogues of α-helical amphipathic png- with the aim to improve its biological properties and a linear analogue of png-r to elucidate the importance of disulfide bridges for its activity. in the second part of the study, we followed the effect of panurgines on the degree of membrane disruption by observing the leakage of fluorescence dye (calcein) entrapped in artificial phospholipids vesicles [ ] . specifically, we investigated membrane interactions of pngs with the vesicles made from negatively charged : dopc/dppg and : dopc/dopg vesicles as a general model of bacteria membrane and : : dopc/dopg/cl as a possible model for a membrane of bacillus subtilis. the membrane interaction of pngs was also investigated on uncharged dopc vesicles as potential model membrane for erythrocytes. pngs exhibited weak dye-leakage activity for neutral vesicles, while they effectively induced dye leakage in the presence of negatively charged vesicles. these results indicate that pngs have stronger potency to disrupt bacteria-mimicking anionic membranes than those which mimic eukaryotic cell membrane. department of biochemistry and toxicology, university "lucian blaga", sibiu, romania a common tool to bias the conformation of linear peptides is the insertion of side-chain modified amino acids or sidechain/main-chain conformationally restricted building blocks. an alternative approach is a simple backbone modification. in this connection, backbone amide replacements with (almost) isosteric surrogates were extensively used. these modifications may impart resistance to enzymatic degradation and better bioavailability to the peptides, but also influence the secondary structure. a thioamide (ψ[cs-nh]) is perhaps the closest structural mimic of an amide. however, it possesses different and attractive features: (i) its nh group forms stronger hydrogen bonds, being more acidic than that of the amide. (ii) its c-n bond undergoes cis/trans isomerization by irradiation at nm (π→π* transition). (iii) it may act as a "minimalist" fluorescence quencher. for all these reasons, we started a programme aimed at exploring how the endothioamide bond affects peptide folding and bioactivity. in this communication, we describe the synthesis and conformational results of the three analogs of the membrane-active peptaibiotic trichogin ga iv listed below: n-octanoyl-aib-gly-ψ[cs-nh]-leu-aib-gly-gly-leu-aib-gly-ile-leu-ome ( / ) n-octanoyl-aib-gly-leu-aib-gly-ψ[cs-nh]-gly-leu-aib-gly-ile-leu-ome ( / ) n-octanoyl-aib-gly-leu-aib-gly-gly-leu-aib-gly-ψ[cs-nh]-ile-leu-ome ( / ) the syntheses of the three peptides were accomplished in solution according to a fragment condensation approach. appropriate thioamide-containing tri-or tetrapeptides were prepared by treating the corresponding all-amide precursors with the lawesson reagent. ft-ir absorption, d-nmr and cd conformational investigations on the three analogs were conducted in comparison with the naturally occurring peptaibiotic. all three analogs maintain the capability to interact with the dope/dopg model phospholipid membranes and exhibit a comparable bioactivity against s. aureus. peptide-peptide interaction of lactococcin g class iib two peptide bacteriocin h. etayash, w.soliman and k. kaur* faculty of pharmacy and pharmaceutical sciences, university of alberta, edmonton, alberta, t g e lactococcin g, a class iib two-peptide bacteriocin, consists of two complementary peptides lcng-α and lcng-β that act as one functional unit with optimal antimicrobial activity achieved by the presence of both peptides in approximately equal amounts. in this study we have investigated the mechanism of pairing of the two complementary peptides as well as explored any specific interaction that could take place between the peptides. molecular dynamics (md) simulation was employed to study the interactions at the atomistic level. four different md simulations with the peptides in a lipid bilayer system were conducted. md results from these simulations confirmed and pointed out that (i) the two putative gxxxg motif, g xxxg in lcng-α and g xxxg in lcng-β, were attracted and came closer to each other, showing the role of these motifs in attracting the two peptides to each other. closer views, however, showed no clear interactions between these two motifs. most likely, nonspecific interactions play a role in bringing the two peptides together; (ii) variations and loss in the secondary structure in both the peptide fragments were confirmed among the four simulations. on the contrary, stability of helical regions was identified between residues w -g and d -q in lcng-α and v -e in lcng-β; and (iii) role of tryptophan at the n-terminal regions in positioning and setting the peptide orientations were confirmed which matched the previous reported results. faculty of pharmaceutical sciences, unesp -univ. estadual paulista, araraquara, sa͂ o paulo, brazil antibiotic resistant bacterial strains represent a global health problem. antimicrobial peptides (amps) are promising novel antibiotics because they have displayed little or no resistance effects. it is well known that the charge, amphipathicity, hydrophobicity and helicity of the peptide are fundamental for the biological activity. in addition, covalent dimerization appears as a new parameter to be studied. in this way, several bioactive sequences were dimerized obtaining pharmacotechnics advantages like enhanced antimicrobial activity, solubility and proteases resistant. however, the effect of this modification is unclear since dimeric versions of some amps are toxic . to evaluate the effects of dimerization on the structure and biological activity of the amp aurein . , the monomeric version (au) and the c-and n-terminal dimers, (au) k and e(au) , respectively, were synthesized. circular dichroism results indicated that dimeric versions showed more defined structures in aqueous solution. e(au) showed "coiled coil" structure while (au) k an αhelix structure. in contrast, au displayed typical spectra for disordered structures. in tfe and lpc, all the peptides acquired a high amount of α-helix structure. the antimicrobial activity against bacteria and yeast decreased with dimerization. however, dimeric peptides promoted the aggregation of c. albicans. hemolytic and vesicle permeabilization assays showed that au has a concentration dependence activity, an effect that can be assigned to a "carpet" like mechanism peptide, whereas this effect was less pronounced for dimeric versions, suggesting that dimerization may change the mechanism of action. in conclusion, our studies showed that the effects of amp dimerization are complex and still unclear. , the first antimicrobial peptide generated in vivo and isolated from the gut contents of the cattle tick boophilus microplus . we have shown that these peptides are equally lethal to candida albicans mdm and practically not active on human erythrocytes . to examine the properties and mode of action of hb - a, we synthesized it and its fluorescently labeled analogue (fam-hb - a) by the solid-phase method at o c, purified them by rp-hplc and characterized their purified forms by lc-esims. at low salt concentration, both peptides were found to inhibit the growth of candida albicans atcc , candida parapsilosis atcc and candida krusei atcc , but hb - a was two-fold more active (mics of . ; . and . μm, respectively). at those concentrations, both peptides also kill the fungi. assays with human erythrocytes showed that, likewise hb - a, fam-hb - a present activity lower than % at μm. apparently, hb - a targets the membrane cell because confocal microscopy analysis revealed that, at the half of mic value, fam-hb - accumulates on the fungal cell membrane. in contrast, fluorescence activated cell sorting (facs) analysis revealed that, at the mic, more than % of the fam-hb - a penetrates into the cell. membrane permeability assay using hb - a, c. albicans atcc and the kit live/dead funga light confirmed progressive membrane damage associated with an increase in peptide concentrations. the use dibac ( ) and facs analysis showed that hb - a alters the plasma membrane potential, leading to cell death. supported by fapesp, cnpq and capes. lasso peptides form a growing class of to residue ribosomally-synthesized and post-translationally modified peptides produced by bacteria. they share a rigid and compact interlocked structure consisting of a macrolactam ring at the n-terminus and a c-terminal tail that is looped back and threaded through the ring, forming a typical [ ] rotaxane , . the macrolactam is formed by condensation of an asp or glu side-chain with the free amino group of a gly or cys . the lasso fold is stabilized either by steric hindrance assumed by bulky amino acid side-chains and/or by disulfide bonds between cysteines from the tail and the ring. given this structure, lasso peptides display a high stability against proteolytic and chemical degradation. they are biologically active on various enzymatic targets, which confer them in some cases an interesting antimicrobial activity. given its characteristics, the lasso scaffold thus represents a promising tool for biotechnological application in the development of bioactive peptides. until now, nine peptides had been structurally characterized as lasso peptides. based on a genomics-based approach, we identified a novel lasso peptide from streptomyces sviceus that we termed sviceucin. it was produced in high yield by heterologous expression in s. coelicolor and submitted to structural analysis by mass spectrometry and nmr. sviceucin is residue long and stabilized by two disulphide bonds. their connectivities were identified mainly from the typical noes between the beta-protons of the cysteines. the lasso structure of sviceucin was obtained by nmr-based molecular modelling. sviceucin was shown to exhibit antibacterial activity directed against gram positive bacteria, while gram-negative bacteria and fungi showed resistant. the penicillium chrysogerum antifungal protein (paf) is a cysteine-rich, cationic protein that inhibits the growth of a variety of filamentous fungi without toxic effect on mammalian cells . although paf is used to be produced in p. chrysogerum or a similar microorganism, preparation of analogues of the protein for structural and functional investigations requires an efficient chemical method. the unsuccessful continuous synthesis of the -mer small protein prompted us to use native chemical ligation . the syntheses of the fragments were performed by solid-phase method applying tboc chemistry. using the acid-labile tboc protecting group, the thioester end of the n-terminal fragment remains intact during the course of the synthesis. the first attempt was the synthesis of peptides with pmethylbenzyl groups on the side chains of all of the six cysteine residues. under no circumstances oxidative folding provided the natural disulphide bridge pattern. the failed attempts led us to orthogonal protection of the sulphydryl groups. different sets of protecting groups were tried and evaluated. our experiments showed that basic treatment triggered rearrangement of the previously formed disulphide pattern. thus, base-labile protecting groups (such as -fluorenylmethyl, fm) have to be avoided in the synthesis of paf. the alarming increase and spread of antibiotic resistance among bacterial pathogens has stimulated the development of new antibacterial agents with innovative mode of action. antimicrobial peptides with broad spectrum activity are widely distributed in nature and play an important role in innate immunity in several species, including humans. tigerinins are a unique family of -to -residue antimicrobial peptides found in skin secretion of the indian frog rana tigerina , . characterized by a disulfide-bridged loop composed of nine amino acids, tigerinins do not show primary structural homology to any known antimicrobial peptides from amphibians. tigerinins could provide novel lead compounds for the design of effective antimicrobial peptides with a new mode of action. the peptide murdp has been identified after the screening of phage display libraries against pseudomonas aeruginosa cell wall biosynthesis murd amide ligase enzyme . murdp is a low micromolar range inhibitor of murd enzyme and showed good antimicrobial activity. composed of nine amino acids, it is also characterized by a nine residues disulfide-bridged loop containing two prolines. this great similarity with tigerinins, led us to investigate if murd enzyme could be a potential target for these peptides. in silico analyses using modelling, molecular dynamics and docking with p. aeruginosa murd showed that murdp and tigerinin- and - make similar interactions in the binding site. these results suggest that murd may be an intracellular target for tigerinin- and tigerinin- . synthesis, murd enzymatic inhibition assay, antibacterial activity evaluation and structure-activity relationships of murdp and tigerinins analogs will be presented. h. etayash, l. norman, t. thundat*, k. kaur* faculty of pharmacy and pharmaceutical sciences, department of chemical and materials engineering, university of alberta, edmonton, alberta t g e , canada listeria monocytogenes is a gram positive bacterium that accounts for about % of the deaths resulting from food borne illnesses in north america. moreover, l. monocytogenes is considered one of the most difficult bacteria to detect in contaminated food products. while standard microbiological and biochemical assays currently used are accurate and sensitive, they are time consuming and often require specialized instruments operated by a trained user making on-site testing difficult. to this end, we propose the development of an antimicrobial peptide (amp) or peptide fragment sensor for the on-site detection of l. monocytogenes. leucocin a, which is a naturally occurring amp consisting of a amino acid sequence, is known to exhibit specific activity against l. monocytogenes at pico to nanomolar concentrations. for this reason, we have synthesized a shorter peptide fragment of leucocin a consisting of amino acids using solid phase peptide synthesis. the peptide was purified by reversed phase hplc and maldi-tof mass spectrometry indicates the desired biological entity was achieved. by including an n-terminal cysteine group, the tailored amp was readily immobilized at a gold interface. the resulting thickness and molecular orientation, determined by ellipsometry and grazing angle infrared spectroscopy, respectively, indicate that the helical peptides were adsorbed on the interface with a preferred orientation parallel to the surface. the bacterial specificity of the anchored leucocin a fragment was tested against three gram positive bacteria and results reveal that the adsorbed amp exhibits a limit of detection of approximately one bacterium/μl which is a clinically useful detection range. faculty of science, university of south bohemia, České budějovice, czech republic during the last few years we have identified three novel defensins from arthropods. two of them, lucifensin and lucifensin ii were purified from various tissues of lucilia sericata and l. cuprina larvae, respectively. larvae of these flies are routinely used in the hospitals around the world for the treatment of non-healing infected wounds in the procedure known as maggot therapy. these amino acid residues and three disulfide bridges peptides differ from each other only in one amino acid residue in position (val-ile). linear precursor of lucifensin was prepared by fmoc-spps chemistry which was then subjected to the oxidative folding yielding a peptide with a pattern of disulfide bridges identical to that of native lucifensin and other insect defensins. this was examined by the identification of the fragments resulting from the thermolysin digestion of lucifensin by means of mass spectrometry. however, this cyclization reaction proceeded via an intermediate having incorrect pairing of disulfide bridges. from the hemolymph of blood sucking tick dermacentor marginatus (d.m.) we purified defensin containing amino acids and three disulfide bridges. its sequence determined by edman degradation and mass spectrometry was identical to that previously determined by molecular biology methods . sequence of d.m.defensin shows no homology to insect defensins. by spps prepared linear precursor of d.m.-defensin was subjected to oxidative folding under the open air. the linear peptide was straightforwardly folded into cyclic one which was identical to the native peptide. in antimicrobial assay using a set of different bacteria all three studied defensins show activity preferentially against gram-positive bacteria including staphylococcus aureus but are inactive against gram-negative ones. the importance of disulfide bridges on tertiary structure of defensins and their antimicrobial activity will be presented. recently, the chemical structure and conformation of pseudodesmin a has been determined through x-ray diffraction and nmr spectroscopic analysis . in this way pseudodesmin a was identified as a new member of the viscosin group of antimicrobial peptides (amps). in addition, it was demonstrated that individual molecules self-assembly in apolar environment into a supramolecular pore-like structure, providing structural support for its biological activity , . to further explore the structure-function relationship, a viable synthesis strategy for pseudodesmin a analogues was developed, based on side-chain attachment of the first amino acid to the solid support, followed by stepwise fmoc solid-phase synthesis of the linear peptide precursor and on-resin head-to-tail cyclization. nmr study confirmed the molecular structure and thus the development of an efficient and successful synthesis of this type of amp's. these results and the synthesis route will be presented. trichogin ga iv, isolated from the fungus trichoderma longibrachiatum , is the prototype of lipopeptaibols, a subclass of short-length peptaibiotics exhibiting membranemodifying properties. its primary structure is as follows: n-oct-aib -gly-leu-aib-gly-gly-leu-aib-gly-ile -lol, where n-oct is n-octanoyl, aib is α-aminoisobutyric acid, and lol is the , -amino alcohol leucinol. this peptaibol is predominantly folded in a mixed -/αhelical conformation with a clear, albeit modest, amphiphilic character . in this work, we synthesized by solution and solid-phase methodologies a set of trichogin ga iv analogs in which the four gly residues, lying on the poorly hydrophilic face of the helical structure, are substituted by one (or more) strongly hydrophilic lys residues. moreover, we synthesized another set of analogs where one (or more) aib residues are replaced by leu. the conformational preferences of these analogs were assessed by x-ray diffraction, cd, and d-nmr techniques . we tested the role played by the substitutions on the peptide bioactivity, e.g. protease resistance, cytotoxicity, and hemolysis. cytotoxicity was tested using three in vitro cell-based assays: (i) human red-blood cells lysis; (ii) cell mortality in total human blood leukocytes and in separate subpopulations; (iii) cell mortality in three tumor-derived stable cell lines (hela, a , and a ). our data show that some of our trichogin analogs are active against tumor cells, leaving the leukocytes unaffected. a convenient post-screening ring opening approach for the decoding of one-bead one-compound cyclic peptide libraries a. girard, e. biron* faculty of pharmacy, université laval and chuq research center, quebec, canada combinatorial chemistry has been widely used as an effective method for the generation and screening of synthetic peptide libraries. amongst the different combinatorial methodologies to discover new bioactive peptide-based compounds, we were particularly interested in the one-bead one-compound (oboc) approach . this powerful approach fully exploit the great molecular diversity accessible with peptides and has been used to identify a great number of ligands and modulators for a wide variety of biological targets. however, its use with cyclic peptides is limited by difficulties in sequencing hit compounds by edman degradation or tandem mass spectroscopy due to the lack of free n-terminal amine and complicated fragmentation patterns, respectively. this problem has been overcome by pei and coworkers by using a bead segregation strategy in which the outer layer exposes the cyclic peptides and the inner layer the linear counterpart for sequencing . more recently, lim et al. reported an elegant method to prepare and sequence oboc cyclic peptoid libraries without encoding by using a ring opening approach with triazine-based cyclic derivatives . unfortunately this method is incompatible with amino acids bearing some functionalized side chains. based on this strategy, we have developed an efficient method to prepare oboc cyclic peptide libraries that does not require encoding by using a simultaneous ring opening/cleavage approach. the procedure is compatible with commonly used amino acids and allows rapid and efficient sequencing of selected hits after on-bead screening. the synthesis of an oboc cyclic peptide library, ring opening methodology and sequencing by mass spectrometry will be presented. cyclotides are a very abundant class of plant peptides that display immense sequence variability around a conserved cystine knot motif and a head-to-tail cyclized backbone conferring them with remarkable stability . their intrinsic bioactivities combined with tools of peptide engineering make cyclotides an interesting template for the design of novel agrochemicals and pharmaceuticals . however, laborious isolation and purification prior de novo sequencing limits their discovery and hence their use as scaffolds for peptide-based drug development . here we extend the knowledge about their sequence diversity by analyzing the cyclotide content of a violet species native to western asia and the caucasus region . using an experimental approach, which we named 'sequence fragment assembly' by maldi-tof/tof-based peptidomics, we were able to characterize novel cyclotides from viola ignobilis. amino acid sequencing of various enzymatic digests of cyclotides allowed the accurate assembly and alignment of smaller fragments to elucidate their primary structure, even when analyzing mixtures containing multiple peptides. using in-source decay and high energy collision induced dissociation of digested cyclotides allowed to distinguish isobaric residues ile and leu. overall this work underlines the immense structural diversity and plasticity of the unique cyclotide framework. the presented approach for the sequence analysis of peptide mixtures facilitates and accelerates the discovery of novel plant cyclotides. glycation is a nonenzymatic reaction occurring between reducing sugars and reactive amino groups of biomolecules. the reaction leads to a formation of a heterogeneous mixture of compounds which are classified as early, intermediate or advanced glycation end products (age). these compounds, especially advanced glycation end products, are involved in many pathological processes, mainly diabetic complications, and could be markers of certain diseases. detection of early products of glycation (amadori products) is a relatively easy task and can be performed by various methods including e.g. ms/ms techniques, isotopic labeling and affinity chromatography on immobilized boronic acid , . however, the diverse structures of ages make detection of these compounds more challenging. the aim of the study was testing a new method of ages identification based on isotopic c labeling. a model protein (hen egg lysozyme) was modified with an equimolar mixture of [ c ]glc and [ c ]glc. then the glycated protein was subjected to reduction of the disulfide bridges followed by enzymatic hydrolysis. the obtained digest was analyzed by lc-ms methods. the glycation products were identified on the basis of characteristic isotopic patterns resulting from the use of isotopically labeled glucose. this method allowed for identification of early maillard reaction products and different structures of the glycation end products. isotopic labeling technique combined with lc-ms is a new and very sensitive method for identification of the advanced glycation end products even if their structures are unknown. this method could be also used as an alternative method of detection of amadori products. in the course of a project aimed to assess the significance of antibiotics for the producing organism(s) in the natural habitat, we screened a specimen of the fungicolous fungus hypocrea phellinicola growing on its natural host phellinus ferruginosus . using a peptaibiomics approach , , we detected -and -residue peptide sequences by (u)hplc/hr-esi-qqtof-ms. structures of peptaibiotics found were independently confirmed by analyzing the peptaibiome of an agar plate culture of h. phellinicola cbs (ex-type) grown under laboratory conditions. notably, h. phellinicola could be identified as a potent producer of -, -, (culture) and -residue (specimen) peptaibiotics of the suzukacillin-type . minor components of the -residue peptaibols, herein named suzukacillins c, are assumed to carry a c-terminal residue tentatively assigned as tyrosinol (tyrol). in addition, the previously isolated suzukacillin b was sequenced and shown to be a microheterogeneous mixture of -residue peptaibols. in order to further investigate the significance of antibiotics for the producing organism(s) in the natural habitat, we screened specimens of the fungicolous fungus hypocrea pulvinata growing on its natural hosts piptoporus betulinus and fomitopsis pinicola . using a peptaibiomics approach , we detected -, -, -(major sequences), and -residue peptide sequences in the five specimens analyzed by (u)hplc/hr-esi-qqtof-ms. structures of peptaibiotics found were independently confirmed by analyzing the peptaibiome of pure agar cultures obtained by single-ascospore isolation from the specimens . major, -residue peptaibols were assigned as deletion sequences of the trichosporins b lacking the ala/aib residue in position . our results corroborate that: i) peptaibiotics are, indeed, biosynthesized in the natural habitat, thus, ii) their membrane-perturbing formation of ion channels may support the parasitic life style of a fungicolous fungus. based on methodology that we have developed in our lab , we identified specific and selective substrates for these serine proteases. we used a , membered pnaencoded peptide library to screen , possible peptide substrates in a single experiment. the library was incubated with the protease of interest and then hybridized on a custom designed dna microarray. microarray scanning and data analysis allowed the measurement of the changes in fam/tamra ratios resulting from the protease activity and the determination of the protease specificity. to verify the predicted activity and specificity, fret peptides were synthesized, incubated with the enzymes and the hydrolysis reaction was followed by monitoring fluorescence emission. specificity constants kcat/km were calculated and the cleavage sites of the peptides were identified. dubs were, moreover, found to be associated with several diseases and as such are emerging as potential therapeutic targets . several directions have been pursued in the search for lead anti-dub compounds. however, none of these strategies have delivered inhibitors reaching advanced clinical stages due to several challenges in the discovery process, such as the absence of a highly sensitive and practically available high-throughput screening assay . in this study, we report on the design and preparation of a fret-based assay for dubs based on the application of our recent chemical method for the synthesis of ub bioconjugates . in the assay, the ubiquitinated peptide was specifically labeled with a pair of fret labels and used to screen a library comprising compounds against uch-l . such analysis identified a novel and potent inhibitor able to inhibit this dub in time-dependent manner with kinact = . μm and ki = . μm. our assay, which was also found suitable for the uch-l enzyme, should assist in the ongoing efforts targeting the various components of the ubiquitin system and studying the role of dubs in health and disease. . more recent work based on rna interference experiments on a mouse model suggested that isoform-specific inhibitors against nmt might be effective anti-cancer agents as a knockdown of nmt inhibits the tumour growth, whereas knockdown of nmt has no effect . if residual nmt activity can compensate for loss of nmt function in healthy cells, potential toxicity may also be minimised. we developed a method to identify peptide or protein substrates of nmt and/or nmt . peptides/ proteins are exposed to nmt and/or nmt and an alkyne-tagged analogue of myristoyl coa. subsequent azide-alkyne "click" cycloaddition allows visualisation of the myristoylated substrates in fluorescence or chemiluminescence, using a fluorescent or a biotin moiety on the capture reagent. this labelling technology was applied to peptide libraries prepared on microarrays to investigate nmt / isozyme substrate specificity using recombinant nmt and nmt . peptides made of the first or amino acids at the nterminus of known myristoylated proteins were functionalised with a biotin moiety at the c-terminus and immobilised on an avidin-functionalised glass plate before being screened for activity. selective peptide substrates will be developed as isozyme-specific inhibitors and applied in cancer cell lines. using chemical proteomics and the labelling technology, a selective nmt or nmt inhibitor could also be used to identify protein substrates of one isozyme. for this purpose computer programs are created which can generate fragments of one compared structure and to reveal homology by their scanning along the amino acid sequence of another. our analysis was performed by comparing the primary structures of all possible protein fragments with the amino acid sequences of all presently known natural regulatory oligopeptides. the oligopeptides were extracted from the erop-moscow database which at the time of analysis contained data on the structures and functions of more than , natural oligopeptide regulators. the structure-function analysis was performed using a specialized software package. the input data were the complete amino acid sequences of the proteins used as a source of fragments with a specified length. then the initial sequence was fragmented in a stepwise manner. for example, in the case of dipeptide fragments, this procedure produced fragments with the following numbers of amino acids from the n-terminus - - , - , and so on until the fragment that started at the second residue from the c-terminus. the cases when the amino acid sequence of a fragment coincided with part of the primary structure of a natural oligopeptide were recorded in the total protein chemical synthesis requires a case by case design and optimization which is governed by factors such as the solubility of the individual peptide segments, their primary sequence and in particular the presence of "difficult" amino acid residues at ligation junctions such as proline or the location of cysteines. usually, a subset of chemical tools are selected among a vast array of methodologies to match the specificities of the target protein. in this context, methods enabling the assembly of three peptide segments in the n-to-c and c-to-n direction play a central role and must be considered as complementary as they can be selected for building subdomains of the target protein. to date, most of the proteins were assembled in the c-to-n direction. only few methods are available for the n-to-c sequential assembly of proteins, whose design is highly challenging. we have recently reported that sea ligation, that is the reaction of a bis( -sulfanylethyl)amido group (called sea) with a cysteinyl peptide, allows the formation of a native peptide bond in water and at neutral ph . in this communication we will show that native chemical ligation and the unique chemical properties of sea group , can be combined in order to design a highly efficient one-pot three segments protein assembly procedure, working in the n-to-c direction amylin is one of the most amyloidogenic peptides, its fibrils are responsible for causing type ii diabetes. amyloid formation mechanism is investigated both to find amyloid inhibitors as potential medical drugs, and to use amyloids as potential self-assembling biomaterials [ ] . amyloid formation of amylin - , its reverse and designed analogue beta-sheets and beta-sheet stacks was studied by molecular dynamics (md), amber . , f force field. md revealed that for amylin - and its reverse analogue both the parallel and antiparallel beta-sheet and beta-sheet stack structures are stable suggesting that this could explain the high tendency of amylin to form amyloid fibrils. parallel amylin - beta-sheet stacks are kept together by two hydrophobic cores, while for the antiparallel system the dominating is the backbone hydrogen bonding between neighbor strands. also the bent form of the amylin - beta-sheet is stable. this is in concordance with transmission electron microscopy (tem) experiments stating that all three peptides, amylin - , its reverse and designed analogues, exhibited significant fibrillar polymorphism [ ] university of gdansk, poland molecular dynamics (md) of two peptides dlsfmkge (mk) and dlsfkkge (kk) not related to any known disease was run to investigate the mechanism of the amyloid formation. the parallel and antiparallel [ ] betasheets of mk and kk peptides were simulated by molecular dynamics (md), amber . , f force field, ntp protocol. it was found that antiparallel beta-sheets both of mk-and kk-peptides show much higher stability than the corresponding parallel beta-sheets. this md result was supported by atr-ftir spectroscopy [ ] . the betasheet stacks built from six ten stranded antiparallel beta-sheets of mk-and kk-peptides: x xmk and x xkk, were subjected to md. it was found that the mk-system, x xmk, is strongly kept together due to hydrophobic core built from two metionines, two phenylalanines and two leucines, but the kk-system, x xkk, which differs only by one mutation m k dissolves already at ns of md run, because the separate beta-sheets don't hold togather in the betasheet stack due to lost hydrophobic core. the hydrophobic core of the mk-system consists of hydrophobic units centered on the two phenylalaninetwo metionine hydrophobic interactions, and two leucines from the both sides stabilize the unit. this mechanism could be used in amyloid based biomaterials. urokinase plasminogen activator (upa) is a serine protease involved in the metastasis of several tumor types. upa is therefore an interesting target in cancer therapy. upain- is a new analogue of a highly specific peptidic inhibitor (upain- ) of upa. the peptide contains twelve amino acids and is cyclized through the cysteines at its termini (s -s cyclo-ac-cswrglenhaac-nh ). upain- inhibits upa with a ki of approximately μm. one method to improve binding affinity is multivalent exposure of the inhibitor, where the local concentration at the binding site is increased. fusion of upain- to the trimeric tetranectin showed improved binding affinity compared to the single peptide. here, we report efforts towards novel chemically linked upain- peptides to allow multivalent display. the ki value of an upain- dimer, linked by a short peg chain through the n-termini, was almost halved compared to that of the single peptide ( μm). this motivated us to explore the role of the site (n-or cterminal) and the size of the linking segment on the binding affinity. additionally, the influence of the number of upain- peptides in the molecule (two vs. four) was investigated by synthesizing a carboprotein that displayed four upain- peptides. we present two novel nmr spectroscopic approaches to study reversible self-assemblies in solution. both methods were applied on the self-assembling pseudodesmin a, a pseudomonas produced cyclic lipodepsipeptide that has the capacity to form pores in cellular membranes. , the first method is based on the dependence of the c α relaxation rate constants on the anisotropy of the assembly. when the monomer conformation is known and the multiple ch bonds in the monomer sufficiently sample all orientations, the rotational diffusion coefficients can be assessed, revealing assembly shape information. in addition, the orientation of the monomer within the assembly is obtained. the second method is based on fitting translational diffusion coefficient data as a function of concentration in a model-free way, i.e. without assuming an oligomer shape beforehand. here, it is assumed that the diffusion coefficient's dependence on the oligomer size behaves as a power law, which dramatically simplifies the expression for the average diffusion coefficient (measured by pfg-nmr) as a function of concentration. the fitted value of the exponent of the power law fully embeds all shape information of the assembly, and may be related to the socalled fractal dimension of the oligomer. moreover, this approach reveals mechanistic information concerning the assembly formation. both methods thus allow structural information of the assembly to be obtained, even when there is little or no prior knowledge available on the mechanism of the selfassembly. nucleotides and α-amino acids are crucial building blocks for living organisms. these chiral molecules are the biosynthetically precursors of two of the most important classes of biopolymers, dna and proteins, respectively. the d-structures of biomolecules are currently studied using a variety of techniques, while helical handedness is routinely detected by means of light pulses of opposite circular polarization. the difference in the uv absorption of these two circularly polarized pulses is called electronic circular dichroism (ecd). in nature, biomolecules explore a wide range of conformations with intrinsically strong ecd signals in the - nm region, but these signals are essentially absent in the visible. nanomaterials such as metallic nanoparticles (depending on their sizes) display absorptions in the visible region but are achiral. as a result, when biomolecules are co-assembled with nanomaterials their chirality is transferred to create a plasmon-induced ecd signal in the visible region. in this work, we present our results which underscore the occurrence of moderately strong ecd bands in the range - nm resulting from a series of appropriately thiolfunctionalized peptide oligomers (based on alternating l-ala and aib residues) covalently anchored to - . nm sized gold nanoparticles. we related the (positive or negative) signs of the ecd plasmonic signal with the oligopeptide length, that in turn is strictly associated with their secondary structure. this latter property was simultaneously monitored via ecd in the - nm range. we believe that in our systems a peptide-tometallic surface chirality transfer would take place. light can be controlled with high temporal and spatial precision. if a specific molecule is made light-sensitive, then a precise spatiotemporal control of some of its properties can be achieved. azobenzene is the most widely used photochromic group due to its propensity to pass reversibly from the cis to the trans state under irradiation with light of the appropriate wavelength. the cisand transazobenzene isomers exhibit different spatial arrangement of the aromatic moieties that give rise to significantly distinct physical and chemical properties. the design of novel azobenzene-based molecules with precisely placed photochromic groups able to induce photomodulation of macroscopic properties is currently attracting much interest. in this work, we explored the behaviour of the conjugate formed by linking each of the four hydroxyl groups of pentaerythritol to the carboxylic function of bis[p-(phenylazo)benzyl]glycine. this c α -tetrasubstituted α-amino acid bears two side-chain azobenzene groups. the resulting system exhibits tetragonal symmetry, with a total of eight azobenzene moieties, and can be viewed as a central core surrounded by a shell of azobenzene groups at the periphery. up to eleven (out of the possible fifteen) discrete states produced by sequential trans-to-cis isomerization of the individual azobenzene units have been observed depending on the time of exposure to uv-light. this process is fully reversible (cis-to-trans) under vis-light irradiation for several cycles. in addition, this compound has been shown to exhibit photomodulated physical properties, such as polarity and hydrodynamic volume. moreover, it shows a high propensity to self-assemble in aqueous solution, giving rise to supramolecular vesicles. light-scattering and electron microscopy experiments confirmed that a conformational reorganization of the vesicles can be triggered under exposure to uv or vis light. the total chemical synthesis of native or modified proteins is gaining increase importance in the study of protein function, but also in the development of protein therapeutics. it is usually achieved by assembling in water unprotected peptide blocks using so-called native peptide ligation methods. recently, our group has developed a novel native peptide ligation method based on a peptide featuring a bis( sulfanylethyl)amido (sea) group on its c-terminus in reaction with a cysteinyl peptide in water at ph . we will discuss in this communication the scope and limitations of sea native peptide ligation. for this, model sea peptides featuring all the possible proteinogenic amino acids were synthesized. their rate of sea native peptide ligation with a model cys peptide were determined in the absence of presence of guanidinium hydrochloride or other additives frequently used in ncl. we will present also experiments intended to clarify the mechanism of sea ligation such as the effect of ph on the rate of ligation, or the ability of the transient thioester sea form produced by in situ n,s-acyl shift to participate in thiol-thioester exchange , . overall, the data show that sea ligation is an interesting method for native peptide ligation at various x-cys junctions, and thus an interesting alternative to ncl. plga copolymers were used as the support for inducing controlled biomarkers releasing system. visualization of the penetration in the hippocampus of mice with confocal microscope was carried out by testing both peptide-free and peptide-bearing nanoparticles, previously labeled with the phthalocyanine fluorescent probe. the encapsulation degree of the larger ( - ) segment was less effective than the others thus stressing the importance of the peptide length to this internalization process. the results showed that all peptide-containing nanoparticles were able to cross the blood-brain-barrier thus indicating improved bioavailability and uptake for peptide delivery into the brain. in regard to the radiolabeling approach, the m tc radioisotope was used to label the peptide sequences at his residues, as previously described . stable metal-peptide complexes were obtained in - - - m peptide concentration range. noteworthy, higher metal labeling yield was achieved with peptide segments bearing his residues at peptide c-terminal position, thus pointing to a positiondependent effect for the m tc coupling reaction. in conclusion, the findings indicate potentials for the proposed encapsulation and radiolabeling strategies applicable for in vitro and in vivo diagnostic assays with these peptides for the study of amyloid plaques. we have used bifunctional short peptides (ac-cg n c-nh , n= , , ) to selectively link gold nanorods in an end-to-end manner. additionally, we have manipulated the gap distance between the rods by changing the length of the peptide linker. the presence of the peptide in the gaps was shown by incorporating a propargylglycine residue in the sequence, which was detected with surface-enhanced raman spectroscopy (sers). the acetylene moiety will allow further chemical modification of the linker in the gaps, opening a wealth of interesting molecular systems to be placed and studies inside self-assembled nanogaps. in this work, the fragmentation pathways of alitame, neotame and andvantame in comparison to those of aspartame and aspartame-d , were studied by negative ion electrospray ionization (esi) high resolution mass spectrometry (thermo orbitrap mass analyzer). accurate mass spectra of the dipeptides allowed proposing specific fragment ions. neotame and advantame, which are the n-( , dimethylbutyl) and n-[ -( -hydroxy- -methoxyphenyl)propyl] derivatives of aspartame, presented similar fragmentation to that of aspartame. for neotame and advantame, the "diketopiperazine'' pathway seemed to be the major one, while a pathway resulting to the formation of a pyrrolidine- , -dione derivative, through the involvement of the side chain carboxyl group of aspartate, was also observed. for alitame, the "pyrrolidine- , -dione" pathway was recorded. similarities in the fragmentation using either orbitrap or triple-quadrupole mass spectrometry have been observed. elucidation of the fragmentation is very useful for the trace-level determination of the artificial dipeptide sweeteners in complex matrices. generation of silver nanoparticles in the presence of oligoproline derivatives p. feinäugle, h. wennemers* eth zürich, switzerland in the last years, the generation of silver nanoparticles (agnps) attracts, due to its unusual physical and chemical properties, more and more attention. agnps offer great opportunities for applications in molecular electronics, catalysis, imaging and for antimicrobial coatings. the characteristics depend on their shape and size. many efforts have been made to optimise the generation process by, for example, varying the reducing agents, which usually are used for the synthesis or using manifold additives which should guide the nucleation and also stabilize the resulting particles. nevertheless, the generation of agnps in defined sizes and shapes still remains a challenge. we address this goal by utilizing functionalized oligoprolines that form a conformationally well-defined and rigid helical secondary structure (ppii) as additives. recently, we showed that by decorating this template with aldehydes which allow for in situ reduction of the silver, they act as scaffolds in the generation process and allow the formation of defined nanoparticles. we will report the results of the generation of agnps with various oligoprolines as additives, which differ in the attached functional groups as well as in the length of the peptides. laser desorption/ionization mass spectrometry (ldi-ms) using specific inert surfaces to promote ion formation has been widely investigated the last decade [ ] . in addition to porous silicon through the original dios technique, different materials were tested as potent ldi-promoting agents. we explored a variety of inert silicon-based uvabsorbing materials that were presenting different physico-chemical properties for the analysis of peptides [ ] [ ] [ ] . both material architecture (amorphous powders, structured particles, structured surfaces) and material hydrophilic/hydrophobic character tuned by specific chemical derivatization (oxidation, silanization) were probed as crucial parameters for achieving efficient and robust detection of an home-made array of model peptides covering a wide structural and mass diversity. through this set of experiments, we were able to compare the performances of all investigated silicon-based supports, especially taking into account peptide detection sensitivity (down to femtomolar concentrations) and reproducibility/repeatability (intra-spot/inter-spot signal variations) as well as the method robustness using conventional maldi-tof/tof instrument. having illustrated the capability to achieve both peptide detection and sequencing on these ionizing surfaces in the same run, high-throughput identification of protein tryptic digests by a rapid ms profiling and subsequent ms/ms analyses was achieved. comparison of the ms and ms/ms data with those obtained with sample conditioned in organic matrix [ , ] showed a great behavior for low mass responses demonstrating the capability of ldi on nanostructured silicon supports to be a complementary method to maldi in proteomic workflow. the dipeptides aspartame, alitame, neotame and advantame are low caloric artificial sweeteners. advantame , which is the n-[ -( -hydroxy- -methoxyphenyl)propyl] derivative of aspartame, is the most recent among them. an application for its approval has been applied in usa, australia and new zealand. such sweeteners are used in food products and beverages and they can help in managing body weight and disorders like obesity and diabetes. in this work, the simultaneous determination of aspartame, alitame, neotame and advantame by negative and positive electrospray ionization (esi), under hydrophilic interaction chromatography (hilic), is presented. advantame, neotame and intermediates were synthesized in our laboratories for the present application. the key-step for the synthesis of advantame and neotame was the reductive amination of h-asp(obu t )-phe-ome with -( -hydroxy- methoxyphenyl)propanal and , -dimethylbutanal, respectively. the chromatographic behavior of the artificial sweetener dipeptides was studied on two hilic columns: kinetex hilic (a fused core silica column) and zic-hilic column (a sulfoalkylbetaine column). the separation of dipeptides was achieved on kinetex hilic using mm ammonium formate buffer ph . / methanol / acetonitrile ( / / ), with a flow rate of μl/min at o c column oven temperature. at this ph, silica is neutral and the dipeptides are in positively charged form. the retention mechanism of all analytes seems to be partition to the water layer as well as hydrogen bonding. département de pharmacologie, université de sherbrooke, sherbrooke, qc, canada plasma and in vivo stability are essential requirements for the successful development of potential drug candidates or diagnostic imaging probes. rapid degradation of compounds in plasma may result in insufficient concentration to produce the desired pharmacological activity or to be used as a diagnostic agent. there are several strategies to improve plasma half life of peptides including pegylation, modification of nand cterminal fragments of peptide, replacement of labile amino acids, and cyclization . we have previously reported on probes which specifically detect matrix metalloproteinase- (mmp- ) activity with magnetic resonance and optical imaging , . mmps are zincdependent endopeptidases degrading the extracellular matrix (ecm) and involved in cancer progression. the main goal of this work was to find more stable probes without sacrificing enzyme specificity. we have selected specific mmp- substrates and their stability was evaluated in three different conditions: in plasma, in plasma with a mmp inhibitor and in a mmp- solution. the samples were analyzed by hplc to detect the degradation pattern of our compounds and by lc-ms to determine the molecular mass of peptide fragments. based on these studies, the most stable peptide was selected and incorporated in a solubility switchable probe with radiolabelled ( )ga-dota. its in vivo stability was estimated up to minutes, making it a suitable candidate for further investigations. cancer of thyroid gland is the most common malignancy of the endocrine system. the treatment improvement could be achieved by early diagnosis. the aim of the study was to identify cancer specific markers using the libraries of artificial receptors immobilized on the cellulose. an array of supramolecular structures formed from n-lipidated peptides attached to cellulose via aminophenylamino- , , -triazine was prone to formation of monolayer of "holes" and "pockets" in dynamic equilibrium. this selforganized structures were found capable of binding small guest molecules very efficiently recognizing the shape, size, and polarity of ligands, thus resembling arti cial receptors . recognition and binding properties of guest molecules by artificial receptors depends mainly on the character of peptidic pockets and structure of the fatty acid. proper construction of the binding pocket allows selective binding components of mixtures of compounds from a living organism . the preliminary data indicates that it is possible to construct an array of artificial receptors with diversified structures of peptidic pockets which are able to distinguish between components of homogenates from tumor and normal tissue. century. most of opioid alkaloids and their derivatives have μ-opioid affinity, while endogenous enkephalins are rather δ-than μ-selective. morphine is still the drug of choice for treating severe pain caused by cancer or surgical operation, but its side effects are the reason for the searching and development of new, selective mor agonists. the aim of our study is to choose within recently published crystallographic structures templates for homology modeling of the human μ-opioid receptor. we generated several models using different templates and all of them were evaluated by docking procedure (gold . ) ligands used in this investigation were synthesized and evaluated for their biological activity in our previous studies. they are enekphalin analogues with substitutions in second position. the best model of the human mu-opioid receptor was chosen according to data obtained from docking and in vitro biological activity of analogues and endogenous enkephalins. acknowledgments: this work was supported by nfsr of bulgaria project dvu / and cost action cm project do - / . . . pneumoniae, h. pylori, proteus sp. are considered as important factor contributory to development of rheumatoid arthritis (ra). the aim of this study was to investigate the level and specificity of antibodies binding to the synthetic peptides corresponding to the bacterial ureases "flap" region sequences in the rheumatoid arthritis patient's sera. for these investigations, peptides with amino acid sequences derived from "flap" regions of different ureases were synthesized using -( , -dimethoxy- , , -triazin- -yl)- -methylmorpholinium tetrafluoroborate (dmt/nmm/bf ) as coupling reagent. peptides were immobilized on a cellulose membrane. the level of antibody binding as well as specificity of them was analyzed by quantitative dot blot method using sera sera from rheumatoid arthritis patients (rap) and sera from volunteer blood donors (vbd). the results of studies suggest that "flap" region may be involved in arising antibodies participating in autoimmunological processes but not to fight infection. this effect indicates that the peptides analyzed by us could be useful for investigation of ra pathogenesis. this suggestion was confirmed by the antibodies absorption experiment which indicates that specificity of antibodies present in rap serum is slightly lower in comparison with vbd serum. it has been found that antibodies present in rap serum recognize not only a specific peptide but also peptides containing fragments with different amino acid sequences. it means that immune system of rap is unstable and may produce a wide spectrum of antibodies recognizing not only a specific epitope but also a set of similar structures. autoantigen-specific t-cells also play a crucial role in the initiation and perpetuation of dsg /dsg -specific t-cell responses. t-cells recognize epitopes from dsg protein and produce different cytokines, e.g. interferon-γ (ifnγ). functional t-cell epitopes of dsg protein have outstanding importance in immunopathological research, development and the design of novel diagnostic tools. our previous studies have shown that certain t-cell epitope peptides are able to stimulate the peripheral blood monomorphonuclear cells (pbmc) of pv patients more effectively than those of healthy donors. our aim was to select a set of t-cell epitope peptides as potential synthetic antigens which are reliably able to distinguish between donors based on the in vitro t-cell stimulating activity. we have prepared synthetic dsg oligopeptides by fmoc/tbu solid phase methodology. after cleaving from the resin with tfa the peptides were purified by rp-hplc, and they were characterized by rp-hplc, mass spectrometry and amino acid analysis. pbmc of pv patients and healthy donors were isolated; and the cultures were stimulated by dsg peptides in a concentration of . mm for hours, and the rate of ifnγ production was determined from the supernatants in sandwich elisa. synthetic dsg oligopeptides induced different in vitro ifnγ production rate on pbmc obtained from pv patients and healthy controls determined by elisa. our approach identified a synthetic antigen set as a promising biomarker for pemphigus vulgaris. [ ] . in particular, cap b (pelyafprvamide) has been shown to elicit antidiuretic activity in the green stink bug acrosternum hilare [ ] , an important pest of cotton and soybean in the southern united states. analogs of cap b containing either an (e)-alkene, cispro or a transpro isosteric component [ ] were synthesized and evaluated in an in vitro stink bug diuretic assay, which involved measurement of fluid secretions of malpighian tubules isolated from a. hilare [ ] . at a concentration of μm, the conformationally constrained transpro analog demonstrated significant antidiuretic activity, whereas the cispro analog failed to elicit any activity. the results provide strong evidence for adoption of a trans orientation for the pro in cap b neuropeptides during interaction with the receptor associated with the antidiuretic process in the stink bug. the work further identifies a scaffold with which to design biostable mimetic cap b analogs as potential leads in the development of environmentally favorable pest management agents capable of disrupting cap bregulated diuretic systems. the enkephalins are pentapeptides (tyr-gly-gly-phe-met/leu) with a proven antinociceptive action. it is believed that the interaction between them and the lipids composing the membranes is important for converting the peptides into a "bioactive" conformation , . using langmuir's monolayer technique the interaction of a synthetic methionine-enkephalin (met-enk) and its amidated derivative (met-enk-nh ) with mixed lipid monolayers composed of palmitoleoylphosphatidylcholine (popc), sphingomyelin and cholesterol was studied. the surface pressure-area (π-a) isotherms with regard to πmin, π max and the hysteresis curve shape of the pure lipid monolayers and after the addition of the respective enkephalins were detected. in addition, by using brewster angle microscopy (bam), the surface morphology of the mixed lipids-enkephalins monolayers were determined. our results suggest that there is a strong penetration effect of the enkephalins studied into the mixed monolayers. moreover, our results demonstrate the potential of lipid monolayers formed in langmuir's through in combination with bam to be successfully used as an elegant and simple membrane models to study lipid-peptide interactions at the air/water interface. acknowledgments: this work was supported by bulgarian ministry of education, youth and science, projects n do - / , drg / and my-fs- / . dept pharmacolgy, temple univ, philadelpha, pa , usa bioinformatic algorithms has predicted the existence of several potential hormone-like peptides transcribed from the ecrg gene . previous publications indicated a highly level of gene expression of ecrg products has been found in the pancreas , choroids plexus, epithelial cells, leukocytes, and macrophages . however, the presence in the hypothalamus and the major form of derived peptides in each tissue haves not been clearly identified. knowingledge of the precise peptide generatesd within a given tissue is essential to understanding its functions. we have generated the peptide specific antibodies to against ecrg -derived pprepro-augurin( - ) and developed a specific ria kit for the quantification of the such peptide in question. a method for the purification of endogenous ecrg -derived peptides from bovine hypothalamus also has been established. using ria to monitor the immunoreactive fractions and maldi-tof to identify the endogenous peptides, we foundhave detected the presence of ecrg derived molecular of bovine preproaugurin( - ) from the homogenates of bovine hypothalamus. immunohistochemistrycal staining by antibody aalso confirmed the presence of thee peptide in some of the hypothalamic cells. of hypothalamus. the amount of prepro-augurin( - ) in the hypothalamus although not soas high as pancreas, but is one third of the augurin level of the pituitary. conclusions: the native peptide derived from augurin preproteinecrg has been discovered.identified. we have confirmed the property of purified peptide,s prepro-augurin( - ), along with the synthetic peptide standards. the present study provides the necessary procedures such as the elution from ( ) c column, ( ) p sizing column, and ( ) a further purification conditions for hplc in order to enhance the immunoreactivity from tissue fractions and yield enough amount for identification. this dsip-related peptide (kn-dsip or knd) differs from dsip by only amino acid residues in positions and . we do not consider the homology between dsip and knd as accidental, bearing in mind functional significance of histone demethylases of the jmjc-group. methylation-demethylation of histones is known as an important mechanism of posttranslational modification playing a prominent role in epigenetic regulation of chromatin structure and gene transcription. dsip is also known as an effective "normalizer" and protector from homeostatic disorders induced by stress related disturbances. we suggest that histone demethylase of the jmjc-group containing dsip-related region can be considered as a possible protein precursor of endogenous peptides with dsip-like activity. in order to test our hypothesis we synthesized knd and studied its biological effects. in a preliminary assay cited below [ ] knd showed similar and probably more pronounced effects than dsip as an agent that stimulates endurance and stress-resistance of animals in the forced swimming test. also knd provided a more active detoxifying action after administration of a semi-lethal dose of the cytostatic agent. in the present work we assessed neuroprotective and antioxidative potency of both peptides in vivo and confirmed the higher efficiency of knd. this study is supported by the moscow government. is a tridecapeptide (pglu -leu -tyr -glu -asn -lys -pro -arg -arg -pro -tyr -ile -leu ) highly expressed in the central nervous system. this peptide elicits an analgesic response following peripheral or central administration. importantly, nt exerts a more potent analgesia than morphine at an equimolar dose, without having the associated side effects of opioid drugs. structure-activity studies have identified the c-terminal fragment nt( - ) as the biologically active minimal sequence. however, nor the full or truncated peptides cross the blood-brain barrier (bbb), thus hampering its clinical development. the substitution of pro by an unnatural amino acid silaproline (sip) increased bioavailability and plasma stability. structural properties conferred by the pro were also retained as determined by nmr and ir. aiming at delineating the mode of action of cl, three new cl derivatives bearing suitable labeling moieties, i.e the fluorescent molecule fitc, the streptavidin-counterpart biotinyl-group and the m tc-radiometal chelating unit dimethylgly-ser-cys, were designed, synthesized, purified, and characterized to be applied in in vitro and in vivo evaluation studies. the structure of the cl derivatives in aqueous solutions was studied with nmr, in parallel and in comparison with the parent molecule cl, in order to examine whether the presence of the labeling moieties has induced changes to the structure of the biologically active part of cl. cell survival assays with cl and the cl derivative bearing the fitc moiety were conducted in the pc cell line in order to explore their rescue effect. in parallel, the cl derivative bearing the dimethylgly-ser-cys moiety was successfully radiolabeled with m tc and its stability was assessed over time in its synthesis reaction mixture and in plasma. this m tc-radiolabeled derivative was subsequently administered to swiss albino mice in order to determine the biodistribution of cl in the living organism and its route of excretion, a study that has not been carried out so far for any peptide of the humanin family. furthermore, the potential interaction of cl with β-amyloid peptide, the hallmark of ad pathogenesis, was explored with circular dischroism. the results of this multifaceted approach to the biological action of cl will be presented. institute of biochemistry and biotechnology, martin-luter university, halle-wittenberg, germany kinins, such as the nonapeptide bradykinin, are important mediators of various physiological and pathophysiological responses including inflammatory disease, asthma, rhinitis, cell division, pain, vascular permeability, allergic reactions, pathogenesis of septic and endotoxic shock. there are two types of receptors for kinins, known as b and b . b receptors are constitutively expressed in wide variety of cells and required entire bk sequence for recognition, while b receptors have normally very limited expression and respond to [desarg ]bk. b receptors gene is turned on following either tissue damage or inflammation. accumulated evidence indicates that most of the clinically relevant effects of bk are functions of b receptors this being the reason why research on their antagonists is a topic of great interest. in our previous study we described the synthesis and some pharmacological properties of four new analogues of bradykinin (bk), designed by substitution of position or of the known [d-arg ,hyp ,thi , ,d-phe ]bk antagonist with l-pipecolic acid (l-pip) (both analogues were also prepared in n-acylated form with -adamantaneacetic acid (aaa)). our results showed that presence of l-pip in position slightly increased antagonistic potency in the blood pressure test, but it turned the analogue into an agonist in the rat uterus test. replacement of thi by l-pip in position also enhanced antagonism in the rat pressure test but preserved the antagonism in the rat uterus test. in the present study we continue our previous investigations to find structural requirements which in the case of bk analogues result in high b antagonistic activity. several new bradykinin analogues modified in their cterminus with d-pipecolic acid were synthesized using spps method. the biological properties of the analogues were assessed by their ability to inhibit vasodepressor response of exogenous bk in conscious rats and by their ability to inhibit the contractions of isolated rat uterus evoked by bk. acknowledgements this work was supported by the university of gdansk (ds/ - - - ). peptides with beta-turn structure in peptide/mhc complexes a. stavrakoudis department of economics, university of ioannina, greece major histocombatibility complex (mhc) molecules interact with small peptides and form complexes. in most of the cases, peptide's structure in these complexes is found in extended conformation. however, notable exceptions exist where the peptide forms a beta-turn structure. this happens mainly in the central part of the peptide in class i complexes [ ] , or at the c-terminal of class ii complexes [ ] . several peptide/mhc complexes were, derived with xray studies, were extensively subjected to molecular dynamics simulations [ ] in order to investigate the stability of this turn-like structural feature and to explore the factors that possibly contribute to this stability. it was found that both intra-peptide and peptide/mhc interactions might be responsible for peptide's conformation. the peptides were found to undergo several structural transitions indicating conformational plasticity and not a completely rigid structure inside the mhc groove. the results might be of special importance in designing defective peptide vaccines and beta-turn pharmaceuticals. the heptapeptide met-enkephalin-arg -phe (merf) with the sequence of yggfmrf is a potent endogenous opioid located at the c-terminus of proenkephalin-a (penk), the common polypeptide precursor of met-and leuenkephalin. our systematic bioinformatic survey revealed considerable sequence polymorphism at the heptapeptide region of different penk prepropeptides among vertebrate animals. four orthologous heptapeptides with single or double amino acid replacements were identi ed among animals, such as yggfmgy (zebra sh), yggfmry (newt), yggfmkf (hedgehog tenrek) and yggfmri (mudpuppy). each novel hepta-peptide, together with the mammalian consensus merf and metenkephalin, were chemically synthesized and subjected to functionality studies, using radioligand binding competition and g-protein activation assays in rat brain membranes [ ] . equilibrium binding af nities changed from good to modest as measured by receptor type selective [ h]opioid radioligands. the relative af nities of the heptapeptides reveal slight mu-receptor (mop) preference over the delta-receptors (dop). [ s]gtpγs assay, which measures the agonist-mediated g-protein activation, has demonstrated that all the novel heptapeptides were also potent in stimulating the regulatory g-proteins. all peptides were effective in promoting the agonist induced internalization of the green uorescence protein-tagged human mu-opioid receptor (hmop-egfp) stably expressed in hek cells. thus, the c-terminally processed penk heptapeptide orthologs exhibited satisfactory bioactivities, moreover they represent further members of the so-called "natural combinatorial neuropeptide library" emerged by evolution. corticotropin releasing factor (crf) exerts most of its physiological and pathophysiological actions by interacting with its type receptor (crf ) and activating different intracellular signalling pathways. the crf is a plasmamembrane protein, which belongs to the family b of g-protein coupled receptors (gpcrs) and like the other gpcrs consists of an amino-terminal extracellular region, a carboxyl-terminal intracellular tail and seven, mostly hydrophobic, membrane-spanning segments (tm -tm ), connected by alternating intracellular (il) and extracellular loops (el). binding of crf and its related peptides, such as sauvagine, to the extracellular regions of crf is associated with receptor activation and subsequent activation of different g-proteins and regulation of diverse signalling pathways. using a mutagenesis approach in combination with a radioligand binding study we found that trp and phe in the second extracellular loop of crf interacted with the amino-terminal portion of crf and sauvagine. interestingly only the interaction of sauvagine with trp and phe is important for crf -mediated stimulation of camp accumulation. in marked contrast the interaction between crf and the residues trp and phe was unimportant for the activation of adenylate cyclase. thus it is possible for trp and phe of crf to regulate distinct signalling pathways, or different sets of them, after their interaction with different peptides. we are now performing experiments to fully elucidate the signalling pathways that are regulated by the interaction of crf and sauvagine with trp and phe . these studies will advance the development of crf -selective selective signalling-specific peptides that would be extremely useful for the elucidation of the role of crf in many physiological and pathophysiological situations, and possibly for the treatment of several crf -related diseases. thymus humoral factor gamma- (thf-γ ), an octapeptide, purified from crude thf, retains essentially all the biological properties of thf [ ] [ ] . it regulates clonal expansion, differentiation and maturation of t-cell precursors, stimulates the production of lymphokine, maitains the normalization of impaired ratios between helper(cd +) and suppressor / cytotoxic (cd +) subsets and augments il- production in spleen cells. thf-γ has a calculated molecular weight of and has the following amino acid sequence: leu-glu-asp-gly-pro-lys-phe-leu. its poor stability towards protein enzyme limits its extensive application. with the inte ntion to promote its bioavailability, bioactivity and develop ideal immunoregulatory drug candidat, four series of derivatives of thf were designed and synthesized: . n-and cterminal acylation. .restitution the flexible segment gly-pro by unnatural amino acids -aminohexanoic acid (aca) in order to shorten the synthetic steps and simultaneity improve the bioavailability and biostability of peptide; . reserve protected group of some amino acid residus as spot mutation. . mannich-based cyclization was carried out on resin [ ] , phe was replaced by tyr serving as the active hydrogen component, a proline was introduced at the n terminal as the amine component and formaldehyde was used as the only component in solution. the bioactivity of synthesized products were detected. the leukocytopenia model in mice was induced by cyclophosphamide intraperitioneal injection. white blood cell count, thymus index and spleen index were detected to evaluate the immune function of compounds in mice. the results show that those compounds play a significant role in improving immune function in mice. the activity of compound lhl and lhl are also better than authentic compound tp- and tα . marine organisms have been recognized as a promising source for the development of new pharmaceuticals. in the course of screening for antitumor substances from marine organisms, we found cyclic peptides containing many nonribosomal amino acids such as hydroxyasparagine, hydroxyleucine, or other supporting a hydrophobic side chain that were shown to be a key element for their biological activity. the laxaphycine b, a cyclic lipopeptide isolated from marine cyanobacteria anabaena torulosa harvested in french polynesia constitutes an example of this peptide class. this compound has attracted our attention because of its micromolar cytotoxic activities on different cancer cell lines as well as its antiangiogenic properties which seems to be due to an interaction with the vegf receptor- - . the synthesis of the non-natural amino acids - and of laxaphycine b analogues will be presented along with their preliminary biological activities. immune response suppressors are used in the medical praxis to prevent graft rejection after organ transplantation and in the therapy of some autoimmune diseases including dermatology. cyclolinopeptide a (cla) c(pro -pro -phe -phe -leu -ile -ile -leu -val -), a cyclic, hydrophobic nonapeptide isolated from linseed, possesses strong immunosuppressive and antimalarial activity. it has been suggested that both the pro-pro cis-amide bond and an 'edge-to-face' interaction between the two aromatic rings of adjacent phe residues in tetrapeptide unit are important for biological activity. this edge-to-face interaction can be influenced when phenyl rings are replaced by naphtyl substituent. in this communicate new analogues of cla modified by naphtylalanine ( -nal) in positions or or both and ( - linear analogues, - cyclic analogues) will be presented. the synthetic strategy and biological activity as well as conformational analysis will be evaluated. the onset of type ii diabetes mellitus (t dm) coincides with the deposition of fibrillar material in the islet of langerhans in the pancreas that is a clinical hallmark of more than % of patients suffering this disease. the main component of the pancreatic amyloid deposits is a -residues polypeptide hormone called islet amyloid polypeptide (iapp) or amylin. in this work we have examined, by means of cd spectroscopy and tht-fluorescence, the conformational polymorphism of both full-length - hiapp, and the related fragment hiapp - , and compared the results with the respective rat counterparts. moreover, the cytotoxic activity was determined toward different pancreatic β-cells lines in the attempt to correlate iapp's fibrillogenic properties with the ability to mediate cell death. together the results suggest that β-sheet conformational transition, that generally preludes to fibril formation, is not a prerequisite for eliciting toxicity toward β-cells cultures. interestingly, confocal microscopy indicated that both hiapp - and hiapp - can enter the cell and might exert their toxic action at intracellular level. acknowledgments: this work was supported by miur, firb-merit project rbne hwlz. due to its physiological functions, s proteasome is considered the target molecule in overcoming several diseases [ ] . its core particle s has three types of active sites: chymotrypsin-, trypsin-and caspase-like. many natural and synthetic compounds were tested for their ability to inhibit proteasome. a recent report describing the inhibition of s by the serine proteases inhibitor -bovine pancreatic trypsin inhibitor -was considered by us with great attention [ ] . our scientific interest is focused on peptide inhibitors and their interaction with serine proteases. sunflower trypsin inhibitor (sfti- ) is the smallest and the most potent peptide inhibitor in the bowman-birk family. owing to its size and the rigid structure (disulfide bridge and "head to tail" cyclisation) sfti- is willingly chosen as the lead structure in the search for new inhibitors [ ] . its sequence is shown below (lys the p residue responsible for specificity): & gly-arg-cys(& )-thr-lys -ser-ile -pro -pro-ile-cys(& )-phe-pro-asp& since native sfti- is not able to inhibit s [ ] , we have designed its monocyclic analogues (with disulfide bridge only) with lys or arg in position (p ) and at least one basic amino acid (lys or arg) in positions (p ') and/or (p '). all analogues inhibit chymotrypsin -(ic at the range of ÷ μm) and caspase-like (ic at the range of . ÷ μm) activities in vitro, whereas their activity towards trypsin-like specificity is much weaker. in several rat tissues, our view on ras has changed. metabolism of the ang-( - ) may represent alternative pathway of ang ii formation, importantly, independent on renin and ace activity , . ahmad et al. have described metabolism of ang-( - ) by human atrial tissues and showed that ang ii is formed mainly by chymase. this renin-inependent ang ii production could explain the "resistance" regarding use of ace inhibitors in patients with hypertension or diabetic nephropathy. noteworthy, the role of ang-( - ) in circulation is still unclear and there are no information about possible pharmacological modulation of its metabolism. in our study, we compared the ex vivo metabolism of angiotensinogen (fragment - ) in hypertensive (shr) and normotensive (wky) rats in organ bath of aorta and heart using lc-ms method . surprisingly, we identified ang-( - ) formed via reninindependent pathway to be a main product of angiotensinogen metabolism in rat aortic tissue and heart. in this setting, ang-( - ) appeared to be not only prevalent metabolite of angiotensinogen, but also served as a substrate for generation of ang i and ang ii. as compared to wky rats, formation of ang ii, from ang-( - ), was much higher in shr aortas but not in the heart. the functional consequences of these findings require further investigation. this study was supported by the grant n n polish ministry of science and higher education. the lysosomal cysteine protease cathepsin c (cat c), also known as dipeptidyl peptidase i (dppi), activates a number of granule-associated serine proteases with proinflammatory and immune functions by removal of their inhibitory n-terminal dipeptides. activity of this protease is associated with several pathologies in human body [ ] . in this work the characterization of cat c specificity using combinatorial chemistry methods will be described. the main goal of this work was to determine of substrate specificity of the prime region of this enzyme. the chemical synthesis and deconvolution of two libraries will be described. the hemostatic mechanism has the crucial role to prevent loss of blood from injured blood-vessels. this loss is prevented by the integrity of the vessel walls, by platelets aggregation or by blood coagulation, which in normal conditions is limited onto the local trauma of the vessel wall. in generally, the blood coagulation mechanism is important for maintaining vascular integrity and thus for the precaution of an organism from bleeding, which may also occur by blood coagulation caused by thrombin production. the diversion rate of this production leads to an expansion of thrombin to the general blood circulation. thus, when thrombin generation is not controlled by the mechanisms of inhibition, a widespread undesirable intravascular thrombosis is occurred. the whole process of platelets adhesion requires the presence of clotting factor viii (fviii), a necessary for the blood coagulation cascade glycoprotein, which takes part in the intrinsic pathway and acts as a coenzyme for the activation of factor ix, a serine protease depended on the thrombin production. the target of the present research is the synthesis of biologically active cyclic, head to tail, peptides, analogs of the sequence - of a subunit of fviii, which are potentially capable to block fviiia-fixa complex, reducing the thrombin production and thus the blood coagulation. the synthesized peptides are investigated for their inhibitory activity and tested for clotting deficiency by measuring the chronic delay in the activated partial thromboplastin time (aptt) and the reduction of the % value of the fviiia, which they generate in samples containing recombinant fviiia, in vitro. the blood coagulation is part of an important host defence mechanism, which under pathological conditions results in inappropriate intravascular coagulation when thrombin is produced. clotting sequence is the result of a cascade of two biochemical pathways, intrinsic pathway, so called because all components are present in blood, and extrinsic pathway, in which tissue factor is required in addition to circulating components. the activated form of factor viii (fviiia) is a key component of the fluid phase of the blood coagulation and plays an important role formatting a trimolecular complex with factor ixa, ca + and negatively charged phospholipids of the cells membrane. this complex is called tenase and participates in activation of prothrombin, which acts on fibrinogen to generate fibrin monomer, polymerized rapidly to form fibrin clot. the fviii is comprised of a heavy (a -a -b) and a light (a -c -c ) peptide chain, both cleaved by proteases at three sites, resulting in alteration of its covalent structure and conformation. its deficiency is known as haemophilia a. our research effort is focused on the synthesis, identification and biological evaluation of peptide analogs, expected to inhibit selectively the increasing of thrombin production. their sequence is based on the regions in which the fviii interacts with fix, specifically on the sequence - of the a subunit. the synthesized peptides are examined for their activity and tested for clotting deficiency by measuring the chronic delay in the activated partial thromboplastin time (aptt) and the reduction of the % value of the fviiia, which they generate in samples containing recombinant fviiia, in vitro. inhibitor with the following structure: ac-llllrvkr-nh , which has potent effects on the proliferation of prostate cancer cells. the potency and stability of this compound was subsequently enhanced by substitution of arg residue in position p with its conformationally restricted mimetic - -amidinobenzylamine (amba). nevertheless, the specificity toward pace was significantly reduced by this modification. thus, in order to improve its selectivity without sacrificing inhibitory potency we decided to use positional scanning approach. in this study we present synthesis of two series of peptide libraries, which were designed by substitution of leu in the p , p position of our control peptide (ac-llllrvkr-amba) with each of nineteen amino acid residues in order to verifying its influence on activity and selectivity of the resulting analogues. all peptides were synthesized by a combination of solid phase peptide synthesis and solution synthesis and tested for their inhibitory potency against furin and pace . the p -p fragments were synthesized by fmoc/tbu spps strategy on hydrazinobenzoyl or acid labile chlorotrityl chloride resin. then coupling of the -amidinobenzylamine · hcl was performed. the best modifications were combined to give as several multipoint substituted inhibitors. we believed that our work, will provide new important information about structure-activity relationship of these class of analogs in order to obtain potent and highly specific pace inhibitor. institute for research in biomedicine, parc cientific de barcelona, barcelona -spain a bacterial toxin-antitoxin (ta) system is composed of two genes organized in an operon encoding a toxin and an antitoxin that regulate the growth and death bacterial cell under various stress conditions. the operon parde encode a ta system formed by pare toxin and its antitoxin pard. pare is a kda protein that inhibits dna gyrase activity and thereby blocks dna replication. however the pare-gyrase interactions and the gyrase activity inhibition mechanism have not been explored. as an approach for understanding of this mechanism and to elucidate the pare region responsible for protein-protein interactions we have designed and synthesized a series of linear analogues of pare and investigated the ability of peptides to inhibit dna topoisomerases activity. so, based on structural data inferred from pare three-dimensional model , peptides were synthesized by solid-phase method. four peptides (parelc , parelc , parelc and parelc ), showed complete inhibition of dna gyrase supercoiling activity, by gel electrophoresis assay , with an ic of to μmol.l - . in addition, intrinsic fluorescence and fluorescence anisotropy assays showed that inhibition process must occur by interaction with the gyra subunit. differently of wild type pare, the peptide analogues were able to inhibit the dna relaxation of topoisomerase iv with lower ic values. interesting was that only parelc displayed inhibition of the relaxation activity of human topoisomerase ii. our results suggest a new class of molecules with simultaneous inhibitory activity in dna gyrase and topoisomerase iv. furthermore, we have obtained the first example of a synthetic peptide from a bacterial toxin with inhibitory activity on human topoisomerase ii. institute of experimental endocrinology, slovak academy of sciences, bratislava, slovakia the renin-angiotensin system (ras) has long been recognized as an important regulator of systemic blood pressure and electrolyte homeostasis. our understanding of ras has experienced remarkable change over the past two decades. besides, angiotensin ii, the new biologically active peptides [e.g. ang-( - ), ang-( - ), ang iv, ang-( - )] and pathways [e.g. angiotensin converting enzyme -ace ] have been described ; some of them, like ang-( - ) may oppose many actions of ang ii. importantly, despite all components of classical ras are found in adipose tissue , the data about fat formation of various angiotensins remain scarce. in our study, we compared the ex vivo metabolism of angiotensinogen, ang-( - ) and ang i in hypertensive (shr) and normotensive (wky) rats in organ bath of retroperitoneal and periaortic fat tissue using lc-esi-ms method. additionally, qpcr measurements of mrna expression of main enzymes involved in ang i metabolism were performed. both in the periaortic and epidydymal fat, the formation of ang-( - ) was higher than production of ang ii. fat tissue formation of two main ang i conversion products, ang ii and ang-( - ), differed significantly between shr and wky rats. compared to wky rats, the formation of ang-( - ) in periaortic fat tissue was decreased in shr. in opposite, in epidydymal fat tissue formation of ang-( - ) and ang ii was higher in shr. interestingly, there were no differences in aorta formation of ang ii and ang-( - ) between shr and wky rats. our results suggest that in hypertension visceral fat production of angiotensin peptides is increased, while generation of "beneficial" ang-( - ) in periaortic fat is decreased. however, the functional importance of such finding require further investigation. department of chemistry and biochemistry, university of washington, usa phospholipases a (pla ) are a superfamily of enzymes involved in various inflammatory diseases. in particular, human secreted giia spla is an attractive target for the development of novel medicines. we have shown that oxoamides based on γ-or δ-amino acids are potent inhibitors of cytosolic giva pla . very recently, we have demonstrated that a long chain -oxoamide based on (s)leucine displays inhibition of human and mouse giia spla s (ic nm and nm, respectively). a combined experimental/computational study was undertaken to further understand the role of the α-amino acid of -oxoamides for the inhibitor-enzyme binding. the crystal structure of giia spla s reveals a highly conserved ca + -binding loop and a catalytic dyad consisting of his /asp . -oxoamides based on hydrophobic αamino acids showed better binding score prediction compared to polar α-amino acid derivatives. a number of new -oxoamides based on α-amino acids were synthesised and tested for their inhibitory activity against giia, gv and gx pla . the -oxoamide based on (s)-valine displayed potent inhibition of giia spla (ic nm) in accordance with the predicted docking score. docking results reveal that (s)-valine-based inhibitor forms key interactions with the active site of the enzyme. the carboxylic group participates in a hydrogen bonding with gly and lys , and -carbonyl group with gly . furthermore, both carbonyl groups are in the proximity with ca + . the side chain of (s)-valine adopts a suitable orientation to interact with tyr and lys . the long aliphatic -oxoacyl chain is accommodated in the hydrophobic region of the active site and creates proximal contacts with leu , ile , his and phe . the search for novel classes of pharmaceutical molecules with enhanced therapeutic power has been the subject of numerous research groups all over the world. moreover, systems of immobilization and controlled release which are adapted to these new classes of molecules, has proven to be an area of extreme importance to provide the same therapeutic efficacy. using solid-phase chemistry a series of ccdb toxin analogous peptides were synthesized and were synthesized and tested against the capacity of inhibition of bacterial enzymes dna gyrase and topoisomerase iv (topo iv). subsequently those peptides were detained in drug delivery systems (dds) to be tested against the inhibition of growth of different bacterial species. in this data we could observed that the analogue ccdbsg could inhibit only dna gyrase and not the topoisomerase iv. in the other hand the analogue ccdbsg presents a hard inhibition potential against topo iv specially because of their structural difference. is possible conclude that topoisomerase iv presents the tertiary structure very similar to dna gyrase, but those mechanisms of action must be clearly distinct . in the in vitro studies, as expected, results revealed that the drug delivery systems are the key to the power efficiency of peptide analogues against the bacterial growth inhibition which cannot be observed when the peptides are free in solution. some of the different lipid compositions of the dds are demonstrating to be more efficient in the membrane cell transverse and this data previously assumes that it is possible to apply different types of dds to promote the peptide molecules transport across the cellular membranes according to several specific therapies. with this studies we have obtained more knowledge about the interaction system of enzyme-toxin and hopes which helps in future studies to development a new antimicrobial molecules class. it is urgent to develop less toxic and more efficient treatments for leishmaniases and trypanosomiases. we propose to target an ancestral form of the proteasome, the hslvu protease, which is present in the parasite's single mitochondrion, essential for the growth of these organisms and has no analogue in the human host. originally discovered in eubacteria, this complex is constituted by two central hexameric hslv protease rings sandwiched between two hexameric hslu atp-ase rings. as hslv shares a similar enzymatic mechanism with the host proteasome, we propose to inhibit the assembly of the complex in order to be selective. according to studies on bacterial hslvu, , the c-terminal segment of hslu is essential in hslv activation and in complex assembly, therefore representing a privileged target. we produced recombinant hslv, which is inactive alone, and showed that a synthetic c-terminal hslu peptide was able to induce the digestion by hslv of a fluorogenic substrate that we developed. with this enzymatic test in hands, we started the characterization of the interaction of the c-terminal portion of hslu with hslv. we will present the results obtained with various series of analogues of the original c-terminal hslu peptide, including truncated forms, ala scan, constrained analogues and multivalent constructions. helped by molecular modelling studies, the aim is to establish structural requirements, which could lead to high affinity and stable ligands able to inhibit the interaction between the hslu and hslv rings, obligatory for the degradation of proteins by the hslvu complex. finally, we checked that hslv was inhibited by classical active-site directed proteasome inhibitors like bortezomib. f. babos a,d , e. szarka b , gy. nagy c , z. majer d , g. saŕmay b , a. magyar a , f. hudecz a,d citrullinated filaggrin peptide (ccp) were detected in ra sera and anti-ccp positivity is widely used for diagnostic purposes. identification of new epitopes of filaggrin would be useful in the diagnosis of anti-ccp seronegative patients. in order to achieve optimal immune recognition of biotinylated epitope peptides it is important to analyse the effect of the labelling moiety on antibody binding. for these studies -as well as -mer peptides with nor c-terminal biotin were synthesised manually by spps, using fmoc/ t bu strategy. biotinylation was performed by using biotin, biotinyl- -aminohexanoic acid or , , -trioxa- , -tridecanediamino succinic acid linker modified biotin. labelled peptides were used in an indirect elisa, on neutravidin pre-coated plates and the binding was detected by anti igg-hrp. to examine the role of the presence/position of biotin in the secondary structure of the peptides, electronic circular dichroism (cd) method was used. we found that the ccp + serum samples specifically recognized the c-terminally biotinylated -mer filaggrin peptides, while showed no binding with the n-terminally biotinylated compounds. in case of the -mer epitope peptides there was no difference between the recognition of nand c-terminal biotinylated analogues. data presented suggest that the position of the biotin in case of the short filaggrin epitope peptides markedly influence the serum antibody binding. upon activation process, they are released from the granules and then involved in immunoresponse of the organism. when out of the cell those enzymes remain in free form or become associate with the cell membrane. the physiological role of this proteases is manifestated in several processes such cytokine and chemokine processing, platelet activation, and degradation of extracellular matrix's proteins [ ] . in this work results of the specificity of two members of nsps pr and hne evaluated using the combinatorial chemistry methods will be presented . both enzymes share primary specificity and to obtain the selective substrate that will be recognized only by one enzyme, the prime sites should be investigated. the general formula of the designed library is as follows: where in positions x ', x ' and x ', the set of proteinogenic amino acids (except cys) was introduced. abz is -amino benzoic acid served as donor of fluorescence and -nitro-l-tyrosine as acceptor. eukaryotic proteasome is a highly organized protease complex comprising a catalytic s core particle (cp) and two s regulatory particles (rp), which together form the s structure. the main function of this large intracellular protease is to degraded ubiquitine labeled proteins. the catalytic particle of the proteasome displays three distinct enzymatic activities: trypsin-like, chymotrypsin-like and glutamyl-like. the increase activity of the proteasome is associated with several disease including cancer [ ] . the main aim of this work is to synthesized the cell permeable fret displaying peptides that will selective cleaved by single proteasome activity. additionally each peptide when independently cleaved by the proteasome subunit, should emit the fluorescence energy in a different spectral region. our intention was designing substrates which would allow to monitor simultaneously (in a single experiment) and independently of three proteasome activities in this report, we will describe the chemical synthesis of several peptides modified at on cand n-termini by synthetic fluorescent amino acids the general formula of these peptides is as follows: where x is a non proteinogenic amino acid that serve as a donor of fluorescence, y amino acid that is a acceptor of fluorescence. the obtained fluorescent peptides were examined for their ability to cross the cell membrane. also kinetic parameters (k cat, km, kcat/km) with proteasome will be presented. approximately dubs are encoded in the human genome and are involved in a variety of regulatory processes, such as cell-cycle progression, tissue development, and differentiation. recently, several groups have introduced various methods for linking ubiquitin to different substrates via nonhydrolyzable isopeptide bonds, which resist the action of dubs. using these methods, one could explore the function and the mechanism of dubs and apply them in activity based profiling. here we present a new and convenient strategy for preparing nonhydrolyzable ubiquitinated peptides and proteins by nmethylating the isopeptide bond. using this method we prepared several nonhydrolyzable ubiquitinated peptides with different lengths derived from ubiquitinated h b and examined their affinity to different dubs. f.i. nollmann, c. dauth, d. reimer, h.b. bode* goethe universität, frankfurt, germany bacteria of the genus xenorhabdus and photorhabdus are gram negative gamma proteobacteria that live in symbiosis with nematodes of the genus steinernema. undergoing their partly entomopathogenic life cycle these bacteria not only produce antibiotics , and insecticides but also several different small molecular compounds and peptides. for the most part the biological benefits of these secondary metabolites have not fully been understood yet. with the help of inverse feeding experiments, hr-ms and nmr as well as molecular engineering we were able to characterize and/or isolate some of these peptides. since they are mostly produced in trace amounts, we synthesized them in order to make them accessible to continuative testing. given that not only linear but also highly methylated or cyclic peptides are produced, the synthesis was quite challenging. nevertheless, we were able to establish in our laboratory a general synthesis route for cyclic peptides and depsipeptides , as well as highly methylated hydrophobic linear sequences. testing several of these peptides has revealed activity against insect cells and against the causative organisms of neglected tropical diseases. cyclotides are a large class of plant peptides defined by a head-to-tail cyclized backbone and three conserved disulfide bonds in a knotted arrangement. these unique structural features confer them with remarkable stability and due to a range of bioactivities they are extensively investigated as templates in drug discovery . based on the use of oldenlandia affinis in traditional african medicine for its uterotonic principle we investigated crude plant extracts and semi-pure cyclotide fractions for the ability to induce uterine contractions using a collagen-gel contractility model . pharmacological analysis of the effects led to the identification of the oxytocin receptor, a representative of the g-protein coupled receptor (gpcr) family, as a molecular target for cyclotides. mass spectrometry-based sequence analysis of 'active' fractions revealed cyclotides with high similarity to the human oxytocin (h-ot) peptide that exhibited weak binding to the human oxytocin receptor. we further analyzed synthetic cyclotide-derived small ot-like peptides and grafted the h-ot sequence into the stable cyclotide frame. these peptides showed increased binding and activation as compared to native cyclotides. these findings may open new avenues for the discovery of gpcr ligands from natural peptide sources. gpcrs are promising drug targets and ~ % of currently used drugs act via binding to these receptors. natural combinatorial peptide libraries are likely to play an important role in identifying novel gpcr ligands . particularly plant cyclotides cover a large chemical space based on their high sequence diversity. together with their range of bioactivities and unique stable structure suggests that cyclotides are of current and future interest for drug discovery and development. acknowledgements: this work is funded by the austrian science fund fwf (p ). drosha and dicer are two key endonucleases for biogenesis of micrornas (mirnas) that regulate target mrna. drosha converts pri-mirna to ~ nucleotide (nt) pre-mirna in nucleus and dicer converts pre-mirna to linear ~ nt single-stranded mirnas in cytosol. even though dicer is potentially important to control availability of mature trans-acting rnas in cytosol, the enzyme itself does not seem to be the suitable target controlling mirna processing due to the lack of its substrate specificity. nature, however, might be intelligent enough to differentiate a variety of pre-mirna, so that a certain specific pre-mirna is converted to mature mirna in case it needs. therefore, other component(s) in the enzyme complex could be involved in recognition of auxiliary proteins from out sources to give extra specificity. we have synthesized trp-containing amphiphilic peptides against several pre-mirna. peptide b showed a picomolar binding affinity and a large specificity against pre-let a- . in vitro mirna processing, dicer activity was also selectively enhanced in the presence of this peptide. on treatment with this peptide on hct colon cancer and p ec cell lines, let a- mirna was more processed than reference mirnas. the toxicity of furan is known to rely on its selective oxidation in the liver by cyt p enzymes transforming it into the very reactive butenedial, which quickly reacts with proximate nucleophiles. this principle was used in our laboratory to develop a high yielding dna interstrand crosslinking methodology. in view of the demonstrated site-selectivity, the method further holds promise for sitespecific crosslinking dna to its binding proteins, which is highly relevant in the study of transient protein-dna interactions. furthermore irreversible dna binding can be achieved through such a covalent linkage, which is potentially useful for new generation therapeutics. the reactive furan moiety can in principle be incorporated either in the dna or in the protein. in the former case, a furan modified nucleotide was built into an oligonucleotide positioning the furan moiety at the periphery of the dna, to avoid interstrand crosslinking. for the latter approach, we initially chose to synthetically access a furan modified dna binding protein mimic. next to a previously described non-covalent gcn mimicking dimer, we have also investigated a new type of steroid-based dipodal dna binders. synthesis of the latter constructs has proven challenging in view of the immobilization of two peptide chains with helix forming tendency at close distance on the template. results, showing the power of microwave assistance will be discussed. in an alternative approach, a full length protein was modified with furan by amber suppression based on the structural similarity between a furan modified amino acid and pyrrolysine. pharmaceutical institute, university of bonn, an der immenburg , bonn, germany human matriptase- is a kda protein with trypsin like specificity. this protein exhibits a domain organization similar to family of membrane-bound serine proteinases known as type ii transmembrane serine proteinases. among many ascribed function in human body, this enzyme is a potent negative regulator of hepcidin, the peptide involved in iron homeostasis [ ] . matriptase- has a similar fold as other tmsp members, however their detailed specificity still remain unclear. the aim of this study was to determine the substrate specificity of this physiological important enzyme using combinatorial chemistry approach. in order to characterize the matriptase- specificity, the tetrapeptide library with c-terminal amide of aminocoumarin (acc-nh ) that serve as a fluorophore, was synthesized. its general formula is given below: x -x -x -x -acc-nh , where in position x -x the set of proteinogenic amino acid residues are present, whereas in position x lys or arg was introduced. deconvolution of such library was performed using iterative approach in solution. the results obtained indicate that matriptase- display diverse p -p specificity as compare to matriptase- . the most efficient hydrolyzed amino acid residue in position p appear to be ile, that is followed by arg in p and ser in p . the arg in position p is % faster hydrolyzed then lys. for selected substrates, the kinetic parameters (kcat, k m ) were determined. amyotrophic lateral sclerosis (als) is a chronic progressive disease. it is characterized by degeneration of upper or lower motor neurons, but its pathogenesis is still unknown and no effective treatment currently exists. it is known that antibodies to gangliosides have been found in some als patients, and these antibodies are also well known to be present in the patients affected by a variety of autoimmune diseases including multiple sclerosis. up to now anti-gangliosides antibodies are detected in clinical immunology laboratories using isolated non consistent antigen mixtures. therefore, we are interested in developing reliable and univocally characterized synthetic antigens for efficient antibody detection. csf (glc) is a family of structure-based designed glycopeptides that we previously developed as multiple sclerosis (ms) synthetic probes. these n-glucosylated peptides are able to detect specific autoantibodies in the sera of an antibody-mediated form of ms. autoantibody recognition was favored because of the exposition of the sugar amino acid on the tip of type ' β turn structures. aim of this study is the introduction, in the type ' β turn peptide structure, of the sugar moiety specific for anti-gangliosides antibody recognition by synthesizing specific building blocks. these building blocks are amino acids carrying glycans mimicking the biological activity of complex oligosaccharides. we selected sialic acids (in particular the n-acetylneuraminic acid -neu ac) because they are involved in a significant number of biological events. neuraminic acid and its derivates are widely distributed in animal tissues and in bacteria, especially in glycoproteins and gangliosides. therefore, we synthesized fmoc-l-asn(neu ac)-oh and fmoc-l-ser(neu ac)-oh. these building blocks will be introduced in the type ' β turn structure for the detection of anti-gangliosides antibodies in als. as a distinct pattern of ms could involve an antibodymediated demyelination, identification of autoantibodies as specific biomarkers is a relevant target. even if interesting data focused on the diagnostic and prognostic role of the detection of antibodies to myelin oligodendrocyte glycoprotein (mog) in adults' serum, its value remains dubious due to many other contrasting results. our research group identified csf (glc), an nglucosylated peptide, able to detect disease-specific autoantibodies in the sera of a statistically significant number of ms patients. , since this synthetic antigen may be considered as a mimic of aberrant post-translational modification (i.e. n-glucosylation) of myelin protein(s) triggering autoimmunity in ms, our goal is to obtain the extracellular domain of mog properly glucosylated thanks to a simplified native chemical ligation approach. for this purpose, the n-glucosylation will be introduced in a synthetic peptide fragment following the building-block approach by spps. the other protein fragment bearing an n-terminal cysteine will be expressed in e. coli after introduction of a selective point mutation into mog. finally, our aim is to test the semi-synthetic protein by sp-elisa to study the ability to detect autoantibodies in ms patients' sera and to find a potential cross-reactivity with csf (glc). this peptide is an endogenous ligand of the opioid receptorlike (orl ), previously referred to as "orphan" receptor, structurally and functionally related to the classical opioid receptors. also the hexapeptide ac-ryyrwk-nh is shown to be a selective ligand for the nop receptor with marked analgesic effect. with a view to developing ligands for the nop receptor with more potent analgesic activity, new series of the ac-rfmwmk-nh and ac-ryyrwk-nh , modified at position and respectively with newly synthesized β tryptophan analogues were synthesized . the aim of the present study was to examine the effects of naloxone (nal) and jtc- (nop receptor antagonist) in the analgesic activity of newly synthesized hexapeptide analogues. all peptides ( μg/kg), nal ( mg/kg) and jtc- ( , mg/kg) were injected intraperitoneally (i.p.) in male wistar rats. antinociceptive effects were evaluated by two nociceptive tests -paw-pressure (pp) and hot-plate (hp) and statistically accessed by anova. the results will be discussed compared to the referent compound in both tests used and mechano-and thermo-receptors are involved. [ ] . socs and socs have many similarities as well as some intriguing differences. both can block signalling by direct inhibition of jak enzymatic activity yet apparently require different anchoring points within the receptor complex. while the primary socs interaction is with a critical py residue within the jak catalytic loop [ ] it interacts also with py residues in the ifnαr and ifn r subunits in a jak -independent manner; the socs -sh domain also interact with y in jak , albeit with slightly lower affinity, but subsequent studies demonstrated a high affinity interaction with py residues located within receptor subunits [ ] . mutagenesis studies identified small regions at the n-termini of the socs and socs -sh domains, and at the c-terminus of the socs -sh domain, which were critical for phosphotyrosine binding. in order to gain insights in molecular discriminants for the interaction of both socs and socs toward jak and tyk we designed and synthesized peptides encompassing regions involved in proteins recognition. we set up a spr assay to evaluate the affinities of complexes formation. then through an alascanning approach we have designed new peptide sequences containing un-natural amino acids that are able to better recognize wild sequences and whole proteins. cellular experiments on stat activation signaling suggest their potential application as modulators of disorders involving socss overexpression. targeting proapoptotic death receptors (drs) to trigger apoptosis in cancer cells is a promising anticancer therapeutic approach. trail (tnf-related apoptosis inducing ligand) is a transmembrane homotrimeric protein belonging to the tnf family that triggers selective tumour cell apoptosis upon binding to its cognate receptors dr and dr . several strategies are being developed to exploit the unique cancer selectivity of the trail-dr pathway in therapy, including the use of recombinant trail targeting dr or dr . [ ] recently, a disulfide-bridged macrocyclic -mer peptide (derived from phage display) that binds selectively to dr has been identified. [ ] oligomeric versions of this macrocyclic peptide display increased binding avidity to the receptor and exhibit the capacity to activate the trail apoptotic pathway both in vitro and in vivo. [ ] however, disulfide bonds are susceptible to reduction and scrambling in vivo potentially resulting in the loss of the desired biological activity. among alternative linkages with increased redox stabilities, lanthionine thioethers, in which one of the sulfur atoms of the disulfide bond is removed have previously been introduced into biologically active peptides with some success. [ ] disulfide bridges can undergo a -elimination in alkaline conditions, followed by a michael addition to give a thioether bridge. optimization of this reaction led to the desulfurized analogue of the dr -binding peptide. the native dr -binding peptide and its desulfurized analog have been compared for their structural (nmr conformational analysis) and biological properties (affinity to dr and signaling pathways). the apelin/apj complex has been detected in many tissues and is emerging as a promising target for a number of pathophysiological conditions. in the central nervous system, apelin/apj was detected in brain regions involved in spinal and supraspinal control of pain, such as the amygdala, hypothalamus, dorsal raphe nucleus and spinal cord. we propose the hypothesis that apelinergic agonists represent a potential new approach to pain modulation and that the synthesis of stable analogues would lead to compounds with antinociceptive properties. there is currently little information on the structure/activity relationship (sar) of the apelin hormone. in an effort to better delineate sar, we synthesized analogs of apelin- modified at selected positions with unnatural amino acids, with a particular emphasis on the c-terminal portion. analogs were then tested in binding and functional assays by evaluating gi/o mediated reduction in camp levels and by assessing β-arrestin recruitment to the receptor. the plasma stability of new analogs was also assessed. several were found to possess increased binding and higher stability compared to the parent peptide. there is compelling evidence that the neuropeptide rfa and its cognate receptor gpr , are involved in the control of food intake and bone mineralization. among the gpcrs whose structures have been solved, gpr exhibits the highest sequence homology with the beta adrenergic receptor. the aim of this work was to experimentally characterize predicted ligand-receptor interactions by site-directed mutagenesis of gpr and design of point-substituted rfa analogs. starting from the x-ray structure of the beta -adrenergic receptor, a d molecular model of gpr has been built. the bioactive c-terminal octapeptide rfa( - ), kggfsfrf-nh , was subsequently docked in this gpr model and the ligandreceptor complex was submitted to energy minimization. in the most stable complex, the phe-arg-phe-nh part was oriented inside the receptor cavity whereas the n-terminal lysine remained outside. a strong intermolecular interaction was predicted between the arg residue of rfa and the gln residue located in the third transmembrane helix of gpr . in order to study this interaction, we have investigated the ability of rfa and arg-modified rfa analogs to activate the wild-type (wt) and the q amutant receptors transiently expressed in cho cells. the platelet receptor αiibβ plays a critical role in the process of platelet aggregation and thrombus formation. upon platelet activation its conformation changes leading to an increased affinity for fibrinogen. the αiibβ activation is regulated by "outside-in" and "inside-out" signaling. among the protein-protein interactions, which contribute to «inside-out» signaling, the most important is that of talin with the β cytoplasmic tail. it has been recently suggested that talin-mediated αiibβ activation relies on the cooperative interaction of the membrane proximal (mp) and the membrane distal (md) β regions with talin f domain and that the -n ply -motif of β , which can be phosphorylated at y , plays a critical role in this process. to evaluate the interaction of talin with the β tail of integrin we designed and synthesized three peptides corresponding to the md and mp parts of β in their carboxyfluoresceinlabeled form (md: cf-r akwdtannplyke -nh , cf-n nplykea -nh and mp: cf-k llitihdrke -nh ). emission and anisotropy fluorescence spectroscopy was used to quantitatively assess the affinity of these peptides for talin. furthermore, to challenge the role of the y phosphorylation in talin-α iib β interaction we also studied the binding of talin to the modified analogues of md, cf-r akwdtannpl(ptyr)ke -nh and cf-n npl(ptyr)kea -nh . our experiments revealed that the md and mp parts of β bind tightly to talin and that y phosphorylation has an inhibitory effect on this binding. functionalized oligoprolines as multivalent scaffolds in tumor targeting p. wilhelm, h. wennemers* eth zurich, zurich, switzerland oligoprolines are known to be structurally well-defined molecular scaffolds. in aqueous media, even short chain lengths of six proline residues adopt a polyproline ii helix (ppii). this secondary structure is a highly symmetric helix where every third residue is on top of each other in a distance of about . Å. [ ] the incorporation of azidoproline (azp) allows facile and versatile functionalization either via copper-catalysed azide-alkyne cycloaddition (cuaac) or an acylation that followed a staudinger reduction. [ ] based on the structural integrity of the oligoproline scaffold, targeting vectors can be conjugated via coppercatalysed azide-alkyne cycloaddition in defined distances. recent studies on radiolabeled oligoproline-bombesin conjugates, to target the gastrin-releasing peptide receptor (grp-r), showed in vitro and in vivo superior internalization in prostate cancer cells compared to the established monovalent ligands. [ ] a facile route to synthesize alkynylated ligands has been developed successfully. we are currently expanding this concept to the integrinligand c(rgdyk) as well as to [tyr ]-octreotide, which binds to somatostatin-receptors. the monovalent analogue of the latter, dota-[tyr ]-octreotide (dotatoc), is well established for diagnosis [ ] and therapy [ ] of somatostatinpositive tumors such as neuroendocrine tumors. the cu i -catalyzed azide-alkyne addition (cuaaa), the useful variant of "click chemistry," has emerged as a powerful technique for specific addition. that chemistry is also commonly used for conjugation, and cyclization of peptides. it is known that cyclization can increase the metabolic stability of peptides, as well as enhance potency or selectivity. another useful application of the cuaaa, which we are reporting, is the n-terminal crosslink of two synergic peptides to gain their potency. cuaaa reaction is performed on solid phase (merrifield resin) where one of the peptide components with azido group on the linker ( azido-hexanoic acid) is "clicked" with second peptide component in solution, made by fmoc strategy in partially protected form containing at n-terminal side alkyne group (fmoc-l-propargylglicine). cuaaa coupling is performed in dmf/t-buoh/h o with presence of cui and sodium ascorbate when reacting mixture was degassed. linked peptides are cleaved finally from resin and purified. as an application example we picked two endothelin active peptide analogues: bq derivative (a highly potent and selective eta antagonist) and irl- derivative angiogenesis is a key step in the transition of tumors from a dormant state to a malignant state. the vascular endothelial growth factor (vegf) is a major contributor to tumor angiogenesis. its pro-angiogenic activity is mainly mediated through binding to two tyrosine kinase receptors located predominantly on the surface of endothelial cells: vegfr- and vegfr- . vegf binding to these receptors triggers the activation of different signal transduction pathways responsible for the proliferation, survival and migration of endothelial cells . vegf/vegfr system constitutes a target to stop tumour growth. an attractive approach is the development of peptides, or small-molecules, with a high affinity for the extracellular domain of the receptors to prevent vegf binding. based on the x-ray structure of vegf and the d domain of vegfr- , cyclic peptides had been developed in our group . such peptides, mimicking simultaneously the - loop and helix · of vegf, can bind to d domain of vegfr- and inhibit receptors phosphorylation and thus map kinase pathway . we describe here our strategies to optimize peptidic antagonists of vegfr- . chemical modifications are made in order to better mimic peptide conformations and to increase their receptor binding affinities. we introduce a hydrophobic functional group at the c-terminal of the original cyclic peptide , some of such modified peptides reveal improved vegfr- binding affinity. otherwise, as the helix · presents most of the important residues in vegfr binding according to alanine-scan in the literature , we try to stabilize the helical conformation by insertion of aib residues or by peptide cyclisation. the peptides affinities are evaluated by an elisa test developed previously . institute of chemistry and biochemistry, freie universität berlin, thielallee , d- berlin, germany new polypeptide was isolated from the azemiops feae viper venom by combination of gel filtration and reversephase hplc and called azemiopsin. its amino-acid sequence (dnwwpkpphqgprpprprpkp) was determined by means of edman degradation and mass spectrometry. it consists of residues and does not contain cysteine residues. according to circular dichroism measurements, this peptide adopts a β-structure. peptide synthesis was used to verify the accuracy of the determined sequence and to prepare sufficient peptide amount for biological activity studies. azemiopsin efficiently competed with α-bungarotoxin for binding to torpedo nicotinic acetylcholine receptor (nachr) (ic . ± . m) and with lower efficiency to human α nachr (ic ± μm). ala scanning showed that amino-acid residues at positions - , - and - are essential for binding to torpedo nachr. in biological activity azemiopsin resembles waglerin, a specific blocker of muscle-type nachr from tropidechis wagleri venom. however the sequences of these peptides are markedly different, and azemiopsin is the first natural toxin to block nachrs that does not possess disulfide bridges. laboratory of peptide science, nagahama institute of bio-science and technology, nagahama, shiga - , japan while neutrophils infiltrate into damaged sites immediately after tissue injury, endogenous factors which induce their acute transmigration and activation have not been thoroughly elucidated. for the candidates, we recently identified two novel neutrophil-activating cryptides, mitocryptide- (mct- ) and - (mct- ), which were hidden in mitochondrial cytochrome c oxidase and cytochrome b, respectively [ ] [ ] [ ] . in addition, the presence of many neutrophil-activating peptides other than mct- and - was observed during their purification. these findings suggest that neutrophils are regulated by many unidentified peptides. here, we purified a novel neutrophil-activating octadecapeptide whose primary structure was identical to mitochondrial cytochrome c ( - ) from porcine hearts. we named this functional peptide as mitocryptide-cyc (mct-cyc). the structure-activity relationships of cytochrome c on β-hexosaminidase release from neutrophilic differentiated hl- cells demonstrated that cytochrome c ( - ) was the most potent cryptide among cytochrome c-derived peptides. since cytochrome c is known to be involved in the apoptotic process, our present results suggest that cryptides produced from cytochrome c play an important role in scavenging toxic debris from apoptotic cells by neutrophils. anthracis spores are very resistant and can remain dormant in soil for decades. therefore, an effective detection system for b. anthracis is urgently needed. recently, it was found that one of the components of the b. anthracis exosporuim is a collagen like protein whose carbohydrate portion is composed of the tetrasaccharide with the highly specific monosaccharide upstream terminal, named anthrose. since anthrose was not found on other bacterial spores, including those closely related to b. anthracis, this monosaccharide is an attractive target for the development of new b. anthracis detection and identification methods. peptide cyclization represents particularly interesting approach for the design of artificial receptors for anthrose, because cyclic peptides provide the possibility of having a spherical lipophilic binding site of appropriate size and shape for a particular carbohydrate substrate. the presence of hydrogen donor/acceptor groups within a three-dimensional structure permits carbohydrate substrates to be encapsulated, thereby allowing their binding in water. in order to determine whether the cyclic peptide receptor can selectively detect the anthrose, we have successfully prepared cyclic peptide combinatorial library (total peptides) by the process of divide, couple and recombine ("tea-bag" technology) using standard fmoc solid-phase peptide synthesis. prepared combinatorial library is screened for anthrose binding in fluorescence-based assay, and individual cyclic peptides with enhanced affinity toward anthrose are identified by the positional scanning deconvolution process. cyclization of linear sequences is a well-known approach used to restrict the flexibility of peptides. cyclization often increases selectivity of peptides towards one specific receptor type, increases metabolic stability and generally increases lipophilicity, which often improves the bloodbrain barrier permeability of peptides. in our previous study [ ] we have reported on the synthesis of a cyclic endomorphin- (em- ) analog, tyr-c(d-lys-phe-phe-asp)-nh , which elicited analgesia after peripheral administration. encouraged by the fact that this analog was able to cross the blood-brain barrier we designed and aliskiren is the first orally active, direct renin inhibitor to be approved for the treatment of hypertension. its structure and conformational analysis were explored using molecular dynamics (md) simulations. for the first time, md calculations have also been performed for aliskiren at the receptor site, in order to reveal its molecular basis of action. it is suggested that aliskiren binds in an extended conformation and is involved in several stabilizing hydrogen bonding interactions with binding cavity (asp / , gly ) and other binding-cavity (arg , ser , tyr ) residues. of paramount importance is the finding of a loop consisting of residues around ser that determines the entrapping of aliskiren into the active site of renin. the details of this mechanism will be the subject of a subsequent study. additionally molecular mechanics poisson-boltzmann surface area (mm-pbsa) free energy calculations for the aliskiren-renin complex provided insight into the binding mode of aliskiren by identifying van der waals and nonpolar contribution to solvation as the main components of favorable binding interactions. adamantyltripeptides and phospholipids in liposomal bilayers . now, we were primarily interested to study incorporation profile of mannosylated adamantyltripeptides. we have demonstrated that the adamantyl moiety, due to its liphophilic properties, penetrates into the lipid core of the bilayer while the hydrophilic part with the mannosyl moiety is exposed on the liposome surface. after concanavalin a (con a), a lectin, which specifically binds α-d-mannosyl residues, was added to the liposome preparation, increase in liposome size and appearance of aggregates has been observed. the enlargement of liposomes was ascribed to the specific binding of the con a to the mannose present on the surface of the prepared vesicles. the afm analysis revealed that the adamantyltripeptide molecules grouped into small domains that raise above the bilayer surface. the molecule size and molecular geometry, as well as the hydrophilic and hydrophobic surfaces in the structure of mannosylated adamantyltripeptides, are responsible for arrangement of molecules in the lipid bilayer. this approach might be a useful model for investigation of specific protein interactions with membrane receptors. also, the adamantyl moiety may be considered as a potential membrane anchor for different carbohydrate or other molecules of interest, which could be bound on it and thus exposed on liposome surfaces and as such used in targeted drug delivery. the assay is carried out in a well format p and images are captured throughout the course of the assay, thus we can not only determine a ligand's propensity to induce internalization, but also its efficacy and internalization rate. addition of test compound, followed by the standard agonist at a later interval, enables differentiation between agonist or antagonist activities. in the positional scanning format [ ] , while the possibility of agonists and antagonists working against each other within a mixture exists, the effects are minimized in screening the whole library as there are as many arrangements of the sub-libraries as there are defined positions. therefore while an agonist and antagonist might be present in a particular mixture in one sub-library they will be in different mixtures in all other sub-libraries. we have used this assay format to simultaneously screen for novel agonists and antagonists against the orexin receptor. assay development and library screening will be presented. [ ] . since the pro residue in position of em is very important in the proper conformational alignment of the two aromatic residues tyr and phe in em molecule at the receptor site, it is possible that structural modification around the pro residue yields compounds with unique biological properties and improved metabolic stability. in the present study, we synthesized seven em analogues containing isopro or constrained residues with oxopyrrolidine or oxopiperadine ring, instead of pro residue in position . all peptide analogues were synthesized solid phase method. incorporation of oxopyrrolidine and oxopiperadine rings were carried out on a solid support by the methods of gellerman, et al. [ ] and mohamed, et al. [ ] , respectively. opioid receptor binding activity for μ and δ-receptors using the development of resistance to mainstay cancer therapies has become a major limitation for the treatment of many cancers. there is an urgent need to develop new antineoplasic agents with innovative anticancer approaches. to overcome resistance to cancer therapies, our attention has turned to proteins that regulate multiple signalling pathways essential for tumour survival. among the few known nodal proteins upregulated in cancer cells and involved in many hallmarks of cancer, we are interested in survivin. an essential regulator of cell proliferation and apoptosis, survivin is sharply overexpressed in cancer cells and plays a major role in resistance. being a small protein, its bioactivity is relies mainly on protein-protein interactions (ppi) with different partners. a critical point for its multiple functions in cancer is its association with hsp , which is required for its stability. a nonapeptide from survivin called shepherdin has been shown to modulate the interaction of survivin with hsp by binding to hsp and to induce death of tumour cells. unfortunately, shepherdin is not cell permeable, has low proteolytic stability and shows poor bioavailability, limiting its use as anticancer therapeutic agent. to improve pharmacological properties of shepherdin, cyclic and peptidomimetic analogs of shepherdin have been synthesized followed by structure-activity relationship studies. in hsp binding studies, some cyclic hexa-and heptapeptidic analogs showed increased affinity compared to shepherdin. the synthesis of cyclic and peptidomimetic analogs and the results from the binding assays and the conformational analyses will be presented. the hexapeptides with formula ac-ryyr/kw/ir/k-nh have been identified as shortest peptide sequence with high nop receptor affinity, selectivity and marked analgesic effect. it was found that the following peptides act as partial or full agonists or antagonists of nop receptor in different in vivo and in vitro systems. these hexapeptides were used as chemical templates in sar studies , . the aim of the present study was the synthesis and the biological screening of new analogs of ac-rfmwmk-nh and ac-ryyrwk-nh , modified at position and respectively with newly synthesized β -tryptophan analogues . these non natural amino acids were prepared using reaction of asymmetric friedel-crafts alkylation of various indoles with a chiral nitroacrylate to provide optically active β-tryptophan derivatives. the four newly synthesized ligands for the nociceptin/orphanin fq (n/ofq) receptor (nop) have been prepared by solidphase peptide synthesis-fmoc-strategy. these compounds will be tested for agonistic activity in vitro on electrically stimulated smooth-muscle preparations isolated from vas deferens of wistar rats. bacterial infections are a common problem associated with dermal wounds. these infections can prolong or impair wound healing. hydrogel materials that display inherent activity against bacteria can be used to directly treat accessible wounds to prevent or kill existing infection. in this work, we describe the design and utilization of injectable gels prepared from self-assembling β-hairpin peptides having a high content of arginine. these gels were found to be extremely effective at killing both gram-positive and gramnegative bacteria, including multi-drug resistant p. aeruginosa. importantly, no added antibacterial agents are necessary since the nanostructure of the gel, itself, is the active agent. using self-assembling peptides for material construction allows facile structure-activity relationships to be determined since changes in peptide sequence at the monomer level are directly transposed to the bulk material's antibacterial properties. structure-activity relationships studies show that arginine content largely influences the hydrogel's antibacterial activity, and influences their bulk rheological properties. these studies culminated in an optimized gel, composed of the peptide pep r. pep r gels prepared at . wt % or higher concentration, demonstrate high potency against bacteria, but are cytocompatible towards mammalian mesenchymal stem cells. the general mechanism by which pep r exerts its action was explored and it is suggested that involves membrane disruption that occurs when cells come in contact with the gel's surface. atomic force microscopy (afm) was used to study the effect of the gel on the cell envelope morphology of e. coli. rheological studies indicate that the gel is moderately stiff and displays shear-thin recovery behavior, allowing its delivery via simple syringe. they are intimately involved in the molecular process leading to the delicate nano-patterned silica shells of diatoms. deciphering the mechanisms of silica-biogenesis in diatoms will inspire the development of novel routes for the biomimetic synthesis of silicon-based materials under mild conditions and expand the scope of biotechnological applications, e.g. for immobilization of enzymes in silica matrices. we synthesized silaffin peptides derived from c. fusiformis that carry posttranslational modifications such as phosphorylation or polyamines linked to lysine side chains. a distinct alteration of silica precipitation activity depending on the particular modifications of the silaffins emerged. these modified silaffin peptides were covalently linked to recombinant proteins by expressed protein ligation leading to stable protein-silaffin conjugates. using egfp as model protein, we could show that egfp-silaffin conjugates can induce biomineralization of silica and ensure an efficient and homogeneous immobilization of egfp into silica particles, superior to simple co-biomineralization approaches. moreover, a significant stabilization of immobilized egfp against denaturing agents was observed. we established a method for controlled immobilization of biomolecules based on covalent attachment of silaffin peptides with well-defined silica precipitation properties. currently this method is applied to the immobilization of biotechnological relevant enzymes in order to test their activity and the stabilization effect. herein, we present the covalent functionalization of multiwalled cnts (mwcnts) with organocatalysts based on proline or proline derivatives carrying either a dipeptide or a sulfonamide moiety. two different approaches were followed, namely, covalent grafting of the organocatalysts either at the tips or at the sidewalls of the cnts. for the former approach, mwcnts were oxidized in order to introduce carboxylic units at their tips and make them easily dispersed in aqueous solutions. then, oxidized mwcnts readily reacted with proline-based derivatives carrying a free amino unit yielding the corresponding hybrid materials. for the latter approach, the functionalization methodology based on in-situ generated aryl diazonium salts was followed. in this context, mwcnts were modified with aryl units carrying free amino terminal groups, which were subsequently conjugated with proline-based derivatives carrying a free carboxylic unit. all newly formed hybrid materials were fully characterized with complementary spectroscopic (atr-ir, raman), thermal (tga) and microscopy (tem) techniques. the catalytic evaluation of the activity of the cnt-based organocatalysts in aldol reactions is in progress. financial support from gsrt/ΕΣΠΑ - ΣΥΝΕΡΓΑΣΙΑ through ΣΥΝ- - -ΝΑΝΟΚΑΤΑΛΥΣΗ project is acknowledged. novel organogels based on self assembly of rationally designed pseudopeptides c. pappas, n. sayyad, a.g. tzakos, i. plakatouras section of organic chemistry and biochemistry, department of chemistry, university of ioannina, ioannina, gr- , greece self-assembly is becoming a rather intriguing way to build an array of nano-and micro-structured materials. low molecular weight organogelators can self-assemble into various architectural types in organic solvents through weak intermolecular interactions. such organogelators have potential applications in the generation of novel materials for nanobiotechnology . herein, we report the synthesis of rationally designed pseudopeptides and the conditions to form organogels. the obtained gels are responsive to temperature, and the sol-gel process is thermoreversible. the architecture of the constructed organogels was characterized via tem and spectroscopic techniques. diffusion ordered nmr spectroscopy (dosy) was further utilized to determine differences in the molecular shape of the different pseudopeptides. applications of the resulted compounds in nanotechnology will be reported. since , organocatalysis has met such a great rate of expansion that is nowadays considered the third major branch of modern asymmetric catalysis along with the transition metal catalysis and biocatalysis. after the seminal work of list, lerner and barbas on the enantioselective aldol reaction between acetone and -nitrobenzaldehyde catalyzed by proline, it became clear that amino acids and peptides could serve as an abundant pool full of potential to develop novel organocatalytic motives. following our recent report that the combination of a prolinamide with a thiourea group having as a spacer a chiral diphenylethylenediamine leads to an efficient organocatalyst for the aldol reaction, we recently considered the possibility to couple the prolinamide unit with an urea moiety. one of our main interests was the substitution of the diphenylethylenediamine spacer by a gem diamine derived from an α-amino acid. the gem diamine is easily synthesized via a curtius rearrengement of the corresponding acyl azide. after synthesis and evaluation of a number of potential catalysts, the prolinamide derivative bearing a gem diamine derived from (s)-phenylalanine and an aryl urea moiety proved to provide the best results in the reaction between cyclic ketones and aldehydes. utilizing mol% of our organocatalyst, the aldol products were obtained in high to quantitative yields (up to %), high to excellent diastereoselectivities(up to > : ) and high to excellent enantioselectivities (up to % ee). peptide self-assembled monolayers are of current interest to study physicochemical properties of modified metal (e.g. au) surfaces. rigid peptide scaffolds could enhance the interaction between gold surfaces and labels by reducing and precisely monitoring the distance between the supported monolayers and gold. the c α -tetrasubstituted αamino acid -amino- , -dithiolane- -carboxylic acid (adt) , which contains a cyclic disulfide system, is interesting in this respect because it may allow the parallel binding of the peptide helical chain to the metal surface. adt occurs in nature and has been utilized in medicinal chemistry and in a model compound of [fefe] hydrogenase. we synthesised a series of constrained helical peptides, based on the ala-ala or the ala-aib sequence, containing one or two adt residues. these peptides were functionalised with spectroscopic or opto-electronic labels. among the large number of reactions involving the formation of carbon-carbon bond, the addition of ketones to nitroolefins is a powerful tool for the synthesis of γ-nitrocarbonyl compounds, useful intermediates for pharmaceutical industry. our recently reported primary amine-thioureas based on tert-butyl esters of natural amino acids exhibit excellent performance for the michael reaction of ketones with nitroolefins providing the products quantitatively and almost stereospecifically (> % ee). , using this methodology, enantiopure baclofen and phenibut (analogs of gaba) have been synthesized. polymersupported organocatalysts constitute a great challenge for the michael reaction. in the current study, we report the immobilization of amine-thiourea catalysts containing ( s, s)or ( r, r)-diphenylethylenediamine and tert-butyl aspartate, on various polymer supports, either directly or through spacer units. the solid-supported catalysts evaluated in the reaction between acetone and βnitrostyrene and highlighted the importance of the choice of the polymer as well as the presence of the spacer or not. the direct attachment of the primary amine-thioureaaspartate to a crosslinked polystyrene-divinyl benzene resin containing a uniform distribution of aminomethyl groups provides a supported catalyst that affords the product of the reaction between acetone and β-nitrostyrene quantitatively and in high enantioselectivity ( % ee a. theodorou, g.n. papadopoulos, c.g. kokotos* laboratory of organic chemistry, department of chemistry, university of athens, athens, greece after the pioneering report that proline can catalyze efficiently the intermolecular aldol reaction between acetone and a variety of aromatic aldehydes, it became evident that amino acids and peptides can afford a plethora of different structural scaffolds for novel catalysts. along the first decade of its life, organocatalysis has grown to such an extend that now it is considered the third major branch of asymmetric catalysis. recently, researchers have paid special attention to other amino acids rather than proline. some primary amino acids have already been applied to a number of transformations with success. usually improved catalytic properties are observed when derivatives of primary amino acids are utilised. we have undertaken a study on the application of simple and cheap primary amino acids and amino acid derivatives, either commercially available or easily obtained, as organocatalysts for the asymmetric α-amination of aldehydes. in the present work, we report that the use of simple derivatives of primary amino acids like phenylalanine and aspartic acid can efficiently catalyze this transformation leading to products in high to quantitative yields and enantioselectivities up to % ee. the majority of the organocatalysts developed up to now for asymmetric organic transformations employ more than one functionalities in the catalytic mechanism that act through either covalent or non-covalent interactions. for example, proline employs the pyrrolidine nitrogen and the carboxylic acid group, while chiral thioureas combine the thiourea functionality with a tertiary or a primary amino group. we have recently shown that an amide of proline with a diamine carrying a thiourea group is a very good catalyst for the enantioselective aldol reaction. trying to improve the activity, we have found that a tripeptide-like thiourea having as building blocks (s)-proline, ( s, s)diphenylethylenediamine and (s)-di-tert-butyl aspartate provides the products of the reaction between ketones and aromatic aldehydes in high to quantitative yields and high stereoselectivities (up to : dr and % ee). a number of structural modifications of the catalyst were undertaken in order to understand the role of the hydrogen bond donors of the catalyst, i.e. the prolinamide hydrogen and the two hydrogen atoms of the thiourea group. we have come to the conclusion that the importance of the hydrogen bond donors of the catalyst follows the order: thiourea hydrogen originated from aspartate › amide hydrogen › thiourea hydrogen originated from diphenylethylenediamine. g eldrug s.a., patras , greece a convenient and facile synthesis and in vitro biological evaluation of n-substituted -butylimidazole derivatives as potent angiotensin ii (ang ii) receptor type (at ) antagonists have been reported in the present study. a series of imidazole based compounds bearing the biphenyl moiety at the n- position, a halogen atom at the c- and polar substituents such as hydroxymethyl at the c- position were synthesized. , these compounds were evaluated for binding to human at receptor and for ang ii antagonism in vitro on isolated rat uterus. in particular, butyl- -[[ ΄-( h-tetrazol- -yl)biphenyl- -yl]methyl]imidazole derivatives complexed with the at receptor and showed high binding affinity. these analogues were also found to be active in the rat uterotonic test. importantly, their binding affinities and potencies were comparable to those of losartan. these results indicate that the hydroxymethyl at the c- position of the imidazole ring is favorable for high affinity binding and antihypertensive activity and in line with the activities of the losartan counterparts. experimental findings are in good agreement with docking studies, which were undertaken in order to investigate ligand/at receptor interactions. z-leu-glu-his-asp-aluc, suc-leu-leu-val-tyr-aluc) are good substrates for bioluminescence assays, for example in the detection of caspase activity during apoptosis . these substrates generally offer significant advantages, such as increased sensitivity, ease of use, and high throughput screening capacity. luciferase-based assays are typically -to -fold more sensitive than the comparable fluorescent assays (rhodamine , -amino- -methylcoumarin (amc) and -amino- trifluoromethylcoumarin (afc)). the synthesis of different type peptide-amino-luciferin conjugates and their precursors have been published and some of them are commercially available. however, because of their high price the in vivo application of these conjugates is limited. to solve this problem we successfully worked out a new, easier and more convenient and economical method for the preparing these derivatives starting from -chloro-benzothiazole. moreover this products have excellent purity (> %) and adequate yield ( - %). major health problems arising from bacterial resistance towards existing antibiotics make discovery of antibacterial drugs with new mechanisms of action pertinent. although proof of concept for a novel antimicrobial approach using peptide nucleic acid (pna) antisense targeting of essential bacterial genes was obtained a decade ago, this technology is still limited by the lack of carriers that facilitate effective bacterial delivery and confer optimal pharmacokinetic properties to the prospective drugs. [ , ] in the past two decades, parallel efforts of exploiting naturally occurring antimicrobial peptides (amps) as drugs have been made. the cationic amp subclass appears to be directly involved in the innate immune response towards microbial infections. [ ] so far only few cell-penetrating peptides, with activity on mammalian cells, and other membrane-active peptides, have been investigated as potential vehicles for bacterial delivery. for instance, cationic amps with an internal target appear not to have been investigated for bacterial delivery of antibiotics. the aim of this project is to develop highly potent genetic antibiotics by exploiting naturally occurring antimicrobial peptides as potential delivery vehicles for antisense peptide nucleic acid oligomers. the amps are chosen from amps reported to act via intracellular targets, and thus must possess an inherent ability to permeate bacterial cell membranes without direct killing of the bacteria. faculty of chemistry university of gdansk, gdansk, poland azt ( '-azido- ' '-dideoksythymidine), a modified nucleoside used in antiretroviral therapy and peptide plant hormone -systemin were used as substrates of , -dipolar cycloaddition (click chemistry). systemin is -aa peptide defense hormone released in response to plant (tomato, tobacco) damage or pathogen attack. we examinated whether systemin's fast movement through plant tissues could be used for cargo (azt) transport. the huisgen cycloaddition also known as , -dipolar cycloaddition is a chemical reaction belonging to the larger class of cycloadditions. reaction between organic azide and alkyne appended substrates allows the synthesis of the desired conjugate in high purity and yields irrespective of the sequences and functional groups on either of the two substrates [ , ] . conjugate of azt-systemin has been synthesized by click chemistry, using systemin modified at n-terminus with propiolic group and azt. the conjugation was catalyzed by cu(i). the reaction was fast, efficient and regioselective. its progress was easily monitored by capillary electrophoresis (ce). ce was also applied for characterization of systemin and azt-systemin stability and movement throughout tomato leaf and stem. despite the fact that systemin moves rapidly through tomato tissues, our calorimetric (itc) studies showed that the peptide does not interact with liposomes-cell membrane model. universitätsklinik für nuklearmedizin, inselspital, bern, switzerland regulatory peptides (e.g., somatostatin, bombesin) have been shown to be suitable vectors for the specific delivery of radioactivity to tumors for diagnostic and therapeutic applications in nuclear oncology. a potential drawback of such vectors is their inherent instability in vivo. thus, new strategies are needed for the stabilisation of radiopeptides in order to improve their bioavailability and, consequently, increase their accumulation in the targeted tissue. it has been suggested that , , -triazoles, readily obtained by cuaac, are suitable amide bond surrogates which are resistant to proteases. in the present study, we report the synthesis and pharmacological evaluation of radiolabelled, triazole-containing analogues of the gastrin releasing peptide receptor (grpr) targeting peptide bombesin (bbn). to study the effect of backbone modifications in the minimal grp-binding sequence, we synthesized a series of analogues of [nle ]bbn ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , in which each amide bond is individually replaced by a , -disubstituted , , triazole. after radiolabelling of the peptidomimetics, their binding affinity and internalization kinetics were determined using pc- cells. metabolic stability was evaluated in blood serum. a number of the novel tumor-targeting peptide analogues presented exhibit both a retained high affinity (nm) towards the grpr and an improved serum stability. first preclinical data on the in vivo evaluation of the most promising candidate will be presented. to the best of our knowledge, this is the first report of the systematic replacement of amide bonds with , , triazoles within the binding sequence of linear, high affinity peptides. the methodology can be applied to a variety of peptide vectors and thus, holds great potential for the development of novel, stabilized peptide-based radiopharmaceuticals. dna is the molecular target for many of the drugs that are used in cancer therapeutics, and is viewed as a nonspecific target of cytotoxic agents. although this is true for chemotherapeutics, other agents that were discovered more recently have shown enhanced specificity. the development of new site-specific dna binders, which are associated with the recognition of the dna major groove, are based on the design of transcription factor mimics that bind the dna as a dimer , and prevent specific genes from being transcribed. these could ultimately result in interesting biomedical applications as designed genome interfering agents or diagnostics. in order to approach this biological constructs, we choose the bzip leucine zipper transcription factor as a model to mimic. as the entire structure cannot be synthesized without expensive, complicated and time-consuming biotechnological methods, the substitution of the dimerization domain by a less complex scaffold is the first step in the design. thus, we consider a steroid based scaffold as a candidate. the specific choice of the steroid scaffold as substituent is inspired by its known ability to enhance proteolytic stability of attached peptides, by its conformational properties ensuring correct positioning of the two appended chains and by its potential to increase bioavailability. this transcription factor binds specific dna sequences by dimerization and inserting short α-helices into the dna major groove. in order to attach the peptides to the scaffold, different strategies were studied. firstly, applying the well-known click chemistry, functionalizing the scaffold with an alkyne moiety, the peptide with an azide and viceversa. secondly, via the unknown resin to resin transfer reaction (rrtr), which has not been applied on peptide chemistry so far. this unprecedent methodology consists on the reaction of a peptide, which is attached on a safety-catch resin, with a second resin bearing a nucleophilic amino terminus resulting in amide bond formation. during the process, the peptide on solid support undergoes cleavage. an hexapeptide was synthesized on a preloaded safetycatch resin. deoxycholic acid derived scaffold with orthogonally protected amines was attached to tentagel resin that acts as acceptor resin. rrtr experiments were performed at both c and c positions of the deoxycholic acid derivative. in addition, this convergent strategy can be applied to other different peptide conjugated systems. we recently described a new kind of cyclized peptide in which the cyclization is performed between the side-chains of two diaminoacyl residues via a diversely substituted guanidine bridge. we showed that the degree of bridge substitution could impact on the orientation of the bridge inside the cycle and therefore the peptide conformation. we prepared two series ( and atoms cycle size) of cyclic enkephalin analogues to assess the potential effect of this kind of bridge on the biological activity. the compounds were synthesized on the solid support via the formation of a thiourea bridge and with the variable substituent being introduced at the last step before cleavage. it is noteworthy that the synthesis afforded at least two stable and separable conformers for each analogue of the shortest cycle series. generally, one major and one minor species were recovered. but in the case of di-substituted compounds with a cyclic moiety (pyrrolidine or piperidine substituents), three significant species were obtained. analogues were submitted to various biological assays (binding to μ and δ opioid receptors and functional assays). we observed a significant variation in affinity and selectivity for the receptors as a function of the degree of bridge substitution. a structural analysis by d nmr has been undertaken and correlated the variation in activity with a variation in conformation. the origin of the multiple conformers observed for the analogue with a pyrrolidine susbtituent was also investigated. this kind of cyclization could represent a useful tool to easily modulate the conformation and biological activity of a unique peptide sequence. the t-cell response is triggered by the formation of the trimolecular complex between the major histocompatibility complex (mhc), the immunodominant myelin protein epitopes and the t cell receptor (tcr). herein, we report the design and synthesis of non-peptide analogues with the ability to mimic the immunodominant epitope - of mbp , . the mimetics were designed to block the formation of the trimolecular complex and therefore the t-cell activation , . more specifically, indole analogues were synthesized with substitution at positions and or . these molecules contain a carboxyl or an ethyl ester group in position and a benzylamino or phenylamino group in position or . the synthesis of the indole ring was achieved by fischer reaction followed by catalytic hydrogenation, reductive amination or arylation and ester hydrolysis. the synthesized molecules were purified using liquid chromatography, and they were identified by mass spectrometry and h-nmr. laboratory of peptide science, nagahama institute of bio-science and technology, nagahama, japan amyloid β peptide (aβ), the main component of senile plaques in the brain of alzheimer's disease (ad) patients, is formed by proteolysis of amyloid precursor protein (app). as β-secretase (bace : β-site app cleaving enzyme ) triggers aβ formation by cleavage at the aβ domain nterminus, it is a molecular target for ad therapeutic intervention. previously, we reported potent pentapeptidic and non-peptidic bace inhibitors containing a substrate transition-state mimic. although these inhibitors exhibited potent inhibitory activities, their molecular-sizes appeared a little too big (mw> ) for developing practical drugs. in this study, we designed a series of small molecular peptides, with bace inhibitory activity, lacking the p -p ' region on the basis of the conformational structure bound in bace . design and synthesis of new '-peptidyl-trna analogues, in particular "hydrolysable" analogues, which represent covalent conjugates of peptide-nucleic acid (pna) with "stop-peptides," were carried out. such compounds are of interest as tools to study the ribosome functioning and as inhibitors of protein biosynthesis. ( aminoethyl)glycine pna models '-end trna sequence cca in designed structures. computer simulations showed the formation of watson-crick pairing of the pna cytosine residues with s rrna nucleotides g and g involved in interactions with peptidyl-trna during its specific binding in p site of the ribosomal peptidyl transferase center (ptc). short "stop-peptides" were planned for conjugation with pna. these peptides form stable complexes with the ribosomal tunnel (rt) that leads to ribosome stalling and translational arrest. structures of "hydrolysable" 'peptidyl-trna analogues that could form peptide bond with amino acid residue of aminoacyl-trna in a-site of ptc included '-deoxyriboadenosine instead of the pna adenine containing residue. such conjugates would permit to identify the chemical nature of specific sites localized in rt and responsible for interactions with amino acid residues of the nascent polypeptide chain. pna and "stop-peptide" as well as pna-"stop-peptide" conjugates were prepared by solid phase synthesis on sasrin polymer using fmoc/bhoc(boc) strategy. synthesis of "hydrolysable" conjugates included modification of the 'hydroxyl of '-protected '-deoxyadenosine by n-blocked "stop-peptide", deprotection of the '-hydroxyl, its conjugation with n-protected pna and removal of protecting groups from the resulted conjugate. the binding of the new '-peptidyl-trna analogues with ribosome will be tested by chemical probing and in the cell free translation system. this study was supported by the russian foundation for basic researches (grant - - -a). a close structural similarity of endomorphin- and another atypical opioid peptide, morphiceptin, which both have a phe residue in the third position, encouraged us to study antinociceptive activity of these two peptides and their analogues. in order to improve the affinity and chemical stability of these opioid peptides, we have designed, synthesized, and analyzed novel analogues. the first modification included endomorphin- and morphiceptin analogues, where halogenated phenylalanines in position or were incorporated as surrogates of the native phenylalanine. another important modifying element is non-protein amino acid canavanine (cav) and its analogue (sarg). it is well documented that cav and sarg exhibit strong analgesic activity. two new morphiceptin analogues were synthesized by introducing cav and sarg in position . we further characterized their antinociceptive activities by the paw pressure (pp) test. the experiments were carried out on male wistar rats ( - g), treated with i.p. doses of mg/kg. e eldrug s.a., patras , greece the renin angiotensin system (ras) has been a prime target for the therapy of cardiovascular diseases. angiotensin ii type (at ) receptor mediates vast majority of biologically detrimental actions. non-peptide at receptor blockers are presently the most specific means to block the ras enzymatic cascade. the dupont group was the first to develop losartan (dup ), an orally effective angiotensin ii receptor blocker, which is metabolized in vivo to the more potent antagonist exp . herein, we report on the preparation of e-urocanic acid based analogs, focusing our attention on the introduction and structural modifications of the substituents on the imidazole ring as well as the modifications on the acrylic side chain. in particular, we have designed and synthesized a series of urocanic acid analogs bearing the biphenylmethyl tetrazole moiety at the n- of the imidazole ring. additionally, the rigid acrylic chain was lengthened by esterification resulting in the ethyl ester and on the other hand the latter was readily converted to the corresponding acrylic alcohol or aldehyde which may proved to be effective structural elements for enhancing biological activity. finally, a lipophilic alkyl chain such as the n-butyl group was introduced at the -position of the ring which may possibly enhance the antihypertensive activity. docking studies and biological evaluation of the synthesized analogs are being undertaken. university of athens, department of chemistry, laboratory of organic chemistry, , panepistimiopolis zografou, athens, greece the backbone modification of bioactive peptides with replacement of a scissile peptide bond in enzymatic hydrolysis is a well-established strategy for developing protease inhibitors. in particular, for zinc metalloproteases, which contain a zinc atom in their active site, several successful modifications have been reported over the past years. phosphinic pseudopeptides are among the best candidates when addressing the challenge to potent and selectively inhibit zinc proteases. a thorough search in the literature revealed the absence of any reference regarding thiophosphinic pseudopeptides. we thought that this class of compounds would add a valuable tool in the field of zincbinding groups. in the present study, we describe in detail the first synthesis of a new class of phosphorous compounds, thiophosphinyl dipeptide isosters (tdis). we prepared several fully protected thiophosphinate pseudodipeptides of the general formula pg-phe-Ψ[p(s)(ox)ch ]-gly-pg' starting from the corresponding phosphinate pseudodipeptide using lawesson's reagent. selective deprotection of these compounds was also studied and the results are disclosed. these compounds can be used as building blocks for the synthesis of longer thiophosphinic pseudopeptides after suitable deprotection and elongation as well as transition transition state-mimicking inhibitors for several zinc metalloproteases. in the last decade, trypsin inhibitor sfti- isolated from sunflower seeds [ ] has become one of the most studied peptidic inhibitors of serine proteases. owing to its small size and a strong trypsin inhibitory activity (ka = . × m - ), sfti- is considered to be a very attractive template for designing proteinase inhibitors with the potential use as pharmacological agents [ ] . it could also serve as an affinity probe for the isolation of trypsin like (sfti- ) or chymotrypsin like ([phe ]sfti- ) proteinases. following this idea, we decided to synthesize a set of cell-permeable monocyclic sfti- analogues with a fluorophore moiety attached at their n-termini. the presence of the fluorophore in the molecule enabled us to show that the analogues can cross the cell membrane. the cell penetration assay was performed using multiple cell lines (hela cells and human fibroblasts cell line ( br. n) was obtained from european collection of cell cultures (ecacc)). for all the obtained peptidomimetics, we determined the association constants with cognate proteinases. selected peptides were also used as a probes for the detection of inhibitor -proteinase complex, which was achieved by the means of gel filtration chromatography equipped with fluorescence detector and acrylamide native gel electrophoresis. the functional reconstruction of folded protein surfaces with peptide-based mimics is an enormous scientific challenge. the majority of proteins show activity through a small area of their folded surface: "the binding site". however, linear peptides are too flexible and seldomly adopt the correct d-structure of the binding site spontaneously. therefore, they show limited or no activity at all . crucial for activity is to control the secondary (αhelix, β-sheet and/or β-turn) and tertiary structure (relative orientation of subdomain structures). we present the development of a new type of watersoluble scaffolds that have the potential to control both secondary and tertiary structure of discontinuous (i.e. double-loop) protein mimics. the new scaffolds contain a first pair of reactive functionalities to constrain the linear peptide conformation via a 'clips' reaction , stabilizing the secondary structure. next to this, a second functionality allows for ligation of two dissimilar constrained peptides to form a discontinuous binding site mimic via oxime-ligation or click-reaction. these ligations offer the ability to position different peptide loops in d, thus mimicking the tertiary structure of the native protein. most unique to our approach is the fact that all chemical conversions are performed in aqueous media, using side-chain unprotected peptides . growth hormone-releasing peptide (ghrp- ) is a synthetic hexapeptide (his-d-trp-ala-trp-d-phe-lys-nh ), which interacts with two kinds of receptors: growth hormone secretagogue receptor a (ghs-r a) and cluster of differentiation (cd ). the latter is a membrane glycoprotein member of the class b scavenger family, and decreases the internalization of oxidized lipids into macrophages, as well as causes inhibitory effects on angiogenesis associated with binding to thrombospondin. to increase activity and selectivity for the cd receptor, different analogues of ghrp- were synthesized. in particular, substitution of trp in ghrp- by aza-amino acids has given selective analogs, due likely to induction of a β-turn secondary structure. for aza-peptide synthesis, a submonomer solid-phase approach has proven effective to introduce side chains onto the semicarbazide residue. studying influences of benzylidene, benzhydrylidene and fluorenylidene residues during the alkylation of the semicarbazide, superior conversion was observed with fluorenone derivative, and mild alkylation conditions employing et noh as base have improved yields and minimized racemisation. our presentation will focus on the improved submonomer synthesis method for optimization of selective and potent cd- ligands with antiatherosclorotic and anti-angiogenic effects. for instance, the integrin αvβ , vitronectin receptor, is expressed in a number of cell types and has been shown to mediate adhesion of osteoclasts to bone matrix, vascular smooth muscle cell migration, and angiogenesis. integrin αvβ also play a significant role in tumor growth, invasion and metastasis, and is a receptor for the extracellular matrix proteins with the exposed arginine-glycine-aspartic (rgd) tripeptide sequence. rgd has been shown to be potent antagonist of the integrin αvβ , and has excellent anti-angiogenic properties including its suppression of tumor growth in animal models. in this context, drug design based on the rgd structure may provide new treatments for diseases such as thrombosis, osteoporosis, and cancer. we designed and synthesized series of short rgdmimetics containing the sequence xaa-gd, where xaa is arg-mimetic. as promising candidates we have chosen canavanine (cav) and canaline (can) instead of the basic residue arg. in order to improve antitumor activity of the parent molecule, c-terminal modifications were also applied. their cellular uptake was determined on human breast (mcf ) cancer cell lines. furthermore, the in vitro cytostatic effect was evaluated by mtt assay on human liver hepatocellular carcinoma (hepg ) and human breast (mcf ) cancer cell lines after , and hours of treatment. in the case with the human tumor cell lines (hepg , mcf ) and c-modified analogues, statistically reliable results were achieved for the most of concentrations used. acknowledgements: this work was supported by bulgarian ministry of education and science, project my-fs- / . microwave assisted solid phase synthesis of urea and urea/amide based foldamers k. pulka, c. douat-casassus, g. guichard* european institute of chemistry and biology, university of bordeaux -cnrs umr , pessac, france foldamers are fully arti cial molecules that structurally and functionally mimic variety of biopolymers. among them, aliphatic n,n'-linked oligoureas with proteinaceous side chains can adopt extremely robust helical folds stabilized by intramolecular three-centred h-bonds. owing to their resistance to enzymatic degradation, diversity of side chains and structural predictability urea-based foldamers represent unique scaffolds to elaborate functional mimetics of α-polypeptides. of note, heterogenous oligo(urea/γamides) backbones obtained by substituting nh groups by ch display very similar folding propensities. in our laboratory we are investigating the solid phase synthesis of urea and urea/γ-amide oligomers. urea bonds are incorporated into the growing chain by reaction of active succinimidyl carbamates. previously we have applied two different strategies involving fmoc-or bocchemistry, but both methodologies suffer some limitations. therefore a new strategy (compatible with the use of tfa sensitive linkers and side chain protecting groups) featuring azide as a masked amine group has been developed. the synthesis of new azido protected succinimidyl carbamate building blocks is reported. they were obtained in steps from α-amino acids ( - % overall yield). the staudinger reduction with pme was successfully applied to restore the amine group after urea formation on solid support. in addition, microwave irradiation has been found to dramatically accelerate the synthesis. overall, this azide-strategy combined with microwave irradiation was found to be very effective for the solid phase synthesis of oligoureas and related hybrids, surpassing previously developed approach utilizing fmoc chemistry. these antibiotics should have a mechanism different from currently used antibiotics to circumvent existing resistance mechanisms . previous results have shown that "genetic" antibiotics operating by gene silencing in bacteria via rna interference may be successful new candidates. efficient silencing requires efficient crossing of cell membrane. this step can be alleviated using cell penetrating peptides (cpp) as carrier of drug candidates, such as peptide nucleic acids (pnas) which inherently have poor internalization properties . the aim of this study is to elucidate mechanisms of uptake in bacteria using pna-cpp conjugates, which previously have shown promising antibacterial effects . the fate of the pna and cpp parts of the conjugates, once inside the cell, is investigated regarding localization and possible degradation within the cell. furthermore, a method for toxicity testing of pna-cpps is being developed using histamine release in rbl- h cells as a quantitative measure of allergenicity of pna-cpps. the prospect of this information is to define boundaries within which cpps can be found, thereby rationally designing novel efficient antibacterial biomolecular drug delivery systems. oxytocin and its fragments have the potential to influence behavioral and cognitive functions, including their disturbances in some brain disorders. therefore, there is an interest to synthesize new peptide-steroids chimeras for potential therapeutic use. oxytocin analogue was synthesized in solution by coupling azido-phenylalanyl residue or p-azidopegylated handle to the n-terminal end of oxytocin molecule. its c-terminal fragment pro-leu-gly-nh (mif- ) was elongated at proline residue by the same type of azido handles as well. both peptides were marked for fluorescent detection of their possible binding on brain slices. peptide chimeras with the suitable steroids were prepared via azide click to the triple bond on the modified steroid counterpart like ( α)- -hydroxypregn- -en- -yn- -one, -norchol- -en- -yn- β-ol. steroidyl-peptides were then used in the trials using rat-brain slices. the sites of the peptide-steroids chimeras bound to the brain tissue were identified with the aid of fluorescent microscopy. the suitable chimeras will be tested for their penetration through blood brain barrier for the pharmacological effects. indicating that the orientation of the n-butyl group is of primary importance. docking studies revealed that the highly active analog affords an additional hydrophobic binding feature compared to losartan which fits to an extra hydrophobic cavity. these results may contribute to the discovery of new biologically active molecules by a convenient and cost effective synthetic strategy. the context of pain research, the co-administration of opioid agonists and nk antagonists previously led to an enhanced antinociceptive potency, and recently largent-milnes and co-workers have shown that a hybrid opioid-nk octapeptide was able to attenuate tolerance development, related to sustained opioid treatment. our group has prepared a compact opioid agonist-nk antagonist peptidomimetic chimera dmt-d-arg-aba-gly-nme- ', '-bn(cf ) that served as a lead structure. we report a solid phase method for the synthesis of the amino- , , , -tetrahydro- -benzazepin- -one (aba) structure, which is used as a central unit in the investigated dual ligands. this method allowed the rapid assembly of new bifunctional ligands containing the aba structure. variations of the d-arg , gly and n-benzyl substituents were made. the introduction of d-cit , a gly → β-ala substitution and the removal of the trifluoromethyl substituents in caused considerable shifts in receptor binding. the obtained structure-activity relationships will be presented. hence, a promising approach for the treatment of dmd is the use of drugs to force ptc readthrough. (+)-negamycin is a dipeptidic antibiotic containing a hydrazide structure. although (+)- was not clinically developed due to some toxicity, it was recently reported that (+)- restore dystrophin expression in the muscles of mdx mice, an animal model of dmd. therefore, (+)- is a promising therapeutic candidate for diseases caused by nonsense mutations. based on our own efficient total synthetic method of (+)- , structure-activity relationship (sar) study was perfromed to discover derivatives with a potent readthrough-promoting activity. we found a derivative, ( r)- -hydroxy- -aminohexanoyl-glycine exhibited not antimicrobial activity but a similar readthrough activity to (+)- , suggesting that the ptc readthrough mechanism can be distinguished from the antimicrobial mechanism. moreover, we synthesized -epi-negamycin and found that this analog exhibited a similar activity to (+)- in in vitro readthrough assay. this result hence prompted us to synthesize a -dehydro-derivative, e.g., -dehydro- -epinegamycin , which is a natural product with little antimicrobial activity. surprisingly, we found that showed a higher in vitro readthrough-promoting activity than (+)- . this result suggests that mother nature independently evolved readthrough-promoting products like suppressor trna, in distinction from aminoglycosides, which show both antimicrobial and readthrough-promoting activities. agricultural university of athens, athens, greece high interest has been paid to synthetic structural motifs that promote specific conformations because of their importance for the development of new therapeutic peptidomimetics. in addition, such motifs may show catalytic activity for asymmetric organic transformations. during the last two decades, various synthetic structural motifs that promote reverse turns have been studied. following our interest on chiral prolinamide-thioureas that present interesting organocatalytic activity, we have undertaken a combined experimental/computational study to understand the structural features that may stabilize a reverse turn in short-length peptidomimetics containing a thiourea functionality. compounds with the sequence r-pro-diphenylethylenediamine-thiourea-asp(obut)-obut (r: boc or fmoc, or boc-ala), were synthesized and studied by nmr spectroscopy (tocsy, h- c hsqc, noesy, roesy spectra) for the sequential assignment and the exploration of the dipolar connectivities. sampling of the conformational space was driven by the noe intensities while molecular dynamics simulations were further applied to the consistent with the experimental data conformers in order to monitor the stability of the formed hydrogen bonding interactions in the course of time. energy refined produced conformers were subsequently modified by applying all combinations of d-and l-amino acids at each site in a stepwise manner. the modelled structures were studied in silico aiming to explore the combinations of heterochiral residues which would promote a folded structure and would favour the potential of β and γ turn motif. the most promising combinations were chosen for synthesis and subsequent nmr characterization. in this research project we will deal with chemical strategies to produce suitable surface modifications in order to induce multidirectional cellular migration along gold surfaces. to achieve this objective we want to use and characterize self-assembled monolayers (sams) of thiolated dna chains (dna-sh) adsorbed on gold surfaces through the hybridization with complementary modified single-stranded pnas. pna is a structural dna mimic obtained by polymerization of n-( aminoethyl) glycine monomers that replace the ribose-phosphate backbone characteristic of natural nucleic acids. it is an achiral, uncharged, and relatively rigid biopolymer of high biological and chemical stability, and it can bind complementary dna strands with higher affinity than the corresponding dna sequences.for all these reasons we have chosen pna as a key molecule to promote and assist the movement of cells. by producing a chemical gradient of dna-sh along a gold surface in the presence of a chemotactic molecule it will be possible to obtain and control a directed cellular migration. the norwegian structural biology centre and the centre for theoretical and computational chemistry, department of chemistry, university of tromso, troms , norway renin is a highly selective aspartic protease which catalyzes the hydrolysis angiotensinogen, a protein secreted from the liver, to the decapeptide angiotensin-i. angiotensin-i is further processed by the relatively nonspecific angiotensin converting enzyme (ace) to give the octapeptide angiotensin-ii, a potent vasoconstrictor and the dominant peptide produced by the reninangiotensin system. renin catalyses the rate determining step in the formation of angiotensin-ii, and has for several decades been an established therapeutic target for drug development in relation to hypertension. in the search for renin inhibitors, substituted piperidine derivatives have been identified as promising, - and piperidines have proven to be efficient scaffolds for the development of novel non-peptide aspartic protease inhibitors, particularly towards renin. [ ] [ ] [ ] we herein describe a series of -triazolyl substituted piperidine derivatives that have been synthesized from n-boc protected trans- -ethynyl- -hydroxy piperidine and tested as novel renin inhibitors. piperidine derivatives containing a -substituted , , -triazol- -yl substituent were found to be most active and molecular docking experiments provides a rank order that is in very good agreement with experimental data. the cxcr /sdf- axis is involved in many biological processes such as hematopoiesis, immune cell migration, as well as in cancer metastasis. cxcr also mediates the infection of t-cells with x -tropic hiv functioning as a coreceptor for the viral envelope protein gp . cxcr , as a pharmaceutical target, is of utmost importance but the lack of synthetic agonists has seriously slowed down drug development. it has been recently described by our research group , that grafting the sdf- n-terminus onto a side-chain of the inverse agonist t . generated high affinity synthetic agonists as well as partial agonists for the chemokine receptor cxcr . to remain stable towards proteases and act as useful pharmaceutical tools, the pk-adme properties need to be improved with a gradual transition to peptidomimetic structures. medicinal chemistry witnessed major advances with the discovery of small synthetic molecules that mimic the natural peptidic substrates. these small molecules do not undergo proteolytic degradation, an advantage they hold over natural counterparts. in order to improve stability against proteases, part of the sdf- chain was replaced with variable lengths of polyethylene glycol and unnatural amino acids at differents positions. here, we have produced a series of compounds, most of which showing nanomolar affinities for cxcr and some are displaying partial agonistic properties. tlrs are the innate immunity receptors that recognize the epitopes found on surfaces of various cells and therefore they initiate and sustain the atherogenic inflammatory response [ , ] . we assume that the use of small stat mrna−binding pna−inhibitors to manipulate the activity and expression of stat could prove an attractive therapeutic strategy in treatment of atherosclerosis. to that end we synthesized a specific stat mrna−binding pna inhibitor as well as a non-specific pna to compare their inhibition of gene expression. in our work we developed effective method of synthesis of pna−peptides conjugates by means of "click chemistry". determination of optimal conditions for conjugation (connection of pna with the peptide) will allow for the design of compounds useful in gene therapy. the specificity of pna hybridization to complementary dna fragment was verified by capillary electrophoresis (ce). as an artificially synthesized somatostatin analogue, tyr octreotate (toca) can specifically bind to somatostatin receptor (sstr), which are usually over-expressed on many tumor cells. carbohydration of n-terminus of toca has resulted in improved pharmacokinetics and tumor targeting ( ) . f is an ideal nuclide for positron emission tomography (pet) imaging; there may be significant uses of f labeled glucitol-toca and its analogues as tumor probes for the diagnosis of sstr-positive tumors. in order to explore a novel pet probe for diagnosis of sstrpositive tumors, we designed a synthetic route to synthesize n-gluc-lys(nota)-toca, which uses , , -triazacyclononane- , , -triacetic acid (nota) as the chelating reagent. n-gluc-lys([al f]nota)-toca is radiosynthesized quickly and efficiently using the chelation reaction of al f complex and n-gluc-lys(nota)-toca. the aim of this study is to develop an efficient method for the synthesis of monomers of triazolic nucleic acid (tna), a new class of artificial nucleic acids. but- -yne- , -diol and nucleobases derivatives will be substrates of the monomers synthesis. tna oligomers could be used as specific inhibitor of tar rna hiv- , the regulatory rna structure crucial for hiv replication. "click chemistry" based on , -dipolar cycloaddition will be used to conjugate an alkyne and azide derivatives of monomers subunits. a ru (ii) complex will be used as a catalyst of internal alkyne (but- -yn-based) cycloaddition. the reaction gives exclusively of , , -trisubstituted derivative of triazole ring . the monomers will be characterized using rp hplc, capillary electrophoresis (ce) and h and c nmr. the resulting monomers containing fmoc-protected amino group and a free carboxyl group will be used for the classical spps method to synthesize tna oligomers. tna sequences will be designed against tar's bulge and an external loop. through the recognition that the repertoire of polypeptide conformations can be greatly expanded by the creation of structures incorporating β-amino acids. moreover, the numerous advantages of hybrid (mixed α-and β-) backbone peptidomimetics with respect to homogeneous ones were quite recently outlined. we describe here various β-amino acid-based β-hphe-β-hphe dipeptide derivatives, also conformationally constrained, and their application to the synthesis and biological evaluation of hybrid analogues of the opioid endogenous peptide endomorphin- (em- ). the opioid system mediates a wide variety of pharmacological and physiological processes, including pain perception and modulation. the amidated tetrapeptide em- has been shown to be μ-opioid receptor (mor) agonist exhibiting a very high μ-receptor affinity and selectivity, and it is an important model in the search towards new analgesics. structural investigation of em- reveals the high conformational freedom of the phe side chains and also the inherent flexibility of the peptide backbone, indicating many probable bioactive conformations, ranging from βturns to extended conformations. with the aim of better clarify the relevant role of the proper spatial orientation of the aromatic rings and in particular of the benzyl side chains at position and , h nmr studies, molecular modelling, and molecular docking to a homology mor model of our hybrid analogues are currently under way. the lantibiotics represent a class of antimicrobial peptides, in which the unusual amino acids dehydroalanine and dehydrobutyrine and the intramolecular thioether bridges (lanthionines) are important structural features for bioactivity.the lipid ii -nisin complex is responsible for pore-formation since the c-terminal part of nisin is inserted into the bacterial cell membrane which ultimately results in cell leakage and collapse of vital ion gradients. in order to increase the metabolic stability of nisin, the oxidationsensitive thioether bridges can be replaced by metabolically stable dicarba moieties, as successfully demonstrated by the synthesis of nisin ab(c) analogs containing alkane/alkene bridges [ ] . to obtain more insight into the importance of the cross-bridged de-ring structure (i→i+ , i+ →i+ connectivity) on nisin's bioactivity, we synthesized a series of all four diastereomers of the crossed alkene-bridged de-ring mimic, using ring-closing metathesis. all four diastereoisomers were obtained by hplc and structurally characterized by nmr spectroscopy. an orthogonal protection scheme was used, to enable the independent n-or c-terminal modification of the bicyclic hexapeptides with azide/alkyne functionalities. via cu(i)-catalyzed cycloaddition chemistries, alkyne-functionalized natural abc-fragments of nisin, which were obtained by tryptic digestion of full length nisin followed by hplc purification, have been conjugated to synthetic de-ring mimics to obtain novel nisin derivatives and their affinity toward lipid ii and pore-forming capacity have been studied. herein, we report on the details of the synthesis and characterization of the geometric isomers of the synthetic de-ring mimics, and their use as synthons in cu(i)-catalyzed click chemistry to obtain newly designed nisin hybrids as potential novel peptide antibiotics. università di ferrara, dipartimento di biochimica e biologia molecolare, ferrara, italy mirnas play an important role in regulation of gene expression, being involved in numerous processes such as cell proliferation, cell differentiation, apoptosis and also in the progress of diseases as cancer and cardiovascular disorders. mirnas associated to diseases recently become targets for the development of new drugs based on antisense oligonucleotides or analogues complementary to the chosen mirna, in order inhibit the binding of the mirna to its mrna target. therapeutic silencing of mirna has been also observed in several animal disease model. in this work we propose a new approach to interfere in the mirna function, based on peptide nucleic acid (pna) oligomers designed to be complementary to selected regions of the mirna precursor (pre-mirna). as the pre-mirna bases belonging to the stem are not perfectly complementary, we hypothesized that the mismatched duplex of the pre-mirna could be opened by pnas inhibiting of its maturation into mirna. two pna sequences, targeting respectively the "sense region" and the " ' end region" of the pre-mir were designed. pnas were conjugated to different carrier peptides, hiv-tat, r , k and two nuclear localization signal (nls and binls), in order to increase their cellular uptake. to verify the ability of the designed pnas to give strand invasion on the pre-mirna, we conjugated also pnas to the thiazole orange, a probe which lights-up upon hybridization the development of privileged molecular scaffolds efficiently mimicking reverse turn motifs has attracted remarkable interest when structural constraints are exploited to increase both binding and selectivity of model peptides. one of the successful approaches to restrict peptide conformation is the disubstitution in the α position of an α-amino acid, leading to a conformational constraint and a stereochemically stable quaternary carbon center. in particular, spirocyclic scaffolds are able to provide, upon the attachment of appropriate functional groups, useful high-affinity ligands, relevant to the field of drug discovery. at present, we are interested to spirocyclic tryptophan (trp) analogues, in order to develop new reverse turn nucleating moieties able to be inserted into pharmacologically relevant peptidomimetic compounds. among peptides sharing a tryptophan-containing β-turn motif of which the trp residue is critical for binding, we looked at the hormone peptide somatostatin, acting in various organ systems as a neuromodulator and a neurotransmitter, as well as a potent inhibitor of various secretory processes and cell proliferation. somatostatin and its analogue octreotide (sandostatin® drug, clinically used for the treatment of endocrine tumors and acromegaly) are thought to interact with the sst - receptors mainly by inserting a β-turn substructure, carrying a lysine (lys) and a trp side chain into a pocket of the g protein-coupled somatostatin receptor. we report here the preparation and structural characterization of a new , , , -tetrahydro-β-carboline (thbc)-based spirocyclic lactam as type-ii β-turn model compound and the application of its core structure to the synthesis of a somatostatin mimetic, whose biological evaluation is under way. the analogues of sfti- modified in the p position by, βand γ-amino acids and n-substituted β-alanines r. lukajtis, a m. filipowicz, a a. legowska, a d. debowski, a a. lesner, a k. rolka a a faculty of chemistry, university of gdansk, - gdansk, poland serine proteinases play very important roles in many physiological processes in humans, such as: food digestion, fertilization of the ovum, blood clotting and dissolution of blood clots, immune response. however, their uncontrolled activity can evoke serious pathological conditions. therefore, serine proteinase inhibitors are considered to be a promising class of therapeutic agents. trypsin inhibitor sfti- , on which we focused our attention in the last decade, is an attractive template for the design of such compounds. its primary structure is shown below: & gly-arg-cys(& )-thr-lys -ser -ile-pro-pro-ile-cys(& )-phe-pro-asp& the inherent feature of natural peptides and proteins is their low stability towards proteases, which seriously reduces their bioavailability. there is a growing need for the development of artificial biopolymers with diverse side chains, capable of mimicing peptide function. β-and γpeptides are an interesting class of peptidomimetics with significant chemical and biological properties. the present communication describes the chemical synthesis and inhibitory activity of a series of trypsin inhibitor sfti- monocyclic analogues (with disulfide bridge only) modified in p position by βand γamino acids and n-substituted β-alanine (β-peptoid units). the following mimetics of proteinogenic lys or phe were used: β hlys, β hphe, γ hhlys, γ hhphe, βhnlys, βhnphe. all compounds were synthesized manually on solid support. β-peptoid monomers were introduced into the peptide structure by two steps method [ ] . newly obtained sfti- analogues modified in p position by β-derivatives of lys and phe were able to inhibit bovine β-trypsin and bovine αchymotrypsin, respectively, whereas the remaining ones (except for [βhnphe ]sfti- ) appeared to be inactive. the notion that early soluble aß intermediates are endowed with cytotoxic effects suggests that a major effort should be directed toward the inhibition of amyloid aggregation at very early stages. inhibiting aß self-oligomerization could, therefore, provide a useful approach to treating and controlling the pathogenic pathways underlying alzheimer's disease (ad). likely, agents that target the basic molecular recognition process preceding the formation of early intermediates are the most valuable candidates. we have conjugated a trehalose moiety to the known ß-sheet breakers pentapeptides lpffd. trehalose has received a special interest because it has been found to be effective in the treatment of neurodegenerative diseases associated with peptide or protein aggregation. the glycosidic moiety was covalently linked to different regions of the peptides' primary sequence, including the n-terminus or c-terminus or the aminoacid side chain. this new class of peptides showed an increased resistance to proteases. in this work, the inherent ability of these peptides to recognize and bind the monomeric form of recently reported a d-amino acid-containing hiv protease inhibitor with a sulfonyl group showed an activity enhancement against drug resistant viruses. x-ray crystallographic study of the derivative revealed existence of four bridging water molecules. we suggest that the additional indirect interactions through water molecules induced the inhibitor's flexibility in binding conformation, keeping the affinity with the mutated proteases. oxalyamide, so-called oxamide, has two carbonyl oxygen atoms as hydrogen bonding acceptor similar to sulfonyl group, which is promising to interact with water molecules. to increase the numbers of bridging water molecules, we built-in two oxamide structures to both terminals of pseudo-symmetric compounds with hydroxymethylcarbonyl-hydrazide isostere. the derivatives were tested for inhibitory activity using wildtype hiv protease and a highly mutated protease with lopinavir resistance. we found that the loss of potency against the mutated protease was relatively small in the oxamide derivatives. the molecular dynamic simulations suggested the ability of bridging water formation of the two oxamide groups. optimization of the pna-synthesis using different bases for fmoc-deprotection s. rawer , k. braun , r. pipkorn life technologies, darmstadt, germany dkfz, heidelberg,germany pna (peptide nucleic acids) are considered as highly sensitive and specific tools for antisense strategies especially conjugated with cell penetrating peptides. individual designed shuttle systems can be applied in cancer diagnostics and possible therapy ( ) . it is, however, undisputed that proper pnas' syntheses prove to be a challenge for coupling and fmoc-deprotection. due to the structure-formation the success of the synthesis depends strongly from parameters, like activator's quality and deproctection kinetics correlating to the length of the pna polymer spps product. using the example of the spps pna synthesis' results of the coding sequence of c-myc human exon ii, different bases, acting as fmoc-deprotection reagents, are compared and analyzed aiming at optimizing the pna synthesis strategy ( ) peptidoglycans are central structural components of the cell wall of bacteria. several plant receptors are known to recognize peptidoglycan fragments. it is believed that these receptors form part of the defense mechanism against bacterial infections in several plant species. peptidoglycans consist of long chains of alternating β( - )linked glcnac and murnac moieties that are crosslinked by short, non-ribosomal peptides. these peptides consist of several d-amino acids and the symmetrical (r,s)diaminopimelic acid (meso-dap). in particular, the latter complicates the synthesis of peptidoglycan fragments due to the requirement for individually addressing the two pairs of functional groups. some chemical syntheses of peptidoglycan fragments have been reported [ ] [ ] [ ] [ ] , hhich involved multi-step formation of an orthogonally protected dap moiety, and elaborate oligosaccharide synthesis. here we present a new and simple approach to peptidoglycan synthesis which is based on the use of commercially available building blocks for the dap and oligosaccharide components. this allows easy access to a range of peptidoglycan fragments for structure-activity studies. the introduction of solid-phase peptide synthesis (spps) and the subsequent refinement of resins, linkers, coupling reagents and amino acid protecting groups allowed access to a wide range of peptides. therapeutic peptides, in particular, have benefitted from the maturation of spps, as complex peptides can be synthesized more efficiently in comparison to conventional solution phase synthesis. however, peptides containing multiple disulfide bonds often still remain difficult to make due to a lack of orthogonal cysteine protecting groups that can be used in routine spps. the cysteine protecting group s-tertbutyl mercapto (s-tbu) is commercial and orthogonal to other cysteine protecting groups. removal of the protecting group is facilitated by reducing agents (e.g. thiols or phosphines) and is stable to tfa and piperidine, hence compatible with fmoc/o-tbu peptide synthesis. however, the protecting group cannot be used in routine spps due to long deprotection times ( - h) . in certain cases it has been shown to be impossible to remove due to proximity of bulky protecting groups and sensitivity to certain sequences. additionally, reports of desulfurization of s-tbu protected cysteine to dehydroalanine, by the use of prolonged exposure to reducing agents, show the limitations of this protecting group. the concept of cysteine protecting groups labile to reducing agents is promising due to orthogonality to other cysteine protecting groups and the limitations of s-tbu initiated an investigation into novel reductive cysteine protecting groups. herein, we introduce s-tmp as a novel cysteine protecting group that is very labile to reducing agents. the increased lability, in comparison to s-tbu, allows utilization of reducing agent labile protecting groups in routine peptide synthesis of disulfide containing peptides. as modern automated spps protocols allow the assembly of larger and increasingly complex peptides, a precise control of the coupling reactions is a crucial prerequisite in peptide synthesis. monitoring the progress of synthesis allows the detection of undesirable products caused by side reactions, incomplete couplings or deprotections. although different methods have been developed for monitoring spps, we observed that the use of colorimetric test or continuous-flow uv absorbance of the reaction column effluent was not informative enough to identify difficult steps in the synthesis. in this study we demonstrate the usefulness of the combination of a mw-assisted mini-cleavage protocol and the uplc-esi-ms analysis for monitoring the quality of the reaction steps. as a proof of concept, based on this strategy, we monitored the synthesis of pthrp( - )nh (synthesised by fmoc/tbu rt-spps, liberty™, cem), characterised by a cluster of arginine residues in the - region. by the use of mw irradiation during the mini-cleavage protocol, we optimized time for mini-cleavages particularly in case of multi-arginine containing peptides, protected by pbf group. the results obtained by uplc-esi-ms showed that the complete removal of the pbf groups from the arginine sidechain residues required h at rt. on the other hand, the mw-assisted mini-cleavage monitoring let us to obtain final results just in min, confirming that the use of microwave irradiation in mini-cleavages is an efficient strategy to monitor also difficult peptide couplings, such as multiarginine peptides. identification of some deletion sequences was helpful to recognise critical couplings in order to adopt more efficient coupling strategies and therefore to optimise the final yield and purity of the crude peptide. development of green sustainable chemistry is currently regarded as a challenge in science and technology to reduce the use of organic solvents and utilize less toxic solvents instead. water and aqueous-based solvent systems represent an increasingly significant choice for replacing traditional solvents in synthetic chemistry. until recently, peptide synthesis in aqueous solution has remained largely unexplored. this is because the most common building blocks are sparingly soluble in water and are considered inappropriate for in-water peptide synthesis. we have developed a method for solid-phase peptide synthesis in water, which utilizes water-insoluble fmoc-amino acids that are converted to water-dispersible nanoparticles. in this way, the solubility problem is overcome. our technology, which uses suspended nanoparticle reactants for the coupling reaction, offers many advantages in terms of reaction efficiency over inwater synthesis using water-soluble or non-disperse reactants. however, there are two main problems with this nanoparticle approach; (i) slow reaction rates compared to general peptide synthesis in ordinary organic solvents (ii) poor yields for the synthesis of long peptides because the protected peptide chains on the resin have a tendency to aggregate in water. mw assisted spps is particularly attractive because of the widespread availability of the new technology, including automated peptide synthesizers. a trial of mw assisted inwater solid-phase synthesis using non-disperse boc-amino acids has been reported by albericio previously. currently, fmoc-amino acids are routinely used as building blocks for solid-phase peptide synthesis. with this in mind, we have developed a mw irradiation procedure aimed at reducing reaction time and increasing reaction yield for in-water solidphase synthesis using water-dispersible fmoc-amino acid nanoparticles. and we demonstrated in-water solid-phase synthesis of difficult sequence peptide with mw irradiation. m. lebl, z. flegelova spyder institute praha, czech republic cotton was shown as a convenient solid phase support earlier - , but did not find wide acceptance by the peptide community. we decided to try its application as (i) a support of choice for the synthesis driven by combination of capillary forces and gravity, (ii) support for synthesis utilizing in situ neutralization boc based protocol, (iii) support for combinatorial synthesis based on easy labeling and physical separability of cotton substrate, and (iv) support for multisupport synthesis. -we have built a simple synthesizer in which the cotton carrier (functionalized thread) is placed inside the capillary tubing and the appropriate reagents are introduced by connecting the inlet with appropriate reagents. the speed of "pumping" the reagents is driven by the difference between the elevation of the inlet and outlet of the capillary tubing. -we have shown that boc solid phase synthesis utilizing in situ neutralization is compatible with cotton substrate and provides high quality products. combining with the fact that cotton by itself acts as the self-association breaking agent, makes cotton a suitable carrier for synthesis of "difficult" sequences. -labeling of individual solid support particles can be easily based on the length of the cotton thread pieces, number and positions of knots, or their attachment to a secondary carrier. in addition, it is possible to synthesize peptides differing by the partial structure (alternative linkers, terminal modifications, etc.) in a mixture of classical resin with labeled cotton carriers, which are easily separable at the end of the synthesis. . use of microwave irradiation provides peptides in a fraction of time compared to conventional methods, and the peptides are also often generated in higher yield and purity. while microwave technology is particularly suited for the synthesis of "difficult" to synthesize peptides, this tool can routinely be used for the synthesis of a wide variety of peptides without the need for extensive method optimization. the focus of this study is to demonstrate how a peptide can be synthesized on a small scale (for example μmol) up through larger scales (> mmol) with ease. as a biologically relevant model peptide the last residues of the human platelet factor protein (hpf - ) was selected due to its significant antimicrobial activity. however, the problem of developing a robust fmoc thioester method is that the deprotection of the fmoc group with base at each cycle is not compatible with an active ester at the c-terminus. many ingenious approaches have been developed to generate the required thioester peptide. , , the most popular has been to use an nacylsulfonamide as a base and acid stable (safety-catch) linker for peptide synthesis. alkylation of the sulfonamide after peptide assembly makes the linker labile to cleavage with nucleophiles. whilst popular, it has been plagued by notoriously low yields which originate from the incomplete acylation of the resin-bound sulfonamide with the c-terminal residue, incomplete alkylation of the sulfonamide and the incomplete thiolysis of the resin-bound protected peptide. in this poster we describe the development of a novel dual linker strategy , involving anchoring of the sulfonamide linker to a standard acid-labile resin. this variation overcomes many of the current limitations of the sulfamylbutyryl linker approach and provides a simpler and scalable method for peptide ligation via fmoc spps. m. ziovas, d. tataraki, p. manousou, n. parveri, f. satoglou, d. gatos and k. barlos department of chemistry, university of patras, patras, greece solid phase peptide synthesis is traditionally performed by the attachment of the c-terminal amino acid through its α-carboxyl function on a suitable solid support and elongating peptide chain towards the amino terminal of the peptide by adding sequentially the amino acid residues in the gradually growing peptide chain. several thousands of publications and patents describe this methodology and its application for the production of peptide pharmaceuticals. in contrary to the attachment of the c-terminal carboxyl function, attachment of amino acids and peptides through an amino acid side-chain on suitable resins and their application in spps is described in a small number of publications and patents. most of these publications describe the attachment of the amino acids through a side-chain carboxyl group of asp and glu. in the present work peptides were synthesized very efficiently in high yield and purity by anchoring of side-chain hydroxyl, amino or thiol groups of amino acids, amino acid amides, amino alcohols or small peptides on resins of the trityl or benzhydryl type. several peptides of pharmaceutical interest, such as exenatide, octreotide, pramlintide, calcitonin, bivalirudin, insulin b-chain and others were produced as examples of this technology, either by the step-by-step procedure or by fragment condensation in solution and on solid phase. step given that some of these diseases are caused by a mutation and/or malfunction of an essential protein, a better understanding of the structure and function of such proteins will allow us to prevent, slow down or even cure these diseases. to increase our knowledge, the synthesis of the target protein, a fragment involved in its activity or interacting peptides that modulate the protein activity is often required. in some cases the preparation of a protein analogue that improves its efficacy is envisage. however, conventional solid-phase peptide synthesis methods have some limitations when attempting to achieve these complex sequences of considerable length. using novel technologies, such as a microwave-assisted solid phase synthesis, commonly found in many peptide synthesis labs, here we performed the step-wise solidphase synthesis of a protein holding more than residues (d-vegf). this synthetic achievement indicates the suitability of this approach for synthesis of long proteins or their analogues. the detailed synthesis of the enatiomeric version of vegf and the selenomethionine substituted analogues of huprp ( - ), proteins involved in angiogenesis and prion protein amyloidoses respectively, are described as study cases, where the use of microwaves allow us to obtain them in a fast and efficient manner. therefore, the development of novel peptide analogues with enhanced in vivo stability could potentially provide therapeutic alternatives. the pharmacological evaluation of a bioactive peptide [des-gly ,tyr (ome),d-leu ,aze-nhet ]gnrh, analogue , is presented herein. in vitro (kidney mouse membranes) and in vivo (clinically relevant pharmacokinetic mouse model) bioassays were coupled to liquid chromatographytandem mass spectrometry. analogue , an agonist of the gnrh receptor with a binding affinity in the nanomolar range, caused testosterone release in mice that was acutely dose-dependent, an effect blocked by cetrorelix. repeated dosing studies in mice demonstrated that analogue was well tolerated and had potency similar to that of leuprolide, based on plasma and testis testosterone reduction and histopathological findings. analogue also shared with leuprolide similar significant antiproliferative activity on androgen-dependent prostate cancer (lncap) cells. on the basis of pharmacokinetic advantages, we expect that analogue or analogues based on this new design will be therapeutically advantageous for the treatment of cancer and endocrine disorders. cortexin is a polypeptide drug isolated from cattle and porcine brain cortex. cortexin is effective in monotherapy and in combination with traditional methods of treatment. cortexin produces tissue-specific, regulatory, and reparative effects on the brain cortex and contains active low-molecular-weight neuropeptides (< kda) penetrating through the blood-brain barrier. cortagen is a tetrapeptide h-ala-glu-asp-pro-oh (geropharm) produces nootropic and neuroprotective effects. oleylcortagen is a lipophylic analog of cortagen c h o-ala-glu-asp-pro-oh was created for increased proteolytic stability and increased penetrating through the blood-brain barrier. the main aim of our investigation is the analysis of psychopharmacological profile of peptide preparations in comparison with piracetam. have been shown oleylcortagen ( mg/kg) and piracetam ( mg/kg) possess activating effect on motor and research components of behavior in «open field» test. two of peptides (oleylcortagen, cortexin) decreased period of immobilisation and demonstrated antidepressant effects on rat behavior in the porsolt's test, on other hand cortagen demonstrated depressant action. therefore, the significant psychoactivating properties are typical for oleyl-cortagen, cortexin. the mechanism of the action of these peptides can be explained from the viewpoint of the regulatory cascade. they produce a direct information impact on cell structures of the brain, and then promote release of the regulatory peptides, which in turn, induce the release of the next group of peptides. neurology, queen square, london wc n bg, uk one of the hypotheses of alzheimer's disease neuropathology involves beta-amyloid (βa) binding with proteins on neuronal cell surface which leads to cell lysis and amyloid plaque formation. according to the latest data α -type of the nicotinic acetylcholine receptor (achr) and the prion protein can be the target for betaamyloid toxicity [ , ] . aggregated βa causes many pathological changes in cultures of mixed neurons and astrocytes such as sporadic cytoplasmic intracellular ca + -signal, activation of reactive oxygen species (ros) production and cell death. in the present work we demonstrated the ability of affinity purified antibodies to synthetic fragment - of α -subunit of the achr (achrabs) or to peptide - of the prion protein (prpabs) to protect cells from βa induced cell death. we also showed that both antibodies did not block βa induced ca + -signal in astrocytes. however, preincubation of cortical co-culture of neurons and astrocytes with achrabs or prpabs significantly reduced the rate of caspase activation and the rate of βainduced ros production via modulating nadph-oxidase. more detailed research of involvement of α -type achr revealed that α-bungarotoxin was also very effective in the inhibition of caspase activation and superoxide production. the observed positive effect of antibodies to α -type achr or to the prion protein gives an additional explanation regarding the involvement of these proteins in ad pathology and provides new approach into an anti-ad vaccine design. capturing and macrophage-aided clearance of amyloid beta by surface modified proteineous particles m. richman, s. rahimipour department of chemistry, bar-ilan university, ramat-gan , israel imbalanced homeostasis and oligomerization of amyloidβ (aβ) peptide in the brain are hallmarks of alzheimer's disease (ad). microglia and macrophages play a critical role in ad progression by clearing aβ from the brain or inducing inflammation. recent evidence suggests that the phagocytic pathway of aβ may be defective in ad microglia/macrophages that contributes to the build-up concentration of aβ in the brain. , therefore, efforts have been directed toward developing treatments that trigger these cells to clear aβ through alternative mechanisms. we have recently demonstrated that protein microspheres modified at their surface with multiple copies of an aβrecognition motif can strongly bind aβ, inhibit its aggregation and directly reduce its toxicity by sequestering it from the medium. here, we describe how the aβ-bound microspheres can stimulate microglial cells and be phagocytosed through a mechanism that is distinct from that of aβ. the phagocytosis was mostly effective with microspheres having diameter size of about . - mm and introduction of polyethylene glycol to the surface of the microspheres changed the kinetics of the phagocytosis. moreover, while aggregated aβ induced a significant inflammatory response that was manifested by the release of tnf-α from the microglial cells, the aβ-bound microspheres dramatically reduced the amount of the released cytokine. our data suggest that surface-modified microspheres could be utilized to detoxify other pathogenic or misfolded proteins that their accumulation may lead to genesis of other diseases. vasoactive intestinal peptide (vip) and its derivatives have been thought to be promising drug candidates for airway inflammatory diseases. however, the therapeutic potential of vips is highly limited because of rapid metabolic degradation and systemic side effects following systemic administration. previously, to overcome these drawbacks, our group developed a novel vip derivative, [arg , , , leu ]-vip-grr (ik ), with improved metabolic stability ( ), and respirable powder (rp) formulation of ik (ik -rp) for pulmonary administration ( ) . these attempts successfully led to enhanced pharmacological effects of ik in the airway system and reduced systemic exposure; however, further chemical modification of ik with a focus on metabolic stability might provide better clinical outcome. the present study aimed to design a pegylated vip derivative with improved metabolic stability and to develop its respirable powder (rp) formulation for inhalation therapy. ik was chemically conjugated with peg ( kda, p k), the physicochemical and biochemical properties of which were characterized by cd spectral analysis, binding assay, and metabolic stability. the rp formulation of pegylated ik (ik /p k) was prepared with a jet mill, and in vitro inhalation performance and in vivo pharmacological effects in antigen-sensitized rats were also evaluated. the cd spectral analysis demonstrated that peg conjugation had no impact on the solution structure of ik . although receptor-binding activity of the ik /p k (ic : nm) was estimated at ca. -fold less than that of ik (ic : . nm), metabolic stability for the ik /p k was highly improved. according to the laser diffraction and cascade impactor analyses, ik /p k-rp had fine in vitro inhalation performance. insufflation of ik /p k-rp ( μg of ik /p k) in antigen-sensitized rats resulted in marked attenuation of inflammatory events, as evidenced by significant decrease of inflammatory biomarkers and granulocyte recruitment in pulmonary tissue at h after antigen challenge. from these findings, pegylation of vip derivative, as well as its strategic application to the rp formulation, might be a viable approach to improve its therapeutic potential for treatment of airway inflammatory diseases. the previous studies have shown that trkb (tropomyocin receptor kinase) acts as an oncogenic agent and its binding to bdnf (brain derived neurotrophic factor) activates signaling angiogenesis of tumor proliferation [ ] . for finding the most stable and potentially effective peptides against the trkb, we applied the following protocol. at the first step of this protocol we designed a peptide library by using sequence tolerance method in rosetta . package, then peptide energy optimization performed by backrub protocol for finding the most stable peptides. the five best peptides in energy optimization selected based on backrup scores by using r package [ ] . d-structure prediction of the selected peptides was performed by using molecular dynamic in hyperchem software. docking of peptides with trkb receptor was carried out in haddock software. we used cyclotraxin, a selective trkb inhibitor as positive control for this protocol. cyclotraxin and the peptides were compared by anova or t-test. the peptides are going to be tested against the trkb in an in vitro model. dirucotide (mbp - ) is a synthetic peptide analog of , that consists of amino acids and tested in a phase trial were failed to reach his tolerance level on previous phase ii in rpms patients. one of the major disadvantages of peptide therapy is the activation of proteolytic enzymes, leading to peptide degradation. to address this problem cyclic peptide analogues have been synthesized. thus we synthesise a linear and cyclic analogues of dirucotide. the two analogues were synthesized by changing the amino acid residue at position from to the synthesis of the linear peptide, as well as of the cyclic one, was carried out by the fmoc/tbu methodology, utilizing the -chlorotrityl chloride resin (cltr-cl). the purification was achieved using semi-preparative rp-hplc and the identification was assessed by analytical rp-hplc and by mass spectrometry (esi-ms). the linear and cyclic peptide analogues will be used in human t-cell cultures to test their immunogenicity in patients versus healthy controls. in the first approach a reporter moiety was introduced to diagnose and treat cxcr related diseases. therefore, an anchor point to attach additional molecules to the ligand was elucidated. using sar studies to optimize the linker from the ligand to the detectable moiety the excellent receptor affinity could be retained. in a final step the reporter moiety was introduced to give a ligand for diagnosis via pet imaging and for possible endoradiotherapeutic applications. originating from the dimeric motif present in many active cxcr ligands several dimers were prepared using a monomeric ligand identified in the prior study. comparison of monomers and dimers yielded a possible subsite binding mode explaining why the dimers exhibit enhanced affinity using a model derived from the origins of the monomer. several peptidomimetic modifications were introduced to the ligand to reduce the peptidic structure. in a conformationally guided approach introduction of a peptoid motif could enforce a single active conformer that was enhanced through subsequent modifications. this yielded a compound times better than the original cxcr antagonist which is the most affine cxcr ligand reported so far. our previous studies have demonstrated that pace represents a potential therapeutic target for the treatment of prostate cancer . moreover, we have developed potent and selective pace inhibitor, ( -fold specificity over furin), known as multi-leu or ml peptide, which has a significant inhibitory effect on the proliferation of prostate cancer cell lines. peptide-based drug candidates can be limited by their poor metabolic stability and low bioavailability. thus, we performed structure-activity relationship studies to improve the pharmacokinetic properties of our ml inhibitor. we have designed and synthesized new ml peptide analogs having various chemical modifications. first, based on our previous results, we combined the most effective modifications of position p (d-amino acids) and p the arginine mimetic amidinobenzylamine (amba) to improve overall properties of our leading compound. second, the n-terminus of the resulting analogs was modified with a fatty acid, in order to enhance their cell permeability properties. third, we modified the inhibitors with a peg moiety to increase their stability and bioavailability. we tested the inhibitory activity, stability in plasma, cellular uptake, and cytotoxicity of each inhibitor. the results of this study demonstrate that the presence of the n-terminal extension (c or peg ) does not affect activity of our inhibitors. on the other hand, we show that introduction of the peg moiety does not increase cytotoxicity of ml analogs. it is interesting to note that the pegylated analog ac-peg -d-leu-lllrvkr-amba has better cell-permeability activity than its counterpart without peg unit. this combination of pharmacological properties makes our new ml analogs promising candidates for the development of potential anti-prostate cancer agents. [ ] peripheral coadministration of rf with opioid analgesics led to confirm the involvement of npff receptors as a part of the antiopioid system. indeed, rf was able to reverse the opioid induced hyperalgesia in rat (randall selitto test). then, a complete structure-activity relationships analysis was performed with rf , assessing the involvement of each moiety for affinity and selectivity towards both npff receptors. a first exploration of the n-terminus part of rf (> synthesized derivatives) led to replace the hydrophobic adamantane moiety by a hindered aromatic group, providing a subnanomolar npff ligand with more than two log-units selectivity against npff . then, the removal of the cterminal amide function led to reduce the dipeptide arg-phe-nh into an arginine derivative. in spite of an initial loss of affinity, optimization of the phenethyl moiety at the cterminus part of arginine led to non-selective nanomolar ligands for both npff & receptors. next, we applied efficient methodologies in order to synthesize non-natural analogs of arginine, leading to various compounds exhibiting selectivity for either npff or npff receptors. in particular, compound rf was identified as a selective npff antagonist (npff , ki = nm; npff : ki > μm). lacking of analgesia properties, oral administration of rf ( mg/kg per os) to rats was able to fully reverse fentanylinduced hyperalgesia. rf is the first orally available npff antagonist capable of reversing opioid induced hyperalgesia at low dose. moreover, this result allows identifying for the first time npff receptor as a key-partner of the anti-opioid system. administration of multiple antiplatelet agents has become the mainstay in the treatment of acute coronary syndromes in everyday clinical practice. we have previously reported significant antiplatelet effects of novel synthetic peptides' single administration on experimental carotid artery thrombosis in rabbits . in the present study we sought to investigate the peptides' effects when administered in marginally effective doses (significantly lower than those utilized in the past), in animals that had previously received low doses of aspirin. the peptides when co-administered with aspirin preserved the carotid artery's blood flow, in contrast to the total artery occlusion observed in animals receiving aspirin and placebo. blood flow at min after electrical stimulation was reduced to . ± . % and . ± . % in the ymesradr and psrcdcr-nh groups respectively (p< . vs aspirin and control). thrombus weight was significantly reduced in animals receiving ymesradr and psrcdcr-nh versus aspirin and control ( . ± . mg and . ± . mg, vs . ± . mg and . ± . mg respectively, p< . ). platelet aggregation was significantly inhibited in the ymesradr and psrcdcr-nh groups by . ± . % and . ± . % for adp (p< . vs aspirin and control), and . ± . % and . ± . % for aa (p< . vs aspirin and control), respectively. blood loss did not significantly differ among the various groups. administration of novel synthetic peptides, even at marginally effective doses, in animals previously treated with low doses of aspirin results in enhanced antiplatelet effects in an experimental model of arterial thrombosis. the study of peptide metabolism is particularly important when examining anticancer peptides; it can provide pivotal clues for the evaluation and improvement of stability of a peptide drug leading to enhanced pharmacokinetics and efficacy. gnrh analogues are used for the treatment of prostate cancer. as with other peptides a drawback is their short half-life due to their metabolic degradation. in order to examine the stability of these analogues we have developed several in vitro peptide stability and metabolism assays using specific tissues, isolated membranes, cancer cells that are analyzed subsequently using lc-ms/ms based approaches. such in vitro studies are followed up with pharmacokinetic studies in mice in order to establish the correlation between in vitro and in vivo approaches. the kidney is the main metabolic organ for peptide metabolism and for that reason we employed a peptide stability and metabolism assay previously described by our group using isolated mouse kidney membranes for the evaluation and comparison of different gnrh analogues. we tested gnrh analogues in other tissues such as mouse plasma, which is the "distributing" tissue for these drugs and mouse brain homogenate, a tissue known for its abundance in peptidases and relevance for centrally mediated effects. stability studies were also performed in cancer cells. in all cases lc-ms/ms based assays were developed for measurement of peptide drug and the resulting metabolites. for triptorelin, and a series of gnrh analogues, degradation and metabolite formation was studied by our mouse kidney membranes assay. studies of coadministering the peptide of interest with inhibitors that are presumed to block the metabolism in mice are ongoing. the vulnerability of gnrh analogues was verified after incubation with plasma and brain homogenate and by metabolite identification we obtained clues about the key cleavage sites. the described in vitro/in vivo protocols provide valuable information that could lead to the synthesis of more stable anticancer peptides with improved anticancer efficacy. in this work, we explored the use of a high-throughput synthesis and screening approach with peptide arrays to identify and structurally optimize shortened hiv- fusion inhibitors. the peptide array technology involves miniaturized synthesis of immobilized peptides, followed by affinity testing with a five-helix bundle, as a mimetic of the fusogenic gp protein. this exercise resulted in the identification of a class of truncated peptides which demonstrates a surprisingly high antiviral potency, despite the absence of the pocket-and the lipid-binding domain. the propensity of these peptides to adopt the bioactive α-helical conformation in solution as determined by circular dichroism, could be the key factor for this unexpected potency. these peptides are promising leads for the treatment and prevention of hiv. the pathological role of platelets in cardiovascular disease (cvd) is well established. platelets secrete adp to recruit additional platelets to a thrombotic site. we have previously identified novel cell-permeable peptide modulators of platelet function by using a bioinformatic screen based on patterns of evolutionary conservation in transmembrane proteins . in this study we further explored peptides derived from cadherin cell adhesion molecules. we explored a range of overlapping peptides derived from different cadherins varying from - amino acids long. peptides are synthesized and analyzed in a high-throughput platelet adp secretion assay. peptides ( . - μm) were assessed in the presence and absence of thrombin receptor activating peptide (trap; μm). we identified cadh- and proteins, but not cadh or in human platelets using western blotting and mass spectrometric analysis. peptides derived from paralogous juxta-membrane, cytoplasmic regions of these cadherins are potent modulators of platelet secretion. by systematically deleting amino acids from c or n-terminus of active peptides, we established that the minimal functional sequence for biological activity was a short six-residue motif, which corresponds to the known catenin-binding region of cadherin tails (kepllp) . peptides alone have no biological activity. however, they potentiate the response induced by the platelet agonist trap at low doses. thus we have identified a cadherin-derived peptide that can modulate platelet secretion events. this highlights a previously unknown role of cadherins in the regulation of platelet function. agents that interfere with cadherin signaling in platelets might have a therapeutic role in cvd. ageing of the brain leads to impairments in cognitive and motor skills, and is the main risk factor for several common neurological disorders such as alzheimer's disease (ad) and parkinson's disease (pd). altered protein handling (proteolysis, repair system, chaperones) forms a basis for a large number of protein conformational disorders. extra-and intracellular, as well as intranuclear accumulation of abnormal proteins, in the form of protein inclusions and aggregates, and dysfunction of the quality control mechanisms are common in all these disorders. alterations in protein homeostasis occur with age, causing molecular changes such as protein misfolding and aggregation. many biologically active proteins lack stable secondary and tertiary structure, these are called intrinsically disordered proteins (idps), some of them (e.g. β-amyloid, α-synuclein) are coupled to neurodegenerative disorders. idps exist as assemblies of rapidly fluctuating structures undergoing coupled folding and aggregation process. protein aggregation is characterized by polymorphism, where a mixture of soluble oligomers, amyloid fibrils and amorphous aggregates is the final product. soluble oligomers are inevitable formed during the self-association process and might initiate the neurodegenerative cascades of ad, pd and similar diseases. the emerging consensus that protein misfolding (leading to idps) is the cause of several neurodegenerative disorders now offers the opportunity to develop a general therapy. soluble oligomers with id regions are potential drug targets. recently short peptide fragments and small peptidomimetic molecules have been found also in our laboratory; these molecules bind to the id regions of β-amyloid and are putative drug candidates. precise control of bleeding is ensured by anticoagulant mechanisms, which under normal conditions prevail over coagulants mechanisms. disrupting the balance between procoagulant and anticoagulant systems due to congenital or acquired defects leading to thrombotic disorders. anticoagulants are substances that inhibit blood clot formation. their action consists in inhibiting the synthesis of prothrombin, the substances forming thrombus as well as some coagulation factors. many peptides and proteins with different molecular weight, such as antistasin (ats), ghilantens, hirudin, etc. showing high anticoagulant activity are isolated from salivary glands of ticks and leeches. they inhibit the action of serine proteinases from blood coagulation cascade. this creates an opportunity for targeted synthesis of low molecular weight analogues of some of these proteins, which can be used in the prevention and treatment of thrombotic disorders. ats is competitive, slow-binding inhibitor of factor xa. it is well known that blood coagulation could be blocked at different stages of the coagulation cascade through inhibition of various enzymes. therefore, it is interesting to determine the place of action of newly synthesized antithrombotic agents in the blood coagulation cascade. this can be done by determining the inhibitory constants of newly synthesized peptides on different enzymes of this cascade. herein we report on our kinetic investigation of newly synthesized peptide amides, analogues of isoform of ats . ki, km, vmax and type of inhibition on the factor xa, thrombin and plasmin were determinate. some interesting differences between type of inhibition of ats, free acids and amide analogues of ats were revealed. to evaluate the relative anti-platelet aggregation activities of each peptide, the lebetins were chemically synthesized and fully characterized. here we described the synthesis, the solution structure of lebetin g -g from the venom of vepera lebetina by h bidimensional nmr and the relation structure-activity. this peptide has been demonstrated to be associated with a potent anti-platelet aggregating activity. the g -g three dimensional structure consists in a compact β-bulged hairpain core from which emerges one loop and the cterminus and the n-terminus. we report on an approach whereby ligands are designed to bind and stabilize the - region of aβ in an α-helical conformation. these ligands reduce aβ toxicity to cells in culture and to hippocampal slice preparations. in addition, when these inhibitors are administered to drosophila melanogaster expressing human aβ ( - ) in the central nervous system, a prolonged lifespan, increased locomotor activity, and reduced neurodegeneration is observed . stabilization of the central aβ α-helix appears to counteract polymerization into toxic assemblies and indicates that this approach holds promise for the development of orally available compounds against alzheimer's disease. encouraged by the above results we are currently developing a second generation of designed ligands. this involves synthesis of different new peptoids and unnatural amino acids. additional support for the concept comes from recent molecular dynamics simulations that also uncover details of the mechanism of unfolding of the aβ central helix as well as retardation of the folding in presence of ligands designed to interact with the native helical conformation . synthesis and methodology for new ligands, which includes synthesis of novel amino acids, will also be presented. triostin a is a well-known natural product with antibiotic, antiviral, and antitumor activities. it inhibits rna synthesis by bifunctional intercalation into dna base pairs through its two quinoxaline units showing cpg selectivity. triostin a must adopt an altered conformation upon dna bisintercalation that is substantially different than its preferred native x-ray or solution conformation. this fact suggests that the destabilizing conformational change in the cyclic octadepsipeptide counteracts much of the gains derived from a second intercalation. nonetheless, the wide range of pharmacological activities exhibited by this compound prompted interests in identifying novel and additionally potent lead compounds with improved pharmacokinetic properties for clinical applications. herein, a library of twelve simplified triostin a analogues has been synthesized by solid-phase peptide synthesis. the introduction of the key quinoxaline units was carried out in solution. the analogues' conformation corresponds to the staple form that bisintercalator cyclic (depsi)peptides adopt when binding to dna and, in addition, some of the synthesized compounds showed improved solubility. our library was evaluated for its antiproliferative activity against four human cancer cell lines and one analogue showed greater cytotoxicity than triostin a and even comparable activity to doxorubicin, a very commonly used drug in cancer chemotherapy nowadays. surprisingly, little is known about its mechanism of degradation in solution or the degradation products. a recent study identified monomeric polysulfides and dimeric degradation products, postulated to derive from β-elimination followed by deamidation and dimerization. we recently reported that degradation of oxytocin and its analogues in aqueous solution at ph . produced monomeric polysulfides with up to sulfur atoms as well as dimeric products. unexpectedly, incubation of ot or of various analogues modified in position resulted in identical dimeric degradants. we concluded that β-elimination via breakage of the c-s bond of cys must be a key step of the process, and that the resulting Δala residue would have to undergo further modifications to yield the same dimeric products independently of the substitution of the n-terminal nitrogen. here we further clarify the degradation mechanism and propose a structure for the dimers. we postulate that hydrolysis of the Δala residue yields an n-pyruvoyl linear peptide, which loses one sulfur atom and subsequently forms dimers, which we found are linked by one disulfide bridge and one non-reducible bond. the putative linear n-pyruvoyl oxytocin intermediate was synthesized and found to degrade to the same dimers as the ones in the incubations of ot. a [u- c]cys ot analogue gave degradation products with c-nmr spectra consistent with a non-stereospecific aldol-type condensation. detailed experimental procedures, structures of the degradants and the postulated mechanism of ot degradation in near neutral solutions will be presented. inserm u paris, france αiibβ is the main platelet integrin and is responsible for platelet aggregation. a lipid-modified peptide corresponding to αiib intracellular sequence - (palmitoyl-k-l eeddeege , pal-k- - ), is platelet permeable and inhibits human platelet aggregation induced by thrombin . ymesradr, a peptide corresponding to the extra-cellular sequence - of αiib, is a platelet activation and aggregation inhibitor . the aim of the present study was to investigate the cooperativeness of the intracellular and extra-cellular peptides on platelet aggregation and their effect on the phosphorylation of fak and erk. pal-k- - together with the extracellular ymesradr peptide, at concentrations lower than their ic values, showed cooperative inhibition of platelet aggregation. the peptide combination inhibited also fibrinogen and pac- binding to activated platelets. fak phosphorylation is a postaggregation event related to outside-in activation of the receptor. the combination of peptides inhibited fak phosphorylation. erk phosphorylation is independent from platelet aggregation, and is enhanced by rgd-peptide inhibitors. the combined peptides inhibited erk phosphorylation. ovarian cancer (oc) is considered a rare disease and represents the fifth most common cause of death from cancer in women. the standard first-line treatment consists of a combination of paclitaxel and carboplatin (ddp) or carboplatin alone. in the case of progressive disease or drug resistance to platinum-based agents, either alone or in combination, investigational compounds should be used ( ) . the mechanisms behind acquired resistance to ddp and its derivatives are not clear yet, although it is evident that the process is multifactorial, including enhanced dna repair processes. some peptides designed from the interface subunit of the human thymidylate synthase (ts) have been identified recently ( ), as effective anticancer agents against sensitive and resistant oc cells. one of them was also able to recover the cellular sensitivity towards cisplatin in resistant oc cells in the μm range. to improve its potency and selectivity structural studies have been performed in combination with cellular assays aimed at understanding the mechanism of action. a label-free quantitative proteomic approach has been undertaken to study the effects of the peptide on the proteins involved in the modulated metabolic pathways, in particular those involved in the folate metabolism. structure-activity relationships (sar) have been performed to improve the lead peptide pharmacodynamics. all the compounds have been assayed and a protein profile set was studied to mark and validate their behavior as inhibitor of oc cell growth. hepatitis c is a liver disease provoked by a virus known as hcv. the disease is insidious. hcv causes anorexia, nausea, vomiting, fever, fatigue and jaundice. in about % of sufferers the disease is short, but others become chronic. in the chronic form in about % of cases the final result is cirrhosis of the liver and in the remaining % it leads to liver cancer. hcv is a very serious problem today. about % of people infected with hcv worldwide, i.e. about million are residents of europe. million people carry the disease as a chronic illness with the potential to develop into cancer in their liver. all these people represent a "reservoir" for storage and distribution of hcv. ribavirin, the nucleoside analog -β-d-ribofuranosyl- , , triazole- -carboxamide, known by the trade name virazole (also known as rebetron in combination with interferon-α), exhibits antiviral activity against a variety of rna viruses (paramyxoviruses, flaviviruses, etc.) as well as some dna viruses. in humans ribavirin is currently used in combination with interferon-α to treat hcv infections. this lack of strict specificity and a broad spectrum of activity are due to its multifunctional mechanism of action against viruses. these characteristics have made ribavirin a drug of substantial research interest. unfortunately, ribavirin shows a significant toxicity, causing bleeding in accumulation [ ] . herein, we report a total synthesis of modified in position ' of ribose residue, ribavirin in order to be further linked to cell penetrating peptides (cpps). in addition the synthesis of some cpps as well as bonding between two parts of final hybrid molecules will be reported. in our case the design of new hybrid molecules is done in order to: (a) vectorize ribavirin into liver cells; (b) transport ribavirin molecule trough cell membrane and (c) decrease toxicity of ribavirin. to obtain oligomeric aβ peptide, our laboratory uses a precursor depsipeptide of aβ. this precursor, termed as "iso-aβ" has an ester bond between the side chain of ser and gly . at physiological ph this ester bond becomes an amide bond via an o→n acyl shift. binding partners by which the oligomeric aβ mediates its toxic effect has not been yet investigated in the proteome or subproteome level. we used protein array technology to study the interaction of oligomeric aβ with recombinant human proteins, immobilized on a protein chip. aβ binding proteins were identified with the aid of a monoclonal aβ antibody. altogether proteins were found to interact with our aβ-oligomers. these proteins were grouped according to their function. one of the major groups contained proteins participating in translation. these proteins were found in ribosomes. to prove our proteomic results ribosomes from rat hippocampus were isolated. elisa experiment revealed that aβ binds to ribosomes in a dose-dependent manner. using the sequence of the aβ-binding proteins a homology search was performed to find oligopeptides, that possibly bind to aβ. based on these sequences a peptide chip containing hexapeptides was prepared. aβ interacting peptides were identified with a monoclonal antibody. several peptides were synthesized and tested on mtt assay. two out of four compounds inhibited the toxicity of aβ on rat hippocampal slices. summarizing our results aβ binding proteins and peptides were identified. knowledge about aβ binding proteins can help to understand the pathogenesis of ad, such us the possible involvement of ribosomes. oligopeptides can be lead compounds of future drug development. huge proteolytic complex named proteasome catalyzes protein degradation in every eukaryotic cell. it consists of subunits forming four stacked rings and one or two regulatory caps. two inner rings of the proteolytic part contain three catalytic β-subunits that possess different substrate specificity. higher vertebrates can express γinterferon-inducible immuno-β-subunits. proteasome plays an essential role in continual turnover of intracellular proteins and in antigen processing. autoimmune diseases such as multiple sclerosis and its murine model eae are believed to rise from breakdown of tolerance of the immune system. it assumed that immunoproteasome could play an important role in autoimmune diseases. several classes of chemicals proved to be inhibitors of proteasome and the most active are boronate peptide derivatives. these inhibitors totally inactivate proteasome and result in full stop of intracellular protein turnover and cell death via apoptosis. another class of inhibitors, epoxy ketones, was shown to be more selective for immunoproteasome and could be used not for full stop of proteasome function, but for fine tuning of altered proteasome functioning. we examined properties of several inhibitors of four different classes, namely peptide boronate bortezomib, peptide aldehyde mg , lactam lactacystin, and peptide epoxyketones epoxomicin, mg ek, uk and pr- . for inhibition experiments we used proteasome isolated from eukaryotic cell lines cho, nso and hek, treated and non-treated with γ-interferon, as a model cells contatinig constitutive and immunoproteasome. the upregulation of proteasome immunosubunits was revealed in cho and nso cells treated with γ-interferon. the ic values for all studied inhibitors were obtained, and ki in some cases were calculated. the epoxyketones were shown to selectively inhibit in submicromolar concentrations the proteasome sample which contain high amount of immunosubunits. in order to find an effective antimalarial, this study refers to some angiotensin ii (aii) analogues which were considered the important physicochemical characteristics described by silva et al. to verify the biological activity against plasmodium gallinaceum and to understand the hydrophobic cluster influence, explained by tzakos et al. these analogues were synthesized and characterized as described by silva , as well as the biological assays and comprises, to verify: the hydrophobic cluster activity -a) drvyhipf; b) drvypr; c) ryhipf and d) fphiyvrd; the importance of these residues in aii molecule -e) rypf; the importance of aromatic residues -f) yhpf and the action of these hydrophobic residues, when interacting with the parasite membrane -g) vipf. it was observed that in a ( % of bioactivity), the phenol group of tyr is close to imidazole group of his that could promote a hydrogen bond formation. besides that, could occur van der waals interactions between ile and phe residues due its proximity and non-polar characteristic. these interactions could not be effective in native aii ( %) , because ile residue promote a steric influence on the organization of his and tyr residues that not exist in b ( %). in c ( %) and e ( %) analogues, the influence of the arg residue could promote a cation-π interaction with tyr residue and the cluster may have suffered slight destabilization and its antiplasmodial activity was compromised subtly. in d ( %), the electrostatic change, obtained with the total inversion can have disordered its interaction with parasite membrane, since it is not related to membrane receptors, because d-aii presented % of biological activity. moreover, hydrophobic and aromatic residues importance was confirmed through the results obtained % and % of activity, with g and f, respectively. we conclude that hydrophobic cluster modifications and interactions of amino acid side-chain influences in the biological activity. closed joint-stock company "vertex", st-petersburg, russia creatine (cr), a small molecule synthesized in the kidney, liver and pancreas plays important role in atp synthesis, replenishing its store even in the absence of oxygen. cr is able to protect brain cells against ischemic damage; however it has poor ability to penetrate the blood-brain barrier without specific carrier protein. thus, synthesis of stable hydrophobic derivatives capable of crossing the bbb by alternative pathway is of great importance for the treatment of different neurological diseases including stroke, traumatic brain injury and hereditary crt deficiency. here we describe the synthesis and biological activity of new hybrid compounds -creatinyl amino acids. originally the title compounds were synthesized by guanidinylation of sarcosyl peptides. however, for large scale synthesis better results can be obtained using direct cr conjugation with amino acid or peptide derivatives by isobutyl chloroformate method. addition of equivalent amount of ptoluenesulfonic acid as lipophilic counterion ensures efficient cr dissolution in dmf along with its simultaneous protection towards intramolecular cyclization. it excludes the application of expensive guanidinylating reagents and permits to simplify the synthetic procedure. purification of final product and its conversion into appropriate salt form can be achieved by iec followed by crystallization from organic solvents. synthesized creatinyl amino acids and peptides exhibited significant biological activity in different assays including platelet aggregation test, ischemic stroke and nano -induced hypoxia model. one of the most effective compounds -creatinyl-glycine ethyl ester increases life span of experimental animals more than two times in hypoxia model and has neuroprotective action in brain stroke model when applied both before and after ischemia. these data evidenced that creatinyl amino acids can represent promising candidates for the development of new drugs useful in stroke treatment. the efficient recognition and destruction of tumor cells via specific cellular markers is a major goal in cancer therapy. various growth factor receptors such as egfr, hgfr, vgfr and their downstream signaling networks have been proven to be effective molecular targets, as they are frequently involved in cancer proliferation and metastasis. downregulation of these receptors and/or blocking their signaling pathways have clear anti-tumoral effects. drugs based on monoclonal antibodies (mab) targeting such cell surface receptors have attracted a lot of attention as a new generation of therapeutics. however, their production is costly and identifying new, variable routes to modified molecules with similar properties is currently a major focus. , here we present an approach to chemically synthesize a molecule that combines the mode of action of antibodies with the advantages of smaller, chemically accessible molecules. these "synthetic antibody" (sab) molecules contain a chemoattractant that activates the innate immune response and resembles the fc domain of a typical antibody. specificity is imparted by two binder peptides that assume the function of the variable antibody domains and bind to a cell surface target. the fc and fab domains of the sab molecules are connected via polyethylene glycol linkers. sab molecules are prepared by solid phase synthesis, a flexible technique that allows fast production, full control of their properties and targeting two different cell surface receptors (bispecific tumor targeting). they are currently tested in vitro and in vivo for their effect on the innate immune system, general toxicity and selective binding to cancer cells. the key enzyme in the processing of polyproteins translated by viral rna genome of sars-cov is a kda protease called c-like protease ( cl protease). sars cl protease is a cysteine protease containing a cys-his catalytic dyad, and cleaves precursor poly proteins at as many as conserved site involved a conserved gln at the p position and a small amino acid (ser, ala, or gly) at the p' position. due to its functional importance in the viral life cycle, sars cl protease is considered to be an attractive target for drug design against sars. recently, we found tetrapeptide aldehyde, ac-thr-val-cha-his-h, showed high inhibitory activity with ic value of nm toward cl-r i mutant protease , . to compare the inhibitory activity of small compounds with those containing active functional groups, we synthesized serine-derivatives within the essential functional groups and evaluated its inhibitory activity. the synthetic scheme was started from fmoc-ser(tbu)-oh, following modification of c-terminal carboxyl group with p , n-terminal amine with p and side chain alcohol with p functionalities. steps overall reaction led to obtain novel serine derivatives for the small molecular inhibitors of sars cl protease. the assay with cl r i mutant protease was examined to evaluate the inhibitory activity of the synthetic serine derivatives. then, molecular docking study of complex of cl protease with the ligand was carried out. docking simulation experiment with r i (pdb id: aw ) and the inhibitor, which has the best activity in the serine derivatives, indicated that p fitting s ' pocket. at the result of assay, p , p and p positions of the inhibitor should be modified by benzoyl group, cyclohexyl group and cinnamoyl group, respectively. their bioactivities are underpinned by their distinctive structure with exceptional stability, thus making cyclotides exciting, not only for agricultural and pharmaceutical purposes, but also as a template in drug design. in all of the reported activities, cell membranes seem to be the primary target for cyclotide activity. to unravel the importance of lipid membranes on the reported activities of cyclotides, a set of cyclotides belonging to möbius and bracelet subfamilies were compared in their mode of action. the lipid selectivity and membrane affinity were compared with their efficiency against different target cells (e.g. red blood cells, bacteria, hiv particles). we have found that the bioactivity of cyclotides is dependent on the lipid composition of the target cell membrane and independent of a protein chiral receptor. in particular, all the native cyclotides tested target the cell membrane through specific binding to phospholipids containing phosphatidylethanolamine (pe)headgroups, but the membrane binding affinity is further modulated thorough non-specific peptide-lipid hydrophobic interactions, which are dependent on the specific cyclotide. in addition, the bioefficiency of cyclotides broadly correlate with their ability to target and disrupt the cell membrane. overall, we have shown that even with a common specificity for membranes containing pe-phospholipids, a fine selection was found across the family. in particular, each cyclotide inserts and disturbs the membrane in a distinct way, which explains the diversity of this family but also their distinct activities. the observation that all the tested cyclotides have a preference for a specific lipid makes this family truly intriguing and brings insights to optimize the use of the cyclotide template in drug design. malaria is a disease that affects around million people causing . - million of deaths annually. based on our previous studies, angiotensin ii (aii) presented antiplasmodial activity against plasmodium gallinaceum, but due to pressure activity, it cannot be used as an antimalarial drug. in an attempt to increase antiplasmodial activity and reduce hypertensive activity, we synthesized by solid phase method, cyclic analogues of aii with i-(i+ ) and i-(i+ ) lactam bridge scaffold using asp and lys residues. the bridge was more effective when inserted next to n-terminal extremity , probably this insertion, on another portion of the peptide, provides a change in the conformation of the molecule and its hydrophobic cluster formed by tyr, ile and his , which may have influence in the peptide-membrane interaction. thus, we have focused in the n-terminal extremity, testing new analogues, using glu/asp/orn/lys residues as bridgeheads components in i-(i+ ) lactam bridge scaffolds, which showed that antiplasmodial activity is increased using glu residue and that larger lactam rings are better to biologically active. therefore, new restrict peptides by i-(i+ ) and i-(i+ ) lactam bridge were designed, using glu residue as bridgehead element, but the same effect was not verified, getting a maximum of % of bioactivity. on the other hand, we promoted an increase in the hydrophobic character of the molecule, replacing the asp residue of aii sequence by fmoc-glu and asp(ofm), in order to improve the interaction of these compounds in the sporozoite membrane. the replacement by fmoc-glu provided a decrease of activity, while that asp(ofm) kept the aii activity, because there are changes of charge in the peptide, which may have modified the conformation in physiological medium. this kind of approach may offer the basis for development of new drugs and chemotherapy against malaria. animal venoms are complex chemical cocktails, comprising a wide range of biologically active reticulated peptides that target with high selectivity and efficacy a variety of membrane receptors such as ion channels or g-protein coupled receptors. venoms can therefore be seen as large natural libraries of biologically active molecules that are continuously selected and highly refined by the evolution process. the vision associated with the venomics project is to investigate in depth the enormous structural and pharmacological diversity of venom peptides through the development, integration and implementation of a novel research paradigm combining cutting-edge "omics" technologies in a high-throughput workflow. this new paradigm enclosed in venomics aims at replicating in vitro the diversity of venoms to generate original peptide banks to be used in drug discovery programs. herein, we show the different strategies we adopted for efficient solid phase synthesis and folding with an easy purification of peptides rich in cysteine and containing posttranslational modifications (ptm). angiogenesis depends on the adhesive interactions of vascular cells. the adhesion receptor integrin av b was identified as a marker of angiogenic vascular tissue. the αν β integrin receptor plays an important role in human metastasis and tumor-induced angiogenesis, mainly by interacting with matrix proteins through recognition of an arg-gly-asp (rgd) motif. inhibition of the αν β integrins with a cyclic rgd peptide impairs angiogenesis, growth and metastasis of solid tumours in vivo. the aim of this study was to investigate the effects of replacement of a-amino acids by aza-β -amino acid analogs in cyclic rgd-peptides as αν β -integrin antagonist on angiogenesis, microcirculation, growth and metastasis formation of a solid tumour in vivo. the selectivity profile of these antiadhesive cyclopeptide is rationalized by a special presentation of the pharmacophoric groups. we synthesized cyclic rgd peptidomimetics that include aza-β -amino acid residues. modifications were added to the rgd skeleton in order to optimize the peptide activity. then, we investigated the pharmacokinetics activity of these pseudopeptides in hek (human embryonic kidney ) and endothelails cells huvec (human umbilical vein endothelial cells) cell by analyzing cell viability and protein involved in the angiogneisis processes. since tenascin c is a factor expressed highly in the tumorassociated matrix, targeting it would be a desirable first step for targeting the tumor-specific microenvironment in fact, a high level of tenascin c expression has been reported in most solid tumors, including lung cancer, colon cancer and glioblastoma. therefore, the targeted binding of tenascin c in tumor stroma would inhibit tumor metastasis by modulating cancer cell growth and migration. we isolated a peptide that bound to tenascin c by phage display peptide library selection, and the selected peptide specifically recognized tenascin c protein in xenograft mouse tissue. we also observed exclusive staining of tenascin c by the selected peptide in tumor patient tissues. moreover, the peptide reduced tenascin c-induced cell rounding and migration. we propose that the tenascin c targeting peptide may be useful as a specific anti-cancer diagnostic and therapeutic tool for most human solid tumors. radiolabeled pansomatostatins are expected to enhance hsst - tumor-uptake and to broaden clinical indications as compared to currently established sst -prefering radioligands. previous experience has revealed [ in-dota ,dtrp ]ss- ([ in]at s) as a true pansomatostatin analog, exhibiting however poor in vivo stability. in order to enhance metabolic stability, we introduced a second disulfide bridge to the at s motif by formation of extra / -amino acid (aa) or / -aa ring generating at s and at s, respectively. the orthogonally protected sequences were assembled on the solid support, deprotected and cleaved from the resin with tfa. the first cys -cys (at s) or cys -cys (at s) cyclization was performed in dmso, while the second was completed with iodine oxidation after in situ deprotection of cys (acm) and cys (acm). during hsst - +-autoradiography, at s showed unexpected total loss of sst - affinity, whereas at s showed high affinity (ic in nm) to all hsst - (hsst = . ± . ; hsst = . ± . ; hsst = . ± . ; hsst = . ± . ; and hsst = . ± . ). consistent with this finding, only at s stimulated sst internalization during immunofluorescence-based internalization assays, showing agonistic properties for sst . furthermore, [ in]at s internalized rapidly and specifically in sst + ar - j and hek -hsst +-cells. hplc analysis of min ex-vivo mouse blood samples revealed that > % [ in]at s remained intact. after injection in scid mice bearing ar - j and hek -hsst + tumors [ in]at s specifically localized in the rsst a+ ( . ± . %id/g vs. . ± . %id/g + nmol tate at h postinjection (pi)) and in hsst + implants ( . ± . %id/g vs. . ± . %id/g + nmol ke at h pi). this study has shown that introduction of an extra disulphide bridge in at s confers high metabolic stability. however, in a / member ring combination it leads to total loss of affinity. the reasons for this effect are currently investigated by nmr conformational studies. transporter compounds are useful tools to solubilise and increase the delivery of therapeutic molecules in the human body. one system to improve the cellular uptake of such therapeutic molecules are cell-penetrating peptides (cpps). these short peptide chains are either polycationic (containing several arg and lys) or show a more amphiphatic structure. it is known that the multivalency effect -the presentation of several copies of a cpp motif on a single molecule -can increase the cellular uptake. peptide dendrimers represent a group of tree-like, multivalent macromolecules, which are synthesized for different chemical and biological applications in our group. we now combine linear cpps with peptide dendrimers to get a well defined branched molecule made up of only natural amino acids. in our systematic study of peptide dendrimers decorated with different cpps we found that the potency of the single cpp as a transporter for small molecules can be increased and that these peptides show usually low cytotoxicity. additionally we designed new dendritic cell penetrating peptides with similar activities like linear cpps. all compounds are covalently linked to fluorescein for visualization with flow cytometry and confocal analysis. the results show that the peptides can transport efficiently a hydrophobic cargo into the cells. chemical stability of esters of acyclovir with amino acid and cholic acids k. chuchkov, r. chayov, i. g. stankova* south-west university "neofit rilski", blagoevgrad, bulgaria amino acid esters of antiviral drugs are a very good solution for improving oral bioavailability of the actual medicine. one of the most effective and tolerant prodrugs is valine ester of acyclovir -valaciclovir. taken orally exhibits three to four times higher bioavailability of acyclovir. the chemical stability of amino acids ( -fphenylalanine) (r,s) and bile acids (deoxycholic acid and chenodeoxycholic acid) esters of acyclovir was studied in experimental conditions simulating some relevant biological medias (ph . and . , °c).the chemical stability experiments revealed that the examined amino acid ester of acyclovir were relatively unstable in acidic ph, but bile acid ester is stable in the same ph. the examined amino acid and bile acid esters of acyclovir in neutral ph are relatively stable. in ph , all of tested compounds are more stable than valacyclovir (t / = h) -the first effective prodrug of acyclovir. in acidic ph acyclovirdeoxycholat and acyclovirchenodeoxycholat are more stable than valacyclovir. acyclovirchenodeoxycholat is the most promising anti-ebv prodrug candidate with high activity and satisfying chemical stability. cell-penetrating peptides (cpp) have become efficient tools for the cellular internalization of bioactive molecules due to their ability to cross the plasma membrane of diverse cells and cell lines. [ ] we recently reported that the cpp sc , which consists of the residues - of the c-terminal region of the cationic antimicrobial peptide cathelicidin (cap ), is an effective carrier peptide for small organic molecules like fluorophors and toxic peptide sequences into various cell lines [ ] . however, in general linear peptides are more susceptible to proteolytic degradation than their cyclic analogs [ ] . therefore, we investigated the cyclization of cpp derived from sc by means of cui-mediated azidealkyne cycloaddition (cuaac) [ ] . furthermore, we examined their conformation and proteolytic stability as well as their internalization efficiency and toxicity against various cell lines, in comparison to their linear equivalent and to other cpp. looking for the proper prodrug: a peptidomimetic approach to identify and inactivate bacterial mono-adp-ribosyltransferase toxins m. beich-frandsen, r. jørgensen division of microbiology and diagnostics, statens serum institute, copenhagen, denmark mono-adp-ribosylation is an endogenous posttranslational modification in eukaryotic cells, simultaneously utilized as virulence strategy by deadly secreted bacterial toxins. many bacterial toxins have been found to act as mono-adp-ribosylating enzymes, targeting anything from g-proteins to the actin skeleton. the diphtheria toxin from c. diphtheriae and exotoxin a from p. aeruginosa, both target the diphthamide-group of a unique modified histidine in eef , inhibiting protein synthesis by ribosome mimicry , . we aim to inactivate these nad+-utilizing toxin enzymes by nad-conjugated peptidomimetics, in a target-specific prodrug-approach. the adp-ribosylation reaction follows a random third-order s n mechanism. in the proposed model for the transition state of the reaction, the cleavage of the n c -nn bond of nad + releases strain and generates a oxacarbenium ion intermediate with a positively charged nicotinamide (n)ribose, subject to a nucleophilic attack from the substrate , , . adp-ribosylating toxins are commonly characterized by a artt-motif involved in substrate recognition . studies suggests conformational rearrangement of the residues surrounding the substrate binding site to be required for optimal geometry of the initial glycosidic nc -nn bond cleavage within nad + . subtype specific nad-conjugated peptides, designed based on previous structural analysis of the adpribosylation reaction, act as substrate for the enzymatic adp-ribosyl-transfer, and hereby attach covalently to and inactivate the nad + -utilizing toxin. relying on previous structural studies, and established ligand-binding and kinetic data, an initial peptide library, designed by bioinformatics and evaluated for specificity of common targets in-silico identifies initial leads. lead-scaffolds are implemented in rational peptide-design, based on high-resolution structural-and biophysical studies of multiple peptide-enzyme complexes, to identify possible prodrug-strategies for enzyme inactivation. nanoparticles play a crucial role in medicine for their potential application as in vivo carriers of active principles [ ] . liposome display unique pharmacokinetic properties slowly releasing drugs loaded in the inner aqueous cavity. in the last years we have developed supramolecular aggregates labeled by bioactive peptides able to recognize overexpressed receptors on tumour cells membrane delivering doxorubicin chemiotherapeutic drug [ ] . neurotensin(nt), a amino acid peptide, has dual functions of neurotransmitter or neuromodulator. the cterminus short fragment - preserve the activity but the half life of wild type form in vivo is very short. nt receptor type (nts ) is overexpressed in severe malignancies such as small cell lung cancer and colon, pancreatic, and prostate carcinomas. we have designed new amphiphilic molecules containing in the hydrophobic moiety two aliphatic chains and in the hydrophilic moiety a the bioactive portion able to aggregates with phospholipid molecules achieving liposome. we have synthesized neurotensin wild type sequence, the truncated form and the tetra-branched neurotensin(nt - ) or a truncated form(nt - ) tetrabranched peptides(nt ) adopting an opportune synthetic strategy on solid phase. all liposome were formulated adding the neurotensin amphiphilic monomer in ratio : with dopc in order to evaluate the capability to recognize selectively receptors overexpress on cell membrane surface. the liposomes size was determined by dynamic light scattering measurements, values for the hydrodynamic radius(rh). the selective internalization and cytotoxicity of fully doxorubicin loaded liposomes as compared to pure dopc liposomes, was tested in ht human colon adenocarcinoma and te human rhabdomyosarcoma cells. recently, small interfering rna (sirna), one kind of rna interference (rnai) technology represent the most common and, to date, the most effective method to inhibit target gene expression in human cells. it is also a common recognition that non-toxic delivery of sirna is an urgent problem for the therapeutic application of sirna. for the efficient gene silencing in vivo, prolonged circulation of sirna with take efficient and non-toxic cellular uptake and resistance against enzymatic degradation are indispensably required. ) telomerase activity has been regarded as a critical step in cellular immortalization and carcinogenesis and because of this, regulation of telomerase represents an attractive target for anti-tumor specific therapeutics. in this paper, we present the efficient and non-toxic cellular uptake of sirna using novel amphiphilic peptides and the application to silencing of htert in human cancer cell lines. in the present study, we investigated the intracellular delivery of sirna using some amphiphilic peptides and the silencing effect of sirna targeting htert mrna in human cancer cell lines, jurkat, hela and k . the complex of sirna and a specific amphiphilic peptide or its hybrid with an intracellular transport signal peptide could be effectively taken up into cells. the complex also showed a high silencing effect against htert mrna. moreover, the combination of sirna-nes conjugates and the amphiphilic peptides improved silencing effects up to . %. the amphiphilic peptides and their hybrids showed almost no cyto-toxicity and protected sirna against intracellular nuclease digestion in % fbs (half life time was over h). tumor targeting with the decapeptide gonadotropinreleasing hormone (gnrh) or its analogues is based on the discovery that gnrh receptors are overexpressed in many tumor cells, compared with their expression in normal tissues. using these peptides as carriers/targeting moieties in a conjugate with therapeutic agents can increase the selectivity and the stability of the conjugates, or eliminate the toxic side effects of the drug. gnrh-iii (<ehwshdwkpg-nh ) is especially favoured as a targeting moiety because of its antiproliferative effect and weak endocrine activity in mammals. the aim of our work was to synthesize novel short-chain fatty acid (scfa) acylated daunorubicin-gnrh-iii bioconjugates with enhanced enzymatic stability, cellular uptake, in vitro and in vivo antitumor activity. ser in position of gnrh-iii was replaced by lys and its ε-amino group was acylated with various fatty acids. gnrh-iii( lys(x), in conclusion, glu - /dglu - substitution in gi-derived radiopeptides enhanced stability and reduced renal values by ~ . fold. on the other hand, removal of the (glu) sequence in mg suppressed renal uptake but undermined stability. both modifications, however, compromised tumor targeting, demonstrating that more efficacious structural changes in the gi motif are warranted to improve the in vivo profile of resulting radioligands. synthesis and characterization of a mtc-labeled rhodamine-conjugated angiotensin peptide as a potential cardiac function imaging agent s.m. okarvi king faisal specialist hospital & research centre, cyclotron and radiopharmaceuticals department, riyadh , saudi arabia angiotensin ii (ang ii) is an -amino acid peptide that has been known to play a vital role in cardiovascular system. the actions of ang ii have been implicated in many cardiovascular conditions, such as coronary heart disease and heart failure. in an effort to develop a cardiac imaging agent with enhanced targeting capacity, we attached ang ii to rhodamine (rh), a lipophilic cation that specifically accumulates in the myocardium. the rh conjugated ang ii was evaluated for its potential as a heart imaging agent. rh-lys-gly-cys-asp-arg-val-tyr-ile-his-pro-phe-conh was prepared by solid-phase peptide synthesis according to fmoc/hbtu methodology. nhs-rh was attached to the ang ii peptide via the free amino group of lys residue by manual synthesis. rh-ang ii conjugate was labeled with tc- m by the stannous-tartrate exchange method. in vitro stability was determined in human plasma and in excess of cysteine. in vivo biodistribution and biokinetics were performed in balb/c mice and sprague dawley rats. the rh-ang ii conjugate radiolabeled efficiently with tc- m (> % labeling efficiency) as determined by hplc analysis. tc- m-rh-ang ii exhibited good chemical stability against cysteine transchelation and sufficient metabolic stability in human plasma. in mice, the bioconjugate displayed efficient clearance from the blood and excreted mainly through the renal route with some excretion by the hepatobiliary pathway. the uptake in the heart was . ± . % id/g as early as min post-injection; whereas, the uptake in the lungs, liver, stomach and kidneys varied between - % id/g. in rats, the bioconjugate displayed relatively better pharmacokinetic characteristics, with low uptake in the major organs (< % id/g). the uptake in the heart ( . ± . % id/g) was found to be higher than the uptake in the blood and muscle, resulting in good heart-to-blood and heart-to-muscle uptake ratios. this initial study towards the development of an effective cardiac imaging agent advocates that the use of hybrid conjugates appears to hold a great promise as a new and attractive approach for rapid and efficient imaging of heart. in humans two isoforms of gnrh are exist, gnrh-i (<ehwsyglrpg-nh , where <e is pyroglutamic acid) and gnrh-ii (<ehwshgwypg-nh ). in contrast to gnrh-i, exact function of gnrh-ii is not well identified. however, it was indicated that gnrh-ii derivatives have more potent antiproliferative activity on human endometrial and ovarian cancer cells in vitro than gnrh-i analogs. some kinds of tumor cells (e.g. breast, prostate, colon) produce gnrh-i and gnrh-ii, and express their receptors. however, the presence of functionally active gnrh-ii receptors on cancer cells is still not clear - . on the other hand, gnrh-ii analogs, both agonists and antagonists, induce apoptosis in many different cell types. in contrast to gnrh-i agonists, gnrh-ii agonist [d-lys ]gnrh-ii does not activate nfκb, therefore does not protect the cancer cells from apoptosis [ ] [ ] [ ] . in the regulation of apoptosis, not only the pro-and antiapoptotic proteins play a role , but other molecules also attend (e.g. ck , casein kinase ii enzyme) . our aim was to synthesize new proapoptotic peptide or enzyme inhibitor containing [d-lys ]gnrh-ii based derivatives and investigate their in vitro cytotoxic, cytostatic and apoptotic effects. the sythesis of [d-lys ]gnrh-ii, which is one of the most potent gnrh-ii agonist, was carried out on solid phase using fmoc/tbu strategy. different proapoptotic peptides or enzyme inhibitors were attached directly to the gnrh-ii derivative. in vitro cytostatic and cytotoxic effects of gnrh-ii conjugates were investigated by mtt-assay using different human breast (mcf- and t -d) and colon carcinoma (ht- ) cell cultures. early apoptotic effect of gnrh-ii conjugates was studied by flow cytometry on mcf- , t- d and ht- cell lines. according to our results, these conjugates might be potential candidates for cancer treatment, because the combination of a targeting moiety (gnrh derivative) with proapoptotic peptides or enzyme inhibitors and drug molecules might have synergistic activity resulting in highly potent multifunctional therapeutic agents. department of pharmaceutical and pharmacological sciences, university of padua, padua, italy melanoma is the most deadly skin cancer with an increasing incidence. its therapy continues to be a challenge since, regardless of the treatment used, longterm survival is quite uncommon. melanoma cells are characterized by high levels of glutathione s-transferase p - (gstp - ) isozyme. this enzyme is also highly expressed in solid tumours and drugresistant cells, where a role for this protein in the regulation of cell proliferation has been described. in the past, lyttle et al. synthesised glutathione (gsh)linked anticancer prodrugs activated by physiological concentration of gsts. structure and mechanistic studies showed that in the gst-gsh complex, the gst tyr residue placed near the s atom of the cys in gsh, is in the phenoxide form to facilitate the deprotonation of the sulfhydryl group in gsh, making it able to react with electrophilic species. by taking advantage of this active-site geometry, we synthesised two tyrosinase activated melanoma prodrugs ( -methoxyphenol and n-acetyl- -s-cysteaminylphenol) linked to gsh. in these compounds an ethoxycarbonyl group was placed between the gsh sulphur, oxidate to sulfone, and the tyrosinase-activated prodrugs. in this way the tyr phenoxide (tyr in gstp and tyr in gstm isozyme) would be able to abstract one of the acidic methylene protons linked to the sulfone moiety. such a deprotonation should trigger a beta-elimination, resulting in the release of melanoma prodrugs. the stability of the obtained putative prodrugs has been evaluated in different ph buffers and in rat liver and kidney cytosolic fractions. synthetic peptides represent first choice biomolecules, with respect to proteins and mab, for the monitoring of population variability and receptor functionality associated to a tumor pathology due to their favorable pharmacokinetic profile. it has been demonstrated that the surface molecule integrin alfa-v beta- could be an ideal target for the interaction with melanoma directed radiolabeled peptides. this integrin is indeed overexpressed both in tumor cells and in tumor vessels endothelium, while it is not expressed in the majority of normal tissues. cyclic peptides with the sequence arg-gly-asp (rgd), are known to bind the integrin alpha-v beta- with high affinity and selectivity and could conveniently target a radiotherapeutic to melanoma cells. we previously demonstrated that the cyclic rgdfk peptide interact with high affinity to a human melanoma cells. the aim of this work was to conjugate the mtc to the peptide by using the [ m tc(n)(pnp)] + system (pnp=aminodiphosphines) to obtain a radiolabeled peptide useful in melanoma imaging. , spectrum. a nh(i)→βch(i- ) cross peak more intense than the nh(i)→βch(i) cross peak is observed when the peptide adopts the . -helixl conformation. by contrast, an opposite trend of intensities of the same noe cross peaks indicates the occurrence of a -helical conformation. by using this method, we were also able to determine that polar solvents, such as mecn or meoh, may induce a helical structure in deg homo-peptides which otherwise adopt the . -helix conformation in cdcl . this finding allows us to rapidly switch these peptide series from "short" ( -helix) to "long" ( . -helix) by changing the solvent only, thus making them extremely attractive tools. school of biological sciences, university of auckland, auckland , new zealand naturally occurring anti-freeze proteins (afps) enable organisms like polar fish to survive the freezing temperatures of their natural habitat. as well as being cryoprotective, afps exhibit properties of ice recrystallization inhibition (ri). the ability of afps to influence the size, morphology and aggregation of ice crystals can be used in food technology, where the growth of ice crystals in frozen foods is of primary concern. chemical synthesis of afps enables access to pure material for structural and functional studies. our new research programme in the 'food and health' area, prompted us to develop tailor made anti-freeze peptides for potential applications in the frozen food industry. 'multiple salt-bridge analogues' of the naturally occurring winter flounder afp were synthesized and evaluated for their antifreeze activity in comparison to the natural sequence. details on the molecular modelling, structural characterization and anti-freeze activity analysis on the synthetic afps will be presented. several disorders are now believed to be conformational diseases caused by misfolding in the structure of specific proteins leading to the gradual aggregation and deposition of these abnormal proteins. attempts to develop effective therapies for these proteopathies (such as alzheimer's, parkinson's and huntington's disease) have been directed toward reducing the production of the proteins, inhibiting their aggregation, or promoting their removal. conformation-dependent antibodies that specifically target misfolded proteins are proving to be useful tools to study the pathogenesis and as possible treatments of these diseases. we have previously shown that active immunization with tetrapalmitoylated aβ - peptide embedded in liposomes (aci- ) was capable of eliciting a conformation-specific immune response in mice. the antigenic peptide was shown to mimic the aggregated β-sheet conformation of the pathological a β peptide on the surface of the liposomes by cd, ssnmr and tht assay. we have also reported the modulation of the conformation of aβ - derived sequences by means of different lipidation patterns, lipid chain lengths and liposome surface charge. here we present the characterization of this panel of liposomal peptide constructs by atr-ir and correlate the results with previous studies by cd. in summary, atr-ir secondary structure analysis of a battery of amyloid-derived lipopeptides allowed to study the key parameters responsible for the adopted conformation on the lipid bilayer. the presented strategy to control the conformation of liposomal peptide immunogens could be applied to the rational design of vaccines against a range of protein misfolding diseases, such as alzheimer's disease. cyclic lipodepsipeptides (clps) are secondary metabolites produced by various bacteria, most prominently bacillus and pseudomonas. these often display potential antimicrobial properties, frequently ascribed to the ability to disrupt cellular membranes or form ion-pores. clps are a structurally diverse group of molecules always consisting of an oligopeptide chain cyclized by a lacton bond and a fatty acid moiety. they are divided into several groups based on sequence similarity. one such group is the viscosin group, which contains clps produced by pseudomonas that all feature nine amino acids and a conserved sequence pattern of hydrophobic and hydrophilic residues. recently, we have extensively described the self-assembly properties of one viscosin group member, pseudodesmin a. , we have shown that in non-polar organic solvents this clp forms anisotropic supramolecular structures of unrestricted size, reminiscent of the ability to form ionpores in the non-polar cellular membrane environment. here, we report on how the most prominent structural variations within the viscosin group affect the conformation and self-assembly properties. since the well-defined monomer conformation of pseudodesmin a is key to this self-assembly, the most striking variation within the viscosin group is the variability of the leu stereochemistry. for the first time, a conformational study will be reported for clps featuring an l-leu residue, such as viscosin and viscosinamide. the results of this study contribute to a deeper understanding of the structure-function relationship of clps in general. effective design of peptide mimicking protein helical segments is a great challenge. an intriguing approach to improve the helical content in a peptide sequence is achieved through the "peptide stapling" strategy using a hydrocarbon cross-linker (staple) . in this study we have investigated the effect of the introduction and location of staples on secondary structure of sequences derived from a reported peptide, mimicking cullin binding region to kctd , , to gain an insight into the designing of new molecular entities able to modulate the kctd oncosuppressor activity. indeed, kctd plays a crucial role in the ubiquitination of hdac by acting, in complex with cullin , as an e ubiquitin ligase, thereby affecting hedgehog activity and medulloblastoma growth. in the designed peptides the stapling has been achieved by ring-closing olefin metathesis between two s- -( 'pentenyl) alanine residues incorporated at i and i+ positions and into different sites within the peptide chains. the stapling position has been chosen on the basis of cullin -kctd complex model. circular dichroism studies have highlighted that the stapled peptides exhibit remarkable and different changes in secondary structure contents in comparison with the linear precursors and the unmodified sequence. a nmr conformational analysis will allow the determination of the conformational preferences of the stapled peptides at atomic resolution with also relevant implications for the kctd-cullin recognition. based on our findings from protease-mediated ligation of cleavage-sensitive peptides and proteins using ionic liquids (ils) as additives and from chemical acylation reactions of peptides in ils, we hypothesized direct and unique interactions between biomolecules and ils as the molecular reason for il-mediated structural effects. to verify this, we performed systematic structural investigations of suc-ala-xaa-pro-phe-pna and ala-xaa-pro-phe model peptides dissolved in aqueous imidazolium-based ils using high resolution magic angle spinning (hr-mas) nmr spectroscopy. highly resolved nmr spectra are obtained that allow for complete h resonance assignments of the peptides as solutes and the ils as solvents. the changes in the proton chemical shifts Δδ( Η) of a peptide on dissolution in ils arise from direct interactions between both, including il-induced structural/conformational changes of the peptide molecules. , the strength, position, and type of these interactions are influenced by the nature of the amino acid residues in the peptide sequence as well as the nature and type of the il anions. downfield shifts indicate strong interactions between the ils and the peptide backbone, whereas upfield shifts point to hydrophobic interactions and stacking effects with the il imidazolium ring. the pattern of Δδ( Η) along the peptide chain reveals the sites and the type of interactions. the extent of Δδ( Η) implies a significant effect on the structure of the peptides. different Δδ( Η) were obtained for cis-and trans-configured peptide isomers and point to a conformer-specific il/peptide interaction. calcitonin is a -amino acid polypeptide hormone, which takes part in calcium metabolism in bones. it belongs to a protein family whose members may form amyloid fibrils. these fibrils (or pre-fibrillar aggregates) are related to serious diseases known as amyloidoses, whereas amyloid-like fibrils may have natural functional roles in many organisms. the amyloid form of human calcitonin is related to medullary thyroid carcinoma. using our online consensus prediction tool for 'amyloidogenic propensity' of protein molecules "amylpred", a novel hexapeptide of human calcitonin was predicted as a novel, possible amyloidogenic peptide. we investigated experimentally its ability to form amyloid fibrils, utilizing tem, x-ray fibre diffraction, atr ft-ir spectroscopy and polarized light microscopy. analysis of the results shows that this peptide self-assembles into amyloid-like fibrils and that amyloid fibrillogenesis is mediated via nuclei of liquid crystalline nature, known as spherulites. we thank the university of athens for the financial support. school of pharmacy, university of norwich nr tj ; inserm u , université de rouen, mont saint aignan rfa, a rfamide neuropeptide, is the ligand of gpr and the system rfa/gpr is involved in feeding behaviour [ ] . a structural study by nmr showed that rfa, in a membrane mimetic medium, possesses a helix p -r -linker k -g -turn f -r motif. sar studies on truncated fragments of rfa indicate that, in addition to the turn motif, additional residues are required to maintain a correct biological activity ( rfa ec = , nm; rfa - ec = , nm; rfa - ec = , nm; rfa - ec = nm) [ ] . our aim is to synthesize new gpr ligands using an iterative process combining rational conception, peptide and modified amino acid syntheses, biological and structural analyses. structural and biological results will allow us to find new modifications and conceive more potent analogues. using nmr, we demonstrated that rfa - encompasses a nascent helix and a turn while rfa - possesses only the c-terminal turn structuration. the destabilization of the helical structure on fragments with a shorter n-terminal region could explain the decrease of the biological activity of rfa fragments. we decided to focus on the importance of a stabilized helix on the biological activity. we chose the hydrogen bond surrogate method which stabilizes a helix by replacing the main hydrogen bond with a covalent link without modifying the side-chains [ ] . syntheses, nmr characterization and molecular modelling of analogues will be discussed. acknowledgments: this work is supported by erdf funding through the is:ce-chem project and interreg iv a france-(channel)-programme. firstly, it induces a constraint by restricting the ı o-dihedral angle to values around - °. secondly, the xaa-pro peptide bond is subject to cis-trans isomerization characterized by an increased cis population and an activation energy that is low when compared to the other amino acids. besides, the five-membered ring of the pro residue can adopt two main distinct conformations (up-puckered or downpuckered) that are almost equally abundant in peptides and proteins. a variety of mimics and analogs have been designed in order to control the conformation of the peptide backbone and/or to alter the cis/trans ratios and the rotational barriers for cis-trans isomerization. in this presentation, nmr studies and theoretical calculations have been performed on model peptides ac-ser(Ψpro)-nhme, (s,s)-ac-ser(Ψh, cf pro)-nhme, and (r,s)-ac-ser (Ψ cf ,hpro)-nhme. their thermodynamic and kinetic features have been analyzed in chloroform, dmso, and water, allowing a precise description of their conformational properties. we found that trifluoromethyl c δ -substitutions of oxazolidine-based pseudoprolines can strongly influence the cis-trans rotational barriers with only moderate effects on the cis/trans population ratio. in chcl , the configuration of the cf -c δ entirely controls the ψ-dihedral angle, allowing the stabilization of γ-turn like or ppi/ppii-like backbone conformations. moreover, in water and dmso, this c δ -configuration can be used to efficiently constrain the ring puckering without affecting the cis/trans population ratio. theoretical calculations have ascertained the electronic and geometric properties induced by the trifluoromethyl substituent and provided a rational understanding of the nmr observations. in contrast to formation of the native state, aggregation of misfolded protein molecules into so-called amyloid fibrils is a strongly irreversible, kinetically-controlled process which may lead to structural variants of fibrils composed of chemically-identical protein chains. this poorly understood aspect of polymorphism of amyloid fibrils has important implications in vivo in the course of amyloidassociated neurological disorders -e.g. in the form of prion strains in creutzfeldt-jakob disease. in this paper, we examine cross-seeding behavior of a model amyloidogenic polypeptide -bovine insulin (bov-ins) and recombinant lys b -arg b insulin (kr-ins). fibrils of bov-ins and kr-ins exhibit characteristic "fingerprint" features in the amide i' infrared region. these spectral traits are transferred to daughter generations of fibrils upon cross-seeding of native bov-ins with kr-ins fibrils and vice versa. this study highlights an interesting aspect of a non-anfinsenian behavior of aggregating insulin: the memory effect. our results indicate that fine infrared features in the amide i region of fibrils from closely related proteins may not reflect different amino acid composition but rather the fact that a particular amino acid sequence favors an amyloidogenic pathway that determines a certain and spectrally-distinct stacking mode of β-strands. an encounter with an alien seed on the amyloidgenic pathway would hijack the yetnative monomers and force them to follow the assembly pattern (and resulting ir characteristics) of the preformed fibril. phosphorylation and oglcnacylation are dynamic intracellular protein post-translational modifications that often are alternatively observed on the same protein serine and threonine residues. phosphorylation and oglcnacylation commonly occur on residues in natively disordered regions of proteins, and often have opposing functional effects. in the microtubule-associated protein tau, hyperphosphorylation is associated with protein misfolding and aggregation as the neurofibrillary tangles of alzheimer's disease, whereas oglcnacylation is stabilizing. a series of peptides derived from the prolinerich domain (residues - ) of tau was synthesized, with the free ser/thr hydroxyls, with phosphorylated ser/thr, with oglcnacylated ser/thr, and with the dietylphosphorylated ser/thr. phosphorylation and oglcnacylation were found by cd and nmr to have opposing structural effects, with phosphorylation favoring polyproline helix (ppii) formation, with oglcnacylation opposing ppii, and with the free hydroxyls intermediate. phosphorylation of serine and threonine residues in prolinerich sequences was found to induce ppii in other proline-rich sequences, with significant conformational restriction at phosphoserine and phosphothreonine residues observed by nmr. notably, serine and threonine residues are common in proline-rich domains of proteins, and ppii is a common conformation observed for phosphorylated ligands in protein-protein interactions. the ser/thr diethylphosphate was found to be a practical mimic of oglcnacylation, as an easily synthesized neutral amino acid with similar sterics to oglcnac and strongly opposing ppii. dept. of biology, university of padova, padova , italy we recently established that designed hairpin peptides interfere with the amyloidogenesis of both human pancreatic amylin and α-synuclein ( α-syn), two 'unstructured' polypeptides associated with human diseases. in the case of α-syn, the most potent 'amyloid inhibitors' act by diverting α-syn to non-amyloid aggregates. the binding sites for these agents and nonhairpin peptides of similar sequence have been determined by d-nmr-monitored titration studies using universally n-labeled α-syn. in the case of the hairpins that produce non-amyloid aggregates from α-syn, initial binding occurs in the non-amyloidogenic c-terminal segment of α-syn and the hairpin peptide is incorporated in the resulting aggregates. nmr studies also serve to define binding interactions and conformational changes that may be essential early steps in aggregation and pre-amyloid state processes. mt-ii is a long acting, non-selective super-agonist of melanocortin gpcr receptors, characterized by lactam bridge between residues i and i+ stabilizing a type-ii β turn structure. the recently introduced cui-catalyzed azide-alkyne , -dipolar cycloaddition, presents a promising opportunity to develop a new paradigm for an orthogonal bio-organic and intramolecular side chain-toside chain cyclization. we introduced this [ , , ]triazolyl-mediated cyclization in a pthrp model peptide, and demonstrated that i-to-i+ side chain-to-side chain cyclization offers a powerful approach for generating stable helix mimetic structures. , , in the current study we explored the potential of the i-to-i+ side chain-to-side chain , -disubstituted [ , , ] triazolyl-bridge to stabilize -turn conformations. we designed a series of [ , , ]-triazolyl containing cyclo-heptapeptides, derived from the model peptide lactam mt-ii, differing in the size of the bridge, the location and orientation of the [ , , ]-triazolyl moiety. conformational and biological studies, performed on this series demonstrate that [ , , ] triazolyl-mediated side chain-to-side chain cyclization results in the conformational stabilization of type-i β turn generating heterodetic cyclopeptides with enhanced in vitro potency and improved receptor subtype selectivity. this study confirms the versatile nature of the [ , , ] triazolyl-mediated side chainto-side chain cyclization. moreover the chemical accessibility, functional compatibility, and metabolic stability suggest that the [ , , ] international associated laboratory samuel de champlain, regional platform for cell imaging of haute-normandie (primacen), institute for medical research and innovation (irib), university of rouen, mont-saint-aignan, france scorpion toxins are polypeptidic molecules capable of selectivity binding to ion channels with the highest affinities and represent natural peptide blockers that are good tools to characterize new or particular types of ion channels. astroglial cells appear to express all ion channels species even voltage-gated ion channels previously suspected to be present only in excitable cells; but these channels are poorly studied and their function are not still completely elucidated.thus, the purpose of the present study was to investigate for the first time, the global effect and the potential protective effect of one scorpion toxin acting on kv channels, the pi ,on h o -induced oxidative stress and cell death in rat astrocytes. pi is a -residue toxin crosslinked by disulfide bridges, isolated from the venom of pandinus imperator and is one of the most potent and selective inhibitor of shaker b and kv . channels . in our study, pi was obtained by solid phase peptide chemical synthesis(spps) and named spi . the identity and homogeneity of spi were also verified by hplc, mass spectrometry analysis(maldi-tof) and an enzyme-based cleavage to determine the assignment of spi half-cystine pairings.the synthetic spi was found to be indistinguishable from the natural toxin. incubation of cultured astrocytes with graded concentrations of h o for h provoked a dose-dependent reduction of the number of living cells as evaluated by flow cytometric analysis and lactate dehydrogenase assay. interestingly, pre-treatment of astrocytes with very low concentrations of spi ( - to - m),dose-dependently prevented cell death induced by h o , and the effect of spi was accompanied by a marked attenuation of ros accumulation. also, spi stimulated sod and catalase antioxidant activities in a concentrationdependent manner,and blocked h o -evoked inhibition of sod and catalase activities. in addition, at low concentration ( - to - m) the toxin exhibits no toxic effect on astrocytes.taken together these data indicate that the spi acts as a protective agent on astrocytes from oxidative assault and then might have therapeutic value in the potential treatment of a number of neurodegenerative diseases. [ ] . in order to select small active peptides, the library was kept at the minimum size and was built using only selected d-amino acids to avoid structural redundancies. to further increase the chemical diversity [ ] a set of different carboxylic acids were coupled to the n-terminus. the iterative screening led to the identification of an n-terminally modified tetrapeptide with a very high radical-scavenging activity. by analyzing several peptide variants, we found that antioxidant properties were strongly sequence-and structure dependent, because scrambled or proline-scan variantswhose secondary structures are deeply altered -are much less effective. data on peptide structure-activity relationship will be presented. an investigation of the in vitro effects produced in cellular models of oxidative stress are also in progress. structural analysis of peptide-analogues of zp proteins with amyloidogenic properties: insights into mammalian zona pellucida formation. n.n. louros, v.a. iconomidou, s.j. hamodrakas* department of cell biology and biophysics, faculty of biology, university of athens, athens , greece a filamentous structure surrounding mammalian oocytes, called zona pellucida, is responsible for species-specific sperm binding. this extracellular porous structure is composed of - glycoproteins, termed zp - , which assemble into filamentous polymers that are cross-linked by disulfide bonds. all zp proteins present a unit with high sequence homology at their c-terminal end, designated as the zp domain. this structural module is found in a family of extracellular proteins with various functions and from a wide variety of organisms (zp domain proteins). it is a binary structure of approximately amino acids, divided into two domains, the n-terminal region named zp-n and the c-terminal region known as the zp-c domain. the zp-c block is associated with protein function, whereas the zp-n domain is responsible for protein polymerization. structural analysis revealed a possible polymerization model based on backbone interactions between specific parts of the zp-n units. peptide-analogues of the aforementioned segments were synthesized, since the ''amylpred'' application, an 'amyloidogenic determinant' prediction algorithm developed in our lab, predicted them as being potentially 'aggregation prone'. our experimental data clearly indicate that these peptides do actually fold and self-assemble into amyloid-like fibrils. therefore, all the derived data suggest that mammalian zona pellucida is a novel functional amyloid, similar to its biological equivalent, silkmoth chorion that has been established to be a natural, protective amyloid. we thank the university of athens for the financial support. biostrucutres and bioimmaging institute of national research council, naples, italy the definition of the molecular basis of human diseases is one of the most important goals of structural biology. nucleophosmin (nmp ) is an ubiquitously expressed protein is a nucleolar chaperon and has a key role in several biological processes from ribosome biogenesis, to cell-cycle regulation and is a frequently target of oncogenic mutations in acute myeloid leukaemia (aml) [ ] . these mutations occur predominantly at the c-terminal domain of npm compromising its stability and causing the aberrant translocation of npm to the cytosol. this domain is structurally characterized by the presence of three alpha helices, and the folding of cter-npm proceeds via an extended nucleus and the denatured state retains significant malleable structure at the interface between the second and third helices [ ] . here, we have further investigated the structural determinants in the folding process of the c-term npm domain and on the basis of available structural and composition data, several peptides covering the three helices were designed and synthesized. their conformational properties were investigated by cd and nmr spectroscopy: these studies demonstrated that once removed from the protein architecture helix and helix substantially miss fully ordered conformations while helix retain a high helical content also in mutated variants. our observations may help structure-based drug-design inserm u university of evry-val d'essonne, bâtiment maupertuis, evry, france sl (stop-like protein with mass of kda) is a neuronal protein with a calmodulin-binding and microtubulestabilizing activity. microtubule binding is inhibited by calmodulin , . the lack of the stops gives rise to defects in synaptic plasticity, as observed in schizophrenia . to study the sl protein's structure and its interaction with calmodulin, we have used a residue peptide derived from sl , containing the microtubule binding motif mn. the structure of the peptide has been analyzed using circular dichroism (cd) spectropolarimetry. the cd spectra showed that the peptide's secondary structure consists in a predominantly β-sheets, while the tertiary structure consists in rather folded d-structure. the temperature dependence of cd indicated that the secondary structure of the peptide remained very stable between °c and °c, while the tertiary structure changed at about °c. the interactions between the sl peptide and calmodulin have been studied using the isothermal titration calorimetry (itc). the results showed that calmodulin binds the peptide with the affinity of m- , in a ca + -independent manner, the reaction being driven by entropic contributions. our results might contribute to understanding the microtubule stabilizing activity of the sl protein. peptides , . based on the structure in polar environment a transition from -helical to an α-helical conformation at the water/lipid interface in bilayer membranes is hypothesized the efficient method development for the analysis of proteins, peptides, and synthetic oligonucleotides with a novel hybrid reversed phase column f. becker * , n. shoji * , a. matsui * , c. yokoyama * , t. sato * , t. takai * , and n. kuriyama * * ymc europe gmbh, * ymc co., ltd. the recent development of bio-pharmaceutical industry has been remarkable and an effective analytical method with higher sensitivity, superior selectivity, and increased speed has been required in the characterization of peptides, proteins and oligonucleotides by highperformance liquid chromatography (hplc). the method development of reversed phase hplc requires optimization of several conditions, such as bonded-phase, column efficiency, solvent type, ph and temperature. especially ph and buffer type is a most important parameter to control retention of biomolecules which have multiple ionic functional groups. furthermore, temperature often becomes a key tool to achieve better peak shapes and resolution for larger molecular-weight compounds. although silica based reversed phase columns have been widely used for separation of biomolecules, they have low stability under alkaline conditions and a limited usable ph range. to improve the chemical stability at an expanded ph and temperature range , we have developed a new type of organic/inorganic hybrid reversed phase column, ymc-triart c and ymc-triart c . the novel technologies of manufacturing particles and surface modification provide outstanding chemical stability and excellent peak shape for many types of compounds under a variety of mobile phase conditions. in this poster, we will show characteristics of this new hybrid column, and some example cases of efficient method development in separation of the biomolecules such as peptides, proteins and synthetic oligonucleotides. the fully-extended peptide conformation ( . -helix) is the longest possible for a single α-amino acid as it is characterized by an axial translation per residue of about . Å. therefore, it is extremely attractive for its application as a molecular bridge. however, it is known that the stability of this type of helical structure (relative to the competing, much shorter -helix) appears to be dramatically governed by the choice of the c-terminal functionality (only esters seem to be appropriate). in this work, we examined the series pyrac-(deg)n-o-(pno )bzl [where pyrac is the fluorophore -pyrenylacetyl, deg is cα,α-diethylglycine, n= - , and o-(pno )bzl is para-nitrobenzoxy] with the deg homo-peptides as fully-extended bridges in a static fluorescence study. to perform a very stringent comparison with our ft-ir absorption and nmr results, the solvent used was chcl . the trend observed in the quenching efficiencies for the shortest members of the series, the (deg) - peptides, steadily decreases from . (n= ) to . (n= ), and . (n= ). this finding is compatible with the conclusion that these three peptides are all essentially fully-extended in chcl solution. however, the quenching efficiencies for the longest deg homo-peptides. . for n= and . for n= , are not further reduced. the inevitable conclusion is that the tetra-and pentapeptides populate at least two different conformations (most probably, the . -helix and the helix), in which the probe dyads are located at different distances. notably, these results fit well with those extracted from our ft-ir absorption and nmr studies in the same solvent of low polarity. we conclude that a combination of terminally aromatic groups, as in the deg homo-peptide series investigated in this work, is not eligible for the fabrication of a stable fully-extended conformation. the surfactant protein c (sp-c) is a -residue highly hydrophobic peptide with two covalently linked fatty acyl chains that adopts a mainly helix structure while associated with the membrane. however, it misfolds into a β-rich structure under certain environmental conditions, which suggests that sp-c is in a metastable state . the slow α→β transitions are likely to be caused by the membranedissociation, deacylation and exposure to the neutral ph of the alveolar subphase, leading to the formation of amyloid aggregates associated with pulmonary alveolar proteinosis. we have been studying the ph-dependent conformational behaviour of both the acylated and deacylated isoforms of sp-c in a membrane mimetics chloroform/methanol/water mixture using a constant-ph molecular dynamics method [ ] [ ] [ ] . our simulations identify the increase of ph as a promoter of peptide-peptide interactions, which may contribute to the α→β transitions observed at neutral and alkaline ph conditions in the experimental results . we also observe that the loss of structure is more prone to begin at the c-terminal region and that the β motifs start to form between the n and cterminal disordered regions of the sp-c. in addition, the presence of the acyl groups results in a slightly higher structural stabilization of the sp-c helix and in a decrease of the n-terminal plasticity at acidic and neutral ph. since a comprehensive conformational analysis and the elucidation of the mode of association between sp-c and the membrane are essential to understand the interactions involved in structure stabilization and in some specific functions of the peptide, we are now studying both sp-c isoforms in a membrane environment. these studies will not only be the most relevant for the in vivo situation but will also answer to some questions left open by the previous work. were rather small. vp plays a very important role in controlling resorption of water by the distal tubules of the kidney and in regulating the osmotic pressure of blood in mammals. vt, while exhibiting both oxytocin and vasopressin activities, has not changed over the last million years until appearance of the mammals. currently, it occurs naturally in the non-mammal vertebrates [ ] . here we report on studies of arginine-vasopressin (cyfqncprg-nh , avp) and arginine-vasotocin ([ile ]avp, avt) in a mixed dodecylphosphocholine (dpc) and sodium dodecylsulfate (sds) micelles. dpc micelles are considered good models of eukaryotic cell membrane [ ] , while the sds micelle improves peptide/micelle solubility [ ] . for these studies we use d nuclear magnetic resonance (nmr) supported with molecular modelling methods. conformation-activity considerations relationships and the role of the peptide-micelle interactions on the structure of the analyzed peptides will be described and discussed. vibrational and chiroptical spectroscopic investigation on diamide peptide models k. knapp, a m. hollósi, a e. vass, a zs. majer a a eötvös lor nd university, institute of chemistry, laboratory for chiroptical structure analysis, budapest, hungary understanding of peptide folding has great importance in many fields of chemistry. since amino acid residues are the simplest building blocks of peptides, the investigation of their conformational properties could help us in understanding the structures of peptides or in designing peptides of specific d structure. describing the conformational building blocks of even the simplest peptide models (e.g., amino acid diamides) can serve as a good starting point to decipher higher-level structural properties of their polymers. among the simplest compounds which include the essential structural elements of polypeptide backbone are the n-methylamides of n'acetylamino acids. these compounds have been the subject of extensive spectroscopic analyses (ftir, vcd, ecd, nmr) [ , ] . the conformational landscape of these model compounds were analyzed by solution phase vcd and ecd spectroscopy and density functional theory (dft) computations. this work reports systematic vcd and ecd spectroscopic studies in different solvents on synthesized diamide models (ac-α/βhomo-xxx-nhch , xxx = ala, pro, phe). vcd and ecd data, combined with quantum chemical calculations performed at the b lyp/ - ++g** and - ++g** level of theory was particularly useful for identifying the preferred dipartimento delle scienze biologiche, università di napoli "federico ii", napoli, italy c dipartimento di scienze ambientali, seconda università di napoli, caserta, italy β-hairpin is an important secondary structural element used by many proteins for biomolecular recognition, and thus is an attractive tool for mimetic design . aβ -hairpin motif consists of two antiparallel strands linked by a turn region. in this work we aimed to stabilize a β-hairpin conformation by an interstrand covalent bridge, made through a click chemistry reaction, which yields , -disubstituted , , , triazole, starting from alkyne and azide reactive groups. in particular, we explored the conformational stability of a series of β-hairpin peptides containing a , -disubstituted , , triazole bridge with variable length. we employed the well structurally characterized trpzip peptide as a convenient b-hairpin model system. as alkyne amino acids were introduced propargylglycine (pra), homopropargylglycine (hpg) or bishomopropargylglycine (bpg), while the following azide amino acids were inserted: lβ-azidoalanine (dap (n )), l-γ-azidohomoalanine (dab (n )), and l-δ-azidoornitine (orn (n )). all peptides were synthesized and purified. successively, the linear unprotected peptides were cyclized in solution by the cu(i)-catalyzed azide alkyne cycloaddition (cuaac) reaction and purified prior to the spectroscopic characterization. linear and cyclic peptides were analyzed by a combination of cd and nmr techniques. have been investigated. their behaviour has been compared with that of proline in homochiral and heterochiral dipeptide sequences including l-or dphenylalanine. nmr and ir studies as well as x-ray diffraction analysis provide evidence that the additional β-phenyl substituent does not disrupt the tendency of proline to occupy the i+ position of a β-turn. moreover, the β-turn conformation is stabilised, with reference to l-pro, when l-cis(βph)pro or l-trans(βph)pro are combined with d-phe, that is, in the heterochiral sequences. the pyrrolidine conformation is significantly affected by the presence of the β-phenyl group. the puckering modes that alleviate most the steric hindrance introduced by the bulky phenyl substituent are preferred. the l-cis(βph)pro residue shows a marked propensity for the cγ-endo/cβexo half-chair arrangement whereas the trans derivative exhibits a higher flexibility, with different pyrrolidine shapes being accessible. interactions between the aromatic ring of (βph)pro and that of the contiguous l-or d-phe residue are observed in all the dipeptides crystallised. in solution, they seem to occur only for the heterochiral sequences. this intramolecular aromatic-aromatic interaction may be responsible for the higher β-turn ratio observed for such sequences and also seems to affect the conformational preferences of the pyrrolidine ring. in fmoc chemistry the trt and acm groups are widely accepted as a protecting group for cys. upon incorporation of these cys derivatives onto the growing peptide chain, however, considerable base-catalyzed racemization of cys is known to always occur at the activating and coupling steps with phosphonium or uronium reagents such as pybop or hbtu, respectively. in addition to this, racemization of the c-terminal cys esterified to resins also occurs during the repetitive n α -fmoc deprotection using piperidine. in order to find an s-protected cys derivative applicable for fmoc chemistry that can efficiently suppress racemization of cys both when its incorporation is conducted by phosphonium or uronium reagents and when it is linked to a resin via ester linkage, we introduced the methoxybenzyloxymethyl (mbom) group into the thiol function on cys. the mbom group was found to substantially supress racemization of cys ( . %) during its incorporation mediated by phosphonium or uronium reagents in comparison with the trt and acm groups ( . % and . %, respectively). even after a -h treatment of the peptide resin, in which its c-terminal cys was linked to a trt-type resin, with % piperidine in dmf, the mbom group caused a significant reduction in the level of racemization with the c-terminal cys ( . %) while trt and acm groups led to a considerable extent ( . % and . %, respectively). next, we demonstrated the usefulness of the mbom group on cys by applying it to ncl chemistry involving the coupling of a peptide thioester and a cys-peptide. in the synthesis of the latter peptide, however, a diastereoisomer with racemization of the nterminal cys tends to be difficult to separate from the intact peptide as its chain length increases. thus, this measure using fmoc-cys(mbom) can facilitate the avoidance of ambiguities in the quality of the synthesized peptides. labeled peptides are important tools in chemical biology to obtain information on their biological function in, for example, cellular communication processes as well as to study the molecular basis of diseases. the functionalization of bioactive peptides with biophysical reporter groups like strongly absorbing chromophores, is most often performed via post-synthetic chemoselective biocojugation reactions, involving amino, thiol, or azide specific reactions. an alternative approach for the installation intense chromophoric organic molecules, is the coupling of nonchromophoric precursor molecules to obtain chromophoric properties in the final product. recently, we have shown that the [ + ] cycloaddition-cycloreversion reaction between peptide-functionalized electron-rich alkynes and electrondeficient cyanovinyl derivatives results in the formation of a new class of π-conjugated peptidic donor-acceptor chromophores. , the chromophoric properties of the final product can be tuned by varying the p-electron conjugation pattern. herein, we describe the synthesis of the versatile building blocks, n-a-( -ethynylphenyl)-n-a-(methyl)-glycine and n-a-( -( , , -tricyanovinyl)benzoyl)-glycine, their subsequent application in spps to functionalize peptides with d-p-a chromophore precursor molecules, and their uvvis absorption characteristics. as a typical example, alkyne-functionalized aβ peptides, val-ala-ile and lys-leu-val-phe-phe-ala-glu, have been reacted with a series of cyanoolefins to obtain far-red intense imaging chromophores for amyloid-rich peptide aggregates. a novel method for regioselective disulfide formation in mini-proteins by kinetic oxidation control t. lindner, u. haberkorn, w. mier department of nuclear medicine, university hospital heidelberg, im neuenheimer feld , heidelberg, germany rigid frameworks are essential for the development of peptide-based alternatives to immunoglobulins. [ ] disulfideconstriction covalently stabilizes tertiary structures, which allows further miniaturization of protein-derived drugs by chemical synthesis. while the amino acid assembly steps in solid phase peptide synthesis are highly efficient, regioselective formation of multiple disulfide bonds is still a challenging and time demanding task. even after applying laboriously optimized folding conditions and extensive purification, a successful synthesis of the correctly folded isomer is not guaranteed. [ ] to circumvent these problems, a procedure that takes advantage of the different reactivity of free vs. acmprotected thiols during iodine oxidation was developed. this protocol to sequentially form two disulfide bridges in a one-pot reaction was applied to different peptides, such as (a) min , a mini-protein scaffold for phage-display assisted drug design; [ ] (b) α-conotoxin imi, a neurotoxic peptide isolated from the venom of marine cone snails; [ ] and (c) endothelin- , a peptide involved in blood pressure regulation. [ ] the presented technique tolerates precarious residues within the to-be-folded-peptide, such as fluorescent dyes or metal chelators as used for bioactivityassays. compared to the individually optimized literature procedures, this novel approach offers significant improvements: overall yields > % are obtained in first attempts and stepwise formation of the disulfide bridges is performed within a few hours instead of days. in recent thirty years, c-terminal modified peptides have been proved to have greater potential as apis (active pharmaceutical ingredients) due to their increased chemical and enzymatic stability and improved pharmacodynamic properties - . a prominent example, octreotide - , an octapeptidoalcohol, has witnessed as a potent anti-cancer agent targeted for gastro-entero carcinomas. in view of synthetic methodology, peptidoalcohol can not be directly prepared by standard spps protocol becouse of the c-terminal structure released from resin are not alcohol but always peptidoacid or peptidoamide. to overcome this problem, a novel protocol of shortened n- coupling cycles on merrifield resin and then the ammonolysis of peptedyl resin by an aminoalcohol as the c-terminal residue getting peptido-alcohol as targetting product has been devoloped in our lab. because of the cleavage treatment of peptidyl merrifield resin is not under acidic condition, such as hf or tfmsa, but ammonolysis, some side-chain producting groups(spg) related to boc chemistry like bzl, clz, tos…; must be avoided in sequence assembly. therefore a hybrid orthogonal protection (hop) of boc/fmoc protocol was adopted for the sake of producing naked peptidyl (without any spg) resin before ammonolysis. fifteen peptidoalcohols with different terminal alcohols were conveniently prepared, most of them released form resin with very good yields. due to its cyclic structure, proline is the coded amino acid with a more restricted conformational flexibility. the incorporation of additional groups into the pyrrolidine ring is a useful means to produce new amino acids that combine the conformational properties of proline with sidechain functionality. this is the case of β-phenylproline, (βph)pro, that can be regarded as a proline-phenylalanine hybrid in which the orientation of the aromatic substituent is dictated by the conformation of the five-membered ring and the cis or trans configuration of the phenyl group relative to the carbonyl moiety. accordingly, cis(βph)pro and trans(βph)pro combine the conformational properties of proline with an aromatic side-chain functionality that is rigidly oriented with respect to the peptide backbone, and this may be useful in the design of biologically active peptides and other applications relying on specificallyoriented side-chain moieties. we have developed synthetic procedures for the preparation of the cis(βph)pro and trans(βph)pro stereoisomers in enantiomerically pure form. the methodology is based on the preparation of racemic precursors of each amino acid and their subsequent hplc resolution on chiral columns. multigram quantities of the target amino acids have been isolated in optically pure form and suitably protected for use in peptide synthesis. the importance of peptide cyclization for studying peptide conformation, creating new structures, or for developing peptide therapeutics is well established. in particular, sidechain lactam bridges linking two amino acid residues that are several residues apart in the linear sequence or headto-tail backbone peptide cyclization enable rigidification of the structure and improvement of in vivo stability. native chemical ligation (ncl) is now an established method for producing backbone-cyclized peptides or proteins. the application of ncl to the synthesis of sidechain cyclized peptides is less frequent. head-to-side-chain cyclization by ligating a c-terminal thioester with a cys residue located on a lysine side-chain was used by few authors. the alternative tail-to-side-chain cyclization mode is rare, probably due to the difficulty of installing a thioester group on amino acid side-chains such as aspartic or glutamic acids the reaction of a bis( -sulfanylethyl)amido (sea on ) group with an n-terminal cysteine residue in water and at neutral ph results in the formation of a native peptide bond. [ ] oxidation of sea on results in a cyclic disulfide called sea off having a , , -dithiazepan- -carbonyl structure. [ ] sea off is a self-protected form of sea on . we show here that bis( -sulfanylethyl)amido side-chain acid(dab), ornithine and lysine were selected as building block; a and n,n'-cbz- -amidinopyrazole ( b) were selected as guanidinylating reagents for specific situation. for synthesis of n-terminus local cyclo-guanidine peptide, designated peptides were assembled on acid labile solid support such as rink amide resin by fmoc strategy. then either fmoc-dab(boc)-oh, fmoc-orn(boc)-oh or fmoc-lys(boc)-oh was incorporated respectively at n-terminus. fmoc was removed followed by guanidinylating by b and then peptide was cleaved by acid. by neutralizing with nmm in acetonitrile solution, side chain amino group and a-guanidine would form , or membered local cycloguanidine. the remaining cbz could be removed by hydrogenation. for synthesis of backbone side chain cyclic peptide, bis-fmoc-daa was introduced in the peptide previously on resin followed by removal of fmoc. selective guanidinylate side chain aminogroup by a followed by peptide assembling with an insertion of orthogonal protected daa at - aa apart from first daa. for synthesis of a-nh sidechain cyclic peptide, first daa should be introduced with orthogonal protected form. b was used to guanidinylate a-nh . after cleavage and neutralization of those two kinds of intermediates, guanidine-bridged marco-cyclic peptide was formed. the resin is also a multipurpose tool for the synthesis of carboxylic acids, esters and thioesters. when the synthesis is completed, the fully protected peptide hydrazide resin is oxidized with either n-bromosuccinimide (nbs) or copper(ii) acetate in pyridine. the resulting acyl diazene resin is then cleaved by peptide displacement at the c-terminus with amine. the fully deprotected peptide amide is finally obtained by treatment with trifluoroacetic acid (tfa). in our approach, we used a -fmoc-hydrazinobenzoyl am novagel resin to synthesize a peptide-substituted amide in the c-terminus. first, the oxidative cleavage was carried out with nbs in pyridine and a nucleophile [a protected (aminomethyl) benzimidamide (amba)]. however, the yield of the reaction was very poor. in the next step, we applied copper(ii) acetate in the presence of pyridine and amba. following optimization, the efficiency of the process was significantly improved. herein we discuss the conditions needed to obtain a reasonably high efficiency of the oxidative cleavage in the synthesis of our c-terminal modified peptides using the aryl hydrazine resin linker. blood vessels on tumor tissues, similarly to integrin receptors. this observation suggests cd as a selective target for targeted delivery of drugs and nanoparticles to tumor neovasculature using ngr peptides as homing motif. in our work, new cyclic-ngr peptides containing a thioether linkage were prepared. the influence of their structure on the speed of succinimide ring formation and deamidation was evaluated and compared with the previously published data on cyclic-ngr derivatives containing amide bond or disulfide bridge in the cycle (c[kngre]-nh and c[cngrc]-nh ). to avoid the deamidation under the conditions used for cyclization, the synthetic routes were optimized. the influence of the ph, ionic strength and temperature of the solution on their chemical stability was investigated. the structure of the cyclic peptides was investigated by circular dichroismand nmr-spectroscopy. receptor binding ability and the influence of the cyclic peptides on the cell adhesion and motility were also evaluated. this work was supported by grants from the hungarian national science fund (otka nk and k ) and the national innovation office (bio_surf, om- / ). clickable peptides and their attachment to oligonucleotides m. wenska, m. alvira, r. strömberg department of biosciences and nutrition, karolinska institutet, novum, se- huddinge methodology for the ready conversion of peptides into "clickable" azido-peptides with the possibility of selecting either n-terminus or c-terminus connection is presented. synthesis of peptide-oligonucleotide conjugates (poc's) include conjugates of oligonucleotides with peptides known to be membrane penetrating and nuclear localization signals. a general procedure, based on a new activated alkyne linker, for the preparation of poc's has been developed. with this linker, conjugation is effective at room temperature in mm concentration and submicromolar amounts. this is made possible since the use of a readily attachable activated triple bond linker speeds up the cu(i) catalyzed , -dipolar cycloaddition ("click" reaction). the main scheme for conjugate preparation involves sequential conjugation to oligonucleotides on solid support of i) an h-phosphonate based aminolinker ii) the triple bond donor p-(npropynoylamino)toluic acid (pata) and iii) azido-functionalized peptides. the method gives excellent conversion of oligonucleotide to the poc on solid support, and only involves a single purification step after complete assembly. the procedure which makes use of a low concentration of copper ions leads to a product with very little copper left (similar or less than in drinking water). the synthesis is flexible and can be carried out in non-specialist laboratories without the need for specific automated synthesizers since it has been designed to utilize commercially available oligonucleotide and peptide derivatives on solid support or in solution. comparison of alternative deprotection reagents to piperidine for the synthesis of a poly-alanine peptide on the tribute® peptide synthesizer m.a. onaiyekan,* j.p. cain, c.a. chantell, m. menakuru protein technologies, inc. tucson, az, usa in peptide synthesis, piperidine is a common agent for fmoc removal. however, piperidine is a controlled substance which requires special handling and cannot be used in some countries. therefore, it would be useful to identify alternative deprotection reagents to piperidine for fmoc removal. it is well known that poly-alanine sequences have a high propensity to aggregate after the fifth residue. in this application, (a) k-oh was synthesized using the tribute®'s intellisynth uvmonitoring and feedback system to compare the efficiency of fmoc removal by piperidine vs. three alternative bases (pyrrolidine, cyclohexylamine, and tertbutylamine) in the last cycles of the synthesis. it was found that pyrrolidine produced a higher purity product with fewer deprotection repeats and shorter deprotection times per cycle than piperidine, proving it to be a highly efficient, viable alternative to piperidine for fmoc removal. the endogenous tripeptide gpe also nammed "glypromate" is made up by the three n-terminal residues (glycine-proline-glutamate) of the insulin-like growth factor (igf ). this tripeptide is a partial glutamate antagonist and showed good results in different neuroprotective in vitro and in vivo experiments. , gpe also binds to glial cells regulating neurotransmitter levels in the brain. , however, gpe suffers from poor lipophilicity and a short half-life in vivo. that's why there is a need for more lipophilic and protease resistant analogues of gpe. in this poster we present the synthesis of trifluoromethylated analogues of gpe based on the or -cf -pseudoproline residues. introduction of fluorine atoms on bioactive compounds is known to deeply modify their physico and biochemical properties increasing lipophilicity and resistance to protease. thus, developing a trifluoromethylated analogues, we intend to increase the bioavailability of gpe, keeping the benefit of its neuroprotective properties. our research team is strongly involved in the synthesis of trifluoromethylated alpha-amino acids. recently we published the synthesis of -trifluoromethyl- , oxazolidines derived from fluoral and (l)-serine and we demonstrated that these five membered ring -cf pseudoprolines are hydrolytically stable and can be considered as proline analogues. that's the reasons why we are interested to replace the proline residue of gpe by those trifluoromethylated compounds. the development of original coupling conditions and the detailed synthesis of two pseudoprolines analogues of gpe will be presented in this poster. modifications. in combination with automated spps, unprecedented access to large peptides and small proteins for biological research has been achieved. we demonstrate the application of this methodology to the synthesis of a variety of peptides on the prelude® peptide synthesizer. exploring the space of fluorine-labeled α-amino acids for solid state f-nmr structure analysis of peptides: rational design, synthesis and applications p. solid state f-nmr is a powerful method to study membrane-active peptides, as it can reveal their conformation, orientation and dynamics when embedded in biomembranes. for this purpose the native peptide has to be selectively labeled with a suitable f-containing amino acid at several different positions. the resulting battery of singly f-labeled analogues is then analyzed by solid state f-nmr. the main limitation to this approach currently lies in the poor arsenal of available f-labels. we have therefore rationally designed and synthesized several specific amino acids bearing a cf -reporter group, which fulfil all strict criteria to a "proper" f-label. , to allow a geometry-based structure calculation, the cf group has to be rigidly attached to the peptide backbone. we thus rigidified the side chain using either a [ . . ]bicyclopentane moiety, a cyclobutane ring, or the intrinsic proline framework. this way, suitable cf -labeled analogues were created as substitutes for bulky hydrophobic amino acids (leu/ile/val/met), for aromatic residues (phe), for polar side chains (ser/thr), and for proline (pro). by now we have applied the developed f-labels for a comprehensive structure analysis of more than ten different membrane-active peptides (gramicidin s, pgla, mag , kigaki, sap, temporin a, bp , etc). recently, several new activators have been introduced into the market, and they were evaluated along with some older activators for their ability to synthesize a range of peptides with shorter and longer reaction times on the symphony® peptide synthesizer. it was found that hdmc, pyclock, comu, hctu, and hatu worked well at shorter reaction times ( x min), but pyoxim and tffh only worked well at longer reaction times. the performance of pybop at shorter reaction times was poor only for more difficult sequences. these results are important for selecting an appropriate activator for fast spps applications. the plant cyclotides form the largest family of cyclic peptides . they contain a signature motif referred to as the cyclic cystine knot, which is derived from the cyclic backbone and three inter-knotted disulfide bonds. intriguingly, cyclotides can be boiled, treated with chemicals or enzymes without disrupting their overall fold. thus, they are sometimes labeled as ultra-stable proteins. in addition, cyclotides are tolerant to mutations, and as a scaffold they can successfully accommodate foreign bioactive epitopes of variable sequences . cyclotides share many of these properties with another disulfide containing cyclic plant peptide, the sunflower trypsin inhibitor (sfti- ) . emerging evidence indicates that cyclotides and sfti- are valuable not only as peptide stabilizing scaffolds; in combination with their cell penetrating properties, these disulfide rich cyclic peptides have significance as intracellular drug carriers. although both peptides are genetically encoded, studies to ascertain the exact mechanisms of their biosynthesis are currently on going. thus, the synthesis of cyclotides and sfti- are currently restricted to chemical means. we have recently adapted a fmoc-spps method for cyclic peptide synthesis, via n-acylurea intermediates with the assistance of microwave irradiation. this method is a safe and convenient alternative to boc-spps and has the ability to be automated conveniently. using this method, parent scaffolds as well as several cyclotide and sfti- analogues with potential antimicrobial and matrix metalloprotease activities were synthesized. with the rising interest in the cyclization concept as a tool to impart stability on unstable peptides, the cyclic peptide synthesis method adapted herein is anticipated to have numerous applications. fixed configuration. the nonnatural oligomers have an extended conformational space and are supposed to adopt non-canonical secondary structures . in addition, the backbone modification makes these molecules more stable towards proteolytic degradation. the majority of proteins in nature are post-translationally modified, and the most abundant modification is the protein glycolysation, which introduces wide structural variety to proteins. glycoproteins have an important role in the biological recognition process, such as immunodifferentiation, cell adhesion, cell differenciation and regulation cell growth . new aza-β -amino acids bearing either an azide instead of amine on lys and orn chain or an alkyne group will be described and used in solid phase synthesis to finally performed a click chemistry to cyclize pseudopeptides or to introduced a glycosylated function . true for a series of peptides that display strong corticotropin releasing factor (crf) antagonistic activity. seminal studies by rivier et al. have shown that the incorporation of a lactam bridge in the crf-sequence resulted in an enormous increase in activity and potency, due to stabilization of the bioactive a-helical conformation of the peptide; and the newly designed peptide was called astressin. based on the astressin sequence, we started a truncation and deletion study to arrive at astressin analogs with a reduced size but still remain active as crf antagonists. this study resulted in the smallest active crf antagonist, astressin( - ). this sequence was further optimized by the introduction of novel covalent constraints, other than the well-known lactam bridge. as a first approach, the alkene/alkane bridge, which can be introduced via ring-closing metathesis via alkenesubstituted amino acid side chains, and as a second approach, the triazole bridge ('click' macrocyclization), via either a cu(i)-or a ru(ii)-catalyzed cycloaddition reaction between azide-and alkyne-derivatized amino acid residues were explored. herein, we will present the details of the synthesis of the alkene-, azide-, and alkyne-functionalized amino acids, their use in spps, and the optimized approaches for macrocyclization. furthermore, the peptides have been characterized by hplc, nmr, lcms, and studied by circular dichroism spectroscopy to obtain insight into the helical propensity of the peptides in relation to the cyclic constraint. the synthesis of a nitronyl nitroxide, c α -tetrasubstituted αamino acid (a class of sterically restricted amino acids that promote the formation of peptide β-turns and helical structures) was achieved by derivatisation of racemic amino- -cyano-indan- carboxylic acid [aic(cn)]. racemic boc-aic(nn)-oh was prepared by bis(alkylation) of ethyl isocyanoacetate under phase transfer conditions with , -(bis)bromomethyl benzonitrile as alkylating agent, followed by acidic hydrolysis, n α -boc protection, and saponification of the ester function. resolution was achieved through formation of the diastereomeric amides of (s)-phenylglycinol with chromatographic separation and mild acidic hydrolysis. reduction of the nitrile group to an aldehyde was carried out with raney nickel in the presence of sodium hypophosphite. condensation with , -diamino- , -dimethylbutane gave the corresponding tetramethylimidazolidine, which was oxidised with chloroperbenzoic acid to the desired nitronyl nitroxide. the uv-vis absorption and epr spectra of the amino acid were recorded and its magnetic properties were examined. in order to develop the synthesis of this peptide using the fmoc solid-phase peptide synthetic methodology, orthogonally protected β-hydroxyaspartic acid was needed. more precisely we wish to dispose of ( r, r)-n -fmoc- -tbdm-silyloxy-aspartic acid α -allyl ester instead of the recently reported dmab ester - indeed, in preliminary assay using this protective group we experienced difficulties during the final cyclisation step . the synthesis was developed starting from inexpensive l(+) dimethyltartrate and extended to the others stereoisomers of the β-hydroxyaspartic acid. structure. for that, we chose to replace proline by silaproline to afford polysilaproline. this study shows the comparison of two polyamino acids: polyproline and polysilaproline polymers. homopolypeptides were synthesized by polymerization of corresponding amino acid n-carboxyanhydride . multicomponent reactions (mcrs) represent a chemical process involving at least three reactants for the formation of several covalent bonds in one operation . by definition mcrs are chemo-and regioselective, convergent stepefficient procedures and take place with high atom economy. the copper(i)-catalyzed , -dipolar cycloaddition of organic azides and terminal alkynes (cuaac) reported by meldal and sharpless has been involved in various fields of chemistry and biochemistry research. however only few reports describe the implementation of cuaac and mcrs. , recently our research focused on a novel threecomponent reaction based on a cu(ii)-triggered aminolysis of peptide hydrazide resin and an azide-alkyne cycloaddition sequence. copper(ii)-induced oxidative aminolysis of hydrazides generates cu(i), catalyst of the azide-alkyne cycloaddition. this feature was exploited to design a solid phase detaching three-component reaction. the mcr process requires a peptide hydrazide resin, an amino azide linker and an alkyne, resulting in the formation of peptide modified at the c-terminus through an amino , , -triazole linker. this method can potentially be applied to the synthesis of a large variety of peptide derivatives starting from fmoc-spps assembled peptidyl resins. furthermore, it is not practical to compare hplc spectra from different resin samples (e.g., before and after reaction) directly. a comparison by analyzing the same (mg) amount of resin would involve tedious sample preparation that is extremely error-prone and would be impractical because factors resulting from the increase or decrease of the molecular weight of the resin-bound compounds may have a significant influence on the results. the use of internal reference compounds allows rapid assessment of reactions performed on solid supports. the internal reference compound is bound to the resin together with the substrate and cleaved with the products after completion of the reaction. commercially available compounds can be used for this purpose, or likewise, the reference compound can be generated from the substrate by partial capping of a functionality. the peak integration of the reference compound in the hplc-uv spectra can be correlated directly to those of the rest of the compounds present in the reaction mixture and therefore a quantitative interpretation of the spectra with respect to conversion and yield is possible. here we demonstrate the proof of principle as well as the accuracy of this method. modifier proteins such as ubiquitin are conjugated to protein substrates in cells and thereby mediate various biological processes. of high interest, is the ubiquitin fold modifer (ufm , residues) which has structural similarity to ubiquitin but has no sequence similarity. unlike ubiquitin, ufm has not been extensively studied and little is known about its biological role. to understand ufm 's biological functions, access to pure, homogeneous natural and modified ufm protein is essential. chemoselective ligation techniques are suitable for providing such proteins. recently, a variation of the α-ketoacid-hydroxylamine (kaha) ligation was developed, which utilizes the chemoselective reaction between a c-terminal peptide αketoacid and a n-terminal -oxaproline. this modified form of the kaha ligation furnishes a native peptide bond and a homoserine residue. this ligation is useful for the synthesis of proteins from two unprotected protein segments in aqueous buffers. for the synthesis of larger proteins, a sequential ligation strategy is necessary. using ufm as the model system we have developed a sequential ligation procedure using kaha ligation with -oxaproline. applying the new sequential ligation strategy we have prepared ufm by total chemical synthesis. we have also prepared a cterminal thioester surrogate of ufm protein, which is suitable for conjugation to proteins of interest. the syntheses required the development of a bifunctional peptide segment bearing an α-ketoacid and an orthogonally protected -oxaproline. the preparation of the protein segments, their intermediates, the deprotection, and sequential kaha ligations towards the syntheses of ufm protein and c-terminal modified thioester ufm protein will be discussed. affinity and biological activity, we have designed and synthesized new analogues by multiple n-methylation of hut-ii( - ) backbone amide bonds. all the peptides were performed by a novel synthetic approach, in which the introduction of n-methyl groups occur during regular solidphase peptide synthesis. on these new ligands we evaluated the binding affinity and biological activity at the ut receptor and performed preliminary nmr conformational studies. since that time a number of different machines have been used to automate peptide synthesis. modern machines are following two general setups; the so called "single approach" and the "parallel approach". in the single approach, the machine is developed to synthesize one or few peptides simultaneously. the user is able to optimize the synthesis conditions on each single peptide and each single coupling step. the maximum product quality regarding purity and yield is the major task of this approach. in the parallel approach, the machine is developed to synthesize a huge number of different peptides in the same single setup and time-frame. the user always has to find a synthesis protocol appropriate for the needs of each peptide to reach the maximum quality, knowing that there will be always a number of failed peptides. as a result you will find both types of peptide synthesizers in laboratories all over the world: the single machine, for the complicated peptides, and the parallel machine, allowing generation of multiple peptides with standardized protocols for each. the tetras is the first instrument combining the advantages of both machine types and allows the user to synthesize up to different peptides in parallel. each peptide can have its own individual synthesis protocols, separate of all others. the user can combine different synthesis scales, peptide lengths, and activator reagents in one run. finished peptides can be removed and new peptides can be started while the tetras is still running. the tetras allows the user to establish an uninterrupted production shop using one instrument only. siemion, i. z.; peptide res the peptides: analysis, synthesis and biology monitoring peptide folding in membrane-active peptides: a time-resolved spectroscopic study e. gatto a cordopatis p. st european peptide symposium sar studies of triazolyl-containing cyclopeptides: a defined -turn structure increases potency and selectivity to melanocortin receptor subtypes c. testa, a,b proc. natl. acad. sci proc. natl. acad. sci usa multicomponent reactions microglobulin: a "difficult" protein s. abel, m. beyermann the protein ß -microglobulin constitutes the noncovalently bound light chain of the major histocompatibility complex class i (mhc) and plays an essential role in the dialysisrelated amyloidosis. [ , ] to examine the amyloid fibrils of the ß -microglobulin (ß -m) via infrared spectroscopy we intended to synthesize -c-labeled ß -m. [ ] due to the two cysteine residues in positions and we used the native chemical ligation (ncl) strategy for assembling the -mer protein. this necessitates the synthesis of three segments which was accomplished on solid phase using the fmoc/t-bu chemistry. the preparation of the segments had to be optimized with respect to aggregation, aspartimide and piperidide formation, trifluoracetylation, and s-tert-butylsulfonium formation. additionally, ncl steps had to be optimized, because of "internal" thioester formation, dimerization and the formation of side-products of the activated n-terminal segment peptaderm inc., krakowskie przedmie cie str. , warsaw, poland immunosuppressors, such as cyclosporine a (csa) and tacrolimus®, are routinely used in prevention of graft rejection after organ transplantation and in therapy of some autoimmune diseases, including skin inflammation. a naturally occurring in linseed oil cyclolinopeptide a (cla, c(-pro-pro-phe-phe-leu-ile-ile-leu-val) possesses a strong immuno-suppressive activity, comparable at low doses with that of csa , but is much less toxic. we synthesized new cla analogs, containing instead of one proline residue its six-membered mimics, pipecolic acid (pip): c(-pip-pro-phe-phe-leu-ile-ile-leu-val) ( ) and c(-pro-pip-phe-phe-leu-ile-ile-leu-val) ( ). the incorporation of pipecolic acid residue led to different conformational behavior of the nonapeptide cycle. nmr experiments in cdcl solution showed that cla analogue with the pipecolic acid residue in position was much more flexible than cyclopeptide . the new peptides were devoid of toxicity up to μg/ml with regard to human peripheral blood mononuclear cells (pbmc), did not inhibit tumor necrosis factor alpha production in blood cell culture, but exhibited dosedependent, anti-proliferative actions for phytohemagglutinin a-activated pbmc. since peptide was more potent it was tested for growth inhibition of l- lymphatic leukemia. the peptide was found to strongly inhibit the cell growth even at low concentration ( % inhibition at μg/ml). hiv- has emerged as the largest and the most devastating public pandemic in our days, affecting approximately million people worldwide . development of an effective, safe and preventive hiv vaccine remains an urgently needed priority. epitopes for hiv-specific antibodies in elite controllers, a subgroup of long term non progressors, encompassing segments of mper of gp and for the v loop of gp were identified using the phage display technology . immunization experiments with epitopes conjugated to an artificial sequential oligopeptide carrier (soc ), formed by four repeats of the tripeptide lys-aib-gly in tandem, or to the palmitoyl group are currently in progress. all syntheses were performed on a rink amide resin following the fmoc technology. conjugation of epitopes to the soc carrier was realized via a chemoselective ligation approach, which generates an oxime bond between the h n-o-groups of the modified lysine residues and the aldehyde group of each epitope institute of immunology and experimental therapy, polish academy of sciences, - wrocław, poland peptaderm inc., krakowskie przedmieście , - warszawa, poland nonproteinogenic amino acids have been a tool to modify the structures of natural peptides since a long time . bioactive peptides involved in a physiological and biochemical processes cannot be applied in the therapy because of their instability in physiological conditions. that's why the synthesis of their stable active analogues is a challenge for medicinal chemistry nowadays. -trans-hydroxyproline (hyp) is an important building block of natural collagen. it is responsible for the stabilization of collagen super helix, forcing the trans amide bonds configuration with preceding amino acids . at the same time the impact of trans- -hydroxyproline on the conformation other than the collagen peptide chains of biologically important compounds is little known. it is known that immunosuppressive activity of cla is comparable with cyclosporine a and is associated with the presence of the tetrapeptide fragment pro-pro-phe-phe containing pro-pro cis amide bond. now we present synthesis, conformation and biological activity of new analogues of cyclolinopeptide a (cla), containing -transhydroxyproline instead of proline residues in position or . we expected that the introduction of the hydroxyl group in the pyrrolidine ring might influence the biological activity and conformation of the native peptide due to its hydrophilic character and hydrogen bonding ability. the linear precursors of modified cla analogues were prepared manually by standard solid-phase procedure "step by step" on wang resin using fmoc/tbu strategy and tbtu as coupling reagent. the cyclizations of linear peptides have been made under high dilution conditions by means of edc/hobt coupling reagents. the biological activity of newly synthesized compounds as well as the conformational study will be evaluated. dip. di scienze ambientali, seconda università di napoli, caserta, italy nmr spectroscopy is a powerful method to perform structural studies on peptides. to completely fulfill the potential of nmr, peptides labeled with stable isotopes ( n, c, h) are essential. peptides are easily prepared on solid-phase but chemical synthesis becomes prohibitively expensive when applied to the incorporation of isotopes. an alternative cost-effective strategy is the recombinant expression of peptides in e. coli as fusion constructs with carrier proteins. the main problem of this approach is the need of chemical reagents or proteases to cleave the target peptide from its fusion partner after purification. proteases may determine the heritage of extrasequence amino acids at the peptide n-or c-terminus, while chemical reagents require harsh reaction condition that may modify target peptides. an interesting solution is represented by the use of inteins as fusion partner. inteins are protein elements that can catalyze their self-excision from a flanking sequences in mild conditions, by adding nucleophilic agents such as thiols or simply by shift of ph and temperature, bypassing the use of proteases or chemical reagents. we used the self-cleaving mxegyra mini-intein as fusion partner for the preparation by recombinant means of two isotope labeled peptides, hplw and qk. , the two peptides target vascular endothelial growth factor receptor (vegfr) and have been described to modulate vegf-dependent angiogenesis. our expression and purification scheme allows to obtain homogeneously isotope labeled peptides. the availability of isotope labeled hplw and qk opens the way to nmr studies aimed to characterize the folding dynamics of the two peptides and their structures in complex with vegfr. an nmr method to discriminate between the fullyextended and different helical conformations in a spacer peptide c. peggion*, m. crisma, f. formaggio, c. toniolo icb, padova unit, cnr, department of chemistry, university of padova, padova, italy the ideal fully-extended, α-peptide conformation, also known as . -helix, is characterized by φ = ψ = ω = °t orsion angles. the repeating motif of this foldamer is a pentagonal (pseudo)cyclic structure (called c ), stabilized by an intraresidue h-bond. the n-h and c=o groups in the . -helix are not involved in intermolecular h-bonds. multiple c conformations were observed in homopeptides made up of c α,α -dialkylated glycines with both side chains longer than a methyl. this is the case for c α,αdiethylglycine (deg), the residue studied in this work. it is known that deg homo-peptides can adopt the . -helix or the -helix depending on environmental factors and nand/or c-terminal moieties. , in this communication, we introduce an nmr method to discriminate between the . -helix and the -helix based on the observation of cross-peak intensities in the noesy human serum amyloid a (saa) is a highly conserved apolipoprotein produced by the liver under inflammatory conditions accompanying e.g. atherosclerosis, cancer and amyloidosis [ ] . it is also known that saa α isoform has the amyloidogenic properties [ ] . till now it is little known about structure of human saa, as it hampers structural studies due to its facile aggregation. the analysis of protein sequence and cd data together with theoretical studies revealed a typical globular structure of the protein [ ] . the c-terminal sequence of saa contains three proline residues, which probably are responsible for the unordered structure. recent in vitro studies involving saa and human cystatin c (hcc) revealed direct interactions between the ( - ) fragment of saa and the ( - ) sequence of hcc. the results of elisa test for the ( - ) saa fragment have shown that it binds to hcc very well. the nmr studies for the wild ( - ) sequence found an unordered structure in phosphate buffer. based on these data we decided to check how the point mutations pro→ala in ( - ) saa fragment could influence the peptide's structures. we synthesized four peptides with pro→ala point mutations and we performed cd experiments at different conditions. the results show that two of them contain disordered structure and two α-helical structures. in this project we analyze the solution structures of these peptides at the atomic resolution using d nmr supported with molecular dynamics. design and conformational analysis of stapled peptides mimicking cullin binding region to kctd . i. de paola, a l. pirone, a e. pedone, a s. di gaetano, a l. vitagliano, a r. fattorusso, b g. malgieri, b l. zaccaro*, a acknowledgement: this study was supported by eu within the european regional development fund (poig. . . - - / - ). model of angiotensin ii bound to the at receptor in the lipid bilayer environment m.t. matsoukas, t. tselios* department of chemistry, university of patras, gr- , patras, greece the renin-angiotensin system () plays a major role in blood pressure regulation. a sequence of enzyme reactions leads to the release of angiotensin ii which interacts principally with the type- angiotensin ii receptor (at ), a -residue, which belongs to the g protein-coupled receptor family. in the present study, the human at d model was constructed using modeler for the sequence alignment and loop refinement tools. on this basis, the crystal structure of bovine rhodopsin, (pdb code u ), was used as a d template. the gromacs software and amber sb forcefield were utilized for molecular dynamics calculations [ ] in order to evaluate the binding mode of angiotensin ii. the role of the critical amino acids of the binding site v , n , l , a , k , s , h , n and y is being studied. moreover, newest information on the role of the nd extracellular loop by unal et. al. [ ] have been implemented on the model, therefore we propose the contribution mechanism of the residues f -q for binding of angiotensin ii to the at receptor for activation and signaling. a. stavrakoudis department of economics, university of ioannina, greece one key step in the immune response against infected or tumor cells is the recognition of the t-cell receptor (tcr) by class i major histocompatibility complexes. it has been found [ , ] that such peptide/mhc complexes can interact with antibodies as well. this happens mainly in the central part of the peptide in class i complexes [ ] , or at the cterminal of class ii complexes [ ] . in some cases, the same peptide/mhc complex has been found to interact with both tcr and antibodies [ ] . in these study a series of supermolecular complexes have been studied with stateof-art molecular dynamics simulations [ ] the dipeptide kyotorphin (tyr-arg, kyo) plays a role in pain modulation in the mammalian central nervous system (cns), and is one of the most investigated neuropeptides. the tyr-arg motif exists widely throughout the brain not only as kyotorphin, but also as the n-terminal part of several endogenous analgesic peptides , . also, this peptide is very rapidly degraded by aminopeptidases . one of the successful strategies in the design of neuropeptides with enhanced stability and improved delivery to the cns is that with the use of non-protein amino acids, like canavanive (cav), a structural analogue and antimetabolite of arginine (arg trichogin ga iv (noct-aib-gly-leu-aib-gly-gly-leu-aib-gly-ile-lol, in which noct is n-octanoyl and lol is leucinol) is an antimicrobial lipopeptaibol, a unique group of membraneactive compounds of fungal origin, characterized by a high content of the nonproteinogenic ca,a-disubstituted glycine aib (a-aminoisobutyric acid). owing to the gem-dimethyl substitution on the c a atom, aib exhibits a strong propensity to induce β-turns and /α-helical conformations in peptides. we have previously reported on a fluorescent analog of trichogin ga iv, the primary structure (and acronym) of which are: fmoc-aib-gly-leu-aib-gly-gly-leu-toac-gly-ile-leu-ome (f t ) where fmoc is fluorenyl- -methyloxycarbonyl, toac is , , , -tetramethylpiperidine- -oxyl- -amino- -carboxylic acid, and ome is methoxy. the double substitution of an energy donor (fmoc) at the n-terminus and an acceptor (toac) in the trichogin sequence enabled us to make use of time-resolved optical spectroscopies, spanning from the nanosecond to the microsecond time regime, to investigate the conformational propensity and the dynamical features of f t . experimental and computational results indicated that the d-structural and dynamical properties of f t are characterized by a transition from an elongated helix to a more compact conformation mimicking a helix-turn-helix motif. to further investigate the role of the flexible gly -gly central motif we synthesized a new trichogin analog having the gly residue substituted by aib: fmoc-aib-gly-leu-aib-gly-aib-leu-toac-gly-ile-leu-ome (f a t ) experimental and computational results indicated that also the f a t peptide populate two conformations, the dynamics of which were studied at different temperatures using time-resolved spectroscopic measurements. this replacement was demonstrated to stiffen the peptide backbone by reducing the flexibility around the crucial -gly -gly -dipeptide unit. the antigen α β , a member of the integrin family, is involved in the migration of lymphocytes through endothelium to the site of inflammation. thus, α β antagonists may be useful tools for the treatment of various inflammation disorders such as asthma and inflammatory arthritis. in addition, recent studies indicate that α β integrin promotes angiogenesis by allowing the invasion of myeloid cells into tumors, while α β antagonists prevent monocyte-induced angiogenesis, macrophage colonization of tumors and tumor angiogenesis. aiming to the discovery of novel α β antagonists, a series of new peptide analogues cyclized through cysteine disulphide bonds were synthesized and tested in vivo against angiogenesis in chicken embryo chorioallantoic membrane (cam model) . sar results indicated that: yr-c(cdpc)-conh promoted angiogenesis at the higher studied concentration and showed slight inhibition at the lower one, sal-r-c(cdpc)-oh, sal=salicylic acid, showed important inhibition of angiogenesis at dose-dependent manner, yr-c(cdpc)-oh and sal-yr-c(cdpc)-oh both showed no activity on angiogenesis. nmr spectroscopy was applied for the sequential assignment as well as for the elucidation of specific conformational features. experimental noe data were further imposed as distance constraints to a thorough conformational search by applying molecular dynamics simulations. energy refined produced conformers were used as template for the generation of the pharmacophore model associated with the antagonistic activity. such studies are intended to drive a rationalized design and development of this class of inhibitors. hynes r. o. cell, , , - . scaffold discovery by phylomers: a novel cd l specific scaffold derived from glycyl trna synthetase s.r. stone [ ] , k. hoffmann [ , ] , n. milech [ , ] , p. t. cunningham [ ] , m. kerfoot [ ] , s. winslow [ ] , y-f, tan [ ] , m. anastasas [ ] , c. hall [ ] , m. scobie [ ] , p.watt [ ] , and r. hopkins [ , ] [ ] drug discovery technology unit, telethon institute for child health research, roberts road, subicao, , western australia [ ] phylogica pty ltd, roberts road, subiaco, , western australia biopanning of phylomer phage display libraries against human cd l yielded a cluster of highly specific overlapping peptide fragments, from three bacterial genomes, corresponding to the highly conserved catalytic domain from the tetrameric gα β class of glycyl trna synthetases. structural analysis of the overlapping peptide fragments described a scaffold consisting of a central βsheet, comprising anti-parallel β-strands, flanked by nand c-terminal α-helices. further structural analysis revealed that these key structural features, which also encompass the crucial atp-binding motifs of the catalytic domain, are conformationally conserved across both tetrameric gα β and dimeric gα glycyl trna synthetases, yet importantly, there is only limited sequence conservation across these classes. given the identical function of the described domain and it's structural conservation, we postulated that members of the dimeric gα class would display similar cd l specific binding as the tetrameric gα β class, despite the sequence dissimilarity. to test this hypothesis, structurally equivalent peptide fragments of representative bacterial, archaeal and eukaryotic genomes comprising the dimeric gα class were tested for cd l binding in a process we termed ortholog scanning. the results showed that both archaeal (p. horikoshii) and eukaryotic (h. sapiens) structurally equivalent peptides bound to cd l with reasonable specificity and inhibited the cd :cd l interaction with comparable ic 's to the primary gα β class sequences. similar results were also observed for the representative bacterial gα class peptides. that the sequentially diverse orthologous peptides display cd l specific binding has important implications to the affinity enhancement strategies to develop the scaffold as a therapeutic agent, and in improving its "drug-like" properties. we have initiated an investigation related to the effect of radical species upon structures of some peptide segments. in the proposed experimental protocol, aqueous peptide solution was submitted to gamma ray irradiation in controlled - kgy doses. the generation of peptide analogues, possibly induced by reactive oxygen species were examined by electrospray triplequadrupole tandem mass spectrometry (collision induced dissociation approach) and amino acid analysis of crude and/or purified by-products. noteworthy, the gamma irradiation process induced, regardless of the peptide sequence, a non-linear and progressive degradation of all peptides assayed. furthermore, these peptides could be classified in some different classes according to their halflife dose. for instance, the vasoactives angiotensin ii (aii), ang ( - ), bradykinin (bk) and some related peptides were more stable than the melanocyte-stimulating hormone α-msh, substance p or the bk 's ( - ) b receptor fragment (lvyvivgkrfrkksrevyqai). usually, the most prominent derivatives generated from this experimental protocol revealed that they are likely induced by oxidation process, yielding a variation of + da in their molecular weight. the main source of peptide modifications seems to lie either on the phe (hydroxyl group insertion at o-, m-or p-positions of its aromatic side chain) or met oxydation. in the former case, only phe and not phe is oxidized in the bk structure whereas substance p generates an analogue bearing metsulfoxide without modifying its phe , residues. thus, collectively, these findings clearly stress the complexity of factors involved in peptide structural modifications induced by gamma ray-type strong electromagnetic irradiation experiment. an additional target of this approach lies indeed, in the production of unusual peptides for further structure-function investigations. university of bern, bern, switzerland linear peptides are typically poor drug candidates due to their low bioavailability and rapid proteolysis. these limitations can be overcome by rigidifying their structure through head-to-tail or side chain-involving cyclizations. cyclic constraints may also increase biological activity by stabilizing secondary structures and by reducing the entropic penalty of binding to a protein target. the use of multiple branching amino acids in a peptide sequence, like diamino acids (as used in peptide dendrimers ) or amino diacids, allows to design peptides resembling polycyclic alkanes, a type of topology only rarely found in nature (e.g. amatoxins and lantibiotics). bicyclic homodetic peptides such as "norbornapeptides" (bicyclo[ . . ]heptapeptides) were prepared using an orthogonal protection scheme: the first cyclization is performed on resin after selective deprotection of a glutamic acid residue, whereas the second ring closure is achieved by amide bond formation at high dilutions. these peptides are structurally well-defined and cover an almost pristine area of peptide topological space. their conformational rigidity was investigated by means of d-nmr and x-ray crystallography and may offer a platform to design drugs tackling protein-protein interactions. the interaction of peptide ligands with protein receptors face peculiar challenge in recognizing binding surfaces due to availability of a multitude of conformations. therefore it is essential to constrain the peptide conformations for the recognition of receptors and thus finding the bioactive conformation. the cell surface receptor protein family integrins recognize "rgd" sequence which is present in different proteins. to determine the bioactive conformation required to bind with receptor αiibβ , the peptide sequence "riprgdmp" from kistrin was inserted into cdr loop region of rei protein (rei-rgd ). it helps out in finding the possible bioactive conformation of peptide by restricting the sampling space. the activity of rei-rgd was studied and found that as the temperature increased rei-rgd showed a higher affinity towards the receptor αiibβ . the proposed mechanisms for the increased activity of rei-rgd at higher temperature were justified in either of two ways. the modified complex forces the restricted peptide to adopt a bioactive conformation or it unfolds the peptide in a way that opens its binding surface with high affinity for receptor. in this study we model the conformational preferences of "rgd" sequence in octapeptide "riprgdmp" at two different temperatures ( o c and o c) using multiple md simulations. we found that at higher temperature "rgd" sequence from "riprgdmp" adopt turn conformation, while a bend conformation was observed at low temperature. the analysis of various pharmacophoric parameters hint that the turn conformation of "rgd" sequences adopted at higher temperature could be the potential bio-active conformation, and helps out in designing of antagonists for cell surface receptor αiibβ . the -residue peptaibol antibiotic trichovirin i- a (tv) of the linear, covalent structure ac-aib-asn-leu-aib-pro-ala-val-aib-pro-aib-leu-aib-pro-leuol (ac, acetyl; aib, α-aminoisobutyric acid; leuol, l-leucinol) has been synthesized and very thin (~ μm) hair-like crystals were obtained from a methanolacetonitrile-water mixture. diffraction data were collected at k at the diamond light source england, using the microfocus beamline i and a x-ray beam focused to a size of μm full-width-half-maximum. two independent molecules (a) and (b) were located in the crystal's asymmetric unit . both chains assume complete turns of a curved right-handed helical conformation stabilized by intramolecular hydrogen bonds. up to now tv represents the longest right-handed -helix of a natural peptaibol sequence complementing those of synthetic, protected homooligo-aib- insulin is a protein hormone that plays a key role in regulation of blood glucose levels and, thus, has widespread impact on lipid and protein metabolism.insulin is known to act through binding to the insulin receptor (ir); however, the structure of the insulin-ir complex is not known. the crystal and nmr structures of insulin represent only inactive storage forms. it is widely acknowledged that insulin must undergo structural changes in the c-terminus of the b-chain upon binding to the ir. in addition, the n-terminus of the bchain may adopt two different conformations in hexamer, known as t-and r-states. the r-state of the n-terminus of the b-chain creates a long b -b central a-helix. the t-state of n-terminus is in an extended conformation. however, the biological relevance of the t/r forms remains elusive . in this study, we have focused on the synthesis of new insulin analogues modified at the nterminus of the b-chain and subsequently correlated their biological activities with their d-structures. the invariant residue glyb seems to be critical for the t/r transition. glycine can adopt wide range of dihedral angles (φ/ψ) and it occupies significantly diverse dihedral angles in t-and r-states. a-aminoisobutyric acid (aib) is an amino acid with a high helical propensity, which often folds into right-or left-handed α-helix. we have introduced aib at position b , b and b with the aim to induce the r-state of the hormone. in contrast, as d-pro and nmeala are not able to adopt the φ/ψ angles of the right-handed α-helix we have introduced these amino acids at position b to obtain the t-state of insulin. peptide dendrimers are tree-like molecules formed by alternating functional amino acids with branching diamino acids such as lysine. unfortunately these molecules have not yielded to structural characterization and little is known about their molecular-level structure. computational methods seem to be an adequate tool to address these issues.herein we present a comprehensive structural characterization of peptide dendrimers using molecular simulation methods. multiple long molecular dynamics (md) simulations were used to extensively sample the conformational preferences of several third-generation peptide dendrimers, including some known to bind aquacobalamine. we used several conformational analysis procedures (clustering, energy landscapes and multivariate analysis) to analyze conformational changes that can be correlated with particular structural trends.the results point to a high conformational flexibility of these molecules, with no clear "folded state", although two markedly distinct behaviours were identified. some dendrimers favour mainly loose conformations, while others prefer more compact configurations. through a series of computational mutations we investigated the influence of the presence and placement of charged residues in dendrimer topology, finding that electrostatic interactions among charged residues are a major determinant in structure acquisition by peptide dendrimers. these conclusions bring new insight into the conformational behaviour of these systems and may provide better routes for their functional design. acid-mediated prevention of aspartimide formation in solid phase peptide synthesis t. michels, a r. dölling, b u. haberkorn a , w. mier*, a a department of nuclear medicine, university hospital heidelberg, heidelberg, germany; b biosyntan gmbh, robert-rössle-straße , berlin, germany aspartimide formation is one of the major obstacles that impede the solid phase synthesis of large peptides and proteins. the main reason for aspartimide formation is the piperidine-catalyzed fmoc cleavage of peptides containing aspartic acid. several side chain protecting groups have been developed but the complete prevention of aspartimide formation can only be achieved using n-( hydroxy- -methoxybenzyl) (hmb) as backbone protecting group. however, hmb-protected building blocks are difficult to synthesize and only the dipeptide containing glycine (fmoc-asp(tbu)-(hmb)gly) is commercially available. until now, no cost effective strategy to suppress this side reaction has been developed. formally, aspartimide is the result of an attack of an amidate species at the carbonyl carbon of the otbu protected side chain carboxylate of aspartic acid, which might be prevented by protonation of the amidates with piperidinium ions. in this work the suppression of aspartimide formation by adding small amounts of organic acids to the deprotection agent piperidine was studied. this procedure was shown to efficiently prevent the formation of aspartimide side products in several peptides, i.e. pres - -y, a -mer peptide derived from the hbv surface antigen and a peptide parathyroid hormone (pth) fragment. the testing of a series of different acids covering a broad range of pka values showed that this effect is virtually independent of the acid strength. since aspartic acid is found in most oligopeptides, the authors recommend to generally add % (v/v) formic acid to piperidine based fmoc cleavage mixtures. decomposition of the resin linkers during tfa cleavage of peptides in fmoc-strategy leads to alkylation of sensitive amino acids . this side product formation is a crucial drawback, especially during the synthesis of biologically important cys-containing peptides on wang support. through a battery of approaches ( h-nmr, uv and lc/esi-ms) we detected an unexpected alkylation of the sulfhydryl group of cysteine side-chain residues by the phydroxyl benzyl group from the wang resin linker. herein, we present the feasibility for s-alkylation of cys-containing peptides from wang linker decomposition. this sidereaction occurs during the final tfa cleavage of the peptide from the solid support, while the position of the cysteine residue within the peptide sequence as well as the resin's substitution influence the extent of cys-alkylation. the stephan angeloff institute of microbiology, bulgarian academy of sciences, sofia, bulgaria influenza viruses cause epidemics and pandemics all over the world. therefore, the development of virus resistance to drugs, leads to search for novel derivatives and approaches to chemotherapy for human influenza infection. antioxidant therapy is known to be one potential approach. the application of combination therapy of antioxidants with antiviral drugs could reduce the complications and lethal effects, caused by an influenza virus . in our study, amino group of neuraminidase inhibitor -oseltamivir, which belong to second generation anti-flu drugs, was covalent conjugated with known antioxidantscysteine, histidine. tryptophan and etc. the study of the role of the modified by antioxidants oseltamivir on proliferation of influenza virus is in progress. recently we reported a short synthesis of or -membered cyclic guanidine via intramolecular reaction of alkyl diamine with n,n'-cbz-methylisothiourea( a). here we report a further application of synthesis two types of cyclo-peptide guanidine-bridged cyclopeptides utilizing this mechanism -n-terminus local cyclo-guanidine peptide and backbone guanidine-bridged marco-cyclic peptide. three n,n'protected diaminoacids (daa) including , -diaminobutyric p . antifreeze glycoproteins (afgps) are found in the deep sea teleost fish in arctic and antarctic oceans. these biomolecules are able to inhibit the growth of ice crystals and depress the freezing temperature of the blood serum in fish enough to keep them from freezing in their sub-zero environments while the melting temperature remains unchanged . despite afgps have been consider as a potent cryopreservation, obstacles to develop afgps as medicinal and industrial application are mainly due to the lack of access to pure form from natural sources and the problem of understanding how afgps inhibit ice crystal growth. as a result, a considerable progress toward the design and synthesis of afgp analogues has been made several groups . in the course of the studies on the structure-activity relationships of afgps, we are interested in peptoids as mimics of α-peptides and synthesized monoglycosylated peptoid analogues by substituting the glyco-thr residue as afgps mimics. in this presentation, we will show our studies on how the insertion of peptoid residue into afgp backbone affects the afgp activity by measuring both thermal hysteresis (th) and ice recrystalliztion inhibition (iri). [ ] , both for diagnosis and endoradiotherapy. for this application the peptides can be attached with chelating agents that bind radioactive metals such as ga, ga or in for imaging or therapeutic radiometals such as y and lu. the chelating agent most frequently applied is the macrocyclic ligand , , , -tetraazacyclododecane-n,n',n'',n'''-tetraacetate (dota), it is commonly introduced as the tris(tbu ester). the cleavage of the tbu protecting groups on dota is known to be sluggish [ ] . several attempts have been made to synthesize dota with protecting groups that can be removed under mild conditions. however, these derivatives have not yet found widespread application. our new approach was to prepare a protecting group for dota-based prochelators that is convergently cleaved under the cleavage conditions of the amino acid protecting groups of the peptide. o-phenylisopropyl (opp) esters are more sensitive towards acid than tbu esters. deprotection occurs with % trifluoroacetic acid in dichloromethane [ ] . therefore, a synthesis of the prochelator dota-tris(opp ester) was developed. the copper-catalyzed azide-alkyne cyclization (cuaac), the most commonly recognized variant of "click chemistry," has emerged as a powerful technique for ligation, conjugation, and cyclization reactions of peptides. it is known that cyclization can increase the metabolic stability of peptides, as well as enhancing potency or selectivity by stabilizing an active conformation. one application of the cuaac that has generated interest is the use of this reaction to replace a disulfide bridge with the product triazole, which among other complementary properties may prevent in vivo redox chemistry. in this poster, we synthesize a new analogue of the cyclic cancertargeting peptide cngrc where we replace the disulfide bond with a triazole linkage using click chemistry and a fully automated, on-resin method using the single-shot delivery feature on the prelude® peptide synthesizer. unnatural amino acids including d-amino acids are manufactured mainly by the enzymatic process. however, one enzyme can produce only one amino acid due to its high specificity and it takes a long time and a lot of expenses to develop the appropriate enzyme itself. arca (alanine racemase chiral analogue) is an organic catalyst which can overcome these drawbacks and can produce almost all kinds of amino acids efficiently. the amine functionality of l-threonine is freely reacted with the aldehyde group of arca to form the corresponding imine, which is easily epimerized in the presence of organic base due to the acidity of the alpha proton of imine. the difference in the stability between the imines of the optical epimers rendered them to be shifted to d-allo-threonine derivative dominantly. once the epimerization reaction reached equilibrium, the reaction mixture was hydrolyzed under acidic condition to give d-allo-threonine and arca, which could be recycled repeatedly without significant loss in yield or purity to produce more d-allo-threonine from lthreonine in excellent yields. optimization of the reaction conditions with various bases and solvents is discussed and mass production of optically active d-allo-threonine including optical purification is described. the manufacuring process for the preparation of arca will be shared as well. our group is interested in the development of efficient synthetic routes for the preparation of enantiopure atrifluoromethylated amino acids (a-tfm-aas) starting from chiral cf -oxazolidines or imines and their incorporation into a peptide chain. these non-natural amino acids are very attractive compounds for the design of biologically active molecules, particularly peptides, due to the unique physical, chemical and biological properties impart by the cf group. as conformationally constrained cyclic amino acids have recently gained considerable interest, we are particularly focused on the preparation of pyrrolidine-type a-tfm aas. , incorporation of proline derivatives is known to restrict the amino acyl-proline cis/trans isomerization, to limit the protein folding and consequently to modulate the biological activity of peptides. based on these observations, mutter's group introduced pseudoproline building blocks (ψpro) into a peptide sequence as reversible protecting groups for ser, thr and cys. the ψpro residues proved to be versatile tools for overcoming the aggregation caused by hydrophobic interactions encountered during solid-phase peptide synthesis (spps). they also turned out to be inducers of βturns containing predominantly cis-amide bond and useful tools in peptide cyclization. here, we report the results obtained for the preparation of various hydrolytically stable trifluoromethylated pseudoprolines (cf -ψpro) as well as the methodological studies developed to optimize the synthesis of various c-and n-terminal cf -ψpro containing dipeptides. rennes, france protein strructure and function rely on a still not fully understood interplay of energetic and entropic constraints defined by the permutation of the twenty genetically encoded amino acids. many attempts have been undertaken to design peptide-peptide interaction pairs and synthetic receptors de novo by using special building blocks. a rational approach starting from hydrazine to create new building blocks based on a tailored metalchelating amino acid analogues was envisaged. to create chemical recognition units, which bind oligohistidine tags with high affinity and stability, several supramolecular entities containing one to three nitrilotriacetic acid analogue (ynta) moieties were synthesized. these new building blocks additionally contained an amino group or an acido group, which can be flexibly introduced into peptide in n or c-termini or into the peptidic chain by solid phase chemistry in fmoc/t-bu strategy. these multivalent chelators were characterized and the corresponding metalchelating peptides could act as metal sensors and synthetic receptors for histidine-tagged proteins. the potential of peptides as drug candidates is often limited by their pharmacokinetic properties. structural modification of the peptide backbone via n-methylation is a powerful medicinal chemistry tool that confers oral bioavailability to these molecules. n-methylation exerts a strong effect on the backbone conformation and, as a result, many n-methylated peptides show enhanced biological activity and higher receptor selectivity. another approach to increase the solubility of peptides is by conjugation of peg to a derivatizable functionality. by combining these two approaches we have developed n-oegylation. this novel form of peptide modification consists of the attachment of oligoethylene glycol (oeg) chains to the amide bonds. many bioactive peptides comprise one or more n-me amino acids which are essential for their activity. thus, we consider that replacement of a backbone n-me group by an oeg chain may imply a minimal structural perturbation and may lead to n-oegylated peptides with preserved biological activity. furthermore, our strategy is a promising way to improve the bioavailability of cyclic peptides that do not have any site where a peg could be attached. as a proof of principle, several n-oeg analogs of two bioactive cyclic peptides were synthesized in spps. first, we performed a full n-oeg scan of the sansalvamide a peptide. next, several analogs of cilengitide were prepared by replacing the n-me of valine by oeg chains of different length. depending on its size, the oeg residue was incorporated by using an n-oeg derivative as building block or, alternatively, using an n-substituted amino acid bearing an attachment site where a peg was conjugated post-synthetically.the biological activity of all the n-oegylated peptides was evaluated. some of the sansalvamide a peptide analogs exhibited cytotoxicity within the same range as the original peptide, which suggests that backbone amide groups may be useful oegylation sites in bioactive cyclic peptides. the modification of peptides is an important step in pharmacology to vary the affinity and the stability of peptidic drugs. whereas a wide range of strategies exists for the functionalization of the n-terminus and the side chains, facile variation of the c-terminus remains an important challenge. we consider peptidyl-phosphoranes as a promising platform to enable orthogonal and mild introduction of a great variety of chemical functionalities at the c-terminus. a convenient method for the synthesis of soluble peptidyl-phosphoranes has been presented by our group recently. in this, -bromo-acetyl bromide was coupled to a wang-resin followed by alkylation of triaryl-or trialkyl phosphine moiety. deprotonation to the phosphorus ylide and subsequent acylation with an fmocamino acid created the basis for assembly of the peptide by spps. final acidic cleavage produced a decarboxylated and unprotected, soluble peptidyl-phosphorane. from this point, a variety of orthogonal modification reactions at the peptides c-terminus is possible, e.g. click reaction with azides allow for the incorporation of triazoles as peptide bond mimetics. the wittig reaction opens up another interesting portal for c-terminal modification, as vinyl ketones are formed by reaction with aldehydes. the described chemistry was applied to modify caspase- inhibitors. in order to address the s site of caspase- , the commonly known devd inhibitor was varied at the c-terminus by introduction of different residues. the devd motif was synthesized as peptidyl-phosphorane and modified in wittig reactions. the resulting c-terminal vinyl ketones were obtained by the reaction of aliphatic and aromatic aldehydes. a small library was generated, and novel compounds were tested for their potential to inhibit caspase- . semmelweis university, department of biophysics and radiation biology, budapest, hungary considering the impact of uv irradiation on the structure and function of proteins , it is a matter of utmost importance to resolve the conditions of photolysis more deeply. we think that a protein, as a complex unit, gives multiple responses to all impacts therefore the analysis of these responses is a rather complex problem. the main goal of our research is the deeper understanding the tryptophan-mediated photolysis of disulphide bridges in bio-active proteins upon near-uv irradiation using cyclic peptide models, as small protein units, to define the caused functional damage. cation -pi interaction is increasingly recognised as an important noncovalent binding interaction which plays a role in establishing the final structures of proteins. within a protein, cation -pi interactions can occur between the cationic side-chains of either lys or arg and the aromatic side-chains of phe, tyr or trp . our earlier results with gla indicate that new covalent bonds are also formed between cys and lys during illumination, which is also a reason why the lys residue is planned to be included in the sequence of the models. our aim is to study whether the cation -pi interaction can have an influence on the ss-bridge splitting in small cyclic pentapeptide models. here we report about the conformational analysis, synthesis and spectroscopic investigation of lys-and arg-containing model peptides. the azide functionality is very popular mainly due to azidealkyne click chemistry used in many peptide ligation strategies. azido-peptides are usually prepared by incorporation of azide containing residues or azide functionalization of aldehyde resins affording c-terminal azido-peptides. alternatively, the n-terminus can be converted into an azide via a cu(ii)-catalyzed diazotransfer reaction using triflyl azide. recently, a number of safer, shelf-stable and easily prepared diazotransfer reagents has been developed, of which imidazole- -sulfonyl azide has been used to introduce azide moieties in proteins under copper-free conditions. typically, it has not been reported to be used on the solid phase. we provide a very easy, fast and efficient method for conversion of amines into azides on a solid phase support which in our opinion has major benefits over earlier reported methods making using of less stable reagents that require a metal ion catalyst. we demonstrate how the diazotransfer reaction can be performed on a solid phase support using the imidazole- sulfonyl azide reagent without the need for a metal ion catalyst. using a model peptide we studied the effect of stoichiometry, added base and solvent. in addition we examined the effect of the nature of the n-terminal residue on the efficiency the diazotransfer reaction. finally, we found that the optimal conditions to perform the reaction also depend on the nature of the solid phase support the reaction is performed on. the novo nordisk foundation center for protein research, university of copenhagen, copenhagen, denmark site-selective strategies for post-translational modification of peptides and proteins are essential tools for many areas of research in the life sciences, yet remain a chemical challenge due to the multiplicity of functional groups present. there are powerful chemoselective reactions, however, they aim at introducing only one functionality at each reaction site. here we present a one-pot, threecomponent dual-functionalization of peptides or proteins based on a , -dipolar cycloaddition between a functionalized malemide, an n-hydroxylamine and a peptide or protein with an n-terminal serine residue at the n-terminus, which is selectively oxidized to a -oxoaldehyde. most common moieties for labeling, e.g. fluorophors and peg-chains, are commercially available as maleimides. nitrones were easily obtained by condensation of peptide-aldehydes and primary nhydroxylamines under aqueous conditions. the chemoselective , -dipolar cycloaddition reaction between the peptide-nitrone, and a functionalized maleimide proceeded in aqueous solution at room temperature or with gentle heating, which provided the stable isoxazolidine product. we envision that this 'one site -two functions' method can be used widely to introduce two separate moieties. the method was used to introduce two separate ligands in a range of other peptides. for example, new multimodal molecular imaging techniques depend on facile chemical methods for site-selective dual-functionalization. we used our new methodology to synthesize a cyclic rgd-peptide for combined pet and optical molecular imaging. finally, the small protein ipb was successfully n-terminally modified, including with a peg-chain, using this new, general method. multiple sclerosis (ms) is the most known chronic, inflammatory, demyelinating disease of the central nervous system (cns), characterized by a progressive neurodegeneration, caused by an autoimmune response to self-antigens in genetically susceptible individuals. it is nowadays known that post-translational modifications may affect the immunogenicity of self-protein antigens, triggering an autoimmune response and creating neoantigens; in particular aberrant glycosylations affect various parts of the immune response and have profound effects on immune tolerance. in previous studies we demonstrated the value of the glycopeptide csf (glc) which, by virtue of the particular type i' β-turn structure, optimally exposes the minimal epitope asn(glc) to autoantibody recognizing in elisa in multiple sclerosis patients' sera . elisa assays allowed to conclude that the ability in detecting autoantibodies in multiple sclerosis sera was stricktly linked to saccharidic moieties and to conformation around minimal epitope of the antigenic glycopeptide. herein, taking advantage of such considerations, we focused our attention on the synthesis of a little library of lysine branched multiple antigen peptides (maps), containing the minimal epitope asn(glc), in an attempt to increase the antigenicity of linear peptide sequences . with this aim, we performed the spps of glucosylated maps via the building block approach, studying the role of different long spacers on the dendrimeric core, and the role of different peptide sequences around the sugar moiety, in order to optimize the synthetic process and to evaluate the influence on the affinity and specificity in sp-elisa. environmentally induced co-or post-translational modifications of autoantigens are hypothesized to break immune tolerance leading to self reactivity in pbc. it has been previously reported that the use of synthetic post-translationally modified peptides, introducing fmoc-l-lys(nε-(±)-α-lipoic acid)-oh, as peptidomimetics of natural neoantigens allowed to detect autoantibodies in the sera of patients affected by pbc, and they might be useful diagnostic tools that can be used in earlier stage patients and possibly to monitor disease activity. only the r-(+)-enantiomer of α-lipoic acid exists in nature and is an essential cofactor of four mitochondrial enzyme complexes. but it remains unclear if the tridimensional structure of the lipoic acid is of any importance in the interactions antibody-peptide during the indirect elisa tests. therefore, it is necessary to synthesize each peptide separately with one absolute configuration of the lipoic acid. herein, we describe the synthesis of the two diastereoisomers fmoc-l-lys(nε -(r)-α -lipoic acid)-oh and fmoc-l-lys(nε -(s)-α-lipoic acid)-oh that have to be used in fmoc/tbu spps as building blocks for the synthesis of post-translationally modified peptides. recently it has been reported the introduction of a new generation of cd diagnostics based on a unique antigen approach, consisting on human ttg cross-linked with gliadin peptides coated on the elisa plates . on the basis of experimental data obtained by mass spectrometry and indicating which are the fragments of these two proteins that are supposed to be involved in the antibody recognition, we were able to select the most representative ttg and gliadin fragments , to design and synthesize by fmoc-spps nine cross-linked eoepitopes. aim of our study was the characterization of autoantigenic epitopes by testing, in celiac patients' sera, the reactivity of these nine synthetic peptides. these neoepitopes were tested in elisa to evaluate the iga and igg response against ttg-gliadin adducts in celiac patients' sera in order to develop a new elisa test based on peptides as an even more powerful diagnostic tool in terms of specificity and sensitivity. more than analogs have already been described, wherein the hydroxy acid and the amino acid constituents were replaced by d-amino acids and/or n-methyl amino acids with preserved or altered side chains. for certain types of cancer cells, several of these analogs were found to be more active than the natural product itself. however, it does appear that many of these compounds have limited solubility in water. b here we report the synthesis of novel analogs of the sansalvamide a peptide bearing an n-oligoethyleneglycyl (oeg) chain attached to the different backbone positions. attachment of this chain is aimed to enhance the hydrophilicity of the original peptide. our synthetic strategy to modify the backbone with the n-oeg group relies on the use of n-oeg amino acids, which were synthesized in solution and then used as building blocks in spps. as expected, couplings to the n-oeg residues were found to require special conditions. methods for the coupling to nmethyl amino acids were applied and this enabled to obtain the different linear pentapeptides, which were cyclized in solution. both the synthetic strategies of these demanding peptides as well as the preliminar evaluation of their biological activity will be deeply discussed. glycoconjugates such as glycoproteins and glycolipids have important roles in cell functions, for example, intercellular recognition, cell proliferation control, and information transmission. in order to study the structurefunction relationship, synthesis of these glycoconjugates is essential. glycoproteins and glycopeptides are classified into two categories: n-and o-glycosylated derivatives. the n-acetyl-α-d-galactopyranosylated ser or thr derivatives [ser/thr(α-d-galnac)] are important intermediates for o-glycopeptide synthesis. however, the synthesis of ser/thr(α-d-galnac) derivatives by chemical glycosylation is difficult because of the decreased nucleophilicity of hydroxy function in the glycosyl acceptor due to an unfavorable hydrogen-bonding pattern between the oh and α-nh groups . several approaches to overcome this problem have been reported , . in addition, the o-glycosidic bond is cleaved easily in acidic conditions. in this study, we assumed that the formation of a cyclic structure containing an α-nh group would increase the reactivity of oh function. thus, we focused on the n, n'isopropylidene derivatives of ser/thr containing dipeptides . we found the reaction of mannopyranosyl trichloroacetimidate and the n, n'-isopropylidene dipeptide in the presence of tmsotf in dichloromethane produced the desired glycosylated dipeptide in good yield. however the selective intermolecular disulfide bond formation is a very difficult and complicated synthetic problem. in this work we report on synthetic approaches for the formation of conjugates with intermolecular thioether or disulfide bonds. for the disulfide bond formation, we use two activation approaches: i) activation of the four cys residues of the carrier testing two activating reagents, in both solid and liquid phase respectively and ii) activation of the cys containing bioactive molecule. as bioactive molecule we selected the r ppleed sequence derived from the intracellular part of the αiibplatelet integrin receptor. this region is critically involved in platelet aggregation and is a target of intervention for developing antithrombotic agents . the ac-[lys-aib-cys(ch co-αiib - )] -nh and ac-[lys-aib-cys(cys-αiib - )] -nh conjugates were synthesized and examined for their ability to inhibit platelet aggregation. the biological assays indicated that the synthesized conjugates penetrate the platelet membrane and inhibit human platelet aggregation, in contrast to the corresponding free peptide analogues. the molecules were reported to exhibit broad-spectrum cytotoxicity against the tumor cell lines. although these peptides contained the novel β-methoxytyrosine, lipton et al. reported the synthesis and cytotoxicity of desmethoxycallipeltin b, in which substitution of d-tyrosine for β-methoxytyrosine did not substantially affect the cytotoxicity of callipeltin b , . however, a structure-activity relationship study of the molecules has not been shown to date in detail. in the course of our recent research regarding the synthetic study of cyclic depsipeptides, we conducted studies on the synthesis of callipeltins supposed to be efficient structures for ccr inhibitors as anti-hiv drugs or anti-cancer agents. in the present study, we report the synthesis of cyclic depsipeptides of callipeltin b analogues consisting of l-, d-amino acids and/or n-methyl amino acids, for a structure-activity relationship study of linear-and cyclic depsi-peptides against hela cells . in the assay of synthetic peptides, all of the synthetic callipeltin b analogues exhibited no cytotoxicity. we supposed that dimethylpyroglutamic acid of callipeltin b was essential structure to show the cytotoxicity against hela cells. monash university, melbourne, australia protein-protein interactions represent a significant portion ( - %) of all interactions within the cell; as such these interactions are ideal targets for drug discovery. while difficult to target using small molecules, these interactions can be disrupted using a small section of the protein's binding partner. these short peptides must retain the defined secondary structure associated with the protein binding interface in order to inhibit their protein targets. as the secondary structure adopted by the parent protein is not always exhibited by its derived peptides, constraints are introduced as necessary to help define the structure of the peptide. inducing secondary structure reduces the energy required for organisation, decreasing the energy of binding and has the potential to increase stability with respect to degradation by proteases. solid phase peptide synthesis was used to make several small peptides corresponding to the structured sections of the binding partners for three protein-protein interactions. these peptides were designed to target heart disease, prostate cancer, and liver cancer respectively. secondary structure was introduced using lactam bridge constraints. for the αhelical peptides, a side chain constraint approach was used to nucleate helix formation. as hydrogen bonding between the c=o of the i th residue and the nh of the (i+ ) th residue stabilises native α-helices, constraints were introduced linking the side chains such that the residues were held in close proximity. for β-pleated peptides, an antiparallel β-sheet arrangement was achieved by introducing turn regions into the peptide in such a way that the β-strands were aligned. constraints were again introduced using lactam bridges between lys and arg or glu side chains. these peptides were characterised by nmr and cd spectroscopy to verify the correct secondary structure had been induced. göttingen, germany different properties can be combined in a single molecule by using a scaffold arranging functional groups in a predefined topology. the tasp (template-assembled synthetic proteins) concept describes templates to reinforce and direct the folding of designed molecules into a predetermined topology. [ ] due to their resistance to proteolytic degradation and their rigid basic structure, cyclic β -tripeptides are suitable carrier molecules for bioactive compounds; they are further known to form tubelike structures by stacking of the peptide rings leading to higher organization of functionalized peptides. [ ] with this scaffold different inhibitory systems were synthesized that feature cell penetrating and fluorescent properties. the signal transducer and activator of transcription (stat ) protein, which has been described as an oncogenic protein, was selected as the first target. [ ] a peptide sequence which targets the sh domain of stat was used in two different approaches. it was either directly attached to the cyclic β-tripeptide via a huisgen [ + ]cycloaddition or the peptide was incorporated into the inhibitor loop of the cystine knot microprotein omcoti-ii, which was also attached to the cyclic-β -tripeptide. [ ] further, sodium channels are addressed usingconotoxines. first, an alkyne functionalized conotoxin siiia was synthesized applying different folding methods. the alkyne linker will be used to attach a fluorophore or to functionalize a cyclic-β-tripeptide. using single molecule imaging the spatial distribution, local concentration and organization of the ion channels in neurons will be imaged. further, the cyclic-β -tripeptide templating effect will be used functionalizing with μ-conotoxines. those proteins are folding helper proteins. together with chaperones, they form receptor complexes. they catalyze the isomerization of prolyl bonds in various folding states of target proteins. indeed, their role has been implicated in refolding of denatured proteins, de novo protein synthesis and the biologically active conformation of proteins . among them, the fkbp subclass comprises the small ppi calstabin and . it is of interest to try to understand the way those proteins act, in order to help the overexpression of various types of membrane proteins, aiming at the renaturation, purification and crystallization attempts of receptors. we chose to work on calstabin because this short ( aa) protein has been described as a sub-family comprising isoforms (from ~ to ~ aminoacids), some of them not being fully described to date. the relative shortness of those proteins together with the fact that the two higher molecular weight ones are catalytically active as prolyl isomerases, facilitate the characterization of the synthetic proteins.in order to obtain the full length calstabin , a native chemical ligation (ncl) approach was chosen . an optimized stepwise elongation allowed the obtention of the c-terminal segment up to the cys . moreover, several methods were compared for the synthesis of peptide - opportunely functionalized at its c-terminus for the ncl.the ligation at thr site between peptide - featuring a bis( -sulfanylethyl)amino the chemical diagnostics of paintings is a relevant topic in the field of chemical sciences applied to the conservation and safeguard of cultural heritage. chromatography is a highly sensitive and suitable technique for accurate methods of analysis of the limited amount of sample material typically available from works of art. paint media deriving from proteins traditionally include egg, milk, animal and fish collagen glue. egg yolk (egg tempera), egg albumin (glair) and casein (a blend of related phosphoproteins commonly found in milk) are traditionally used as pigments binders. we propose the uplc-based amino acid analysis as diagnostics technique on non pre-treated or submitted to extraction processes model samples, showing that good results can be achieved with very scarce sample manipulation and great advantage. we applied the amino acids analysis carried out by the accq•tag™ ultra performance liquid chromatography to the standard and model samples. in particular, after protein hydrolysis ( h, °c, m hcl) of the samples, the amino acid derivatization by -aminoquinolyl-n-hydroxysuccinimidyl carbamate allowed a reproducible amino acids analysis characteristic of the protein type. the results obtained confirmed the reliability of the data achieved and demonstrated that the accq•tag™ ultra uplc method could be a powerful technique to be applied to the relevant field of protein binders diagnostics for paintings conservation. moreover a multivariate analysis that offers a wide variety of tools and methods mainly concerned with mathematical models for the representation of multidimensional data has been proposed and the high model efficiency has been established for sample containing mixture of proteins. reactions performed on solid supports, such as resin, are commonly monitored by hplc-uv after cleaving the products from the support. however, uv-absorption coefficients may differ between compounds, and therefore the relation of the area percentage values of the peaks may not directly reflect the molar concentrations of the corresponding compounds. it is for this reason that, for example, in solid-phase peptide synthesis it is difficult to calculate the yield of the coupling of a fmoc-amino acid or the removal of the fmoc-group because of its high absorbance. recently, we reported the identification of minimal phosphopeptides that specifically interact with the pbd of human plk , but not those of the closely related plk and plk . comparative analyses of the crystal structures of the plk pbd in complex with the minimal phosphopeptides revealed that the c-terminal spt dipeptide functions as a high-affinity to the interaction. in an attempt to obtain the adequate cellular permeability and stability in vivo, we have accomplished the peptide-peptoid hybrid or peptomers cyclization using various methods like formation of amide, thioether and triazole and screened the plk inhibition activity on the first cyclic peptomers liibrary using pbd-binding assay. based on our first screening results, we also carried out the detailed investigation to further increase the activity and also to understand the significance of peptoid mimics as plk inhibitors. the mode of interaction between the cyclic peptomers and pbd might provide a template for designing therapeutic agents that target plk . a synthetic amino acid long peptide corresponding to the minimal metacaspase catalytic domain induces cell death in leishmania major c. servis, h. zalila*, i. gonzalez, l. lozano, n. fasel department of biochemistry, university of lausanne, epalinges, switzerland despite a lot of controversy during the last decade, there is increasing experimental evidence that cell death (cd) is genetically programmed in lower eukaryotes.in the cd proteolytic cascade of plants and protozoa, caspases are likely replaced by metacaspases that are cysteine peptidases recognizing arginines or lysines in p position. metacaspases have been found to control cell death in plants. the human protozoan parasite leishmania major expresses a single metacaspase (lmjmca) harboring a central domain with the catalytic dyad histidine and cysteine as found in caspases. metacaspase could therefore be one of the executioners of the death pathway in leishmania.in this work we showed that, in stress conditions, lmjmca precursor forms were extensively processed into soluble forms containing the catalytic domain and this domain was sufficient to enhance sensitivity of parasites to hydrogen peroxide by impairing the mitochondrion function. we tested different lengths of the lmjmca catalytic domain and found that the overexpression of the polypeptide corresponding to amino acids - was sufficient to sensitize l. major mitochondria to oxidative stress.we synthetized an aa long peptide corresponding to the minimal metacaspase catalytic domain (aa - ) and showed that it has specific metacaspase activity in vitro.we are currently investigating its activity on possible target proteins, which have been identified in a yeast two-hybrid screen. identifying proteins involved in the metacaspase signaling pathway will shed light on the understanding of cd in leishmania and open new perspectives in drug target investigation to fight leishmaniasis and other major infectious diseases. s. alasibi, g. ashkenasy department of chemistry, ben-gurion university of the negev, beer-sheva, israel various factors can affect the conformations and folding states of protein molecules and as a consequence their activity. these factors include amino acid mutations, interactions with other macromolecules, binding to regulatory molecules, and also external changes such as ph jump or shining light. in order to control the folding states and to modulate the functions of peptides and proteins by light, photocleavable groups are usually incorporated into specific residues to mask critical interactions. for example, introducing caging groups into coiled-coil proteins recognition interface affects complex formation and template-assisted ligation reactions, in which the coiled-coils serve as templates to catalyze the condensation reactions between two short peptide fragments . our research group has been studying peptides replication networks, which were made of coiledcoil peptides and analyzed the response of such networks to light as external trigger . it was shown that even replicating networks made up of a small number of molecules can possess complex behavior, considering the wealth of catalytic pathways and transformations. hence, boolean logic operations can provide valuable means to analyze and interpret their behavior . herein, we describe the use of chemical inputs and uv light to manipulate peptides folding and functionality within new synthetic networks. these networks perform complex behavior and, as a result, selective product formation is used to implement boolean operations that have not been achieved before. institute of bioorganic chemistry of ras, moscow, russia earlier, we have shown that n-acylated amino acid nitriles and amides react with ethylene derivatives forming the amino-and -hydroxypyridines and pyrroles [ ] . a possible reaction mechanism is the geterodienic condensation of aminooxazole derivatives to dienophiles. the higher yields were observed when used the dicarboxylic acids as dienophiles and -amino or alkoxyoxazole as geterodienes. while the same reactions with the fullerene derivatives, as dienophiles, gave low yields. the nitrile groups of specified pyridines possess ability to react with amino groups of peptides and proteins even at room temperature. in view of high activity of nitrile groups such pyridines can form tetrapyridotetrazoporphyrins and self-condensation products giving appropriate dendrimers (possible due to mobile hydrogen in the th position). high molecular weight dendrimers were identified by massspectrometry, gel electrophoresis and dynamic light-scattering. catalytic oxidizing properties of tetrazaporphyrin derivatives and phtalocyanin were used in synthesis of cyclic peptides and for the s-s bonds formation. the transformation of peptides into heterocycles via an intramolecular reaction of nitrile groups was used to determine the sequence of some peptides, which favored the resistance of transformed compounds to hydrolysis and to the electron impact at mass spectrometry. diazotization of peptides and their derivatives facilitates identification of amino acid sequence by mass spectrometry due to the peculiarities of their fragmentation. in addition, an amino acid analysis of the diazotized peptide makes it easy to determine the n-terminal amino acid. we present here a multi-disciplinary approach combining x-ray crystallography, computational analyses, and immunological tests to identify epitopes of the oligopeptide-binding protein a (oppa bp) from the gramnegative pathogen burkholderia pseudomallei, the etiological agent of melioidosis. computational analysis on oppa crystal structure was used to design potential consensus epitopes, that once synthesized as free peptides (comp - ) were found to be immunoreactive against sera from melioidosis patients. notably, one of the predicted peptides allowed to distinguish between seropositive, seronegative and recovered groups, underlining its potential for diagnostic purposes. parallel experimental epitope mapping, based on proteolysis and mass spectrometry, allowed us to identify linear peptide epitopes (exp - ) localized in similar protein regions as comp - . moreover, the match between theoretical and experimental mapping of epitopes was improved by expanding our computational approach, i.e. including an energy based decomposition procedure to divide oppa bp into separate fragments. overall, our results illustrate the successful development of a novel integrated structurebased approach for the discovery, design and preparation of epitopes. nonetheless, given antigen crystal structures, our method is expected to be broadly applicable in the design and generation of new epitope candidates, as being confirmed by on going experiments on different antigens. the application of peptide thioacids as reactive intermediates and building blocks has received considerable attention recently. the chemical ligation reaction between thioacids and azides has been reported for the synthesis of small to larger peptides as well as for the modification of proteins. fmoc based methods for the preparation of peptide thioacids have to our knowledge not been extensively researched and a facile approach to their synthesis is desirable.we have recently shown that t-butyl thioesters are robuster than previously reported, and can be used for the fmoc based solid-phase preparation of peptide thioesters being also easily cleavable with thiolates. peptides attached via a -mercapto -methylpentanol (mmp) resin can be cleaved using -mercaptopropionitrile to obtain protected thioacid peptides with a ß-elminable cyanoethyl group.the thioacid peptides could then be obtained in situ after treatment with dbu ( . % in dmf) and further reacted with sulfonyl azides in the presence of , -lutidine in a one pot reaction. by treating the cyanoethyl peptide thioesters with (nh ) s in a sodium phosphate buffer (ph = ), various model penta-peptide thioacids could be obtained cleanly at room temperature in up to % overall yield based on initial coupling. these peptides were then further ligated with electron deficient sulfonyl azide functionalized peptides.larger peptide thioacids could also be obtained using this protocol. a mer derivative of penetratin- , a cell-penetrating peptide from the third helix of the homeodomain of the antennapedia protein, was prepared as a peptide thioacid in a % yield (based on coupling of the first amino acid). in this report, a sensitive, selective and rapid uplc-ms method was developed for the determination of the [lys-gly] -mog - peptide in order to control the conjugation of mannan with the [lys-gly] -mog - peptide. the separation was performed on an acquity uplc system with a beh c column packed with . μm particles. the total run time was min. calibration curve based on peak area ratio was linear at the concentration range of - μg/ml, with a detection limit of μg/ml. the method showed satisfactory reproducibility and confirmed the entire conjugation between oxidized mannan and peptide sequence. the development of simple, low-cost and fast methods for protein purification is of increasing importance both for academic and industrial applications. a very promising approach is inverse transition cycling (itc) that exploits the temperature dependent aggregation properties of elps. elastin-like polypeptides (elp) are artificial polypeptides composed of pentameric repeats (val-pro-gly-xaa-gly) derived from mammalian elastin. elps are characterized by a specific transition temperature (t t ) that depends on the amino acid composition of the pentarepeat; they are water-soluble below and aggregate reversibly above this narrow temperature range (t t ). these properties are transferred to target proteins by n-or cterminal fusion with elps. during itc these fusion proteins precipitate, while other components remain in solution. repeated cycles of heating and cooling allow simple recovery of the target protein.we synthesized various elps consisting of to pentameric repeats and including different guest residues. the transition temperature of all synthetic elps was determined using photometric assays and measuring turbidity. in order to test if elp properties can be efficiently transferred, we fused elp to a small recombinant protein (ras-binding domain, rbd) by expressed protein ligation. this approach will allow the incorporation of elps with unnatural amino acids and other chemical modifications into target proteins. currently we are focusing on biotechnologically relevant enzymes that constitute a major cost factor in industrial processes. the authors thank süd-chemie/clariant for their financial support. department of pharmacy, university of patras, rio, greece peptides penetrating the cell membrane, known as cell penetrating peptides (cpps), as well as their mimics, used as delivery agents to cells have been reported , . cpps can be natural sequences or artificial constructs designed to capture the features of natural formations. cpps are particularly important in the delivery of peptides, proteins, nucleic acids, small molecule drugs or imaging agents. incorporation of a heterocyclic motif into a peptide or peptide-like backbone introduces conformational constraints and/or latent reactivity related to the heterocycle's structural profile. heterocycle-based cpp mimics are, thus, promising candidates for therapeutics protected synthetic non-ionic peptides, which are for example synthetic intermediates for the production of api's, are often very hydrophobic and not soluble in most common solvents. they are thus difficult to purify by preparative rp-hplc, classically used for industrial production. it is then challenging to develop alternative purification chromatographic processes using suitable solvents and providing good yields, high purity and sufficient productivity. the technique of support free liquid-liquid chromatography , including both its hydrostatic (centrifugal partition chromatography or cpc) and its hydrodynamic (counter-current chromatography or ccc) declensions, are mainly involved in phytochemical studies but has also been applied to peptide purification . the previously developed biphasic solvent systems are not adapted to the purification of highly hydrophobic protected peptides. to overcome this problem, two new scales of biphasic solvents systems and a ternary biphasic solvent system were developed to overcome solubility problems often encountered with those peptides. the new systems composed of heptane/thf/ch n/dmso/water, heptane/me-thf/nmp/water, and cmpe/dmf/water were efficiently used for the cpc purification of a mer protected exenatide and a mer protected peptide intermediate of bivalirudin synthesis. the developed scales show a wide range of polarity and should be useful for general use in cpc for the separation of hydrophobic synthetic free or protected peptides. the progressive aggregation of β-amyloid peptide (β-ap) into insoluble amyloid fibrils ultimately leading to formation of toxic amyloid plaques is widely considered to be the central pathogenic cause of alzheimer's disease. in the last decade accumulating evidence suggests that soluble oligomeric non-fibrillar forms of β-ap are neurotoxic as well. consequently, inhibiting the aggregation of β-ap is one of the therapeutic strategies against alzheimer's disease and a number of small molecules have been identified as inhibitors of β-ap aggregation and neurotoxicity. among these, curcumin, the phenolic yellow pigment and active ingredient of the turmeric herb, is receiving special attention because of its rich pharmacology that includes in vitro and in vivo inhibitory action against alzheimer's disease insults. in the current work the interaction of β-ap( - ) with curcumin is investigated with fluorescence, cd, and nmr spectroscopies in water and water-methanol mixtures and at various β-ap( - ):curcumin ratios. in nmr studies in % methanol curcumin behaves like a macromolecular species with a change in the sign of its noe signal providing direct indication of its association with β-ap( - ). in % methanol the presence of β-ap( - ) results in great broadening of the h peaks of curcumin, indicative of a complete change in its solution state. additionally, the fluorescence of curcumin in % methanol shows a blue shift with enhanced intensity, observations consistent with a hydrophobic modification of curcumin environment upon interaction with β-ap. finally, in water the induced circular dichroism spectrum of curcumin in the near uv region provides clear evidence for the loss of symmetry of curcumin molecule due to changes in its microenvironment generated by interaction with β-ap( - ). our experimental findings support the direct interaction of β-ap( - ) with curcumin and establish its importance as a potential aggregation inhibitor of β-ap. [ ] . based on its sequence, we synthesized h-tyr-d-trp-nh- -ada ( -adamantane) (yo- ) and h-tyr-d-trp-nh- -ada ( -adamantane) (yo- ) and reported they had potent antiproliferative activity on cancer cells (a- and sw ), which were comparable to tt- and cycloheximide. a structure-activity relationship analysis revealed that lipophylicity of yo- and - could be responsible for their antiproliferative activity. now, we described the substitution of tyr of yo- and - by tyr(bzl), phe, -nal( -naphthylalanine), -nal ( -naphthyalanine) and the anticancer and dna polymerase inhibitory activities in order to explore the effect of hydrophobic substituent. among the compounds, yo- and - had the highest lipophilicity judging from their retention time and lipophilicity index (yo- : . min, . ; yo- : . min, . ). yo- and - exhibited strong dna polymerase inhibitory activity as well as antifroliferative activity on hct cells at m. these activities were greater than those of yo- and - . antiproliferative activity of the compounds containing -ada such as yo- , - , - , - and - , was comparable to that of the compounds containing -ada such as yo- , - , - , - and - . these findings suggest that the lipophilicity well correlates with dna polymerase inhibitory activity and antiproliferative activity on hct cells. further structureactivity relationship study is progressing in our group. multiple sclerosis (ms) is a chronic autoimmune disease of the central nervous system (cns) , . our aim was to immunologically control the attack of the myelin sheath in ms patients without the total suppression of the immune system. anthraquinones (mitoxantrone, ametantrone) are widely used in cancer therapy as immunosuppressants.mitoxantrone is also used to treat several forms of advancing ms, including secondary progressive ms, progressive relapsing ms, and advanced relapsingremitting ms . more specifically, mitoxantrone is an inhibitor of the type ii topoisomerase, which disrupts dna synthesis and dna repair in both healthy cells and cancer cells. herein, we report the synthesis of an anthraquinone type compound conjugated to the immunodominant - myelin oligodendrocyte glycoprotein (mog - ) for the selective immunosuppression of the encephalitogenic t cells in ms patients. the anthraquinone was synthesized by a friedel-crafts acylation of hydroquinone from phthalic anhydride, followed by reduction of the resulted quinizarine to its leuco form, addition of the appropriate diamine and air oxidation . the synthesized molecules were purified using liquid chromatography, and they were identified by mass spectrometry and h-nmr. the synthesis of the mog - was performed under microwave irradiation and its conjugation with the anthraquinone was performed in solution. the final analogue was purified by rp-hplc and identified by esi-ms. benzopyrans, diketopiperazines and , benzodiazepin- , -diones are well-known and widely investigated scaffolds, e.g. the latter showing anxiolytic and antiarrhythmic effects. now, we propose a new potential "privileged structure" containing a triazole moiety mimicking the cis-amide bond within the , -benzodiazepin- , -dione motif .molecules based on this [ , , ]-triazolo [ , -d] benzo- , diazepin- -one scaffold are synthesized and decorated via a modular approach on wang resin using α-amino acids, -ethynylaniline building blocks and n-alkylating agents resulting in five points of diversity. the methodology involves the attachment of α-amino acids onto a solid support, subsequent removal of the fmoc group followed by an optimized diazotransfer reaction of the resulting amine yielding a resin-bound azide. conversion of the latter into a , -disubstituted , , -triazole moiety is achieved quantitatively by addition of a range of -ethynylaniline building blocks using a ru(ii)-catalyst. the desired scaffold can be obtained in high crude purities (> %) in solution via an acid catalyzed one-step cyclisation-release strategy. solution-phase n-alkylation finally affords the fully diversified scaffold. interestingly, n-alkylation induces atropisomeric effects which can be studied via h nmr spectroscopy.taking into account future screening results of the synthesized libraries, a well-thought decoration of this scaffold leading to discovery of new lead molecules is within reach. peptide symposium in the wonderful small seaside town of porto carras. maurice manning and lajos balaśpiri were nominated as captains of the two teams, the rest of the world and europe. similar matches were organized at subsequent european peptide symposia. now, in greece, the two captains would like to hand over their roles to younger scientists [professors gabor mezö(hungary) and laśzlóÖtvös (usa)] to continue this tradition at the coming european and possibly american peptide symposia. it seems best to play in the free time (in the evenings after the excursions). necessary conditions: good weather; a nice large soccer field; a soccer ball, preferably new; and jerseys and shorts (different colours), organized as always by the organizer commettee. the captain of the winning team will receive a trophy at the end of the nd european peptide symposium. the teams will remember two earlier excellent referees: professor lajos kisfaludy in porto carras [ ] and the soccer professor + ferenc puskaś (hungary) in budapest ( ). in the poster session, the results from the past years will be presented in about - pictures. these pictures may possibly be bought free, % can be saved at the poster session. all participants are welcome at the new party in athens. conclusion will be presented by the players and fans in athens. the human lactoferrin-derived peptide, hlf - , was proven to be highly active against antibiotic-resistant bacteria . however, the clinical use of this antimicrobial peptide (amps) is hampered by the peptide low stability due to fast degradation or to peptide aggregation, as the use of higher peptide concentrations results on higher toxicity levels. amp immobilization onto a biomaterial surface could be the pathway to overcome these difficulties . the aim of this work is the development of an antimicrobial surface by covalent immobilization of hlf - onto the surface of chitosan thin films. chitosan ultrathin films were prepared through the spincoating of a . % chitosan solution in gold substrates. hlf - immobilization was performed through an ss bound between hlf - terminal cysteine and an n-acetyl cysteine previously coupled at chitosan films. surfaces were characterized using ellipsometry (thickness), infrared reflection absorption spectroscopy (irras) and x-ray photoelectron spectroscopy (xps). bacterial adhesion studies were performed using methicillin-resistant s. aureus (atcc ). chitosan films were incubated with this bacterial suspension at ºc for h and h. the viability of the attached bacteria was evaluated using live/dead® bacterial viability kit (baclight tm ) and fluorescence microscopy. hlf - peptide was successfully covalently immobilized onto chitosan thin films. both soluble and attached peptide presented a higher antimicrobial activity than the control chitosan. identified as a potent vasoconstrictor that binds with high affinity to ut receptor. the cysteine-linked cyclic region, hut-ii( - ), is responsible for the biological activity and has been widely used to elucidate the structure-activity relationship of hut-ii. with the aim to investigate the role of hydrogen bond and the effects of a peptide backbone constraint on binding key: cord- -w gc nx authors: nan title: poster presentation abstracts date: - - journal: j pept sci doi: . /psc. sha: doc_id: cord_uid: w gc nx nan background and aims: homodimerization of myd adapter protein is essential for nf-kb activation in the inflammatory pathway triggered by il- and tlr [ ] . we designed a peptidomimetic of the myd tir domain consensus peptide arg-asp-val-leu-pro-gly-thr [ ] , named st . here, we report its synthesis and biological activity. we also report the synthesis and biological activity of its enantiomer, st , and its diastereoisomers, st and st . methods: the structure of the myd tir domain consensus peptide is subdivided into three distinct portions, the most important of which is a b-turn. in the peptidomimetic design we changed the b-turn with a tricyclic spirolactam [ ] , already known [ ] . we synthesized this building block, its enantiomer and two of possible diastereoisomers by "in solution" synthesis. based on semiempirical calculation of heat of formation [ ] , we could predict the right stereochemistry of the products selectively obtained in the last cyclization step. results: these four compounds were tested for their biological activity by reporter gene assay (rga). some coimmunoprecipitation experiments were also carried out and we report their results. conclusions: the results show the activity of st and its isomers on our target, with limited specificity towards their stereostructure. introduction of a methylene bridge between the cα(i+ ) and the n(i+ ) atoms in an open peptide (i) to mimic simultaneously the cαh(i+ ) and hn(i+ ) protons (β-lactam scaffold assisted design -β-lsad) has proven to be a practical tool for the preparation of monotopic β-turn peptidomimetics (ii, r = r = h), according to the principle of separation of constraint and recognition elements . in this work we report a short, general, and stereocontrolled synthesis of multitopic β-lactam scaffolds of type vi. α-alkyl serinates iii are converted into the corresponding enantiopure nnosyl-aziridines iv which undergo "in situ" ring-opening with amino acids v. subsequent base-promoted cyclization affords the n-protected α-alkyl-α-amino-β-lactams vii. incorporation of the novel scaffolds into linear and cyclic peptides and their conformational features are also presented, most of them showing stabilized β-and γ-turn conformations. poly(amino acids) are emerging as promising therapeutic carriers finding widespread application in the field of drug delivery. in this context, polyproline polymers have been used to solubilize poorly water-soluble proteins, in affinity chromatography for the purification of platelet profilin, and more recently, in the design of dendrimers. poly(amino acids) are most conveniently synthesized by polymerization of the corresponding amino acid n-carboxyanhydride (nca). in spite of the interest of polyproline, the preparation of proline n-carboxyanhydride (pro-nca) renders poor synthetic yields. in this work a new method for the preparation of pro-nca in high yields and purities is described. amino acid n-carboxyanhydrides are obtained by the method described by fuchs. but, in the case of proline, the n-carbamoyl chloride does not cyclise spontaneously as it takes place with other amino acids, and the use of a non-nucleophilic base is required for the cyclisation. a tertiary amine, such as triethylamine, is commonly used but it renders a low conversion of the n-carbamoyl chloride to the expected pro-nca, together with the presence of the pro-pro diketopiperazine byproduct. in the present work, polymer-supported bases have been used instead of triethylamine. higher yields of pro-nca, and very low percentages of diketopiperazine have been obtained. in addition, no tertiary amine contamination was observed. polymer-supported bases could also be recycled and pro-nca yields were reproducible. in conclusion, we have developed an efficient method for pro-nca preparation with polymer-supported bases. the introduction of novel nonproteinaceous heterocyclic amino acids into peptides results in new compounds with interesting structural, physicochemical and biological properties. the transformation of amino acid side chains after the peptide assembly is a convenient method of generating such modified peptides. taking into account the biological activity and complexing abilities of nitrogen-containing heterocycles, we investigated the formation of imidazole, benzimidazole and quinoxaline moieties using condensation with various aldehydes and α-dicarbonyl compounds after classical peptide synthesis on solid support. the imidazole synthesis utilizes the n-terminal or side chain amino group of amino acids, whereas a derivative of phenylalanine, β-( -amino- -nitrophenyl)alanine, was developed for benzimidazole and quinoxaline synthesis. the modified peptides were purified by preparative hplc and characterized by esi-ms, uv and nmr. in conclusion, we developed a straightforward method of synthesis of peptides with specific ion affinity and spectral characteristic. the broad range of commercially available aromatic aldehydes and dicarbonyl compounds makes possible the synthesis of combinatorial libraries of modified amino acids and peptides. part of this work was supported by a grant no. t a from the ministry of education and science. nmda receptors belong to the ionotropic group of glutamate receptors. the activity of the receptor can be altered by compounds acting at binding sites. the (r,s)-(tetrazol- -yl)glycine (tg) has been shown to be a highly potent nmda (n-methyl-d-aspartic acid) receptor agonist with exitotoxic effects [ ] . the aim of our studies was to investigate the chelating ability of tg towards copper(ii) ions. copper is widely distributed throughout the body with a distinct concentration in the brain. copper enters cells as complex and seeks out targets requiring it to function. for these reasons it was interesting to evaluate stability and structure of tg -copper(ii) complexes. the equilibrium and structural properties of complex species were characterized by ph-metric and spectroscopic (uv-vis and epr) methods. in the system, polymeric species are dominant at acidic ph range having { nh , coo-} coordination with possible ntetr bridging elements. monomeric complexes were found at physiological ph. the two tg molecules are bound to copper ion via four nitrogen donors. the formation of two {nh , ntetr} donor sets results in very strong metal-ligand interactions and the complex species are very stable over a wide ph region. we have also performed an investigation on similar tetrazole compounds in order to compare the chelating ability of the tetrazole moiety . the targets of our studies were , -diamino- h- , , , -tetrazole [ ] and tetrazole aspartic acid. references continuing work in that field, we synthesized oxytocins containing tetrazole analogues of amino acids. the -tetrazolyl group is widely used in medicinal chemistry as an isostere of the carboxyl group. compounds containing tetrazole ring appear to be metabolically more stable than their carboxylic analogues and have comparable acidity. we synthesized derivatives of aspartic, glutamic, and alpha-aminoadipic acids containing h-tetrazole ring in side chains. these derivatives were then used for syntheses of oxytocin analogues substituted in position . apart from above we also obtained two analogues with tetrazole analogue of glycine in position . the first one contains h-tetrazole ring, the second one has tetrazole ring substituted with methyl group in position . oxytocin analogues possessing amino acids with tetrazole ring in side chains were synthesized on amide resin using fmoc methodology. in the case of analogues with c-terminal tetrazole ring, fragments - were synthesized on resin and then coupled with suitable dipeptides in solution. all obtained peptides show no pressor and rather low uteronic activity. however, for some analogues the uterotonic activity when measured in the presence of magnesium ions was several times higher. in humans, two classes of defensins, α-defensin and β-defensin, have been identified on the basis of tissue specificities and structural features including their modes of disulfide pairing. in general, particular combinations with disulfide bonding in cysteine-containing peptides are critical for expressing their intrinsic biological activities. in the case of human α-and β-defensins, however, disulfide isomers without the native pairing were demonstrated to exhibit similar antimicrobial activity to that of the native defensins. therefore, to assess the biological activities of defensins as well as defensin-based therapeutics, extreme care is required in the chemical synthesis of these peptides to avoid ambiguity in quality. in the present study, we synthesized human α-defensin- , - and - , and human β-defensin- , - , - and - by employing boc chemistry, and determined the optimal conditions for folding the respective reduced peptides preferentially into a native conformation. among the factors affecting the oxidative folding in the presence of reduced and oxidized glutathione, the buffer concentration and reaction temperature were essential. all the synthetic human α-and β-defensins were confirmed to have the respective native disulfide pairing by sequential analyses and mass measurements with cystine segments obtained by enzymatic digestion. all the human α-and β-defensins could be efficiently oxidized to the α-and β-defensin-type disulfide structure, respectively, under several conditions determined in the present study. these synthetic peptides of high homogeneity were used to accurately assess the antimicrobial activity. native chemical ligation is based on the reaction of a peptide bearing a c-terminal thioester group with an n-terminal cysteinyl peptide, leading to the formation of an amide bond at the aa-cys junction. the key starting materials for native chemical ligation are unprotected c-terminal thioester peptides. thioester peptides are often prepared using boc/benzyl solidphase peptide chemistry. however, the widespread use of the fmoc/tert-butyl chemistry for peptide synthesis, over the boc/benzyl method, has stimulated the development of methods allowing the preparation of thioester peptides that are compatible with the basic treatments used to remove the fmoc alpha-amino protecting group. we report here a novel method for thioester peptide synthesis that is based on the use of the sulfonamide safety-catch linker. once the peptidyl chain is assembled by fmoc/tert-butyl chemistry, the thioester function is generated on the solid-phase through an intramolecular n,s-acyl shift. the procedure seems to be insensitive to the bulkiness of the amino acid directly attached to the sulfonamide linker. the thioesters were successfully used for native chemical ligations in solution or on the solid support. we optimized the recognition sequence of the substrate and the reaction conditions with respect to the yield. the sortase-mediated ligation was successfully applied to the synthesis of cellpenetrating peptide-pna conjugates which showed enhanced activity in antisense experiments compared to pna alone. this ligation strategy was also employed for the coupling of a chemically synthesized construct of the extracellular loops of the crf-receptor with the corresponding n-terminal receptor domain, which was expressed in e. coli. this kda protein behaves like an artificial receptor, binding specifically natural ligands. linear gramicidins represent the most investigated family of antibiotic peptides forming ionic channels. gramicidins produced by bacillus brevis are hydrophobic peptides composed of amino-acids with d and l configuration strictly alternate. the presence of d-amino acids in the sequence of gramicidin a (hco-val-gly-ala-dleu-ala-dval-val-dval-trp-dleu-trp-dleu-trp-dleu-trp-nhch ch oh) should possible make the peptide highly resistant to proteolysis [ ] . striking features like ethanolamine group in c-terminus, the n-terminal n-formylated valine and the high hydrophobicity of the peptide sequence, make the solid-phase synthesis of gramicidin a very tricky. therefore, we followed a new synthetic strategy for peptide chain elongation assisted by microwave energy. in fact, microwave energy has been demonstrated to produce highpurity compounds with more rapid reaction times, enhancing coupling rates and efficiency in difficult syntheses [ ] . however, microwave-assisted solid phase peptide synthesis (mw-spps) has not been yet extensively investigated. in this context, we synthesized gramicidin a by mw-spps in high yield and purity, enhancing reaction rate compared to the traditional spps. thermal disruption of peptide aggregation, induced by microwaves, is possible favorable for obtaining this particularly difficult sequence. gramicidin a was incorporated in synthetic lipid bilayers, self-assembled on mercury electrodes, characterized by hydrophilic spacers interposed between the metal and the lipid bilayer. we tested the behaviour of gramicidin a in biomimetic membranes using electrochemical impedance spectroscopy (eis), ac voltammetry and other electrochemical techniques [ ] . [ csf (glc) is an n-glucosylated peptide to be produced in large scale by peptlab because it is the active molecule of the first specific diagnostic/prognostic test for monitoring disease activity and guiding therapeutic treatments of multiple sclerosis patients [ ] . in order to develop a synthetic protocol by an automated instrumentation, increasing yield, purity of the crude, and reaction time, a microwave-assisted solid phase peptide synthesis was validated comparing the use of the new generation of triazine-based coupling reagents (tbcrs) with a series of commonly used ones. activation of carboxylic acids by tbcrs is particularly effective because of formation of triazine "superactive esters". the usefulness of tbcrs as coupling reagents has been recently confirmed in the synthesis of z-, boc-, and fmoc-protected dipeptides, sterically hindered amino acids, in the synthesis of esters, in manual and automated spps of difficult peptide sequences, and head-to-tail constrained cyclopeptide analogues [ ] . moreover, we also demonstrated tbcrs efficient in a microwave-assisted solution synthesis of the n-glucosylated building block fmoc-asn(glcoac )-oh using a manual monomode microwave instrument [ ] . this building block was used to obtain csf (glc) comparing the efficacy of a monomode microwave automatic instrument with the traditional solid-phase peptide synthesizers such as the manual and automatic in batch systems, as well as the continuous-flow one. it is known that enzymatic peptide synthesis is more advantageous than chemical synthesis in many aspects; it is highly stereoselective, racemization-free and requires minimal side-chain protection. the method is, however, limited to the use of amino acid derivatives which meet the enzymatic specificity as a coupling component. this problem may be solved using enzymes which have wide specificity of substrate. but in this case, secondary hydrolysis of the resulting peptide may arise from the inherent nature of the protease. in this matter, ficin and ficin-like enzymes were used as cysteine protease to analyze the diminishment of specificity for the substrate. the cysteine protease-catalyzed peptide coupling reaction has been studied by using synthetic fourteen boc-amino acid phenyl and naphthyl esters as acyl donor. the reaction conditions were optimized for organic solvent, ph, and concentration of acceptor. the coupling reaction was carried out by incubating an acyl donor ( mm) with an acyl acceptor (ala p-nitroanilide, mm) and enzyme ( . u) in a mixture of gta buffer ( mm, ph . ) and dmso ( : ) at ْc. the progress of the coupling reaction was monitored by rp-hplc. the products were obtained in satisfactory yields. non-enzymatic glycosylation, also called glycation, is a common modification in living organisms formed by the reaction of carbohydrates with free amino groups of peptides and proteins. it is a slow chemical reaction yielding amadori products undergoing further oxidation and degradation reactions finally leading to advanced glycation end-products (age). amadori products are early markers for ageing, diabetes mellitus and alzheimer's disease. despite the clinical importance of these amadori products, universal protocols to synthesize amadori modified peptides are still missing. here we describe a solid phase strategy for the glycation of specific amino groups on partially protected resin bound peptides using a global post synthetic approach. the peptides were synthesized by standard fmoc/tbu-chemistry using carbodiimide activation. the lysine position to be modified was incorporated with a methyltrityl protected ε-amino group, which can be selectively cleaved after completion of the peptide synthesis with % tfa in dichloromethane. the partly deprotected peptide was glycated in methanol using a ten-fold molar excess of , - , -di-o-isopropylidene-aldehydo-β-d-arabino-hexos- -ulo- , -pyranose and nabh cn for h at °c. after cleavage the overall yields were in the range of - % for the tested octapeptides. all byproducts were well separated by rp-hplc allowing a simple purification strategy even for medium-sized peptides. thus the general strategy presented here allows routine synthesis of amadori peptides at reasonable yields and purities using standard protocols established in most laboratories synthesizing peptides. -chlorotrityl chloride resins are recommended for the synthesis of c-terminal proline peptide acids to overcome diketopiperazine formation during chain assembly. however, we have found these (and similar) resins to be unsuitable for the synthesis of peptides greater than residues. for example, the chemokine guinea pig eotaxin, ( residues c-terminal proline) assembles poorly if not at all on a -chlorotrityl resin. we sought to circumvent these problems in the chemical synthesis of peptides and proteins, through the development of a resin-swap procedure. whereby the initial c-terminal protected tripeptide is assembled on a -chlorotrityl resin, liberated from the solid-support, then reattached to a resin that is suited for long chain peptide / protein synthesis. using this approach, the synthesis of guinea pig eotaxin is reported. the tripeptide fmoc-thr(but)-lys(boc)-pro-oh was assemble on -chlorotrityl resin, cleaved with % tfe in dcm and attached to wang resin using standard protocols. peptide assembly gave the gp eotaxin in % overall yield (as determined by uv monitoring). fmoc-on cleavage, purification and tag removal followed by folding gave the native chemokine in good yield. choice of resin is one of the most critical factors in ensuring a successful peptide synthesis, we have shown the superiority of wang resin over chlorotrityl resin in the synthesis of medium and long peptides and developed a method for the synthesis of c-terminal proline containing peptides which overcomes the problem of diketopiperazine formation. the technique is being applied to the synthesis of other c-terminal proline peptides e.g. human eotaxin and ip . dimerization of cell receptors, involved in antigen presentation, is an essential step in several cellular signal transduction processes, therefore substances that are able to modulate such a process are of potential therapeutic value. dimeric peptide ligands could represent useful tools to cause dimerization of such receptors. a similar strategy applies dimerization of ligands, interacting with dimeric proteins or proteins with multiple binding sites, to design molecules with enhanced affinity. dimeric analogs of the immunosuppressory hla class ii fragments were synthesized using suitably modified, standard fmoc solid-phase protocols and mbha-resin. the dimerization was achieved by crosslinking n-terminal amino groups of the peptides with the commercially available mixture of poly(ethyleneglycol)biscarboxylic acid (average mw , length range - Å), activated by esterification with pentafluorophenol. the same procedure was applied to synthesize a series of dimeric analogs of c-terminal fragments of plexin-b, consisting of two undecapeptides, linked by the polyethyleneglycol spacers. other biand polyvalent linkers were also investigated. our results demonstrated that the amino-terminal dimerizations of the tested hla-fragments resulted in enhanced immunosuppressive activities, whereas interaction of pdz dimer with the plexin fragments led to about -fold increase in affinity, as compared to their monomeric counterparts. [background and aims] elucidation of alzheimer's disease (ad)-related aß - dynamic events is a difficult issue due to uncontrolled polymerization. [methods] based on the "o-acyl isopeptide method" (chem. commun. , ; j. am. chem. soc. , , ), we have developed a novel photo-triggered "click peptide" of aß - ( ), e.g., " -n-nvoc- -aiaß ( )", in which a -nitroveratryloxycarbonyl (nvoc) group was introduced at ser in -o-acyl isoaß - ( -aiaß , ). [results] i) the click peptide did not exhibit the self-assembling nature under physiological conditions due to one single modified ester; ii) photo-irradiation of the click peptide and subsequent o-n intramolecular acyl migration afforded the intact aß - ( ) with a quick and one-way conversion (so-called "click"); and iii) no additional fibril inhibitory auxiliaries were released during conversion to aß - ( ). [conclusions] this method provides a novel system useful for investigating the dynamic biological functions of aß - , such as the self-assembly and aggregation processes in ad. several insulin analogues have recently been introduced clinically for improved treatment of diabetes. industrial productions of such insulins are based on microbial expression systems, which are highly efficient, but generally limited to the proteogenic amino acids. also, some sequences form inclusion bodies or fail to express. the total chemical synthesis of insulin in research scale was a landmark achievement in peptide science. however, the most commonly used method relies on recombination of a-and b-chains under "random" folding and pairing of the three disulfide bridges. this folding/oxidation step is difficult and low yielding. a general approach using a removable auxiliary which can direct correct formation of disulfide bridges is highly desirable. in the pancreas as well as in microbial expression systems, insulins are prepared and folded as single chain precursors, with a c-peptide connecting the a and bchains. the c-peptide helps direct the orientation of a and b-chains in obtaining the correct disulfide pairing and overall peptide folding. upon folding, the c-peptide is removed enzymatically. we report here a new method for total chemical synthesis of insulin by use of fmoc-based step-wise solid-phase synthesis of single-chain precursors followed by cpeptide directed folding and cleavage of c-peptide, thereby allowing total chemical synthesis of novel insulins with unnatural substitutions. -chloro- -methoxy- , , -triazines a-c anchored on cellulose, silica or wang resin were prepared by the treatment of , -dichloro- -methoxy- , , -triazine with appropriate solid support in the presence of a base. immobilized, environmentally friendly triazine coupling reagents a-c were obtained by treatment of a-c with n-methylmorpholinium p-toluenesulfonates in the presence of hcl acceptor. the loading of the solid carriers were calculated from n, s contents, determined by microanalysis. all prepared immobilized n-triazynylammonium toluenosulfonates a-c have been found stable at room temperatures. activation of carboxylic components afforded triazine activate esters a-c connected to the support. treatment of a-c with appropriate amino components gave amides or peptides. the final products, chromatographically homogenous amides and peptides, were isolated by filtration or extraction from the solid support. mutter's pseudoproline dipeptides and sheppard's hmb derivatives are powerful tools for enhancing synthetic efficiency in fmoc spps. they work by exploiting the natural propensity of n-alkyl amino acids to disrupt the formation of the secondary structures during peptide assembly. their use results in better and more predictable acylation and deprotection kinetics, enhanced reaction rates, and improved yields of crude products. however, these approaches have certain limitations: pseudoproline dipeptides can only be used for sequences containing serine or threonine, and the coupling of the amino acid following the hmb residue can be extremely difficult. to alleviate some of these shortcomings, we have prepared fmoc-ala-(dmb)gly-oh and fmoc-gly-(dmb)gly-oh. these dmb-dipeptides can be incorporated into peptides in place of ala-gly and gly-gly, resulting in peptides containing structure breaking (dmb)gly residues. by introducing the (dmb)gly residue as part of a dipeptide unit, the need to acylate the highly hindered secondary amino group of (dmb)gly is avoided. on treatment with tfa the dmb group is cleaved regenerating gly. to test the efficacy of our new derivatives in expediting the synthesis of hydrophobic peptides, we undertook the preparation of the challenging neurotoxic prion peptide - <b> </b>; this peptide reportedly can not be made using fmoc spps methods. the dipeptides marked in bold were systematically substituted with the appropriate dmb peptides. the effects of the substitution were evaluated using conductivity monitoring and lc-ms analysis of the crude peptides. h-lys-thr-asn-met-lys-his-met-<b>ala-gly</b>-ala-ala-ala-<b>ala-gly</b>-ala-val-val-<b>gly-gly</b>-leu-gly-oh <b> </b> th efficient dipeptide production form unprotected l-amino acids with the novel enzyme l-amino acid α-ligase. k. tabata , h. ikeda , m. yagasaki , s. hashimoto background and aims: application of α-dipeptides has been limited due to the lack of cost-effective manufacturing methods. the known methods require the protection of amino acid(s) to fix the order of the amino acids ( fig. ) . furthermore, they usually accompany the formation of longer peptides. to establish the costeffective manufacturing method, a novel activity which synthesizes α-dipeptides from two unprotected l-amino acids was screened. methods and results: a gene was found in the genome of bacillus subtilis by in silico screening based on a putative reaction mechanism. the purified protein coded on the gene, i) catalyses α-dipeptide formation from unmodified l-amino acids with a specific order in an atp-dependent manner, ii) never forms tri-or longer peptides, and iii) takes a wide variety of l-amino acids but no d-amino acids. the enzyme was tentatively named l-amino acid α-ligase (lal). the whole cell reaction of a recombinant e. coli strain expressing lal and polyphosphate kinase (ppk) with two l-amino acids and polyphosphate (polyp) enable the efficient production of many dipeptides with a certain order of the constituent amino acids through the coupling reaction of lal and ppk (fig. ) . conclusion: a novel enzyme, lal, enables to synthesize dipeptides cost-effectively directly from unmodified l-amino acids. t. ye marine organisms continue to provide rich sources of structurally unique and pharmaceutically active compounds. due to the difficulties in the isolation of significant quantities of these natural products, synthetic chemistry serves an important role in their structural assignment and biological evaluation. antifungal agents have received considerable attention recently since the spread of hiv has left many people open to fungal infections, and there is a rapidly growing number of drug resistant strains of fungus emerging. ll- g gamma is a cyclodepsipeptide isolated from the marine fungus hypoxylon oceanicum and structurally assigned in by schlingmann. the structure of ll- g gamma was determined by a combination of chemical degradation, chiral chromatography and spectroscopic analysis. ll- g gamma uniquely combines a beta-ketotryptophan and a polyketide portion within a macrolactone ring. ll- g gamma has exhibited potent activity against fungal strains and as such, is an attractive compound to develop as a future therapeutic agent. to date, there have been no reported studies towards the synthesis of ll- g gamma. we have completed the total synthesis of ll- g gamma by employing the macrolactamization followed by a c-h oxidation as the key step. aspartimide (aminosuccinimide, asu) formation is the first step in the degradation of asp/asn containing peptides and proteins. the reaction is especially prevalent at asx-gly sites and results in a variety of rearranged and racemized products. the bases used in fmoc-tert-based spps promote the formation of asu and related products. we recently found that the dmb backbone protection efficiently prevents secondary structure formation at gg sites and is orthogonal with respect to standard fmoc spps. here we explore the use of dmb, tmb and nbzl groups (z) for the synthesis of "difficult"/asu-prone peptides, in three different schemes: a) fmoc-asx-(z)gly-oh dipeptide building blocks; b) fmoc-(z)gly-oh monomer building blocks, and c) two steps "submonomeric" approach for synthesis of substituted n-benzyl glycines on the resin. we tested the new methods on two model peptides vkd/ngyi and ha - hiv-tat - (h-g lfgaiagfi engwegmidg grkkrrqrrr -oh) fusion peptide. the yield and purity of the products reach and even exceed the level in control experiments obtained with hmb protection and the peptides were found free of asu/piperidides. the acid removal of the dmb protection is ~ % faster than that of hmb. the submonomeric route (strategy c) is especially simple, efficient, cost effective and it allows the use of different amines for halogen-displacement. the backbone protecting groups used were in many respects superior to the commercial reagents and applicable for synthesis of both peptide acids and peptide amides. the use of nbzl-nh for halogen displacement represents a new method for preparation of backbone-caged peptides. alkyl bonded silica gels historically have been the standard in reversed phase (rp) purification of biomolecules such as synthetic peptides, small proteins, and oligonucleotides. silica gels provided the resolving power needed for challenging separations and the mechanical stability required to be operated industrially under high pressure conditions. the chief disadvantage of silica gels is poor chemical stability under alkaline conditions, which limits their capability to withstand rigorous clean and sanitization -in-place (cip/sip) protocols. as a result, polymeric media have gained recent market attention because of their excellent chemical stability, which enables full compatibility with modern cip/sip protocols. however, first generation polymeric gels lacked both the resolving power and the mechanical stability to be compatible with industrial high pressure dynamic axial compression (dac) hardware. rohm and haas' advanced biosciences division recently introduced a new, monospheric, micron, high performance polymeric rp material. unlike existing softer polymeric gels, this product has higher mechanical stability which enables it to be used effectively with industrial dac / hplc hardware. in addition, this material provides high resolving power for the most challenging industrial separations, because of its unique and selective pore structure, as well as its small monospheric particle size. finally, because of its excellent chemical stability, the media is not limited in the range of ph that can be used. the combination of mechanical stability for high throughput, chemical stability for long lifetime in use, and high resolution for high yield, together translate to an effective cost-in-use solution for industrial polishing processes. we have developed new types of peptide nucleic acids with improved water solubility by introducing ether linkages and pyrrolidine rings in the main chain; pyrrolidine-based oxy-pnas (popnas). in this work, cellular uptake and endosomal release of the trans-l-popna oligomers, one of stereoisomes of the popna, were investigated. the cellular uptake was achieved by combining the popna oligomer with an n-terminal -mer peptide of an influenza virus hemagglutinin protein (ha ) that is labeled with a rhodamine fluorophore at the n-terminal and covalently linked with a hepta-arginine unit at the c-terminal (rho-ha -r ). the fluorescence images of the cho cells after incubation with fam-po( ) [fam-o-cag tta ggg tta g-gly-nh ] in the absence and presence of rho-ha -r were observed with confocal laser-scanning microscopy. incubation with fam-po( ) alone, no internalization of the oligomer was observed. in the presence of rho-ha -r , however, fam-po( ) was successfully internalized into cho cells and, more importantly, the fluorescence spread over the whole cell. the fluorescence image indicates that the popna oligomer in combination with the ha -r peptide was transferred into cytoplasm within h. since both the red (rho) and green (fam) fluorescence spread over the cytoplasm, the popna oligomers that were taken up into endosomes together with the rho-ha -r were released into cytoplasm as the disruption of the endosomes by the ha peptide. in summary, the popna oligomers were readily taken up into cytoplasm of cho cells, when combined with a ha -r peptide. most of functional rnas have post-transcriptional modifications, some of which are quite important for their structure and function. thus, for studying such rnas, it is necessary to use purified raw rnas obtained from living organisms. isolation of native rna is necessary also in the case of analyzing the sequence and modifications of mature rna, which may be different from simple transcript of its gene. therefore, rna isolation method is required. many previous reports demonstrated isolation of rnas, especially trnas. most common and traditional purification methods are based on successive column chromatographies. it seems difficult to apply such method to every trna because effective combination of columns varies among individual trnas. to overcome the difficulty, a sequence-specific selection method using a solid-phase dna has been devised. in this method, a trna can be purified from rna mixture by a single step. however, this method needs high temperature treatment, which might assist hydrolysis of rna strand and might impair heat labile modifications. pna-rna hybrid has been known to be much more stable than dna-rna hybrid. thus pna-based rna purification method seems to be possible for wider variety of rnas in lower temperature, in comparison with dna-based method. in this study, we attempted to purify a single rna, such as a trna and a noncoding rna, from rna mixture by using immobilized pna. r. pipkorn , w. waldeck , h. spring , j. jenne , k. braun background and aims: safe drug delivery technologies are pivotal for genetic interventions, but viral vectors baer the risk of inflammatory reaction. questions concerning the efficacy of delivery of the genetic substances, the desired topical gene activation and targeting must be answered. therefore we attempted to develop a membrane non-perturbing delivery system for transport of inactive functional genes into cells and tissues. genes can be subsequently activated at the target site. our concept bases on the use of peptide-nucleic-acids (pnas) resistant against proteases and nucleases, oligonucleotide derivatives, in which the phosphate-backbone has been replaced with ethylen-amin connected alpha-amino-ethylglycine-units. methods: peptides conjugates were composed and synthesized according to the solid phase synthesis and protecting group chemistry strategies. pna sequences were conjugated covalently, non cleavable, with a capronic acid spacer to the nls, pkkkrkv. pnas have gained broad attention in antisense/antigene experiments and as diagnostic tools. in principal, they can be synthesised with several activating reagents known from peptide synthesis. namely, hatu or pybop are often used. synthesis with hatu is more laborious, because preactivation is needed in order to avoid guadinylation of the n-terminus of the growing pna-chain. we wanted to use pybop, because preactivation should not be needed in this case, which is especially useful in automated synthesis. surprisingly, in the pybop-mediated syntheses of mer pnas we obtained products showing molecular masses approx. da above the expected ones. detailed analysis revealed, that the modification occurred at the only guanine residue in the sequence. in order to further characterise the side reaction, a short pna fragment was synthesised using hatu and pybop activation, respectively, and cleaved from the resin with and without the n-terminal fmoc-group. while synthesis with hatu gave the desired products, pybop partly activates the aromatic carboxy group of the guanine residue, which is substituted by piperidine during subsequent fmoc cleavage. the modified sequences could be further characterised by ms/ms-fragmentation. our results show that care must be taken when synthesising pnas with pybop activation. on the other hand, this reaction possibly opens an opportunity to synthesise guanine derivatives. the opioid receptor system in the central nervous system (cns) controls a number of physiological processes including pain, reward, gastrointestinal and cardiovascular functions. as a consequence, most pain modulating compounds currently available cause a variety of side-effects. the endogenous ligands for the opioid receptors are a series of peptides that includes endomorphin- . endomorphin- has been shown to elicit potent anti-nociception through the highly selective activation of µ-opioid receptors. it is this receptor that mediates supraspinal analgesia and thus, selectivity for this receptor results in analgesia without affecting other processes. therefore, endomorphin- is considered a promising lead compound for the development of a new, safer pain medication. we have synthesized a large number of lipid-and carbohydrate-modified endomorphin- analogues and screened these compounds for their binding and activation of µ-and δ-opioid receptors in sh-sy y cells as well as caco- cell monolayer permeability and plasma stability. compounds conjugated with either a lipoamino acid or sugar moiety on the c-terminus lost binding affinity by several orders of magnitude, whilst n-terminal conjugations resulted in minimal loss of binding affinity. a number of analogues showed pm binding affinity and high apparent permeability, and of these compounds, one has been selected for assessment in nociceptive and neuropathic pain models. in addition to these pre-clinical studies, internalization and tolerance formation of these compounds has also been measured in an effort to synthesise a non-tolerant opioid agonist. endomorphin- analogues with a high degree of amphiphilicity cause increased receptor internalization and subsequently less tolerance formation. a. marcinkowska , l. borovičkova , j. slaninowá , z. grzonka carbohydrate moieties of glycopeptides and glycoproteins play different decisive roles in various biological phenomena. conformation and solubility of proteins are influenced by the oligosaccharide chains, which can also inhibit the proteolytic degradation. as a result, the synthesis of glycopeptides is an attractive field that contributes to understanding of mutual interactions between both moieties and for their biological interest. the synthesis of glycopeptides requires a combination of synthetic methods from both carbohydrate and peptide chemistry. moreover, this synthesis needs stereoselective formation of the glycosyl bond between a carbohydrate and a peptide (amino acid) part, and also an appropriate protecting group methodology that allows selective deblocking of only one functional group in these polyfunctional molecules. in the present work we modified the oxytocin and vasopressin structure with glycoamino acids. transformations of fmoc-protected serine and threonine derivatives into appropriate o-glycosylated precursors suitable for solid phase peptide synthesis were worked out. the -and -o-glycosides were synthesized from fmocserine and fmoc-threonine allyl esters and appropriate glycosyl bromide using hanessian's modification of the koenigs -knorr reaction. these n--fmoc-protected glycosides were used in synthesis of glycopeptides. eight analogues of oxytocin modified in position were obtained. we have also prepared two types of lysin-vasopressin analogues modified with glycoamino acid, in which the glucuronic acid was attached to the ω-amino group of lysine in position through the amide bond. glycosylated analogues of oxytocin and vasopressin display an increased stability towards enzymatic degradation, and retain some hormonal activities. supported by grants: ds/ - - - (zg) and z (js) according to many authors the formation of amadori products is a key stage in the glycation process. glycated proteins may show allergenic properties and potentially initiate autoimmunological processes. they may also serve as the markers of diabetes. to our best knowledge, all procedures concerning the synthesis of peptide-derived amadori products reported in literature are based on "in solution" approach which makes them tedious and time consuming. a modified method of the solid phase synthesis of peptide-derived amadori products based on direct alkylation of the deprotected ε-amino groups with , : , -di-oisopropylidene-β-d-arabino-hexos- -ulo- , -pyranose in the presence of sodium cyanoborohydride was proposed. isopropylidene groups, protecting the sugar moiety in the obtained conjugate, were removed with trifluoroacetic acid containing % water. studies on optimization of the reaction performed on the model peptide attached to a wang resin, fmoc-lys-leu-leu-phe-(resin), showed that the best yield of the product is attained with a two-fold excess of , : , -di-oisopropylidene-β-d-arabino-hexos- -ulo- , -pyranose and a five-fold excess of sodium cyanoborohydride. the identity of the product was confirmed by high resolution ms. the several side products were isolated and their structures will be discussed. our results prove that the synthesis of glycated peptides in the solid phase is feasible. the lack of homogeneous glycoproteins in sufficient quantities is an ongoing challenge in glycobiology. in order to solve this problem researchers have turned to a variety of approaches ranging from mutant eukaryotic strains to the highly demanding total synthesis of glycoproteins. [ ] using rnase b as a model nglycoprotein [ ] we have searched a path to assemble this enzyme employing a combination of chemical and recombinant methods. native chemical ligation [ ] allows the coupling of protein segments of unrestricted size in a chemoselective manner. we have developed solid phase methods to produce the required thioester building blocks - -sr (a) and glycopeptide thioester - -sr (b) containing an n-glycan at asn on a dual linker pega resin. [ ] the remaining segment - (c) was expressed in e. coli as a fusion protein and released by intein mediated protein cleavage. [ ] sequential coupling of the three rnase segments requires the use of a protective group at the n-terminus of segment b compatible with the oligosaccharide part. dysfunctional mutations of antitrypsin can result in a loss of elastase inhibitory activity or allow self-aggregation to occur and cause emphysema and cirrhosis, respectively. insights of the mechanism of disease provide strategy to cope with the aberrant protein aggregation and may bring potential therapeutic agents. in the present work, we describe our effort to identify effective anti-protein polymerization ligands by the employment of combinatorial technology. antitrypsin from human plasma was purified by glutathione sepharose and mono q-sepharose column chromatography. both ala-scanning and peptide shortening were carried out systematically to explore the structural requirements necessary for binding. combinatorial chemistry was then employed to conduct the library screening experiments. assessment of peptide binding was achieved through an unique gel electrophoresis assay. the structural requirements and the minimal peptide length required for binding were revealed by our systematic approach. this information was critical for the design of combinatorial library and the discovery of antitrypsin binding peptides with much improved affinity and specificity. there is currently no effective cure for z antitrypsin related cirrhosis and emphysema. the synthesis and screening of combinatorial libraries offer avenues to increase throughput and ultimately lead to the discovery of inhibitory peptides to the polymerization of pathogenic antitrypsin. with the rapidly increasing number of biopharmaceuticals in the industrial pipeline the need for efficient and expedient purification procedures is growing ever greater. affinity chromatography is one of the most promising technologies in this regard, as it offers very high selectivity and can often replace lengthy and expensive traditional chromatographic procedures. the use of combinatorial split-and-mix libraries is a powerful tool for discovering new affinity ligands but the technique has been limited by the laborious spectroscopic and chemical analysis needed to identify the binding ligand. we have previously introduced a novel bead encoding technology based on a -dimensional image recognition of patterns made by fluorescent particles randomly distributed inside larger beads. [ ] the beads are read prior to each chemical transformation by an instrument featuring three fluorescence microscopes at a rate of , beads per hour. we here present the development of small peptidomimetic affinity ligands for the human growth hormone (hgh) by the use of this technology. the library was sought enriched prior to synthesis by in silico screening of a virtual combinatorial library using a large number of diverse building blocks. binding ligands were identified by incubation with fluorescence tagged hgh. [ the cinnamic acids and their derivatives have been found to possess a variety of biological effects, including antiviral, antimicrobial, antitumor and antioxidant activity. for example, several hydroxycinnamic acid conjugates with amino acids, isolated from plant sources showed enhanced antioxidant activity. the synthesis of cinnamic acid amides and their opioid activity was also cited in the literature. however the synthesis and pharmacological properties of sinapoyl-peptide amides continues to be virtually unexplored. on the other hand, the synthesis and opioid activity of analogs of tyr-mif- has been well documented by us. herein we present a synthesis of a series of sinapoyl -peptide amides where sinapic acid were attached consecutively to both c-and n-end of the tyr-mif- peptide chain: sa-pro-xaa-gly-nh ; sa-tyr-pro-xaa-gly-nh ; pro-leu-gly-nh(ch )nnh-sa sa=sinapic acid; xaa=leu, unusual aminoacid; n= , to obtain the sinapoyl-peptide-amides, both fmoc-and boc-based spps approach were used. analgesic activity was determined by the randall-sellitto paw-pressure test. the antioxidant effects were examined by dpph test as well. studies to establish the importance of introducing the sinapoyl moiety in the tyr-mif- molecule for the antioxidant and opioid activities are underway. several proteins are involved in the transcription of dna to mrna, among which the basic leucine zipper (bzip) proteins. these transcription factors bind specific dna sequences by dimerization and inserting short alpha-helices into the dna major groove. because the dimerization domain is only required to obtain the correct geometrical positioning of the alpha-helices, we will replace it by a dipodal steroid scaffold with defined stereochemistry. due to orthogonal protecting groups, a unique feature of this scaffold is the possibility to design not only homodimers, but also heterodimers. therefore this strategy allows for the construction of both major/major groove and major/minor groove binding peptides, either mimicking naturally occurring proteins or designing peptides with new binding properties. native chemical ligation and staudinger ligation are both suitable for the construction of these peptide dimers. moreover, a combination of solution-and solid-phase chemistry allows for the generation of combinatorial libraries. the increasing number of antibiotic-resistant bacteria is a global health problem. therefore the development of new highly efficient drugs is one of the major tasks of this century. as an example of peptides, which inhibit the growth of e. coli, we demonstrate an easy and rapid method for finding peptides with optimized antimicrobial properties. as a first step we built a modular construct. this construct consists of a constant cationic and a variable module. the cationic module was choosen to achieve cellpenetrating properties. the variable module was expected to act as the virtual active part of the peptide. to increase the proteolytic stability of the peptide we synthesized them in cyclic form. in the first step we used the combinatorial approach to screen approximately . . peptide sequences in the variable region in order to find highly active peptides against e. coli. to optimize the identified sequence, we substituted all amino acids of the sequence with other amino acids and building blocks. additionally, in order to increase stability we modified the bridging. in this way we were able to uncover peptides with high antimicrobial activity as well as proteolytic stability and reasonable solubility. a series of melanocortin active core tetrapeptide hfrw nonpeptide imitations has been prepared using a combination of solution and solid phase synthesis. most of them included residue of -( -imidazolyl) propylamine or histamine as substitutes of histidine. phenylalanine residue, which is included in melanocortins was replaced by residues of derivatives of , '-disubstituted isopropylidenedicyclohexane, , '-disubstituted bicyclohexane, , -disubstituted cyclohexane, , -disubstituted cyclooctane, and , -, , -or , disubstituted benzenes. instead of arginine, residues of oligomethylene diamines, -butyl- -ethyl- , -pentanediamine, , '-methylene-bis(cyclohexylamine), and , '-diaminodiphenylmethane were introduced. -naphtyloxyacetyl-, ( - h-indol- -yl)-butyryl-, -phenyl-ethanesulfonyl-and naphthalene- -sulfonyl-groups served as replacement of tryptophan residue. tested on binding assay on melanocortin receptors, active core imitations exhibited a micromolar affinity to them. isopropylidenedicyclohexane and bicyclohexane derivatives showed about fold higher affinity compared with corresponding derivatives of cyclohexane, cyclooctane or disubstituted benzene. obestatin is a novel endogenous ghrelin-associate peptide, which is involved in the regulation of food intake and weight gain. it was shown to be anorexigenic, able to decrease food intake, gastric emptying and jejunal motility. although obestatin and ghrelin originate from a common prepropeptide of residues, they are reported to exert opposing physiological roles, by binding distinct receptors belonging to the subgroup of type a gpcrs [ ] . obestatin was found to be the natural ligand of the orphan gpr receptor, a gpcr, expressed in jejunum, duodenum, stomach, pituitary, ileum, liver and hypothalamus. as many other peptides involved in the obesity process, it is a new and interesting drug target for the discovery of new anti-obesity molecules. in particular, the first step for the design of new molecules with potential improved anti-obesity activity, is the elucidation of the obestatin conformational features. here, we present the synthesis and the conformational analysis by nmr and cd spectroscopies of obestatin and its related -mer c-terminal sub-fragment, in aqueous solution and in membrane mimicking environment. the data outline the obestatin c-terminal portion as the region characterized by significant conformational features potentially opened to interesting future developments. [ a total of isolates of rhizobium were collected from root nodules of medicago sativa and melilotus officialis plants in different regions of isfahan province .all of isolates on ty medium formed white ,slimy colonies with smooth margins and their inoculation on to roots of young alfalfa plants produced spindly nodules . the nodules developed with some of the isolates were big and pinkish ,although the rest of isolates produced small and white nodules .the speed of nodulation for all the isolates was almost similar and the related nodules were appeared within two weeks . the production of brown pigments on aged colonies of some isolates on ty or ty supplemented with l_tyrosine and copper sulfate revealed that these isolates of s. meliloti are melanin-producing rhizobia.based on the motility and sensitivity to antibiotics tests ,all of the isolates formed a reasonably homogenous group .however a few of them were able to produce an anti-microbial compound which was found to inhibit a number of isolates of s. meliloti .the compound did not suppress the growth of other bacteria . partial purification and spectrophotometery of the compound suggested that it likely belong to the antimicrobial polypeptides .considering on their physiological and biochemical properties ,none of the isolates were selected as a superior and competitive strain ,although based on nodulation efficiency , melanin and antimicrobial compounds production capability the isolate s. meliloti sm and sa were nominated to investigate in details. cyclotides are a fascinating family of plant-derived peptides characterized by their head-to-tail cyclized backbone and knotted arrangement of three disulfide bonds. this conserved structural architecture, termed the cyclic cystine knot, is responsible for their exceptional resistance to thermal, chemical and enzymatic degradation. cyclotides have a variety of biological activities but their insecticidal activities suggest that their primary function is in plant defense. in this study we determined the cyclotide content of the sweet violet viola odorata, a member of the violaceae family. we identified cyclotides from the aerial parts and roots of this plant, of which are novel sequences. the new sequences provide information about the natural diversity of cyclotides and the role of particular residues in defining structure and function. as many of the biological activities of cyclotides appear to be associated with membrane interactions, we used hemolytic activity as a marker of bioactivity for a selection of the new cyclotides. the new cyclotides were tested for their ability to resist proteolysis by a range of enzymes and, in common with other cyclotides, were completely resistant to trypsin, pepsin and thermolysin. the results show that while biological activity varies with the sequence the proteolytic stability of the framework does not, and appears to be an inherent feature of the cyclotide framework. the structure of one of the new cyclotides, cycloviolacin o , was determined and shown to contain the cyclic cystine knot motif. this study confirms that cyclotides may be regarded as a natural combinatorial template that displays a variety of peptide epitopes most likely targeted to a range of plant pests and pathogens. furthermore, the inherent stability of the framework makes it an excellent scaffold for protein engineering applications. warfarin is the most widely prescribed anticoagulant drug for the prevention and treatment of arterial and venous thromboembolic disorders.because of large interpatient variability in the dose-anticoagulant effect relationship and a narrow therapeutic index careful dosage adjustment based on inr is essential. warfarin is available as a racemic mixture of two enantiomers,(s)-and (r)-warfarin. in contrast to (r)-warfarin, which is metabolized by multiple cytochrome p s(cyps), including cyp a and cyp a ,(s)-warfarin, is predominantly metabolized to -hydroxywarfarin by polymorphic cyp c . since the potency of (s)-warfarin is much higher than that of (r)-warfarin, about -to -fold,any change in the activity of cyp c gene is likely to have a significant influence on the anticoagulant response. previous in vitro findings revealed that certain variants in the cyp c gene are associated with large interindividual differences in the pharmacokinetic and pharmacodynamic outcomes of warfarin therapy. three major alleles have been found to date in humans:arg /ile , and cys /ile and arg /leu , and arg /leu , which have been designated cyp c * (wild-type), cyp c * , and cyp c * , respectively. we have investigated this polymorphism in iranians that has not been described previously. genomic dna was isolated from whole blood. for detection of cyp c * , and cyp c * variants, a protocol based on pcr technique and endonuclease digestion with kpni, ava ii was used. in this research work, we have studied a group of patients, in which warfarin therapy was initiated. recently new -residue antimicrobial peptides -arenicins were isolated from coelomocytes of marine polychaeta arenicola marina and their sequences were determined [ ] . there are two isoforms of arenicins which differ only with single amino acid. these peptides have no structure similarity to any previously identified antimicrobial peptides. we have synthesized and estimated the antibacterial properties of arenicin- : rwcvyayvrvrgvlvryrrcw. the linear peptide was prepared by solid phase method using boc-technology without any problem. however the cyclization caused the appreciable difficulties. the following methods of oxidation were used: oxygen of air, k fe(cn) and hydrogen peroxide in aqueous or organic media. the best results were obtained by using hydrogen peroxide in methanol, but and in this case the yield of the aim peptide did not exceed %. synthetic arenicin had the same hplc profile and maldi-tof spectra as a natural molecule. the peptide showed an antimicrobial activity against gram-positive bacteria: peptidergic hormones and neurotransmitters are known to be produced by the specific cleavage of their precursor proteins that per se have no biological functions. the neutrophil-activating peptides we recently identified, however, are the peptides cleaved from mitochondrial proteins by proteolysis. therefore, we named them "functional cryptic peptides" because they are hidden in protein sequences. some of these peptides activate gi type of g proteins directly, and neutrophils are suggested to be stimulated by the direct (i.e., not via gpcrs) activation of g proteins. these peptides had features, in common, in their distributions of charged and hydrophobic amino acid residues, but homologies in their primary structures were not apparent. in the present study, we predicted functional cryptic peptides that activate g proteins, based on the distribution of charged and hydrophobic residues. receptors for these peptides were also investigated by the direct cross-linking experiments between peptides and their targeted proteins. the finding of functional cryptic peptides is expected to lead to the identification of novel signaling mechanisms where such peptides are involved in the regulation of bio-functions. the fragment - of the precursor of human interleukin- alpha (pil- α) (gk-vlkkrr) appeared to have more than % homology with corticotropin fragment - (gkpvgkkrr). we have previously synthesized the octapeptide gkvlkkrr (referred to as leucocorticotropin, lct) and found its high affinity binding to corticotropin receptors on various immunocompetent cells in human and mouse. in this study we investigated the interaction of lct with rat adrenal cortex membranes and the effects of lct on the level of -oxycorticosteroids (cs) in rat adrenal glands and plasma in vivo. lct was labeled with tritium by the high-temperature solid-state catalytic isotope exchange reaction to specific activity of ci/mmol. receptor binding studies revealed that tritium-labeled lct bound with high affinity and specificity to corticotropin receptor on rat adrenal cortex membranes (kd = . nm). lct at concentrations of . - nМ was found to have no influence on the adenylate cyclase activity in adrenal cortex membranes, while intranasal injection of lct to rats at doses of - microg/kg was found to inhibit the secretion of cs from the adrenals to the bloodstream. thus, lct is an antagonist of corticotropin receptor. comarin derivatives such as warfarin are prescribed widely for treatment and prevention of thrombosis. warfarin is the widespread oral anticoagulant drug employed, but its required dose is highly variable both inter-individually and inter-ethnically. so it is desirable to develop strategies to predict the warfarin dose response in patients before initiation of anticoagulation. the vitamin k-dependent γ-carboxilation system, consists of the vitamin k-dependent γ-carboxylase, which requires the reduced hydroquinone form of vitamin k as a cofactor and the warfarin sensitive enzyme vitamin k , -epoxide reductase (vkor), which produces the cofactor. warfarin exerts its anticoagulant effect by inhibiting the vitamin k epoxid reductase enzyme complex (vkor) that recycles vitamin k , -epoxide to vitamin k hydroquinone. a component of the vkor termed vkorc , has now been identified as a therapeutic target site of warfarin. point mutations were identified within the gene encoding vkorc in individuals who required large doses of wafarin to maintain therapeutic anticoagulation. however the relationship between the primary structure of vkorc and the mechanism of action of warfarin is poorly understood. in previous works we have shown that naturally occurring functional protein fragments affect cell proliferation [ ] . their mechanisms of action involve receptors of "classical" regulatory peptides, or are non-receptoric [ ] . among protein kinases involved are pka, camkii, mapk [ ] . in organism bioactive functional protein fragments could participate in maintenance of tissue homeostasis. in present work, homeostatic potential of functional protein fragments was studied in compare with classical regulatory peptides. the panel of test substances was formed, including signal transduction modulators (pka, pkc, ca +-channel activators), classical regulatory peptides (bradykinin, somatostatin, met-enkephalin, endothelin, neurotensin) and fragments of functional proteins (β-actin fragments from ( - ) and ( - ) segments; valorphin (β-globin ( - )); neokyotorphin (α-globin ( - ); short acidic peptides from multiple precursors). their activity was tested in cultures derived from similar sources but differing in transformation degree (e.g., mouse embryonic vs. tumor fibroblasts) and/or culturing conditions. the factors most affecting cell sensitivity to the test substances were (in order of the importance decrease): ( ) cell type; ( ) transformed vs. normal phenotype; ( ) cell density; ( ) serum supply. activity of fragments of functional proteins, showing general correlation with other test substances, was more influenced by the culturing conditions (i.e., cell population status). thus, fragments of functional proteins could be regarded as partners of "classical" homeostasis regulators, playing role of finer tuners of tissue proliferative status. the study was supported by ras presidium programme "molecular and cell biology" histone-like proteins in bacteria are small basic proteins that contribute to the control of gene expression, recombination and dna replication. they are also an important factor in compressing the bacterial dna in the nucleoid. among the hlps, hu protein is attracted to dna containing structural aberrations such as four way junctions or single stranded lesions. this protein plays an important role in binding as a dimer and bending dna. it also contributes to the beginning of the dna replication. in this study we showed that a -kda protein, probably hu exists in halobacillus karajiensis which is a novel gram positive moderate halophile bacteria that was recently isolated from surface saline soil of the karaj region, iran. since hu is purified and characterized in e.coli we used this bacteria as the control in this study. the -kda protein extraction was carried out by using pca % which is normally used for extracting histones from eukaryotic cells. the results of running the protein extracts on sds-page demonstrated a band around -kda which was seen in protein extracts of this protocol. these results supported the hypothesis of the existence of a -kda protein in halobacillus karajiensis. sufficient oxygen and nutrient delivery is a necessity for tumors. when oxygen supply decreases, tumors initiate growth of new blood vessels. low grade astrocytomas, a class of malignant brain tumors, grow along the existing vessels in a process called co-option. hypoxia is induced in the progression from grade iii to grade iv astrocytomas (glioblastoma multiforme, gbm) which in turn triggers the formation of a new and distinct tumor vasculature. the new vessels formed by tumor-triggered angiogenesis differ by molecular composition from their normal vascular counterparts. we are utilizing phage displayed peptide libraries to identify peptides that specifically home to either co-opted or angiogenic brain tumor vessels. furthermore, we aim at characterizing differentially expressed endothelial markers (receptor molecules) to get a better understanding of the molecular changes in the vasculature. several rounds of in vivo biopanning was performed in mouse models of astrocytomas to isolate a phage pool that has up to a hundred-fold homing to low grade tumor lesions. out of the selected pool we discovered peptides capable of homing and accumulating to the tumor islets and co-opted vasculature. the homing potential of our newly identified peptides has shown to be highly specific for clusters formed by the tumor cells and co-opted "early" vessels within these palisades. these homing peptides represent promising candidates to selectively target co-opted vessels and tumor lesions in the brain and act as lead compounds in identification of surface molecules (receptors) differentially expressed by co-opted tumor vessels. the αvβ integrin receptors play an important role in human tumor metastasis and growth. the inhibition of these receptors by antibodies or by cyclic peptides containing the arg-gly-asp( rgd) sequence may be used as selectively treatment to suppress the disease. our research group has previously described that the formal introduction of a single carbon atom to bridge the cα (i) and n(i+ ) contiguous residues of a linear or cyclic peptide leads to α-amino-β-lactam peptidomimetics containing predictably placed β-turn and γ-turn motifs, respectively. the combination of these results with the well-known capacity of rgd tripeptide for inhibition of the biological answer in integrin led us to the design of the following cyclic peptide. the adhesion and cell-growth "in vitro" assays using human umbilical vein endothelial cells (huvec), as well as "in vivo" assays with xenograph mice revealed that the rgd peptidomimetic was active to micromolar concentrations, slightly better than the reference compound in this field: cilengitide®. whole saliva is composed of secretions from parotid, submandibular and sublingual glands, and smaller ones from saliva of minor salivary glands (e.g. palatal and labial). saliva contains a variety of proteins and polypeptides. one of them is statherin, a multifunctional -amino acid residue, phosphominiprotein. this peptide is present in human parotid and submandibular saliva . the aim of our study was to investigate the stability of statherin in extracts of the major salivary glands. submandibular, sublingual and parotid gland tissues were obtained at autopsy h after death. samples of the gland tissues were homogenized, centrifugated ( , g, min., c) and the supernatants were frozen and stored at - c prior to analysis. synthetic statherin was added to the supernatants before analysis ( microgram/ml). the samples were analysed for the presence of the peptide by the matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (maldi-tof ms}technique and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds page). statherin has been found to be decomposed in extracts of parotid and submandibular glands and also in the extract of sublingual glands. the dramatic increase in research for new anthrax therapeutic approach was prompted by potential use of the causative agent of anthrax bacillus anthracis as a biological weapon. anthrax toxin consists of three proteins, the protective antigen (pa) and the two enzymes lethal factor (lf) and edema factor (ef) that are carried through the membrane of the target cell upon binding to specific site on the membrane receptor-bound pa. lethal factor and edema factor were found to cooperate to promote immune evasion of the bacterium. here we describe the production of peptide inhibitors of pa-lf binding, obtained by selecting pa-binding peptide by a competitive panning of a phage peptide library, using recombinant lf. we selected several mer peptides, which were synthesized in tetra-branched multiple antigen peptide (map) form, inducing resistance to proteolytic degradation ( ) and maintaining biological activity of phage peptides. lead tetra-branched peptides were systematically modified by progressive shortening and residue randomization, to obtain an increase of peptide affinity and inhibitory efficiency. affinity maturation of lead sequences enabled selection of a peptide which has an ic at least one log lower that any other lethal-toxin-inhibiting peptide described so far and is effective for in vivo neutralization of anthrax toxin activity ( ) . the same peptide can also efficiently inhibit the binding of ef to pa and ef-induced camp increase in different cell lines. microtubules are dynamic polymers that have important roles in eukaryotic cellular processes such as signal transduction, cell polarity, vesicular transport and chromosomal movement. the dynamic behavior of microtubules has been studied both in vivo and in vitro. the effect of arsenic trioxide on microtubule polymerization has been studied under in vivo experimentation shown that it inhibits formation of mitotic bundles. we studied the mechanism of arsenic trioxide effect on polymerization of microtubule protein purified from sheep brain in vitro. microtubule polymerization has been conducted by adding mm gtp to purified tubuline in pem buffer at oc for minutes and simultaneously followed by measuring turbidity ( nm). the results shown that lag time of polymerization (nucleation step) is affected by increasing concentrations of arsenic trioxide from - micromolar. moreover the rate of elongation step was decreased exponentially by increasing arsenic trioxide concentration. electron micrographs also showed microtubules length decrement due to arsenic trioxide. the results have shown the inhibitory effect of arsenic trioxide on microtubule polymerization via its effect on nucleation step as well as elongation rate. background and aims: alzheimer's disease (ad) is the major cause of dementia among the elderly. the increase in life expectancy worldwide demands new therapies for ad urgently. self-association of the amyloid ß-protein (aß) into neurotoxic assemblies, a seminal event in the etiology of ad, is considered to follow interactions of the c-terminus of the -residue form of aß (aß ). we hypothesized that molecules with high affinity for the c-terminus of aß will disrupt aß oligomerization. a series of c-terminal fragments (ctfs) of aß , aß(x- ) with x = - , has been prepared to study their potential to inhibit aß oligomerization and neurotoxicity. methods: attenuation of aß assembly by ctfs was studied by quantitative analysis of oligomer size distributions using a photo-cross-linking assay followed by sds-page. biological activity of ctfs themselves and as inhibitors of aß -induced neurotoxicity was assessed by mtt reduction assay using differentiated pc- cells. the structure of the ctfs was studied by circular dichroism (cd) spectroscopy and ion mobility spectrometry-mass spectrometry (ims-ms) coupled with molecular dynamics (md) simulations. results: ctfs were found to inhibit aß oligomerization in a length dependent manner with minimal or no toxicity of the ctfs themselves. certain ctfs were found to inhibit aß -induced neurotoxicity. cd spectra indicate that increasing peptide length results in growing ß-sheet content. structures based on experimentally determined cross-sections support the existence of a previously proposed turn around residues gly -gly . the data suggest that aß ctfs can serve as lead compounds for development of peptidomimetic drugs for treatment and prevention of ad. background and aims: human kallikrein hk is a prostate specific serine protease, which expression level is elevated in aggressive human prostate cancer suggesting a possible role in a tumour growth and spreading. since hk protease is highly prostate specific, inhibition of its activity is a possible method to prevent tumour growth without interfering the function of the other proteases. we have identified hk specific linear peptide inhibitor by using phage display techniques. in order to design peptide for in vivo studies we tested the protease stability of the linear and the cyclic forms of the peptide. methods: the prerequisite of the binding was studied by using conventional ala-replacement method and the most optimal sequence was selected for further studies. the stability of the original linear form, acetylated form, peptide with cystein bridge and head-to-tail cyclic peptide was tested with modified trypsin (sequencing grade) and with human plasma. results: both linear versions and peptide with cystein bridge were unstable and were degraded during the first minutes in both stability tests. head-to-tail form of the peptide was stable in both tests during the first minutes. conclusions: since our peptide contains arginine there was a possibility that our peptide is sensitive to trypsin and other serum proteases. indeed both linear and one cyclic from degraded in our tests. only head-to-tail peptide was stable during the first hours suggesting protease resistant folding. background and aims: a large number of anticancer agents has been developed in recent years. however, these agents have very little or no specificity which leads to systemic toxicity. among them paclitaxel is considered to be one of the most important drugs in cancer chemotherapy; however, this agent has also lack of selectivity to the tumor tissue. therefore, development of tumor-targeting prodrug is highly promising. methods: to activate cytotoxic agent specifically at the tumor tissue, we developed a new prodrug strategy based on o-n intramolecular acyl migration, which is a well-known reaction in peptide chemistry, and photodynamic therapy. results: we synthesized a prodrug which has a coumarin derivative conjugated to the amino acid moiety of isotaxel (o-acyl isoform of paclitaxel). the prodrug was selectively activated by visible light irradiation ( nm) leading to cleavage of coumarin. finally, paclitaxel was released by subsequent o-n intramolecular acyl migration. conclusion: we synthesized and evaluated a novel type of paclitaxel prodrug. this prodrug showed promising kinetic data. therefore, we believe that photoactivation can be promising novel strategy for design of tumor-targeting prodrugs. the search of new immunosupressants, exhibiting the mechanism of action characteristic for cyclosporine a (csa) and fk- is an important challenge for medicinal chemistry. cyclolinopeptide a (cla) natural cyclic nonapeptide [cyclo(leu-ile-ile-leu-val-pro-pro-phe-phe)] possesses a strong immunosuppressive activity comparable with that of csa in low doses. the possibility of practical application of cla as a therapeutic agent is limited due to its high hydrophobicity. it has been suggested that the tetrapeptide sequence pro( )-pro( )-phe( )-phe( ) is responsible for the interaction of the cla molecule with the proper cellular receptor. in order to evaluate the role of this tetrapeptide unit for biological activity of native peptide, we decided to modified this fragment. in this communication we present linear and cyclic cla analogues in which phenylalanine residues in position and/or have been replaced with amphiphilic; alphahydroxmethylphenylalnine or homophenylalanine . the synthetic strategy and biological activity will be evaluated. resistance to currently used small molecule antibiotics develops at an alarming rate. while resistance to β-lactams in clinical isolates is primarily due to hydrolysis of the ring by β-lactamases, when bacteria develop resistance to fluoroquinolones or aminoglycosides, the sequences of the target biopolymers are altered. earlier we developed a family of antibacterial peptide derivatives that kill bacteria by inhibiting protein folding and are active in animal models of infection. in the current study we examined the synergy between antibiotics acting by different modes of action. inhibition of properly folded active resistance enzymes was completely efficacious to recover the activity of amoxicillin, a β-lactam antibiotic against strains that were originally resistant to this molecule. some activity of ciprofloxacin was also recovered by reducing the load of the induced self-defense dnak protein, but the synergy between the antibacterial peptide and the fluoroquinolone did not yield full bacterial killing. the mode of action of the synergy is indeed inhibition of protein folding because no such effect could be observed with kanamycin where resistance involves changes in the target protein sequence. as opposed to current β-lactamase inhibitors and combination therapies that work against only a limited number of strains, inhibition of all protein folding in bacteria is a universally applicable treatment option. elimination of resistance to β-lactams by proline-rich peptide derivatives may represent a viable avenue to give second life to these antibiotics for which large stockpiles are available for pharmaceutical companies in both patented and generic forms. the integrin αiibβ is the major integrin-adhesion receptor on platelets. in unstimulated platelets αiibβ is present in a resting conformation state. upon platelet activation by agonists, αiibβ receives intracellular signals (inside-out signaling) that allow its rapid conversion to a high-affinity state capable of binding soluble ligands, resulting in platelet aggregation. the intracellular signals include proteins that bind to the cytoplasmic tails of the two subunits α and β of the integrin, or integrin-associated membrane proteins. in vivo charge swapping mutation studies suggested that αiib and β tails have a direct site of interaction between αiib (r ) and β (d ). peptides derived from the cytoplasmic tail sequences can specifically induce or block αiibβ activation in platelets. the aim of this study is to develop peptide analogues based on the cytoplasmic tail sequences of both αiib and β subunits that could inhibit platelet thrombus formation by specifically disrupting the inside-out signaling pathway. peptide analogues of the αiib and β subunits spanning the sequences αiib- - , αiib - - , αiib - - , αiib- - and β - - , β - - , β - - were synthesized in their free state, palmitoylated and/or tagged with the tat fragment - and carboxyfluorescein-labeled, in order to investigate their membrane permeability, as well as their inhibition of the platelet aggregation. inflammatory pain begins when noxious stimuli (thermal, chemical or mechanical) excite sensorial neurons called nociceptors. the activation of nociceptors leads to the opening of some ionic channels and depolarization of the cell membrane. one of these channels is trpv , which is directly implied in thermal hyperalgesia associated to inflammation. in previous work it has been found that peptoid h-arg- - c ( fig. ) inhibits the activation of trpv by blocking the pore entrance. however, this compound showed toxicity in vivo. the aim of our work is the design and synthesis of new compounds, based on the structure of h-arg- - c, with better therapeutic properties. we synthesized some new non-competitive antagonists of trpv that exhibit notable anti-inflammatory and analgesic activity in vivo. th. skarlas, e. panou-pomonis, d. krikorian, m. sakarellos-daitsiotis, c. sakarellos erf is a transcriptional repressor with tumor suppressor activity regulated by the ras/erk signaling pathway. it has been shown that erf interacts with, and is phosphorylated by erks in vitro and in vivo. this phosphorylation determines its subcellular localization and biological function. erf exhibits a high degree of specificity and sensitivity for erks. the major objective of our study is to provide proof of principal for a specific anti-cancer approach targeting the ras-pathway, which is commonly activated in human tumors, via the stimulus of the downstream effector erf. this will be attained by modeling specific peptide inhibitors that block the erf phosphorylation and inactivation by the ras/erk signaling pathway. we present the design and synthesis of peptide inhibitors incorporating the fsf and fkf motifs, known to play a critical role in the erf/erk interaction, in their free forms or conjugated to a carrier. ubiquitinium is a well known mechanism in protein degredation of eukaryotic cells ,in which many obsolte and corrupted three dimentional structure protein ,become marked by covalent attachment of ubuquitin through a multi-step enzymatic pathway.ubiquitin is a small , . kda peptide of amino acid residues that targets such substrtes for proteolysis in proteasome .recnt studies showed that an extra cellular ubiquitination process also taking place in the epididymes of humans and other animals marks protein on the surface of the defective sperm .it appears that structurally and functionally defective sperm become surface ubiquitinated by epididymal epithelial cells. a certain portion of ubiquitin -labeled sperm is phagocytosed and the remaining is ejaculated .hence ubiquitin on the sperm surface could be a good marker of semen quality control in men. the aim of present study is to purify ubiquitin from packed blood cells , to produce and purify antiubiquitin antibodies,to design an immunofluorescence assy for detection of defective sperm, to compare the percentage of ubiquitinated sperm in oligoasthenotertozoospermia and normozoospermia and finally to determine correlations between sperm parameters and sperm ubiquitination. p. vakalopoulou, ch. anastasopoulos, g. stavropoulos, s. yiannis c-terminal analogues of substance p (sp) have been studied for their ability to prevent tumor growth or the proliferation of several cancer cell lines. the incorporation of damino acids into the sequence of sp and n-methylation of peptide bonds have shown to protect sp from the action of plasma and tissue peptidases. aiming to design and prepare more potential antagonist of cancer cells proliferation and taking into account that all the metabolites of the c-terminal hexapeptide analogue [arg , d-trp , , mephe ]sp - (antagonist g) possess the n-me group and d-trp residue, we proceeded to the synthesis of peptoid-peptide hybrids. they are oligomeric peptido-mimetics containing the residue [-Ν(bzl)-ch -co-]=(nphe). the incorporation of n-substituted glycine in peptide chains has been proved to improve their stability against proteases and give biologically active peptides. thus, the tetrapeptoid-peptide hybrids h-arg -d-trp -nphe -d-trp -oh and h-d-trp -nphe -d-trp -leu -oh, corresponding to metabolites of antagonist g and also the hexapeptoid-peptide hybrids glp -d-trp -Νphe -d-trp -leu -glu(obzl) -ΝΗ and glp -d-trp -Νphe -d-trp -leu -glu(obzl) -oh have been synthesized. the latter have incorporated the amino acid residues glp at the n-terminal and glu(obzl) at the c-terminal of the analogue, which have shown to give to the analogues increased resistance and biological activity. all the products were purified (hplc), identified (esi-ms) and set about for study their biological properties and activity against cancer cells proliferation. a chemokine receptor cxcr has multiple critical functions in normal and pathologic physiology. cxcr is a gpcr that transduces signals of its endogenous ligand, cxcl (stromal cell-derived factor- , sdf- ). the cxcl -cxcr axis plays an important role in the migration of progenitors during embryologic development of the cardiovascular, hemopoietic, central nervous systems and so on. this axis has recently been proven to be involved in several problematic diseases, including hiv infection, cancer cell metastasis, leukemia cell progression, rheumatoid arthritis (ra) and pulmonary fibrosis. thus, cxcr is a great therapeutic target to overcome the above diseases. fourteen-mer peptides, t and its analogs, were previously found to be specific cxcr antagonists that were identified as hiv-entry inhibitors, anti-cancer-metastatic agents, anti-chronic lymphocytic/acute lymphoblastic leukemia agents and anti-ra agents. cyclic pentapeptides, such as fc [cyclo(d-tyr-arg-arg-l- -( -naphthyl)alanine-gly)], were previously found as cxcr antagonist leads based on pharmacophores of t . in this symposium, we would like to report the development of low molecular weight cxcr antagonists involving fc analogs and other compounds with different scaffolds including leaner-type structures. erythropoietin (epo) controls the proliferation and differentiation of red blood cells. it activates epor by inducing dimerization and reorientation of two receptor chains. peptides mimicking the action of epo, epo mimetic peptides (emp), have been discovered by phage display, interacting with the receptor on the active site and competing with the hormone [ ] . another peptide, epor derived peptide (erp), was reported to activate the receptor through an alternative site distant from the hormone binding site, and to have synergic action with epo [ ] . we report the design of new synthetic epo-r agonists by dimerization of active peptides. pegbased polyamide linkers of precise length were used to link the molecules, using oxime chemistry [ ] . these peptides include emps that have been homo-dimerized through their n-or c-terminus. a hetero-dimer of one emp and one erp peptides was also created. biological characterization of the molecules is currently under investigation. [ the envelope spike (s) glycoprotein of the severe acute respiratory syndrome associated coronavirus (sars-cov) mediates the entry of the virus into target cells. recent studies point out to a cell entry mechanism of this virus similar to other enveloped viruses, such as hiv- . as it happens with other viruses peptidic fusion inhibitors, sars-cov s protein hr derived peptides are potential therapeutic drugs against the virus. it is believed that hr peptides block the six-helix bundle formation, a key structure in the viral fusion, by interacting with the hr region. it is a matter of discussion if the hiv- gp hr derived peptide t (enfuvirtide) could be a possible sars-cov inhibitor given the similarities between the two viruses. we used fluorescence spectroscopy techniques to test the possibility of interaction between both t (hiv- gp hr derived peptide) and t- with s protein hr and hr derived peptides. our biophysical data show a significant interaction between a sars-cov hr derived peptide and t . however the interaction is only moderate (kb = ( . ± . ) × m- ). this finding shows that the reasoning behind the hypothesis that t , already approved for clinical application in aids treatment, could inhibit the fusion of sars-cov with target cells is correct but the effect may not be strong enough for application. [ ] were used to investigate the structure, dynamics and thermodynamics of the known complex between erythropoietin mimetic peptides (emp) and erythropoietin receptor (epor). with gromacs . bioinformatics software, we have obtained from the known emps about the key functional amino acids required for effective epo mimetic action. then we systematically altered the amino acids in those peptides, and simulated the complex to observe the differences between the altered peptides with the original ones. based on these results, we designed new emps of potential significance. in order to fast identify the mimetic action of these new peptides, we synthesized these peptides and labeled the epor binding peptide (ebp) with quantum dots [ ] , to study the binding of these new emps to epor. our results illustrate a principle for fast identifying receptor-specific sites importance for receptor internalization, and for enhancing sensitivity to hormones and other agonists. blood vessel formation largely contributes to the pathogenesis of numerous diseases, including ischemia and cancer [ ] [ ] . in this regard therapeutic strategies aim to stimulate vascular growth in ischemic tissues and suppress their formation in pathologies like in tumour and diabetic retinopathy. placental growth factor (plgf), an homolog of vascular endothelial growth factor (vegf), ( % amino acid sequence identity), stimulates angiogenesis and collateral growth in ischemic heart and limb. whereas vegf exerts it biological function through the binding to both vegf receptor- (vegfr- or flt ) and vegfr- (or kdr) plgf binds specifically to flt . the complex plgf/flt constitutes a potential candidate for therapeutic modulation of angiogenesis and inflammation [ ] . the binding between plgf and flt- has multipunctual features [ ] and potential antagonist must have a sufficient molecular surface to spatially distant contact points. we have used an elisa-like screening assay to select antagonists of plgf/flt- complex from a large random library of tetrameric unnatural peptides (complexity: ^ = . molecules) identifying two active molecules with an about m ic . the relative stability of identified peptides were assessed in human serum and their inhibitory properties were tested in a capillary-like tube formation assay performed with human umbilical vein endothelial cells (huvec the αvβ integrin is a cell adhesion receptor involved in angiogenesis and tumor cell invasion. the tripeptide motif rgd is the αvβ minimal recognition sequence and many rgd-containing peptides have been investigated as radiopharmaceuticals for targeting angiogenesis and tumor metastatic phenotype. since rgd sequence binds also to other integrins, the aim of the present study was to develop and characterize a selective αvβ ligand suitable for imaging. a novel peptide containing the rgd loop covalently linked to an echistatin domain (crgdechi) was designed, synthesized and then tested for selective binding to αvβ integrin. a panel of peptides were used for comparison. adhesion assays showed that the novel peptide was able to inhibit adhesion of αvβ overexpressing cells but not αiibβ and αvβ overexpressing clones. in conclusion the novel peptide showed a high affinity and specificity for αvβ integrin. the design of new molecules, based on the lead compound presented here, is currently ongoing with the aim at developing novel anticancer drugs and/or new class of diagnostic noninvasive tracers as suitable tools for αvβ -targeted therapy and imaging. background and aims: short peptides like leu-pro-phe-phe-asp (lpffd) and leu-pro-tyr-phe-asp-amide (lpyfda) can influence the structure and aggregation of ß-amyloid peptides. soto's pentapeptide lpffd has been published as a ß-sheet breaker (bsb). it is necessary to gain more information about the nature of the interaction of aß and the pentapeptides mentioned above for the understanding of their action and for the possible development of future therapeutic agents. methods: in this study radioligand binding assay, diffusion ordered nmr spectroscopy, dynamic light scattering, circular dichroism and ft-infrared spectroscopy was used. results: it was shown by radioligand binding assay and diffusion ordered nmr spectroscopy that both pentapeptides bind to aggregated aß. dynamic light scattering, circular dichroism and ft-infrared spectroscopy revealed, that after the treatment of the aß with the pentapeptides aß fibrils are still present. conclusion: both peptide can bind to aß and can cause small conformational changes of aß, however, they cannot prevent completely the formation of aß fibrils in - micromolar concentration using : molar ratio of aß and the bsb peptide. peptide arrays are convenient tools for the analysis of antibodies, protein binding domains and to address other biological questions. here we present a new method to produce identical copies of arrays on microscope slides. the peptides are synthesized on modified cellulose-discs, using a variation of the spot-method introduced by ronald frank more than years ago [ ] . the new array format overcomes several limitations of the spot-method, e.g. the low throughput with only one copy of the library and the large sample volumes that are needed for membrane incubations. for the presented arrays modified cellulose discs with covalently bound peptides are dissolved after synthesis. the resulting solutions can be spotted onto glass slides by conventional spotting techniques. three dimensional layers of cellulosepeptide molecules are formed on the surface of the supports used for spotting. a virtually unlimited number of identical arrays can be printed and assays are performed with a sample volume of µl or less. as application example we show mapping experiments of the streptavidin recognition site with a peptide library containing histidine-proline motives. because of the much higher peptide loading compared to conventional arrays, the formed -dimensional structure might be superior for protein-interaction studies with even low binding constants. [ the interaction between the cap binding protein, eukaryotic initiation factor (eif) e, and the scaffolding protein eif g is critical for the formation of the heterotrimeric eif f translation initiation (ti) complex (eif e/eif g/eif a). elevated levels of eif e and eif g found in several human solid tumor cancers and the induction of malignant transformation in animal models by overexpression of eif e and the reversal of this phenotype by treatment with anti-sense rna, suggest the importance of the eif e/eif g interaction in the excessive translation of oncogenic proteins. eif g binds to eif e through a conserved eif e-binding motif yx l (non-specified (x) and hydrophobic ( ) amino acids) that interacts with an hydrophobic hot spot on eif e. we report here the identification of a putative eif e anionic exosite that is distinct from the hot spot and contributes to the binding of eif g-derived ligands. our strategy focuses on in situ eif e-templated click reaction-mediated assembly of hybrids comprised from an anchoring minimal eif g-derived peptide fragment, which binds to the hot spot, and a series of complementing positively charged fragments targeting the anionic exosite. we synthesized a training set of [ , , ] triazole-containing hybrid peptides that are potent inhibitors of eif e/eif g interaction. moreover, we achieved in situ eif e-templated assembly of these hybrids from the corresponding fragments via click reaction in the absence of cu(i) catalysis. as such, we demonstrate a proof-of-concept for a new paradigm in the development of inhibitors of protein-protein interaction merging click reaction with fragment-based and in situ target-templated approaches. goodpasture disease is an autoimmune pathology caused by the accumulation of reactive autoantibodies against the alpha- of collagen iv. goodpasture antigenbinding protein (gpbp) is a ser/thr protein kinase that phosphorylates the alpha- chain and might be important in human autoimmune pathogenesis [ ] . we are carrying out in our laboratories the biophysical and functional characterization of gpbp protein. in the presence of some proteins and at specific experimental conditions, gpbp participates in structurally ordered intra-and inter-protein aggregation processes. structure prediction programs identify four different domains for gpbp: an n-terminal domain showing pleckstrin homology (ph domain); a central domain with high tendency to form coiled-coils; a domain with ww features; and a c-terminal start domain ('star-related lipid-transfer'). using the tango algorithm [ ] , we have identified several aminoacid sequences in the gpbp start domain of vertebrates with high tendency to participate in protein aggregation. in this work we present the synthesis and structural characterization of a collection of peptides derived from the sequences described above. we recently developed a combinatorial library screening protocol to identify hpq-containing cyclopeptides that bind streptavidin more tightly than its linear analogues. the relative affinities in ic of these structurally constrained ligands and its linear counterparts were measured by a captured enzyme-linked immunosorbent assay. however, their intrinsic binding kinetics remained to be elucidated. in this work, surface plasmon resonance (spr) was employed to directly determine the kinetics and thermodynamics of the ligands binding to a streptavidin chip. solid-phase peptide synthesis was carried out using standard fmoc chemistry. spr experiments were carried out using biacore optical biosensor. streptavidin was immobilized onto a cm sensor chip using the standard amine coupling procedure. the equilibrium dissociation constants and kinetic on/off rates of n-to-side chain and n-to-c cyclopeptides were deduced by scatchard analysis and computational simulation, respectively. it was found that both cyclopeptides exhibited similar binding kinetics and bound streptavidin far more avidly than its linear form ( -fold). in addition, the reversed (qph) linear and cyclic peptidyl ligands were hardly recognized by streptavidin. not only the binding specificity was distinguished qualitatively, but also the entropic advantage acquired by the pre-organized conformation over its linear analogues was demonstrated quantitatively by spr in this study. the mutation of tumour suppressor genes in the progression of cancer is well characterized. for example, p is found to be mutated in approximately % of cancers and the loss of this proteins activity has been shown to lead to the deregulation of cell growth and apoptosis. the potential of peptide aptamers to inhibit protein/protein interactions in a highly specific manner makes them very attractive as research reagents or as target validation tools in anti-cancer drug discovery. more interestingly, these molecules have the potentially to inhibit the activity of proteins which are key regulators of cancer cell growth and therefore could act as synthetic tumour suppressor proteins. we used peptides based on known protein/protein interactions, as well as peptides isolated using display technologies, for the design of protein aptamers that were used to analyze pathways critical in controlling cancer cell growth. a range of scaffolds were used to present these peptides in an effort to optimize the peptides activities. data relating to the activity of these peptide aptamers in vitro as well as in cellular systems will be discussed. the cyclic undecapeptide urotensin ii (u-ii) is the endogenous agonist for the u-ii receptor (ut), a gq coupled gpcr. current views suggest that binding by agonist, but not antagonist, leads to induction of stabilization of an active receptor conformation. we have previously probed the interactions of urotensin ii with rat ut (rut) using a series of photolabile u-ii analogues containing p-benzoyl-l-phenylalanine (bpa). it was found that the c-jun n-terminal kinases (jnks) are important mitogen-activated protein kinases. these ser/thr protein kinases are activated by various growth factors, cytokines, and cellular stresses. jnks have been shown to play a key role in phosphorylation of proteins in signal transduction of different diseases including cancer, neurodegenerative, cardiovascular, and inflammatory diseases. therefore, these enzymes are considered as important therapeutic target proteins. the interactions of jnk with peptides are of special interest for development of novel specific atp-noncompetitive inhibitors. interactions of this kinase and its mutants with various substrates were demonstrated in vivo using yeast ras-recruitment system. bioinformatical tools have been developed to predict optimized binding peptides as well as to correlate sequence position and amino acid with binding effiency to extract binding determinants. biomolecular interaction analysis have been performed for selected peptide sequences using surface plasmon resonance (spr) technology. real time measurements of the binding of peptides to the different isoforms jnk and jnk resulted in the determination of affinities as well as kinetic constants for association and dissociation. experimental results and their bioinformatic analysis are discussed with respect to critical features of potential atpnoncompetitive inhibitors. the b-domain of is one of the five nearly homologous domains of staphylococcal protein a. this domain contains three alpha-helices which are assembled in an anti parallel three-helix bundle. the b-domain binds the fc region of mammalian immunoglobulins through the n-terminal fragment that contains two alpha-helices. the c-terminal helix does not interact with fc but it is necessary for the correct folding and immunoglobulin recognition of the b-domain. to search for new peptide analogues of the c-terminal helix that bind the n-terminal fragment, a ¨one-bead one-compound¨ library of peptides was designed based on the sequence of the c-terminal helix. active peptides were obtained after incubation of the library with the n-terminal fragment and rabbit immunoglobulin g labelled with fluorescein. new peptides were found and their sequences identified by maldi tof-tof mass spectrometry. the synthesis of the two most active peptides was carried out and the binding with the n-terminal fragment was confirmed by cd spectroscopy. the nterminal fragment peptide showed an increase in helicity when the c-terminal wild type peptide or some analogues were present in solution. the complete domains with the c-terminal fragment mutations were synthesized and structurally characterized by cd and nmr spectroscopy. the wild type and the new mutants adopt predominantly an alpha-helical structure. the interaction between rabbit immunoglobulin g and the wild type b-domain and the new analogues was investigated using surface plasmon resonance. although compared with the wild type, the mutants exhibited different kinetics, they were able to bind the immunoglobulin with high affinity. a. jaśkiewicz, e. bulak, h. miecznikowska, k. rolka sfti- , a strong trypsin inhibitor, was isolated in from seeds of sunflower. it is homodetic -amino-acid-residue peptide containing a disulfide bridge. because of its small size and strong trypsin inhibitory activity, this inhibitor became an interesting model for studying enzyme -inhibitor interactions. sfti- possesses one reactive site located at the lys -thr peptide bond and therefore is able to interact with the enzyme in a : stoichiometry. in this report we describe chemical synthesis and kinetic studies of a series of sfti- analogues containing double sequences of the wild inhibitor. their structures contain combinations of disulfide bridges and/or head-to-tail cyclization. each of these analogues contains two trypsin-specific reactive sites. we expect that kinetic studies should answer the question whether such dimeric analogues are able to interact simultaneously with more then one trypsin molecule and how this fact affects their inhibitory potency. in addition, we alsopresenttwo analogues in which we substituted the disulfide bridge with a carbonyl one. since carbonyl bridge has not been previously introduced into molecules proteinase inhibitors, we decided to check its impact on the activity and proteolytic stability of such modified analogues. ubiquitination, the covalent attachment of one or multiple polymerized ubiquitins is a post-translational modification of proteins, which has manifold functions. it mainly determines the protein for degradation, but also activation, deactivation or substrate alteration. due to its ubiquitinous distribution in all eucarionts no high-affinity antibodies could be originated. highaffinity ligand peptides are of interest to study ubiqutination. based on bioinformatical considerations and investigations of ubiquitin-interacting proteins short peptide sequences were selected. by using a peptide array specific ubiquitin binding was monitored and quantified with label free detection based on reflectometric interference spectroscopy (rifs). the results from rifs were confirmed by detection of binding in solution with fluorescence correlation spectroscopy (fcs) using carboxyfluorescein and s -labelled peptide amides. binding constants were determined by isothermal calorimetry (itc) and rifs. finally h, n-nmr chemical shift analyses of the peptides with the highest affinity were carried out, which allowed the localisation of the interaction site of ubiquitin with the peptide the results from all four methods correlated very good. they showed fast equilibria within s and binding constants down to the low micromolar range. nmr results revealed hints for discrimination possibilities between lys and lys polymerized ubiquitins. ( f is a potent neutralizing mab that binds the human respiratory syncytial virus (hrsv) f protein and is a promising candidate for clinical development. the majority of neutralizing antibodies to hrsv f protein map to two regions of the protein designated site ii and site iv,v,vi. to further characterize the f epitope, we employed a trypsin digestion of a hrsv f protein- f mab complex, followed by mass spectrometry analysis of the resulting recovered mab bound peptide. one peptide at m/z was captured by the f mab. sequence assignment was based upon mass and matched with the database from a virtual digest. this peptide was assigned as residues - [tkctasnknrgiiktfsngcdyvsnk] of the hrsv f protein which spans antigenic site iv,v,vi. to further delineate the epitope, the binding of f mab to a series of peptides corresponding to antigenic site iv,v,vi in the hrsv f protein was determined. based on the peptide elisa data, the f-binding region could be reduced to - sequence [ctasnknrgiiktfs] . as demonstrated by the substitution analysis, r and k significantly contribute to epitope binding, but another positively charged residue, k makes a minor contribution to the binding. both, the peptide elisa and proteolytic digestion of the mab-antigen complex approaches identified the same region of hrsv f protein as being critical for the binding of f. furthermore, these data confirm the results obtained using complementing genetic approaches using a panel of mutations in recombinantly expressed f protein and selection of antibody escape virus mutants (data not presented). the recently identified uracil-dna specific nuclease (ude) is the first representative of a new family of nucleases. the protein sequence has no detectable homology to other proteins except a group of sequences present in genomes of other pupating insects (vertessy et al, submitted). to analyze the physiological function of this protein, peptide conjugates were prepared to serve as synthetic antigens for the generation of antibodies against isoforms of dutpase, an enzyme inherently involved in preventing the synthesis of uracil-dna [ , ] . we used poly[lys(seri-dl-alam)] (sak) as a synthetic branched polypeptide [ ] or bovine serum albumin (bsa) as a natural macromolecular carrier. peptides were prepared by solid phase method utilizing syro (mulltisyntech gmbh, germany) peptide synthesizer, using fmoc chemistry and dipci/hobt-mediated coupling on rink-amid mbha resin. a c-terminal cys(acm) was added to the native sequences for incorporation of sh group into the peptide. in case of sak choroacetylated polypeptide was conjugated with sh-peptide to form thioether linkage. the maleimidobenzoyil-n-hydroxyszukcinimid (mbs) derivative of bsa was used to introduce the peptide into the macromolecule. antibodies have been developed as diagnostic tools and therapeutics for many different diseases. however, the isolation and preparation of intact specific antibodies is often very tedious or even unfeasible. recent studies have shown that single paratope peptides might be well capable to mimic corresponding antigen ligands [ , ] , suggesting that paratope peptides from a native antibody might have many advantages, e.g. for molecular vaccine design and targeting. we have developed a new method for identification of paratope-containing peptides by proteolytic affinity-proteome analysis in combination with high resolution fticr-mass spectrometry (fticr-ms) [ ] . in the present study we used hen eggwhite lysozyme (hel) and a polyclonal rabbit anti-lysozyme antibody (helpab) as a model system. the direct determination of paratope peptides was obtained by selective binding of a dtt-cleavage mixture of the anti-lysozyme antibody to immobilised hel, followed by proteolytic digestion of the antibody-antigen complex (paratope excision, parexprot). two specific paratope peptides were identified by maldi-fticr-ms, and the corresponding peptide sequences were identified by database search within a - ppm threshold. additionally, the identified paratope peptides were synthesised and characterised by affinity mass spectrometry, which ascertained their full binding specificity to lysozyme. the propeptide blocks the active site of inactive zymogen of cathepsin d and is cleaved off during maturation. we have designed a set of peptidic fragments derived from the propeptide structure and evaluated their inhibitory potency against mature cathepsin d using kinetic activity assay. the mapping localized two segments in the propeptide involved in the inhibitory interaction with the enzyme core: n-terminus of propeptide plays a major role and the active site anchor plays a minor role according to their respective ki values. in addition, a fragment derived from the mature n-terminus of cathepsin d displayed inhibition, which supports its proposed regulatory role. the mechanism of interaction of both propeptide segments was characterized by the mode of inhibition and by spatial modeling of propeptide in cathepsin d zymogen. using fluorescence polarization measurements, kd in nanomolar range was determined for the n-terminal propeptide segment. the inhibitory potency of the active site anchor segment was modulated by ala val mutation that was reported to be associated with cathepsin d pathology. . by comparing the resulting low-energy conformations using different sets of atoms, specific conformational features common only to the high/medium affinity compounds were identified. they included the spatial arrangement of the three most important pharmacophoric side chains tyr , arg , and nal as well as the orientation of the xaa -arg amide bond, which together represent a "minimalistic" d pharmacophore model for binding of the cyclopentapeptide antagonists to cxcr . this model rationalizes the data for the cyclopentapeptides as well as for the peptidomimetic cxcr antagonist krh- . automated docking of the pharmacophore model to the d structure of the tm region of cxcr revealed that the pharmacophoric groups of the cyclopentapeptide ligands were involved in favorable interactions with their counterparts in cxcr . for instance, the hydroxyl group of tyr formed a hydrogen bond with lys , the guanidino group of arg formed a salt bridge with glu , and the backbone carbonyl of xaa -arg formed a hydrogen bond with lys . this finding gives additional support for the suggested d pharmacophore model, and also provides opportunities for rational design of cxcr mutants to map potential contacts with peptide ligands. with the successful completion of the human genome project, the next challenge is to assimilate enormous amount of genetic information generated and to assign functions to a large number of proteins encoded. although the dna chip technology to detect the abundance of mrnas has been established, it is known that the abundance of mrnas and proteins does not correlate. thus, protein detection methods for reproducible and quantitative investigation of protein networks are strongly required. we attempted to establish a novel protein detection system based on a fluorescent measurement that does not require labeling of target molecules and preparation of secondary antibodies. we focused on a steric hindrance caused by the interaction between a target protein and a specific capture agent. when a target protein interacts with a specific capture agent immobilized on solid surface, we assumed that a steric hindrance in the vicinity of a capture agent increases. in order to detect the differences in the steric hindrance, we utilized a fluorescent system with the staudinger reaction. this reaction is a chemical ligation between a phosphine and an azide group. these two functionalities are unreactive with protein surfaces under biological conditions. we incorporated an azide group into an immobilized capture agent and investigated the efficiency in the staudinger reaction between the azide and an external triphenyl phosphine derivative. it was found that a target protein bound to the capture agent immobilized onto the solid support interferes with the efficiency in the staudinger reaction. the major histocompatibility complex (mhc) has a crucial role to initiate the immune response via the binding of the peptide fragments (epitopes) of foreign antigens and their presentation to the t-cell receptors (tcr). the co-receptor molecule cd enhances the binding between tcr and mhc ii. small molecules that mimic surfaces of mhc-ii may lead to blockage of the autoimmune response and the development of drugs for immunotherapy. hla-dqa * /dqb * (dq ) and hla-dqa * /dqb * (dq ) are highly correlated to autoimmune diseases as sjogren syndrome (ss) and systemic lupus erythematosus (sle). the non polymorphic β regions of the modelled hla-dq , which are exposed to the solvent and may disrupt the interaction of dq with cd + t lymphocytes were determinated using the getarea program. it was found that the regions - (arg-asn-asp-gln-glu-glu-thr-thr) and - (glu-tyr-trp-asn-ser-gln-lys-glu) display the highest solvent accessibility. peptide analogs of these regions were synthesized, by the fmoc/otbu solid phase strategy, purified by rp-hplc and characterized by mass spectrometry esi-ms. the dimeric analogs of the peptides, designed to mimic the superdimeric nature of the immunosuppressory fragments of hla class ii molecules were also synthesized and investigated. conformational studies were performed with cd spectroscopy and biological experiments are in progress. background and aims: aggregates of β-amyloid peptide (aβ) play central role in the etiopathology of alzheimer's disease (ad). short peptides like c. soto's pentapeptide lpffd and lpyfd-amide synthesized in our laboratory are neuroprotective agents against aβ assemblies both in vitro and in vivo. however, the mechanism of their neuroprotective effect has not yet been fully understood. methods: transmission electron microscopy (tem), cd, ft-ir, diffusion ordered nmr spectroscopy, dynamic light scattering, and radioligand binding assays were used. results: all the methods applied showed that the pentapeptides mentioned above do not break the fibrillar structure of aβ, that is these molecules are not real β-sheet breakers (bsb). the pentapeptides bind to aβ fibrils and cause small structural changes by intercalating into the aβ assemblies. fibrils of aβ survive one week treatment with the pentapeptides using them in to -time molar excess. conclusion: all the results in our laboratory show that the short peptides have long-term interaction on aβ-assemblies. in the first step they bind tightly to the aβ surface and prevent further interaction of aβ fibrils with the neuronal membranes. after this step the short peptides can be built into the structure of aβ-assemblies with intercalation causing a less ordered β-conformation. proteolytic enzymes (neprilisin, ide) could cleave and hydrolyze aβ peptides after this structural change, therefore the short peptides are good drug candidates for the treatment of ad. cellular processes in normal and pathogenic cell states are regulated by external stimuli via complex networks of catalytic and non-catalytic protein-protein interactions. we have developed methodology for the synthetic variation of peptides and peptidomimetics using polymer reagents including linker reagents enabling polymer-supported cacylations. [ ] in combination with the virtual screening of protein subsites, we have demonstrated the application of the novel synthetic methods to inhibitor optimization for various proteases including plasmepsin ii, hiv protease, and sars coronavirus main protease. [ , ] moreover, multivalent peptide polymers have been developed for the intracellular targeting of proteins. [ ] this methodology was now extended to the inhibition of peptide-protein interactions by small molecules. for this purpose, we have composed a library of , small molecules by algorithmic searching of a database of bioactive molecules with virtually designed substructures (fragments). high throughput assays were developed on the basis of fluorescence and fluorescence polarization detection. despite the scepticism regarding the inhibition of protein-protein interactions with small molecules, efficient hit molecules have been developed for several intracellular targets and were subjected to synthetic variation and cellular follow-up assays. the essential event in platelet adhesion to the blood vessel wall after injury or in thrombosis is the binding to sub-endothelial collagen of plasma von willebrand factor (vwf), a protein which interacts transiently with platelet glycoprotein (gp) ibalpha , slowing circulating platelets to facilitate their firm adhesion through other collagen receptors, e.g. integrin alpha beta and gpvi. to locate thevwf-binding site in collagen iii; we synthesized overlapping triple-helical peptides which comprise the whole native sequence of collagen iii . peptide # (gpogpsgprgqogvmgfogpkgnd (o is hydroxyproline)) alone bound vwf, with an affinity comparable to that of native collagen iii. immobilized peptide # supported platelet adhesion under static and flow conditions, processes blocked by an antibody which prevents the vwf a domain from binding full-length collagen. truncated and alaninesubstituted triple-helical peptides derived from # either strongly interacted with both vwf and platelets, or lacked both vwf and platelet binding. thus, we identified the sequence rgqogvmgf as the minimal vwf-binding sequence in collagen iii. the present work completes our understanding of the collagen-vwf interaction, providing information on crucial sequences in collagen that perfectly complements our existing knowledge of the collagen-binding site in vwf and may assist in targeting the collagen-vwf interaction for therapeutic purposes solid phase assay systems such as enzyme-linked immunosorbent assay (elisa), surface plasmon resonance (spr), and overlay gels are used to study processes of protein-protein and protein-peptide interactions. the common principle of all these methods is that they monitor the binding between soluble and surfaceimmobilized molecules. following the use of bovine serum albumin (bsa)-peptide conjugates or isolated synthetic peptides and the above-mentioned solid phase assay systems, we were able to demonstrate that positively charged peptides, which would be expected to repulse each other, can interact with each other. both the elisa and spr methods showed that the binding process reached saturation with kd values ranging between and nm. no interaction was observed between bsa conjugates bearing positively charged peptides and conjugates bearing negatively charged peptides or with pure bsa molecules, strengthening the view that interaction occurs only between positively charged peptides. however, interactions between the same peptides were not observed in solution when was monitored by nuclear magnetic resonance (nmr) or by native gel electrophoresis. thus, it appears that for positively charged molecules to interact one of the binding partners must be immobilized to a surface, a process that may lead to the exposure of otherwise masked groups or atoms. the relevance of our findings for the use of solid phase assay systems to study interactions between biomolecules will be discussed. the hematopoietic progenitor kinase (hpk ), a mammalian hematopoiesis-specific ste kinase, contains a cluster of four proline-rich sequences called p , p , p and p located after the kinase domain. these pro-rich regions play an important role in the interactions of this kinase with different adapter proteins. previous studies showed that p , which contains the canonical pxxpxr motif, and p and p with the canonical pxxpxk motif interact with the c-terminal sh domain of hematopoietic lineage cell-specific protein (hs ) even if with different affinity. hs protein shares a high amino acid sequence and structural similarity to cortactin although their functions differ considerably. here we report the results of our investigation on the interaction between the c-terminal sh domain of cortactin and the four proline-rich motifs of hpk . these interactions were analyzed by non-immobilized ligand interaction assay by circular dichroism (nilia-cd). upon peptide addition, the binding was monitored by the cd changes of the trp side-chains of the conjugate gst-sh cort. the dissociation constants kd were determined analyzing the cd data at nm using a nonlinear regression method. the results demonstrate that gst-sh cort displays an affinity binding higher than that found with the corresponding hs domain and that the four hpk pro-rich regions are not equivalent. p appears to bind with the highest affinity (kd= . µm), followed by p (kd= µm) and p (kd= µm), whilst p does not interact at all. the generation of a fibrin clot is mediated by the regulated activation of a series of serine proteases and their cofactors. factor viii in its activated form, fviiia, acts as a cofactor to the serine protease fixa, in the conversion of the zymogen fx to the active enzyme fxa. both fviii and fix are essential for normal coagulation, deficiencies of either are associated with the bleeding diatheses hemophilia a and b, respectively. the role of fviiia is to bind factor ixa, generating the phospholipid-dependent intrinsic factor xase complex. at least two interactive sites have been identified for the enzyme-cofactor interaction. the ser -gln region within the a subunit has been shown to be crucial for viiia-ixa interaction. in an attempt to study this interaction, we synthesized a series of peptides of - loop of the a subunit. the syntheses of these peptides were carried out by using spps and fmoc/but methodology. the synthesized compounds were purified by rp-hplc and lyophilized to give fluffy solid, identified by ft-ir, nmr and es-ms spectra. these compounds were tested for inhibitory activity on human platelet aggregation in vitro, by adding common aggregation reagents to citrated platelet rich plasma (prp). the aggregation was determined using a dual channel electronic aggregometer by recording the light transmission. and ci/mmol, respectively. both tritiated aβ peptides were used in cat brain( in vivo) experiments and it was found that the peptide aggregates enter the neurons within min (electronmicroscopic autoradiography). this transport is most probably an endocytotic pathway. aβ aggregates could interact also with cytoplasmic proteins such as -phosphoglyceraldehyde dehydrogenase etc. we suppose that aβ assemblies can interact both with membrane receptors (nmda, ampa, ach) and with cytoplasmic proteins triggering neuronal dysfunction and death. background and aims: ww domains are the shortest known protein domains and contain a stable three-stranded b-sheet, which presents the binding site for prolinerich ligands. this interaction is mediated by hydrophobic interactions between aromatic and hydrophobic residues of the domain, and the polyproline core of the ligand. as part of our ongoing efforts aimed at synthetically mimicking conformationally defined protein binding sites ( ), we have designed and synthesized linear and cyclic peptides covering the binding site of the ww domain of human yes-associated protein (hyap-ww), whose structure in complex with a proline-rich ligand had been solved by nmr spectroscopy ( ) . methods: peptides were synthesized by spps, purified by hplc, and characterized by d-nmr spectroscopy, as well as by molecular dynamics calculations. affinities of the peptides to a hyap-ww ligand were determined in direct and competitive binding assays. results and conclusions: a cyclic peptide covering the sequence stretch of hyap-ww that contains its primary contact residues for proline-rich ligands, was found to compete with the domain for binding to a hyap-ww ligand. long-ranging noes identified in the nmr spectra of this peptide indicate a conformation, in which sequentially distant residues are brought into spatial proximity, likely through formation of a beta-sheet. these result demonstrate the feasibility of functional, as well as structural, mimicry of conformationally defined protein binding sites through synthetic peptides. the rockefeller university, new york, ny, usa integrins constitute a family of transmembrane cell surface receptors. they are involved in cell-cell and cell-extracellular matrix interactions. thus, they participate in many physiological and pathophysiological processes and are of crucial importance for the living organism. integrins possess two non-covalently bound subunits, &alpha; and &beta;, that jointly participate in ligand binding. these dimeric proteins show very high specificity in recognition of natural ligands. for example, α β integrin recognizes vcam- (vascular cell adhesion molecule ) and fibronectin through binding amino acid motifs tqidspln and ldv, respectively. on the other hand, fibronectin is a classical ligand for α β integrin with the recognition motif rgd. as shown, identification of the integrin ligands occurs through small recognition amino acid sequences (mostly tripeptides). thus, small cyclic peptides possessing a recognition motif in the appropriate three-dimensional conformation are able to interfere with the integrin-ligand interactions and act as inhibitors. the aim of this investigation is the characterisation of small cyclic peptides containing the rgd motif and the determination of the selectivity and specificity of these inhibitors. two new pentapeptides with -amino-cyclopropane- , -dicarboxylic acid monomethyl ester ((+/-)acc) were synthesized and tested. peptides were characterized in biological assays with living cells (k and wm ) and in surface plasmon resonance binding studies. experiments have shown that cyclic peptide cyclo-(arg-gly-asp-(+)acc-val) is a very potent inhibitor (ic -value in nm range) of interaction between vitronectin and αvβ or αvβ integrins. when preparing biotin-labelled peptides as ligands for avidin-based assays, it is chemically most expedient to locate the biotin label on the n-terminal group of the peptide. this is done without any regard to how this may affect peptide-target interactions, biotin-avidin binding, and the solubility properties of the resultant peptide. in many instances, the products are poorly soluble, have little biological activity, and poor affinity for avidin. problems can also arise during the synthesis of such nterminally biotinylated peptides due to the poor solubility and reactivity of many of the reagents used for biotin introduction. to overcome these limitations, we have developed an extremely simple method for synthesising peptides c-terminally with biotin. peptides can now be easily prepared by standard solid phase techniques either n-or c-terminally labelled, and screened to determine the optimum presentation for the biotin. in cases studies using protein-protein interaction and kinase assays, peptides c-terminally labelled with biotin gave better sensitivity. y. yang , j. eble , n. sewald many bacterial pathogens bind and enter eukaryotic cells to establish infection. invasin is an outer membrane protein required for efficient uptake of yersinia into m cells. invasin mediates its entry into eukaryotic cells by binding to members of β integrin family that lack insertion domains (i domains), such as α β ,α β ,α β ,α β , and αvβ . this type of peptide-protein interaction is an ideal subject for the rational design of inhibitors. the integrin binding motif consists of one loop region with a conservative asp residue and two synergistic regions. the aim of this project is to synthesize cyclic peptides based on the invasin binding epitope sdms. this sequence has to be positioned in a β-turn with asp in i+ position for optimal activity of the peptide. also the arg and asp residue, which are about . Å and . Å respectively away from the asp residue of the sdms loop in invasin, should be investigated. peptides that mimic these recognition sites have been synthesized and tested as ligands for the integrin peptide-dna cross-linking is a very powerful tool for studying peptide-dna complexes. it transforms non-covalent complexes into covalent complexes, which renders characterization of the adduct by classical techniques (mass spectroscopy, nmr,…) much easier. the aim of our research is to develop a new method for peptide-dna cross-linking involving the incorporation of a furan moiety. the strategy is inspired by the naturally occurring process of oxidative furan ring opening by cytochrome p . the resulting cis-butene- , -dial has been shown to react with amino-and sulfhydryl groups of macromolecules such as proteins and dna. in our research, dna binding peptides are modified with a furan moiety and then chemically oxidized into a reactive enal. this enal can react with dna to form a covalent peptide-dna complex. previous attempts to selectively oxidize furan modified minor groove binding peptides consisting of n-methylpyrrole building blocks failed. we are now applying the same strategy on major groove binding peptides consisting of natural amino acids. currently the oxidation conditions are being optimized so that the furan moiety undergoes selective oxidation. these optimized conditions will be applied to known dna binding peptides, in order to obtain a peptide-dna cross-link. we coupled octanoyl or palmitoyl group to the n-terminus of an analogue of sv nuclear localization signal (nls) peptide, sv - (ser ) to investigate the effect of fatty acid chain length on the conformation of the lipopeptide-antisense oligodeoxynucleotide (odn) complexes and to establish the optimal peptide/odn molar ratio (rm) for the effective delivery of odn into the cells. the odns used in this study were targeted towards either the green fluorescent protein (gfp) mrna and the junction sequence between ews and fli genes. the conformational change of odn at different rm values was followed by circular dichroism (cd), attenuated total reflection-fourier transform infrared (atr-ftir) spectroscopy and atomic force microscopy (afm). the sv peptide-mediated odn transfer into nih/ t cells was studied by epifluorescence microscopy. the interaction between the hiv- regulatory protein rev and rev responsive element (rre) of hiv- mrna has emerged in the last decade as an important target in antiviral therapy. the rev-rre interaction is essential for the replication of hiv. the rev protein binds to the rre site located in the env coding region of the full length viral mrna and facilities the export of the rna from the nucleus, while protecting it from the cell's splicing machinery. in the published nmr structure of the rre/rev-derived peptide complex, an -helical segment of rev binding domain recognises a specific region of rre. an approach is described to design a new class of -hairpin peptidomimetic ligands for hiv- rev protein, which inhibit its binding to the rre rna. a model -hairpin peptide served as a scaffold to pre-organise side chains into a geometry similar to that seen in a helical peptide. a library of peptidomimetics was prepared by grafting sequences related to the rna recognition element in rev onto a hairpin-inducing d-pro-l-pro template. the electrophoretic mobility shift assay (emsa) revealed that all of the designed peptidomimetics bind to rre and the best examples show affinities (kd) in a nanomolar range. these new ligands show a novel approach to designing rev peptidomimetics, represent interesting leads for the development of more potent hiv rre/rev inhibitors and permit more detailed studies of the mechanism of binding to rna. a. napiorkowska, a. sawula, m. olkowicz, p. mucha, p. rekowski tat (trans-activator of transcription) is the protein which controls the early phase of hiv- replication cycle. it is a potent viral trans-activator containing from to amino acid residues which binds to tar rna. the fundamental role of tat is promoting effective elongation of viral mrna (vmrna). binding of tat to tar is mediated by a -amino-acid, highly basic arg -lys-lys-arg -arg-gln-arg-arg-arg sequence of the arm (arginine rich motif); the key role in these interactions is played by arg . the goal of our research was to investigate the interaction of -nucleotide tar rna with synthesized tat peptide analogues using capillary electrophoresis (ce), a powerful analytical technique of biochemical studies. changes in electrophoretic mobility of the tar peak are employed for monitoring tar-tat complex formation. ce experiments were performed using lpa-coated capillary and a physical gel containing buffer. native arm fragment tat( - )nh , its analogues ac-tat( - )nh and ac-[lys ]tat( - )nh and analogues substituted in position with alanine-, homoalanine and lysine-derived amino acids containing nucleobases (adenine, guanine, cytosine, uracil, thymine) and nucleosides (adenosine, guanosine, uridine and cytidine) in the side chain were studied. specific interactions and complex formation were observed for both the native arm peptide fragment and selected tat analogues. the research is aimed at improving our understanding of the molecular mechanism of peptide-nucleic acid interaction, as well as evaluating the usefulness of selected nucleobase-containing amino acids as point probes for investigating peptide-rna interactions. interactions between proteins and dna are important to all living organisms. the goal is to investigate the molecular recognition between dna and the transcription factor phob of e. coli on the single molecule level and to identify amino acids required for dna binding. phob is composed of a transactivation domain (amino acids - ) and a dna binding domain (amino acids - ) that binds to specific dna sequences (pho boxes) containing a tgtca sequence. [ ] chemical synthesis of peptide epitopes present in the dna binding domain of phob and isolation of the whole dna binding domain of phob was performed. the protein was purified using intein mediated protein purification. an additional cysteine residue was ligated to the protein using intein mediated ligation reactions and will be used for immobilization and labeling. in single molecule force spectroscopy (afm) experiments it has been shown that both a peptide with a native phob-sequence and the recombinant protein bind to dna. competition experiments were performed to prove specific dna binding. [ ] mutated peptides and proteins where strategic amino acids were replaced by alanine have also been examined to reveal the contributions of single residues to molecular recognition. the binding contribution of the proteins is determined by surface plasmon resonance, electrophoretic mobility shift assays and fluorescence correlation spectroscopy. we investigated the biophysical characteristics and the pore formation dynamics of naturally occurring and synthetic peptides forming membrane-spanning channels by using isolated rod outer segments (os) of reptilia and amphibia recorded in the whole-cell configuration. once blocked the two os endogenous conductances (the cgmp channels by light and the retinal exchanger by removing one of the transported ion species from both sides of the membrane, i.e. k+, na+ or ca +), the os membrane resistance (rm) could be > gΏ. therefore, any exogenous current can be studied down to the single channel level. macroscopic currents of amplitude of ~ pa were recorded in symmetric k+ or na+ (> mm) and ca + ( mm) from the commercially available alamethicin mixture, the synthetic alamethicin f / (a major component of the natural mixture), and selected analogues applied at µm concentration at - mv. once applied and removed the peptide, the current activates and deactivates with a time constant of about ms. the synthetic analogues [glu(ome) , , ] and [glu(ome) , ] produce a current of about pa at µm concentration, and they show a strong activation by hyperpolarization as alamethicin f / itself. clear single channel events were observed when the concentration of all of the alamethicin peptides is reduced to < nm.<br> these results indicate that the three gln residues at positions , and of alamethicin f / are not a key factor for pore formation and its conduction properties. in general, the pore assembly and disassembly are very fast and cooperative events. the translocation mechanism of penetratin (rqikiwfqnrrmkwkk) is not clear, but the involvement of cell membrane was supposed. recent studies with phospholipid model membranes have shown that penetratin interacts only with negatively charged liposomes. we aimed to analyse the effect of penetratin on liposomes composed of different phospholipids (dppc/dppg : - : ) by fluorescence spectroscopy. in the first set of experiments, liposomes labelled with fluorescent markers (dph, ans and tma-dph) were incubated with penetratin and the fluorescence polarisation was determined as a function of the temperature. in the range of - mol/mol phospholipid/penetratin ratio, no change in the transition temperature was observed indicating that penetratin has no influence on the membrane structure. next, we have analysed the interactions between phospholipids and penetratin through changes in the intrinsic fluorescence of the peptide due to the presence of two w residues in its sequence. comparing the emission spectra corresponding to penetratin in aqueous media or in presence of vesicles one can clearly appreciate a blue shift. this indicates that that tryptophan residues are mainly exposed to a hydrophobic environment. analysis of the main band shows low values of polarization suggesting a free motion of the peptide chain. on the contrary polarization measured for penetratin mixed with liposomes results in higher values. this indicates that hydrophobic residues, like trp, are inserted into the bilayer and their motion is restricted. these data suggest the presence of interation sensed strongly by trp properties. cyclopeptide antibiotic gramicidin s (gs) has antimicrobial activity against gram-positive and gram-negative bacteria and some fungi. but non-specific action of gs and its high lytic potential limits therapeutic application of gs. we attempted to elucidate in which way gs molecule could be modified to lose its haemolytic side effects. gs molecule interacts directly with membrane phospholipids due to electrostatic and hydrophobic interaction. naturally, changes in the state of a lipid bilayer cause changes in the gs molecule binding to a bilayer. we studied the effect of gs on human blood platelets and the effect of platelet membrane state on the gs-induced disaggregation of cells with the help of turbidimetric and microscopy techniques. we modified the membrane state by temperature, osmotic stress, ionizing irradiation, lipid oxidation. depending on concentration gs causes platelet shape change and activation. when added to preliminary aggregated (in response to physiological agonists -thrombin, epinephrine, adp) platelets, gs causes crumble of cells aggregates. the rate and extent of platelet disaggregation under the effect of gs non monotonously depends on temperature (range of - °c) and irradiation dose (up to gy). parameters of the gs interaction with membranes are determined by the mobility of membrane lipids. factors modifying the lipid bilayer change the degree and the speed of the gs interaction with platelet membranes. results obtained permit to use gs for testing the state of membrane lipids and on the other hand allow to suppose ways of gs molecule modifications to achieve its tolerance to blood cells. g. bai , p. gomes , r. seixas , m. hicks , m. prieto , m. bastos eukaryotic antibiotic peptides (eaps) have been widely studied for the past years as an alternative to conventional antibiotics due to emergence of multi-resistant microbial strains, and significant efforts targeting increasingly potent and specific antimicrobial peptides are being made. one interesting approach in peptide antibiotics is based on hybrid sequences derived from natural eaps, with ca ( resistance to conventional antibiotics has stimulated a search for alternative therapeutics for microbial infections, a possible source that has gained much interest in recent years are antimicrobial peptides. antimicrobial peptides target the cell membrane directly, which is a key feature as evolution has shown bacteria have had difficulty in altering their membrane composition and organization to mount a suitable defence against these peptides. a common theory is that peptides that bind strongly exhibit high biological activity, but our real-time quantitative binding studies via surface plasmon resonance (spr) have shown that this correlation does not always hold. as more information on the molecular details of membrane disruption is required, we have used atomic force microscopy (afm) to visualise peptide insertion and changes in membrane morphology by a range of antimicrobial peptides in situ. interaction studies were performed with a series of phospholipid mixtures that mimic either mammalian cells (high in phosphatidylcholine and cholesterol) or microbial cells (high in phosphatidylethanolamine and phosphatidylglycerol). the present study may assist in the design of new specific antimicrobial peptides with high antimicrobial activity and low host toxicity. proportions of popc and popg as models. very high molar ratio partition constants (( . +- . )x ^ and ( . +- . )x ^ ) were obtained for the bacterial models (popg:popc : and : , respectively), these being about one order of magnitude greater than the partition constants obtained for the less anionic mammalian model systems (( . +- . )x ^ for the % popc system). at low lipid:peptide ratios there were significant deviations from the usual hyperbolic-like partition behavior of peptide vesicle titration curves, especially in the case of the most anionic systems. membrane saturation was shown to be related to such observations and mathematical models were derived to further characterize the peptide-lipid interaction under these conditions. the calculated peptide-to-lipid saturation proportions, together with the determined partition constants, suggest that the minimal inhibitory concentrations of omiganan pentahydrochloride could represent the conditions required for bacterial membrane saturation to occur. the hemolytic pore-forming toxin sticholysin ii (stii) produced by the sea anemone stichodactyla heliantus belongs to the actinoporin protein family. the n-terminal domain of these proteins is required for interaction with membranes. to investigate the role of stii´s n-terminal domain in membrane binding and in the molecular mechanism of hemolysis, peptides corresponding to residues to , or shorter fragments from this region, were synthesized. in some peptides leu was replaced by trp. all peptides exhibited hemolytic activity, albeit to a lesser extent than the whole protein. moreover, peptides lacking the - hydrophobic stretch were less active. the longer peptides were also able to permeabilize phospholipid vesicles. conformational studies were performed in aqueous solution and in membranemimicking environments. cd spectra showed that, while the shorter, more hydrophilic peptides, displayed a random conformation, the longer peptides underwent aggregation with increasing concentration, ph, and ionic strength. in the presence of trifluoroethanol and upon binding to detergent micelles and phospholipid bilayers, the peptides showed a propensity to acquire -helical conformation, as expected for the sequence comprising residues to . fluorescence spectra demonstrated that the first residues of stii´s n-terminus penetrate more deeply into the bilayer, whereas residues - are located more superficially. this is in agreement with the predicted amphipathic nature of the helix formed by these residues and corroborates the existing hypotheses for the role of the n-terminal domain in the process of membrane insertion and pore formation. among a great number of antibacterial peptides a group of trp-rich peptides is of special interest. taking into consideration, that in most of proteins tryptophan is not frequently occurring amino acid, the biological meaning of a high content of tryptophan in structure of these antimicrobial peptides is particularly interesting. in the present study we carried out the investigation of antimicrobial and hemolytic activities of selected trp-rich peptides and their action on microbial membrane: ilpwkwpwwpwrr-nh - indolicidin (i) pitwpwkwwkgg-nh - b (ii) plswffprtwgkr-nh - gsp- a (iii) fpvtwrwwkwwkg-nh - puroindoline (iv) vrrfpwwwpflrr-nh - tritrypticin (v). sunflower trypsin inhibitor sfti- is the smallest and the most potent known peptidic trypsin inhibitor from the bowman-birk class of proteins [ ] . this head-to-tailcyclized -amino-acid peptide contains one disulfide bridge and a lysine residue (lys ) present in the p position, which is responsible for inhibitor specificity.as was reported by us and other groups, sfti- analogues with one cycle only retain trypsin inhibitory activity. very recently we have shown [ ] that introduction of nsubstituted glycine residues mimicking lys and phe (denoted as nlys and nphe) in the p position of monocyclic sfti- with disulfide bridge yielded potent trypsin and chymotrypsin inhibitors, respectively. in this novel class of proteinase inhibitors contains completely proteolytic resistant p -p ' reactive site. in the present communication we report chemical synthesis and determination of trypsin and chymotrypsin inhibitory activity of a series of ten sfti- analogues modified in the p position by these peptoid monomers (nlys and nphe). each of the synthesized peptomeric (peptide-peptoid hybrid polymer) sfti- analogues contains one of the following cycles: head-to-tail, disulfide bridge formed by cys, by pen and by cys/pen residues. the impact of the different cycles introduced into sfti- structure on proteinase inhibitory activity will be discussed. s-protein contains a proteolytic processing site and two interacting heptad repeat regions denoted as hr-n and hr-c. following processing of s-protein mediated by host cellular protease/s, the c-terminal s -fragment fuse with host cell membranes via its hr-n and hr-c domains that form coiled coil -helix bundle (trimeric of dimers)-crucial for its receptor-mediated viral fusion. our objective in this work is to study the proteolytic site using model peptides and also to examine the interaction of hr-n and hr-c domains using fluorescence microscopy and other techniques. thus we synthesized an intramolecularly quenched fluorogenic peptide containing the proposed cleavage site [abz-eqdrntr evfatyx, abz= -amino benzoic acid and tyx= -nitro tyrosine] and showed by kinetic measurements that this cleavage is mediated most efficiently by furin, followed pc and pc . other potential substrates were also tested and compared. above cleavage can be blocked by specific-pc-inhibitors in a dose-dependent manner. in addition using fluorescent-labeled peptides derived from hr-n and hr-c domains, circular dichroism spectra and surface assisted laser desorption mass spectral interest has grown to develop specific and potent inhibitors of this enzyme. our objectives in this study are to generate soluble recombinant human (h)ski- enzyme, design potent inhibitors and study its dmodel structure. we have successfully expressed hski- enzyme lacking its transmembrane domain in hek- cells and purified the enzyme via chromatography. in addition we developed ski- inhibitors by using pseudo-and multi-branch peptide approaches. in first approach we inserted dipeptide isosteres amino oxy acetic acid (aoaa) or -amino , dioxa octanoic acid (adoa) at scissile p -p ' position ((r l) of hski- . a typical example is gryssrrl(adoa)aip . other dipeptide isosters were also incorporated at the cleavage site of either ski- prodomain or lassa virus glycoprotein. in second approach we prepared and -branch peptides containing hski- - segment. these peptides inhibit ski- in competitive manners with varying degrees ranging from low m to high nm ic . circular dichroism spectra indicated strong interactions of inhibitors with ski- consistent with observed inhibition profile. a d-model structure of catalytic domain of ski- indicated a broad catalytic pocket cysteine proteases are of great importance in biochemical processes and these enzymes are used in biotechnology, food industry and agriculture. in this connection synthesis of high selectivity and high specificity substrates for cysteine proteases is of importance. enzymatic synthesis of peptides is a good tool for obtaining different biologically active peptides. immobilized serine proteases, subtilisin carlsberg and α-chymotrypsin immobilized on poly(vinyl alcohol) cryogel (pvacryogel), proved to be a convenient biocatalyst for such kind of syntheses. the high specific chromogenic substrate for cysteine proteases assay glp-phe-ala-pna was obtained with high product yields (up to % in h) using subtilisin and chymotrypsin immobilized on pva-cryogel. the reaction was carried out according to the following scheme: glp -the residue of pyroglutamic acid, pna -p-nitroanilide. the influence of initial concentrations of components, the reaction mixture composition, the biocatalyst content and time on product yield was studied. it was shown that the optimal conditions are: dimethylformamide-acetonitrile mixture : (v/v), initial concentrations - mm, and enzyme-to-substrate ratio - : . this approach was used in order to synthesize analogous substrates, containing different fluorogenic and chromogenic groups as well as other amino acids in p position. the obtained substrates were tested for the papain assay. peptidyl-a-ketoaldehydes represent attractive lead compounds and intermediates in the development of potent protease inhibitors due to their structural similarity with peptide aldehydes, previously known to be excellent inhibitors of serine-and cysteine protease. recently, we demonstrated the application of polymer cyano methylene-and carboxylato methylene phosphoranes in the assembly of a-hydroxy-b-amino esters (norstatines), a,b-diketoesters, and a,b-unsaturated ketones. [ , , ] we now present a further development of our reagent linker approach employing peptidyl-a-ketoaldehydes and diamino propanoles . carboxylato methylen phosphoranes derived from bromo acetic esters which are readly acylated without racemization, play the key role in our synthetic concept. herein we show the oxidative cleavage to peptidyl-a-ketoaldehydes using dimethyldioxirane (dmd) in acetone as oxidant, after saponification and decarboxylation on the solid support. diamino propanoles were furnished via the reductive amination of resin-bound peptides. over the past few years nuclear magnetic resonance has emerged as a powerful means for lead molecular identification and optimization.on the other hand, the f nmr has been used succesfully in several structural studies, protein folding studies and for the identification of active compounds, using a very similar methodology that the one used in the present work. the methodology required the labeling of the substrate with a cf moiety. the enzymatic reaction is performed with the cf substrate and quenched, using an enzyme inhibitor. f nmr is then used to monitor the evolution of both substrate and product. only two peaks are observed, the starting substrate and the cleaved substrate. this nmr method has some advantages: fluorine nmr is very sensitive, , times that of the proton. there are no spectral interference from protonated solvents, buffers or detergents typically present in the enzymatic reactions.the f isotropic chemical shift is extremely sensitive to small structural perturbations resulting in different chemical shift for the signals of the substrate and product. isotopic labeling of the protein is not required. as a model, caspase- , which play a critical role in the initiation of apoptosis process and hiv- protease were chosen. two different kind of libraries were screened: one based on natural products from plant and animal extracts used in tradicional chinese medicine and a second one corresponding of a synthetic library with two sublibraries of and compounds.with this methodology it has been possible to identify some compounds with very promising inhibitory properties. background and aims: human kallikrein (hk ), a prostate specific serine protease, regulates the activity of several factors that may participate in proteolytic cascades promoting tumor growth and metastasis. thus, inhibition of its enzymatic activity is a potential way of preventing growth and metastasis of prostate cancer. moreover, specific ligands for hk have potential use for targeting and in vivo imaging of prostate cancer and for development of novel assays. methods: to find peptide ligands we panned several phage display peptide libraries against active recombinant hk captured by a monoclonal antibody exposing the active site of the enzyme. alanine scanning and amino acid deletion analyses were performed to elucidate the motifs required for hk inhibition. results: from libraries expressing and amino acid long linear peptides we isolated six different hk -binding peptides. three of these peptides are specific inhibitors of the enzymatic activity of hk . amino acid substitution and deletion studies indicated that motifs of amino acids are necessary for the inhibitory activity. conclusions: we have developed specific hk inhibitors by phage display technology. these novel hk specific peptides are potentially useful for treatment and targeting of prostate cancer. peptidylarginine deiminase iv (padiv) catalyzes the citrullination of arg residues in various peptides and proteins, such as histone, resulting in the production of citrullinated proteins in granulocytes [ , ] . the citrullination mechanism of histone subunits and its functional effects in cells are not well known yet in detail. recently, it has been reported that the protein deimination/citrullination by pad iv plays a role in rheumatoid arthritis [ ] . this implicates that the citrullination of histone may be related to rheumatoid arthritis. in order to further study the citrullination mechanism of histone, we explored the citrullination sites of histone h a and h by pad iv using a series of synthetic peptides. recently, hagiwara et. al. reported that pad iv only citrullinates the arg of histone h a as well as the arg in histone h [ , ] . in order to investigate the citrullination mechanism, the n-terminal peptides of histone h a and h were chemically synthesized and examined the citullination by pad iv. the n-terminal acetylation effect of the n-terminal synthetic peptide was also estimated on the citrullination by padiv. the velocity of each arg residues in the n-terminal peptides were estimated in vitro. the results indicated that padiv recognizes the specific arg residues in the synthetic peptide, and that the n-terminal acetylation of the histone peptides dramatically affects on the substrate recognition of padiv. in addition, the cd spectra of the n-terminal peptides were measured to elucidate the structural specificity for the recognition of pad iv. background and aims. prolyl oligopeptidase (pop) is a serine peptidase that cleaves oligopeptides after prolyl residues. it has been associated with cognitive disorders. pop inhibitors have been shown to enhance cognition in monkeys ( ) and to improve performance in verbal memory tests in humans ( ) . in the present study, the p l-prolyl residue of pop inhibitors was replaced by two l-proline mimetics, the -t-butyl-l-prolyl group and the (r)-cyclopent- -enecarbonyl group. the effect of the mimetics on in vitro potency, lipophilicity and binding kinetics were studied. methods. the l-proline mimetics were synthesized according to the published procedures ( , ) with minor modifications. the ic and ki values and the binding kinetics were determined for porcine pop. the log p values were determined with the shake-flask method. results. the replacement of the p l-prolyl residue by the l-proline mimetics gave compounds which were equipotent with their parent structures. both l-proline mimetics increased lipophilicity but the effect of the -t-butyl-l-prolyl group was more pronounced. while the -t-butyl-l-prolyl group increased the dissociation half-life of the enzymeinhibitor complex, the (r)-cyclopent- -enecarbonyl group decreased it. conclusions. both l-proline mimetics perfectly mimicked l-proline at the p position of pop inhibitors. these mimetics can be used to modify the lipophilicity and the binding kinetics of pop inhibitors. the proteasome is an essential multicatalytic protease of the ubiquitin proteasome pathway. as a prime executor of regulated proteolysis, the proteasome controls almost all aspects of cell metabolism from signal transduction to cell cycle and differentiation. pharmacological intervention into proteasome activity leads to cell apoptosis. this observation was applied to successfully treat multiple myeloma, since the cancer cells exhibit substantially higher sensitivity to competitive inhibition of proteasome than normal cells. however, the complete shutting down of the proteasome catalyzed proteolysis leads to serious side effects resulting from the disruption of proteolytic homeostasis even in noncancerous cells. here, we show an alternative approach to control the proteasome activity using peptide based noncompetitive regulators. the cathelicidins derived peptides rich in proline and arginine (pr) residues have been found to affect activity of all the proteasome complexes both in vivo and in vitro, likely by binding to the face of the enzyme. mechanism and structural constrains of the pr peptides dictating their influence on the proteasome remain elusive. our results indicate that there are three sequence related properties of the pr peptides controlling their effectiveness as proteasome regulators: length of the peptide, distribution of a set of positive charges at the peptide n-terminus, and positioning of proline residues. far uv cd spectroscopy demonstrates that these properties also correlate with the structure of pr peptides. in particular, it seems that structural propensity of the pr peptides to form beta-turns are required to bind to proteasome as regulatory competent molecules. our work is focused on the search of selective, low-molecular cathepsin b peptide inhibitors acylated with the (e)- -(benzylsulphonyl)acroyl group (bsa). the double bond, embedded in the bsa moiety is activated by two electron-withdrawing groups and may be a good target for the michael-type addition of the catalytically active -sh group. three series of peptide derivatives possessing general structures: bsa-phe-asn(r)-oh, bsa-ile-x(oh)-n(ch ) and bsa-x-pro-oh were synthesized in solution and characterized by enzyme kinetic studies against papain, cathepsins b and k. it should be noted that all the investigated compounds were competitive and reversible inhibitors of the enzymes examined. using d h nmr (tocsy, cosy, roesy) and c nmr spectroscopy along with theoretical calculations (analyse program) we determined the conformational properties of two most potent and selective cathepsin b inhibitors. this work was supported by grant ds/ - - - . background and aims: we have developed peptides inhibiting human kallikrein- (hk ) activity. as hk is overexpressed in prostate cancer tissue, these peptides are potentially useful for treatment and diagnosis of prostate cancer. two of the potential physiological substrates for hk are proform of prostate specific antigen (propsa) and insulin-like growth factor-binding protein- (igfbp- ). both of these might participate in the regulation of prostate cancer growth: igfbp- by inhibiting igf-dependent tumor growth and psa by degrading extracellular matrix. we aimed to study whether our hk -inhibiting peptides inhibit also hk activity towards natural protein substrates, i.e. activation of propsa and degradation of igfbp- . methods: the effect of the peptides on the activation of propsa by hk was studied by preincubating the peptides with hk , followed by addition of psa and specific psa substrate. igfbp- degradation was studied by two specific immunoassays, one detecting only native igfbp- , while the other one also detected proteolytically cleaved forms of the protein. results: hk -inhibiting peptides were found to inhibit propsa activation and igfbp- degradation by hk in a dose dependent fashion. conclusions: we have developed new peptides inhibiting hk activity towards natural substrates, like propsa and igfbp- . the peptides might be useful for treatment of prostate cancer and other diseases associated with increased hk activity. from the seeds of garden four-o'clock and spinach we isolated two serine proteinase inhibitors (mjti i -mirabilis jalapa trypsin inhibitor and soti i -spinacia oleracea trypsin inhibitor), which are probably representatives of a new family of inhibitors. the purification procedures of these inhibitors included affinity chromatography on immobilized methylchymotrypsin in a presence of m nacl, ion exchange chromatography and/or preparative electrophoresis and finally rp-hplc on c column. their primary structures (fig. ) differ from those of known trypsin inhibitors, but showed significant similarity to one another, as well as to the antimicrobial peptides isolated from the seeds of mirabilis jalapa (mj-amp , mj-amp ), mesembryanthemum crystallinum (amp ) and phytolacca americana (amp- and pafs-s) and from hemolymph of acrocinus longimanus (alo- , and ). the equilibrium association constants (ka) of mjti i and soti i with bovine -trypsin were found to be about - m- . mjti i and soti i have been synthesized using solid-phase method. the synthesized inhibitors and inhibitors isolated from plants have similar properties. the disulfide bridge pattern in both inhibitors was established after digestion with thermolysine, followed by the maldi-tof: cys -cys- , cys -cys and cys -cys . s. cosgrove, l. rogers, c. hewage, j.p. malthouse aspartyl proteases are required for the multiplication of the aids virus and for producing the amyloid protein which causes alzheimer's disease. hiv protease inhibitors have been highly effective in treating aids patients and it is hoped that potent inhibitors of the beta secretases will also prove effective in treating alzheimer's disease. therefore inhibitors of the aspartyl proteases have great therapeutic potential. we have shown that the peptide glyoxals are potent inhibitors of the thiol protease papain and of the serine proteases subtilisin and chymotrypsin. using c-nmr we have been able to show that glyoxal inhibitors react reversibly with an active site nucleophile in these enzymes to form a tetrahedral adduct which is tightly bound by the enzyme. in the present work we synthesise c-enriched peptide glyoxals, we assess their inhibitor potency, and use c-nmr to examine how the inhibitors interact with the aspartyl protease pepsin. z-ala-ala-[ - c]phe-glyoxal was synthesised from [ - c]phenylalanine which was converted to its methyl ester. this was then coupled with z-ala-ala to give z-ala-ala-[ - c]phe-ome which was hydrolysed to the free acid. this was converted to the diazoketone and transformed into z-ala-ala-[ - c]phe-glyoxal using dimethyldioxirane. nmr spectra at . t were recorded with a bruker avance drx standard-bore spectrometer. we show that peptide glyoxal inhibitors can be potent inhibitors of pepsin and that pepsin only binds one of the four glyoxal forms (one non-hydrated, one fully hydrated and two partially hydrated forms). alzheimer`s disease (ad) is the most common cause of dementia in older people. a major factor in the pathogenesis of ad is the cerebral deposition of amyloid fibrils, consisting of amyloid β peptides (aβ), as senile plaque. the -to amino acid long aβ is generated by the proteolysis of β-amyloid precursor protein (app) by β-and γ-secretases. since bace , a unique member of the pepsin family of aspartyl proteases initiates the pathogenic processing of app by cleaving at the n-terminus it is a molecular target for therapeutic intervention in ad proteolytic activity was found to occur, to a variable degree, in digestive organs of all studied organisms over the entire ph range. the common feature was the existence of two activity peaks, in the acid (ph . - . ) and alkaline (ph . - . ) zones, as well as a similar protease set containing e and d cathepsins, a trypsin-like enzyme, elastase, and collagenolytic proteases. proteolytic activity in the hepatopancreas of crab and sea star was found to be an order higher than in other study objects. high protease activity in crab hepatopancreas is an evolutionary mechanism compensating for a poor differentiation of digestive system, low substrate specificity of enzymes, and cold environment. trypsin activity in digestive organs of invertebrates suggests that a trypsin-like enzyme is a genetically old one, an evolutionary origin of all serine proteases. a difference of kind between vertebrates and invertebrates is that the latter have cathepsine activity (absent in vertebrates) and no pepsin activity. it is of interest to develop enzyme inhibitors containing a light activated switch that can be used to control binding and inactivation of an enzyme. several inhibitors containing the azobenzene photoswitch group have previously been developed and have shown changes in activity of around two times on photoswitching. this study aimed to improve this switching by more extensive derivatisation of azobenzene to closer resemble the peptide substrates of proteases. a series of peptidomimetics containing the azobenzene photoswitch group were synthesized and assayed against the protease alpha-chymotrypsin. these compounds contained azobenzene, linked to a known chymotrypsin inhibitory group (either a trifluoromethylketone or boronate ester), and otherwise designed to be peptide-like. in some cases both ends of the azobenzene moiety were derivatized in order to increase the impact of photoswitching on the shape of the compound and thus its enzyme binding strength. assays showed that most compounds were reversible inhibitors of chymotrypsin, with low micromolar inhibition constants (ki or ic ). up to four times increase in enzyme inhibition on light activated switching of the azobenzene group conformation was obtained. a number of peptidyl derivatives structurally based upon the inhibitory sites of cystatins has been synthesized. these compounds are prone to proteolytic degradation, are rapidly excreted and poorly bioavailable. the majority of this problems might be overcome by use of peptidomimetics with structures resembling those of previously synthesized peptidyl derivatives. among the peptidomimetics are azapeptides, in which alpha-ch group of amino-acid residue is replaced by a nitrogen atom. the azapeptides have recently been demonstrated as potent and selective inhibitors of cathepsins b and k. it was shown that azapeptide inhibitors bind along the active site cleft of cathepsin b in a bent conformation. this bent structure is likely to result from the mobility of the bonds in the vicinity of the inserted azaamino acid residue as well as from the interaction with enzyme. in our present work we have studied the peptide of a sequence: z-arg-leu-arg-gly-ile-val-ome, which is characterized by one major and three minor conformations. the replacement of alpha-ch group in the gly residue of peptide chain of z-arg-leu-arg-gly-ile-val-ome by the nitrogen atom likely results in rigid conformation. our aim was a comparison of structure of the parent peptide z-arg-leu-arg-gly-ile-val-ome and a selective cathepsin b inhibitor z-arg-leu-arg-agly-ile-val-ome by using h-nmr. severe acute respiratory syndrome corona virus associated main protease (sars cov mpro protease), alternatively known as chymotrypsin-like protease ( clpro), is a mediator of virus infection cyclus and from there a therapeutic target. a peptide aldehyde library targeting the sars corona virus main protease (sars-cov mpro, alternatively known as clpro) was designed on the basis of three different reported binding modes and supported by virtual screening. a set of peptide aldehydes were prepared by a newly developed methodology and investigated in an inhibition assay against sars-cov mpro. [ ] protected amino acid aldehydes furnished by the racemization-free oxidation of amino alcohols with dess-martin periodinane were immobilized on threonyl resins as oxazolidines. following boc-protection of the ring nitrogen yielding the n-protected oxazolidine linker, peptide synthesis was performed efficiently on this resin releasing deprotected products under mild hydrolysis conditions. the library was tested in a new fluorimetric enzyme assay for sars cov mpro. via immobilization of the fluorophor, -( -amino- -methyl- -coumarinyl)-acetic acid, the substrate actsavlq-amca was prepared, surprisingly displaying a higher affinity than the native substrate. several potent inhibitors were found with ic values in the low micromolar range. interestingly, the most potent inhibitors seem to bind sars-cov mpro in a non-canonical binding mode. currently, the initial screen is extended towards the discovery of small molecule inhibitors of sars corona virus main protease. literature: a method of bromelain cleavage of surface glycoprotein hemagglutinin (ha) from the influenza a virions was initially employed for ha ectodomains crystallographic study [ ] . the remaining spikeless subviral particles were used by us earlier for ha c-terminal fragment extraction and mass spectrometric (ms) investigation [ ] . now sds-page analysis of the subviral particle preparations revealed several additional bands in a range of - kda together with major viral proteins comparing to intact virions (figure, m matrix protein, f -f -m protein fragments, np-nucleoprotein). maldi-tof ms analysis of the in-gel trypsin hydrolyzates has shown that the additional bands are fragments of М protein. this was confirmed by n-terminal sequencing of the protein fragments electroblotted from the bands. concentration of sh-reducing reagent in bromelain digestion reaction influenced on the m fragment bands intensity. we conclude that due to membrane destabilization during ha spikes removing, m protein localized under viral membrane inside intact virions becomes accessible to limited proteolysis by bromelain. [ dipeptidyl peptidases (dpp's) sequentially release dipeptides from polypeptides. among those enzymes, dppiv, fapα, dpp , dpp and dppii cause the release of n-terminal dipeptides containing proline or alanine at the penultimate position. they are all members of clan sc, a group of serine proteases that contains proline-specific peptidases. dipeptidyl-peptidase iv (dppiv) is the best studied member of this group of enzymes and has become a validated target for the treatment of type diabetes over the last years. the development of inhibitors for the related enzymes (i.a. dppii) has only started recently. this poster presents selected products synthesised to further elaborate the structure-activity relationship for dpp ii inhibitors with a , -diaminobutyrylpiperidine basic structure. this class of compounds was described earlier by our group as the hitherto most potent and selective inhibitors of dpp ii. starting from n -p-chlorobenzyl-substituted uamc , our lead compound, two types of modifications were proposed: • the synthesis of n -(di)alkyl-and arylalkyl analogues; • the synthesis of -methyl analogues. in our previous study, we reported potent and small-sized bace inhibitors containing phenylnorstatine [( r, s)- -amino- -hydroxy- -phenylbutyric acid; pns] at p position as a transition-state mimic. in developing more active compounds, we focused our attempts on the p position, where we replaced the pns by its thioderivative. herein, we present the synthesis of a novel phenylthionorstatine [( r, r)- -amino- -hydroxy- -(phenylthio)butyric acid; ptns] as a p moiety with hydroxymethylcarbonyl (hmc) isostere, and then an application to the bace inhibitors design. we have synthesized ptns starting from readily available n-benzyloxycarbonyl-serine and after multistep reaction (including weinreb amide formation, thiophenyl group introduction, through cyanohydrin derivative the transformation into the -hydroxy ester and then acid). purification was done by column chromatography and rp-hplc. peptides were synthesized by the fmoc based solid phase method and characterized by maldi-tof ms. the peptide inhibitors were adopted to enzyme assay using a recombinant human bace and a fluorescence-quenching substrate. bace inhibitory activity was determined based on the decrease% of the cleaved substrate by the enzyme.we have synthesized ptns and then the ( r, r)-enantiomer was applied to spps (solid phase peptide synthesis). we synthesized octa-and pentapeptide-type inhibitors of bace containing pns or ptns at the p position. these compounds were enzymatically tested and showed high bace inhibitory activity. a novel derivative of pns, ptns, was synthesized, and evaluated in comparison to corresponding pns. the inhibitors with ptns exhibited a slightly higher inhibitory activity against bace comparing to those with pns. this study suggests possibilities of the application of ptns to design other aspartyl proteases inhibitors. the αν β integrin receptor plays an important role in human metastasis and tumor-induced angiogenesis, mainly by interacting with matrix proteins through recognition of an arg-gly-asp (rgd) motif. inhibition of the αν β integrins with a cyclic rgd peptide impairs angiogenesis, growth and metastasis of solid tumours in vivo. the aim of this study was to investigate the effects of replacement of amino acids by aza-β -amino acid analogs in cyclic rgdpeptides as αν β -integrin antagonist on angiogenesis, microcirculation, growth and metastasis formation of a solid tumour in vivo. the selectivity profile of these antiadhesive cyclopeptide is rationalized by a special presentation of the pharmacophoric groups. thr rgd motif resides in position i to i+ of a regular γ-turn. we synthetized linear and cyclic aza-β rgd-peptide with the purpose to examine the effect on the conformation and the activity. are aza-β amino acids γ-turn mimetics? the preferred conformations were determined by nmr. prostaglandins are involved in a large number of biological activities mediated by their g-protein coupled receptors (gpcrs). the prostaglandins pgf alpha receptors are found specifically in uterine muscle, where they initiate parturition and labor. the pgf alpha receptor plays a key role in preterm labor, for which medical and social costs are estimated at $ billion per year in the usa (the highest per patient cost of any disorder). peptide mimics have been developed in our laboratory ( , ) , that serve as allosteric antagonists of the pgf alpha receptor. the importance of the turn geometry of the central residue in these peptide mimics has been investigated using enantiomeric indolizidin- -one beta-turn mimics which can respectively induce type ii and ii' geometry. our presentation will discuss the synthesis and biology of these novel allosteric modulators of prostaglandin pgf alpha receptor activity. it was shown that luteinising hormone -releasing hormone (lhrh) receptors are overexpressed in the most of adenocarcinoma cells in contrast to their low content in normal tissues. these data create the basis for lhrh analogues application in therapy of breast, ovary, prostate, lung, intestine, liver and kidney cancers. both agonists and antagonists utility for the targeting of cytotoxic moiety to the tumor cells is well documented. however, the number of lhrh analogues possessed their own cytotoxic activity is still very limited. we nicotianamine (na) that was first isolated from the leaves of nicotiana tabacum l [ ] , is known as a key biosynthetic precursor of phytosiderophores. various studies have proved that nicotianamine plays a significant role in plants as an iron, nickel, zinc ... transporter [ ] . the aim of our study was to synthesize unnatural analogues of na via peptide intermediates, to investigate the mechanisms of metal transport and accumulation within the plant. we found that the strategy developed for na synthesis could not be applied when the azetidine ring was changed for pyrrolidine ring and we investigated a new route to synthesize such analogue. these synthetic pathways will be discussed. the primary physiological roles of arginine vasopressin (avp), [cycle - (h-cys -tyr -phe -gln -asn -cys -pro -arg -gly -nh )], involve vasoconstriction of vascular smooth muscles, via v a receptor, and antidiuretic action in kidney (blood osmolality regulation) via v receptor. binding of avp to the v a receptor subtype also stimulates glycogenolysis in the liver and promotes platelet aggregation. in addition, activation of the v b (also known as v ) receptor causes adrenocorticotropic hormone release from the anterior pituitary. v b receptors are also present in the brain where avp functions as a neurotransmitter. in the recent years by the salivary glands of several bloodsucking animals like, teaks, leeches, vampire bats and so forth are isolated plenty of proteins and peptides with different molecular weight and well established anticoagulant activity. many of the strongest anticoagulants isolated by bloodsucking animals are found in the extract of salivary glands of different kinds of leeches. such leech is the haementeria officinalis, from which is isolated the most active inhibitor of factor xa -ats. in order to study the role of some amino acids in the process of interaction among peptides mimetics and the active site of serine proteinases, some fragment analogues of ats's active site by replacement of some amino acids with the other with similar structure or with unnatural amino acids were synthesized. in the present work the synthesis and the anticoagulant activity according to the aptt and ic of the newly synthesized peptides and structure-activity relationship will be discussed. rational design of peptides is a challenge which would benefit from a better knowledge of their rules of sequence-structure-function relationships. peptide structures can be approached by spectroscopy and nmr techniques but data from these approaches too frequently diverge. structures can also be calculated in silico from primary sequence information using three algorithms: pepstr, robetta and peplook. the most recent algorithm, peplook introduces indexes for evaluating structural polymorphism and stability. the method uses a de novo search of energy minima by an iterative boltzmann-stochastic procedure and using a combination of phi/psi couples derived from the structural alphabet for protein structures proposed by etchebest et al. for peptides with converging experimental data, calculated structures from peplook and, to a lesser extent from pepstr are close to nmr models. the peplook index for polymorphism is low and the index for stability points out possible binding sites. for peptides with divergent experimental data, calculated and nmr structures can be similar or, can be different. these differences are apparently due to polymorphism and to different conditions of structure assays and calculations. the peplook index for polymorphism maps the fragments encoding disorder and the mean force potential score indicates which residues will be most available for interactions with partners. this should provide new means for the rational design of peptides. several diseases like cancer metastasis, rheumatoid arthritis and chronic lymphocytic b-cell leukemia are linked to the interaction of the cxcr chemokine receptor to its natural ligand, the amino acid protein stromal cell-derived factor- α (sdf- α). [ ] one strategy for the treatment of these diseases could be to block the interaction between cxcr and sdf- α with small cxcr antagonists. furthermore, radiolabeling of suitable compounds with appropriate radioisotopes could provide agents for imaging of cxcr expression in vivo via pet. previous studies by fujii et al. on cxcr antagonists led to a high affinity cyclic pentapeptide with the sequence cyclo[gly-d-tyr-arg-arg-nal]. [ ] to further improve this structure, different approaches have been chosen with respect to metabolic stability, bioavailability, conformational rigidity and chemical versatility for radiolabeling. first, an n-methyl scan of the backbone amides was performed to influence conformational freedom and to increase metabolic stability and bioavailability. n-methylation of arginine residues yielded peptides with moderate affinity (ic<sub> </sub>-values: nm (n-me)arg<sup> </sup> and nm (n-me)arg<sup> </sup>, resp.) whereas n-methylation of other amino acids significantly decreased the affinity (ic<sub> </sub>> nm). by substitution of arg<sup> </sup> by ornithine, the affinity was mostly retained. [ ] the amino group of orn can be alkylated or acylated via radiolabeled groups containing short lived isotopes. moreover, the bioavailability should be improved as the high basicity of the two guanidino groups could be reduced. first ornithine-acylated derivatives showed ic<sub> </sub> values between - nm enabling for the first time <sup> </sup>f-radiolabeling of small cxcr antagonists for pet imaging in vivo. binding of ligands to integrins plays a major role in cell adhesion, migration, and signal transduction of cells. these interactions are important not only for normal cell functions, but also in pathogenic processes. the v integrin for example is involved in tumor cell adhesion and osteoporosis. the association of ligands is specific and requires minimal recognition sequences. therefore, suppression of integrin activity using competitive inhibitors bears great pharmacological potential. the tri-peptide sequence rgd is a prominent recognition sequence of integrin ligands. two new cyclic pentapeptides were synthesized containing the tripeptide sequence rgd as well as -amino-cyclopropane- , -dicarboxylic acid monomethyl ester (acc) and valine varying only with respect to the stereochemistry of acc. both the (+) (all r) and (-) (all s) isomers of acc were incorporated. acc is a cyclic -amino acid as well as a cyclopropyl analogue of aspartic acid. biological tests with cell lines expressing mainly v and v integrin show a higher inhibitory activity of cyclo-(-arg-gly-asp-(+)acc-val-). in order to derive a structure-activity relationship of these two isomers, solution structures in dmso-d were investigated by nmr spectroscopy. subsequently, structural information was obtained by applying distance restraints derived from the nmr spectra in distance geometry/simulated annealing and molecular dynamics calculations. due to the rigidity of the cyclopropyl unit in acc, the structure of the cyclopeptide is significantly influenced by the integrated propane ring, thus explaining the different biological properties. integrins are an important family of cell adhesion molecules. currently, members are known. among other functions, integrin α β is involved in inflammatory processes, leukocyte migration and tumor angiogenesis. the structure of its natural ligand vcam- , including the binding loop sequence tqidspln, has been determined by x-ray crystallography. therefore, it is possible to apply the concept of spatial screening: using small cyclic peptides with structure inducing building blocks, the binding motif is presented in different well-defined structural arrangements. for this study, a series of cyclic penta-and hexapeptides based on the tqidspln sequence has been synthesized. β-homoamino acids, i.e. β -amino acids with proteinogenic side-chains, have been incorporated as structure inducers for spatial screening. although β -amino acids are supposed to prefer the central position of Ψγ-turns, less data exist than for e.g. d-amino acids. apart from the structural characterization of potential high affinity ligands for integrin α β , a major goal of this work is to provide a better understanding of the influence of β -amino acids on the structure of cyclic peptides. the structures of the peptide library have been investigated by nmr spectroscopy, followed by dg/sa and md calculations. the results substantiate the γ-turn inducing capability of β-homoamino acids, but also prove the formation of different turn structures in certain cases. a comparison to the x-ray structure of vcam- shows that the structure of the binding sequence has been successfully approximated by some of the peptides. biological activity tests should lead to meaningful structure-affinity relationships. neuropeptide y (npy) is a -amino acid peptide amide and binds to the so-called y receptors. its most dominant element is the c-terminal alpha-helix spanning amino acid residues - . residues - form a polyproline helix with highly conserved proline residues at positions , and , followed by a loop structure. the importance of the polyproline helix strongly varies between different receptor subtypes. it obviously plays no role in ligand binding at the y receptor subtype, whereas the n-terminal segment is of importance for ligand binding at y and y . in order to further study the importance of the polyproline helix we introduced a conformationally constrained pyridone dipeptide mimetic at different single positions by solid phase peptide synthesis using fmoc/tbu strategy. the resulting peptides have been investigated in cell lines that selectively express y and y receptor, respectively. different methods including radioactive competitive binding assay, cd and nmr have been applied to investigate conformation and interaction of receptor and ligand. loss of affinity at the y receptor is independent of the position and about -, -and -fold, respectively, when introduced once, twice and thrice. introduction of the building block in position / leads to the most reduced affinity at the y receptor subtype but, surprisingly, affinity can partially be regained by introduction of the dipeptide at two additional positions. the position of the dipeptide is of greater importance at y . these novel peptides clearly indicate the importance of proline residues and the structure of the n-terminus for ligand binding. interactions of src homology (sh ) domains with phosphotyrosine (py) containing ligands is critical for regulating cellular processes. the cytosolic protein tyrosine phosphatase shp- contains two sh domains. an intramolecular interaction of the n-terminal sh domain with the catalytic (ptp) domain renders the enzyme inactive in the native state. binding of a py-ligand to shp- n-sh leads to a conformational shift and the dissociation of the sh -ptp complex [ ] . in previous studies we investigated the topographical and conformational preferences of the n-sh domain of shp- using conformationally restricted linear and cyclic peptides derived from the natural interaction partner ros py [ ] . we identified peptides that showed an increased binding affinity for the n-sh domain and partially inhibited ros-mediated shp- activity. on the basis of these results we hypothesized that an imperfect fit of the py+ and py+ side chains might be responsible for the inhibitory effect. in order to confirm this hypothesis we synthesized a new series of peptides and evaluated their biological activity. to better understand the role of each individual sh domain in the activation process we also determined the binding affinity against the c-sh domain and the activation profile of different shp- mutants. pull-down assays of the interactions of the py-ligands with full length shp- confirmed the results obtained for the binding to the individual sh domains. proteins are targets for photo-destruction due to absorption of incident light by endogenous chromophores. mass spectroscopic data presented evidence that structural modification observed upon irradiation of goat alpha-lactalbumin at nm results from tryptophan (trp) mediated cleavage of disulfide bonds [ ] . the aim of the recent studies is to define structural elements that direct the destructive influence of near-uv light on the disulfide bridges of proteins. most of the proteins of the immunoglobulin superfamily contain a so called triad, consisting of two s atoms, forming a disulfide bridge, and a single trp in their close vicinity [ ] . we have indications that this arrangement gives rise to a photolytic degradation similar to that described in our earlier studies for goat alpha-lactalbumin [ ] . we therefore investigated the influence of uv light on the single chain variable fragment (scfv) of a monoclonal antibody ( d a ) [ ] which contains two triads. the results showed that after irradiation of the wild type scfv (i) new bands (degradation products) appeared in electrophoresis experiments and (ii) the affinity for its antigen, von willebrand factor decreased. by site-directed mutagenesis, we modified the critical trp-residues to perform a parallel study on these mutants. background and aims: it is known that thrombomodulin has important function which prevents thrombus. we found kmylcvckn (m, n >= ) peptides derived from thrombomodulin had strong anti-thrombus activity in our recent studies. these peptides formed two structures, parallel and anti-parallel, as dimers, we examined the relation between structure and activity. methods: two peptides of kkkylc(acm)vckkk and kkkkylcvc(acm)kkkk were synthesized by fmoc chemistry. dimer peptides were made by removing acm with iodine, after dissolving in . m tris hcl buffer (ph . ) and oxidizing the mixture of these synthesized peptides spontaneously. then three peptides shown in figure were separated using rp-hplc. the peptide concentration in normal human pooled plasma was micro moles / l when measuring aptt (activated partial thromboplastin time). results: the anti-parallel formed peptide, peptide b, was prolonged aptt approximately . times, although two parallel formed peptide, peptide a and c, were not significantly different from the aptt of normal plasma. conclusions: these peptides have structure-activity relationship, we observed that the anti-parallel formed peptide had strong anti-thrombus activity. insect kinins share a highly conserved c-terminal pentapeptide sequence phe-xaa-xbb-trp-gly-nh , where xaa can be tyr, his, ser or asn and xbb can be ala but is generally ser or pro. they are potent diuretic peptides that stimulate the secretion of primary urine by malpighian tubules, organs involved in the regulation of salt and water balance ( ). the insect kinins preferentially form a cis-pro, type vi β-turn. insect kinin analogs containing tetrazole ( ) and -aminopyroglutamate ( ), both cis-peptide bond, type vi β-turn motifs, demonstrate significant activity in the in a cricket diuretic assay. in this study, we compare the diuretic activity of insect kinin analogs incorporating the four stereochemical variants of the -aminoglutamate (apy) motif. three of the insect kinin analogs incorporating the stereochemical variants, ( the need for new effective and to mammalian cells non-toxic antifungal agents increases in parallel with the expanding number of immunocompromised patients at risk for invasive fungal infections. in our laboratory we have produced a serie of low-molecular peptide derivatives of the general structure: x-arg-leu-nh-ch(ipr)-ch -nh-y (where x and y were acyl groups with aromatic carbocyclic system). we have found and earlier reported that some of these display high antimicrobial activities against several clinically important gram-positive pathogenic bacteria. in this study we have by solution methods synthesized a group of low-molecular compounds and investigated their antifungal activity. the study included both candida and aspergillus species. we have found that some of the compounds were highly fungicidal. we also made a conformational study in which the residues were separately replaced by selected hydrophobic amino acids and their equivalents. the conformational study showed that the desirable stable intramolecular structure could only be formed in the presence of some vital components. this work was supported by grant ds/ - - - . increased resistance of bacterial pathogens to currently employed antibiotics has resulted in efforts to develop antimicrobial compounds with new mechanisms of action. previously, we have synthesized some high potent antimicrobial compounds based upon the n-terminal binding fragment of human cystatin c. some derivatives of the general structure: x-arg-leu-nh-ch(ipr)-ch -nh-y ( ) (where x and y were acyl groups with aromatic carbocyclic system) have displayed the broad antibacterial spectrum and high activity against several clinically important gram-positive pathogens, including multi-resistant staphylococci. herein, the synthesis and structure -antibacterial properties relationship for two series of analogues of are presented. the x and y groups in were replaced by selected substituents with various geometry and distance between aromatic moieties and carbonyl. we have established the general structural features which the discussed class of peptide derivatives should possess in order to displaying the particular antimicrobial activity. this work was supported by grant ds/ - - - . we have synthesized beta-endorphine-like decapeptide immunorphin sltclvkgfy which corresponds to the - sequence of the heavy chain of human igg. immunorphin was found to be a selective agonist of non-opioid (naloxone-insensitive) beta-endorphin receptor. the purpose of this study was to prepare [ h]immunorphin and characterize by its using the non-opioid beta-endorphin receptor on mouse peritoneal macrophages and membranes isolated from various rat organs. by use of tritium-labeled immunorphin ([ h]sltclvkgfy) with specific activity of ci/mmol, non-opioid beta-endorphin receptors were revealed and characterized on mouse peritoneal macrophages and rat myocardium, spleen, adrenal, and brain membranes. since dehydroamino acids are quite reactive and various thiol nucleophiles are known to add to their double bonds [ , ] , we hoped that these compounds might act as alkylating inhibitors of cathepsin c (dipeptidyl-peptidase i). its main function is protein degradation in lysosymes, but it is also found to participate in the activation of neuraminidase and proenzymes of serine proteinases (leukocyte elastase, cathepsin g, granzyme a) [ , ] . it is well known, that phosphonodipeptides structural analogues of synthetic substrates of cathepsin c are the model substances in designing the new inhibitors of this enzyme. for that reason we have undertook the synthesis, theoretical and structural investigations of phosphonic analogues of dehydropeptides. gly-∆zphe-abupo(ome) gly-∆zphe-alapo(oet) gly-∆zphe-leupo(ome) gly-∆zphe-valpo(oet) gly-∆zphe-glypo(ome) gly-∆zphe-nbupo(oet) the structure and conformational preferences in this group of peptides had been investigated by mean of nmr techniques. in order to find the interactions between compounds-enzyme (cathepsin c) and interpret the results of biological test, the molecular modelling methods had been used. the interaction of v integrin receptor with its ligands is selectively implicated in various processes, like angiogenesis, bone-formation, tumor genesis and tissue-genetic migration of embryonic cells. several cyclic rgd pentapeptides are known as selective ligands for v integrin receptor. the aim of this study was to prepare a new conjugate, composed of the cyclo[rgdfc] derivative and a branched chain polycationic polypeptide, poly[lys(dl-alam)] (ak). the cyclopeptide was prepared on -cl-trityl chloride resin by fmoc/tbu strategy. the "head-to-tail" cyclisation was achieved in a diluted solution of dmf in the presence of bop and hobt coupling reagents and diea base. coupling of the cyclopeptide to ak polymer was carried out by thioether linkage. adhesion properties of soluble cyclic rgd peptides and their plate-immobilized forms were studied. free cyclopeptides evoke aggregation of cultured primary neural and cloned neural stem cells, while their plate-immobilized forms fail to support cell adhesion. on the contrary, in case the newly synthesized ak-c(rgdfc) conjugate such induction of cell aggregation was not observed. whereas immobilizing this derivative to either glass, or plastic was found to support cell-attachment in case of various cell types. in addition, all cell lines investigated -including also the primary neural cells -attached to ak-c(rgdfc) coated surface and survived, grew or differentiated even in the absence of serum. our data suggest that cyclic rgd -polypeptide conjugates represent a new tool to investigate selective cell adhesion and may provide a novel scaffold-material for directed cell-seeding. in the ph-induced channel closure in combination with the pip interactions. however, their detailed regulations are still remaining unclear. therefore, in the present study, we investigate these crucial residues with electrophysiological recordings and rationally designed mutagenesis based upon our structural analysis of kir . tetramer. lys- is located fairly close to the intracellular channel gate and protrudes its long side chain positive charge into the pore. this may interfere with the potassium flow by providing repulsion charge while ph is lowered, which pushes the channel towards its closed state. mutation to met- therefore reduces such ph-sensitivity. on the other hand, arg- is supposed to be responsible for the maintenance of channel opening in the presence of pip . loss of positive charge at this site may lead to the enhanced ph-sensitivity due to an abolished or reduced pip interaction. more interestingly, the double mutant for both sites reveals a compensation scenario. in combination with the discussion for the role of previously known r-k-r triad, our data provide very clear structural explanation for the exact functional roles of these basic residues in the regulation of ph-sensitive channel gating. mouse obese cart peptides are neurotransmitters involved in feeding, stress and endocrine regulation. leptin, a long-term adiposity signal, upregulates expression of cart in the hypothalamus. recent findings of co-localization of cart and cholecystokinin (cck)-a receptor (responsible for satiety effect of cck) in brain and gastrointestinal tract suggest a neurochemical link between cart peptides and cck. in normal fasted mice, cart( - ) peptide decreased food intake after intracerebroventricular (icv) administration in a dose-dependent manner. anorectic effect of cart peptide was enhanced by peripherally administered cck- , while cck-a receptor antagonist, devazepide blocked the effect of cart peptide on food intake. we used two mouse obesity models in this study: monosodium glutamate (msg) and diet-induced obese (dio) c bl mice. both dio and msg mice had substantially increased fat to body mass ratio compared to their controls and were hyperleptinemic. msg mice were hypophagic and neither cart peptide nor cck- and devazepide had any effect on food intake of these mice. dio mice fed high-fat diet showed slightly decreased sensitivity to central administration of cart peptide, effect of cck- on food intake was preserved. in conclusion, cart peptide and cck- showed a synergistic effect on feeding in control mice that pointed to their probably integrated action in the central nervous system. analogously, devazepide suppressed cart anorectic effect. in msg obese mice, effects of both cart peptide and cck- on food intake were diminished due to disrupted signaling in hypothalamus. in dio mice, additive effects of cart and cck- were partly preserved inspite of hyperleptinemia and increased adiposity. b. chini , s. stoev , l.l. cheng , m. manning , were subsequently shown not be selective for the rat v b receptor [ ] . peptides a-d served as excellent leads to the design of selective agonists for the rat vp v b receptor [ ] . replacement of the arg residue in a-d by lys, orn, dap and dab, led to the first potent and selective agonists for the rat v b receptor [ ] . we now report that three of these; d the aim of the de novo peptide synthesis and the incorporation of cofactors is the construction of artificial protein models. these model systems can be used for understanding the structure-function relationship of native proteins and might open a way for possible applications. protegrin- (pg- ) is an -amino acid peptide with an amidated c-terminus, which forms an antiparallel beta-sheet, constrained by two disulfide bridges. the native sequence of pg- is highly cationic, containing six positively charged arginine residues. it was found that the structural features such as amphiphilicity, charge and shape are important for the cytolitic activity of pg- . in this study we investigate the sar (structure activity relationship) of two pg- analogues: rglcycrgrfcvcvg-nh (bm- ) and rglcyrprfvcvg-nh (bm- ). our antimicrobial activity studies of these peptides show that the bm- peptide is active against microbe species as well as the native pg- , whereas the bm- is completely inactive. the bm- analoque is shorter than native pg- and contains only three arginine residues, therefore is much cheaper in the chemical synthesis, what could be an advantage of this antimicrobial peptide. the conformational studies of both analogues were performed by using d h-nmr technique (in dmso-d ) and molecular dynamics studies. the d solution structure of both analogues was established using interproton distances and torsion angles. for simulated annealing calculations the xplor program was used. our conformational studies show that the bm- forms a regular beta-hairpin structure, which is very similar to that of the native pg- peptide, whereas the bm- analogue is very flexible, what could be a reason of the antimicrobial inactivity. copper amine oxidases (ec . . . ) catalyze the oxidative deamination of primary amines to the corresponding aldehydes, ammonia and hydrogen peroxidase. these enzymes are ubiquitous, occurring in micro-organisms, plant and animals. activity of this enzyme increases under various stress conditions including thermal and water stresses. although lsao is not a thermostable enzyme, it is in maximum stability and activity above physiological temperatures. in this study we have investigated the kinetics of thermal denaturation of lentil seedling amine oxidase (lsao) by measuring its denaturation constant (kden) at various temperatures from to degrees centigrade in mm phosphate buffer, ph . . the results of thermal inactivation curves as well as measuring of a at various temperatures were used to calculate kden. moreover, activation energy (ea) for denaturation reaction was obtained from corresponding arrhenius plot. our results showed that unfolding process started to occur at degree centigrade and ea of denaturation was changed at degree centigrade proving a dominant conformational change of the enzyme at this temperature. the results of the kinetic study are coincident with previously reported equilibrium studies denoting the optimum and melting temperature of the enzyme are and degree centigrade, respectively. development and advancing of enzymatic processes used for production and modification of natural polysaccharides are now major biochemistry challenges. the paper investigates enzymatic systems in invertebrates, in particular, an enzymatic complex obtained from the hepatopancreas of red king crab paralithodes camtschaticus, and clarifies its effect on the mechanism of chitin and chitosan hydrolysis. chitinolytic activity was estimated with spectrophotometer using -(dimemylamine)-behzaldehyde method by the concentration of n-acetyl-d-glucosamine which is educed under chitinolysis. total glycolytic activity was defined by the sum of n-acetyl-d-glucosamine and d(+)-glucosamine in the reaction with potassium hexaferricyanide (iii). content of d(+)glucosamine in the hydrolysates of chitin and chitosan was estimated by highly effective reverse-phase liquid chromatography (helc) of aminosaccharides with ortho-phthalaldehyde. the paper studies the process of chitin and chitosan glycolysis and the effects of different factors (ph, temperature and time of incubation, enzyme/substrate ratio) on the total glycolytic activity of the enzymatic complex from crab hepatopancreas, which is compared with a previously studied proteolytic and exochitinase activities. a mechanism of enzymatic hydrolysis of chitin and chitosan is suggested. study results allowed the following conclusions concerning glycolytic and deacetylase activity of ep: ) ep induces the formation of a monomer (n-acetyl-d-glucosamine) and oligomers (chitin and chitosan) with low deacetylation. thus, ep is characterised by a marked endochitinase (endochitosanase) activity; ) n-acetylglucosamine deacetylase and, apparently, exochitosanase activity was not revealed; ) it was found that chitinase and protease activities of ep are associated with different enzymes. [background] in opioids, the n-terminal amino acid ', '-dimethyl-l-tyrosine (dmt) enhances bioactivity by orders of magnitude. c-terminal modification of the dmt by a methyl group, h-dmt-nh-ch , exhibited µ-opioid receptor affinity (kiµ = . nm) equivalent to that of morphine; however, antinociception was only . - . % [ ] . dmt plays an important role in the message domain to anchor opioid ligands into the active site of opioid receptors, specifically to trigger biological activity by the µ-opioid receptor. [methods] dimerization of dmt through diaminoalkanes [ ] or , -bis-(aminoalkyl)- ( h)-pyrazinone produced potent opioidmimetics with high affinity for µ-opioid receptors (kiµ = . - . nm), agonism (gpi, ic = . - . nm), and antinociception in mice after systemic and oral administration, which verified passage through the epithelial membranes of the gastrointestinal tract and blood-brain barrier [ ] . ( -aminocycloalkane- -carboxylic acid) . in this case the ring consists of five atoms. knowing that acylation of the nterminus of several known b blockers with a variety of bulky groups has consistently improved their antagonistic potency in the rat blood pressure assay, the apc substituted analogues were also synthesized in n-acylated form (with -adamantaneacetic acid (aaa)). the activity of eight new analogues was assayed in isolated rat uterus using a modified holton method in munsick solution and in rat blood pressure tests. the results clearly demonstrated the importance of the position in the peptide chain into which the sterically restricted apc residue was inserted. apc at positions led to preservation or reduction of antagonistic qualities, respectively. acc at position enhanced antagonistic qualities in blood pressure test and led to preservation of activity in antiuterotonic test.. in most cases acylation of the n-terminus led to enhancement of antagonistic potencies. our findings offer new possibilities for designing new potent and selective b blockers. background: during the course of developing opioidmimetic analgesics, data revealed that the n-terminal residue ', '-dimethyl-l-tyrosine (dmt) plays an important role in anchoring opioid ligands in the active site of opioid receptors. as a single residue c-terminally extended with an aminomethyl group exhibited µ-opioid receptor affinity (kiµ = . nm) similar to morphine; however, antinociception was only . - . % [ ] . in order to develop potent µ-opioid agonists, the dimerization of tyr or dmt through diaminoalkanes [ ] or , -bis-(aminoalkyl)- ( h)-pyrazinones [ ] resulted in production of unique opioidmimetics with high receptor affinities and potent biological activities [ ] . methods: the synthesis of opioids and opioidmimetics and the determination of their receptor binding characteristics were performed as described previously [ ] [ ] [ ] . results and conclusion: newly synthesized -(tyr-nh-butyl)- -(tyr-nh-propyl)- ( h) pyrazinone and -(tyr-nh-propyl)- -(tyr-nh-butyl)- ( h) pyrazinones (i and ii) exhibited fairly high binding affinity towards µ-opioid receptor (kiµ = . and . nm, respectively). replacement of tyr with dmt in i and ii gave opioidmimetics iii and iv (kiµ = . and . nm, respectively); they exhibited -and -fold higher binding affinity than the tyr derivatives. while iii is a dual µ-/δ-opioid agonist, iv is only a µ-opioid agonist. these findings pave the way to design additional µ-opioid receptor agonists and antagonists for therapeutic application. divalent cations have been known for a long time to influence significantly binding to receptors and biological activity of the peptide oxytocin (ot). there is very low binding of h-ot to the receptors in the absence of these ions. it has been speculated where the divalent cations work. recently an article appeared showing formation of a complex divalent cation-ot and stressing the importance of n-terminal amino group for binding and activity [ ] . however deamino analogues of ot are also very active and their binding is also influenced by divalent cations. we have studied ot, deaminooxytocin (dot) and an ot antagonist (antag) by means of electrospray ms and we have observed that all these compounds form molecular adducts with zn +, mg +, mn + and ca +. in binding experiments using -i antag, the quantity of tracer bound to membranes of hek cells having stable expressed human ot receptor strongly depends on the character and concentration of divalent ions. displacement curves using unlabelled antag do not change in the absence or presence of mm of tested divalent ions. on the other hand, displacement curves using unlabelled ot and dot are shifted to the left in the presence of mg + and mn +, and to much lesser extent by zn + and ca +. all this points to the idea that the divalent ions do work on the site of membrane receptors. biologically active peptides exhibit multiple conformations in solution. thus, the synthesis of conformationally restricted analogues is a valuable approach for determining structure -activity relationships. restrictions can be imposed e.g. through the formation of cyclic structures within the peptide framework by disulfide bridges, or by substitution of chosen amino acid residues that limit conformational freedom, thus forcing the peptide backbone and/or side chains to adopt specific orientations. in recent years, conformationally constrained analogues of bioactive peptides seem to be a feasible approach to providing useful informations concerning threedimensional structure of such compounds which, in turn, could rationalize our knowledge about structure-biological activity relationships and thus help to design peptides with desired pharmacological properties. steric restrictions can be introduced by the formation of cyclic structures within the peptide backbone or by incorporation of amino acids with limited conformational freedom, which in turn results in specific orientations of the peptide backbone and its side chains. another approach to reduce the flexibility of the analogue is substitution of chosen amino acids with various types of pseudopeptides prepared trough short-range cyclizations. the present work is a part of our studies aimed at clarifying the influence of sterical constraints in the n-terminal part of arginine vasopressin (avp) and its analogues on the pharmacological activity of the resulting peptides. we describe the synthesis of four new analogues of avp substituted at positions and or and with two diastereomers of -amino-pyroglutamic acid and four peptides in which we combined the above modification with the placement of mercaptopropionic acid (mpa) at position . all the peptides were tested for their in vitro uterotonic, pressor and antidiuretic activities in the rat. different strategies to modulate shp- activity protein tyrosine phosphatase shp- consists of two sh domains n-terminal to the catalytic (ptp) domain and a short c-terminal tail. the binding of a py-ligand to the n-sh domain is required for an efficient activation of shp- phosphatase activity. the specificity of the shp- sh domains is determined by the py-residue (position ) and residues at positions - , + and + . combinatorial peptide library methods revealed different classes of consensus sequences for both sh domains [ , ] . in addition, the importance of residues c-terminal to py+ (+ to + ), in particular for binding to the n-sh domain, has been demonstrated [ ] . together with investigations of the determinants for optimal sh domain binding and stimulation/inhibition of shp- activity [ ] , these informations were useful for the generation of different strategies for effectors of shp- activity. peptides cyclized between different positions of the general consensus py- to py+ were synthesized and evaluated with respect to n-sh domain binding and stimulation of phosphatase activity. structure-activity studies have revealed that the specificity of an integrin towards its rgd-containing ligands can be evaluated through the distances between the cβ atoms and/or the distance between the charged centers of arginine and aspartic acid as well as, the pseudo-dihedral angle (pdo), composed by the r-cζ, r-cα, d-cα and d-cγ atoms, which defines the relative orientation of the arg and asp side chains. in a previous study [ ] , the antiaggregatory activity of rgd peptide analogues, i.e. their ability to act as fibrinogen receptor αiibβ antagonists, was correlated with the above structural criteria. our results suggested that the fulfillment of the criterion - ο < pdo < + ο is a prerequisite for an analogue to exhibit activity. in the present study, we examine the above criteria to rgd-containing peptides, derived from the active sites of the ecm proteins fibrinogen, fibronectin and vitronectin, as well as, from the cryptic rgd site of von willebrand factor. the correlation of the structural data with the biological activity of compounds, are in good agreement with the previously mentioned - ο < pdo < + ο criterion. furthermore, our results show that the differences in activity of compounds, which display similar distances between the charged centers of arg and asp, can be better evaluated by the pdo structural criterion. acknwolegments : this work was supported by grants from eu and the hellenic ministry of education ( heraklitos). references : the gpiib/iiia receptor, which is a member of the integrin family, is the most abundant receptor in the surface of platelets and can interact with a variety of adhesive proteins including fibrinogen, fibronectin and von willebrand factor. fibrinogen binding on gpiib/iiia is an event essential for platelet aggregation and thrombus formation. mapping of the fibrinogen binding domains on gpiib subunit suggested the sequence - as a putative binding site [ ] . this region was restricted to sequence gpiib - (ymesradr) using synthetic octapeptides overlapping by six residues [ ] . the ymesradr octapeptide inhibits adp stimulated human platelets aggregation and binds to immobilized fibrinogen. in this study we present the conformational analysis of three synthetic analogues yaesradr (a ) ymesaadr (a ), and ymesraar (a ), using nmr spectroscopy and distance geometry calculations. common structural characteristic of peptides a and a is the interaction between the side chains of arg and glu , however in a the guanidino group of arg seems to form salt bridges with both glu and asp . peptide a is stabilized only by a week interaction between arg and glu side chains. the interactions between the residue side chains provoke different overall shape of the three molecules. the most populated structural family of a exhibits a π backbone shape, a a turn around -s a -, while a an almost extended shape. background and aims: endomorphin- (em- : h-tyr-pro-phe-phe-nh ), endogenous opioid peptide isolated from bovine and human brain, has high affinity and selectivity for the mu receptor and produces potent and prolonged analgesia in mice [ ] . in this presentation, the incorporation of ethylene-bridged phe-phe unit (eb[phe-phe]) or piperidine carboxylic aid (pic) in position was carried out to obtain more potent agonist or antagonist with stability against dipeptidyl peptidase iv (dpp iv). methods: the synthesis of eb[phe-phe] unit was achieved according to the procedure of lammek b. et al. [ ] protected peptides were synthesized by a solution method using boc-chemistry. the final products were identified by maldi-tof mass spectrometry and elemental analyses. the receptor binding affinity of peptides was assessed by radio-ligand receptor binding assay using mu and delta opioid receptors from rat brain membranes or cos- cell membranes expressing each opioid receptors. muc glycoprotein, produced by the epithelium of the colon, built up mainly of repeat units of ptttpitttttvtptptptgtqt , can be underglycosylated in colon carcinoma. we have been studying the epitope structure of the muc repeat unit with the mucin peptide specific mab monoclonal antibody. this antibody recognizes the ptgtq sequence as minimal, and ptptgtq as optimal epitope. our interest lies in the modification of this epitope with maintained or enhanced specificity, and we aim to clarify the effect of different epitope modifications on mab antibody binding: a) amino acid changes in the flanking region, b) glycosylation in the epitope core and in the flank. for this we have prepared a) libraries of ax( )ptgtqaa and atptgtqx( )a peptides, and x( )ptgtqx( ) heptapeptides based on the antibody binding properties of the libraries; and b) glycopeptides pt(galnac)ptgtq, ptpt(galnac)gtq and ptptgt(galnac)q. the peptides were prepared by solid phase synthesis; after purification, esi-ms and amino acid analysis characterisation their antibody binding properties were studied by competitive elisa. our results show that a) although all amino acids in positions x( ) and x( ) resulted in antibody binding; in position x( ) hydrophobic, in x( ) aromatic residues provided stronger binding than that of the native peptide; b) glycosylation on thr( ) did not influence the binding of mab , but on thr( ) the presence of n-acetyl-galactosamine, interestingly, slightly increased the antibody recognition. these findings could be useful in designing synthetic peptide vaccines for tumour therapy. histidines play essential role in binding of biological metal ions, either in small or macromolecular chelating molecules, e.g. in metalloenzymes. therefore the low molecular weight polyhistidine type ligands are of potential importance as model substances. continuing our investigations on a novel branched oligopeptide type ligand -(his) (lys) lys-nh -prepared by solid phase peptide synthesis, we investigated the metal ion binding properties with zinc(ii) and copper(ii). the eight primary metal-binding sites are the four imidazole and four ammine groups on the ligand. phpotentiometric titrations revealed, that up to ph all these donor atoms loose their protons on increasing ph. the competition between the protons and the metal ions results the decrease of pka values to about - in the case of copper(ii) and to about - in case of zinc(ii) ion. this reflects the higher stability of the complexes formed with copper(ii) in spite of the weak axial coordination that seems to occur in zinc(ii) complexes. combined potentiometric, spectrophotometric, cd and nmr spectroscopic methods were utilized to investigate the speciation and the structure of the complexes formed in aqueous solution. the prepared cu(ii) complexes cleaved dna, but it is not known whether in oxidative or in hydrolytic manner. because of this ambiguity further studies with zn(ii) complexes will be undertaken. this work has received support through sapstclg nato collaborative linkage grant and from the hungarian science foundation (otka t ). lgr . further studies have shown that in both male and female gonads, insl and lgr represent a paracrine system important for meiosis induction in the ovary and male germ cell survival in the testis. thus insl may have clinical applications in fertility management. we undertook to determine the key structural elements responsible for its unique actions. methods: alanine-scanned analogues of human insl and mimetics of the b-chain alone were prepared by solid phase peptide synthesis. each was subjected to cd spectroscopy for secondary structure analysis and assayed for in vitro lgr binding and activation activity. the tetrapeptide h-dmt-d-arg-phe-cbp was found to be a selective µ agonist [ic (gpi) = . ± nm] with -fold lower potency than the corresponding, highly potent tiq -tetrapeptide, but with still -fold higher potency than leu-enkephalin. in conclusion, we developed selective, cbp-containing δ antagonists and µ agonists with significant potency. recently, we described the syntheses and biological activities of several opioid peptide analogues that contained the n-terminal sequence - , common to dermorphin and deltorphin. some of them showed very high agonist potency both in the gpi assay and in the mvd assay [ , ] . in this work, we designed new analogues in which the sequences were elongated at the c-terminal to obtained the full sequences of dermorphin (a) and deltorphin (b). the syntheses of compounds and their biological activity profiles will be discussed. background and aims: endomorphin- (em- : tyr-pro-phe-phe-nh ) is very potent endogenous opioid peptide, which exhibits high affinity and selectivity for the mu-opioid receptor [ ] . previously, we had reported that [ac c ]-em- containing -aminocyclopropane- -carboxylic acid (ac c) exhibited higher affinities than em- for the mu-opioid receptor [ ] . in order to clarify that the substitution of -aminocycloalkane- -carboxylic acids (acnc: n indicates the number of carbon atoms in a ring) for pro in position of em- is efficient to obtain higher affinity for the mu-receptor, we synthesized . therefore, the replacement of pro to ac c and ac c will be efficient to make these analogs adopt bioactive conformation and exhibit high affinity for mu-receptor. in the past few years, many attempts have been made to prepare a synthetic insulin. the biological activity of insulin is known to be closely related to the c-terminal octapeptide fragment of its b-chain.it was found that b gly and b phe were present in all insulins so far obtained from various animal species indicating the significance of these two residues.it would therefore seem desirable to study the effect of each of these two amino acid residues or both on biological activity of the octapeptide fragment of the b-chain. a heptapeptide arg-phe-tyr-thr-pro-lys-ala-och , corresponding to (b -b ) insulin des gly -phe , and an octapeptide arg-phe-phe-tyr-thr-pro-lys-ala-och , des gly were synthesized using the solid phase method. the c-terminal ends of both peptide were converted to methyl ester by transesterification cleavage from the resin. the side chain protecting groups were removed by hf. manual counter current distribution method was used for purification of the free peptides. the way to solve the evaluation of tyrosine containing peptide was studied. the free methyl ester peptides were administered for insulin-like activity test by glucose metabolism in the rat fat cells technique in vitro. aim of this study is to develop peptides as useful tools for degradation of synthetic dyes, which are often pollutants. we focused our interest in peroxidases, a class of enzymes reported to efficiently degrade azo and anthraquinonic dyes. in particular, the fungus versatile peroxidase (vp) of pleurotus eryngii can perform this degradation. therefore, our goal is the synthesis of a peptide based on this peroxidase able to emulate its biological function. the linear and cyclic peptide sequences were derived by the theoretical model of vp [pdb: a ], which determined the amino acids fundamental for the desired function of the active sites. in particular, the residues instrumental for the coordination of the heme, the mn binding site, and the long range electron transfer pathway [ ] , were pin-pointed. moreover, we calculated the radius of the heme cavity. the next step was the synthesis of these peptides in order to verify the coordination of the heme and optimize their sequences. the syntheses were carried out by solid-phase following the fmoc/tbu strategy. because of purification difficulties of the fully-protected peptide, we undertook an alternative synthetic pathway, based on a solid phase head-to-tail cyclisation strategy, following the fmoc/tbu/allyl three-dimensional protection scheme [ ] . next steps will be to test the coordination properties of the synthetic peptides, with respect to the heme, and further computational studies based on the new model of pleurotus the calcium plays an important role in biochemical pathways. it binds to enzymes and proteins in a different process. aspartic (asp, d) and glutamic (glu, e) acid side chains are the main ligands of calcium, but the contribution of the backbone carbonyl groups in the binding is also important. generally the binding places in the proteins are an unstructured loop between two helixes ( -or alpha-helix). the common sequence is the so-called ef-hand motif, which contains amino acids [ ] . it is already known that some proteins also bind calcium with a non-ef-hand loop. for example alpha-lactalbumins have a ten amino acid long sequence for binding [ ] . it is an asp rich sequence where asps are closer to each other than in ef-hand motif (-k fldddltdd -) but only asps side chains take part in calcium coordination. we constructed a series of cyclopeptides to mimic the loop structure of alpha-lactalbumin [ ] . in this study we focus on determining the importance of conservative amino acids within the ca + binding loop of this protein, using microcalorimetry (itc). the itc measurements were performed in different organic solvents and at different temperature. the synthesis of fatty acids in adipose tissue. in this article, we present the solution structure of gip in water and tfe/water determined by nmr spectroscopy. the calculated structures are characterised by the presence of an -helical motif between residues ser -gln and phe -gln respectively. the helical conformation of gip is further supported by cd spectroscopic studies. six gip( - )ala - analogues were synthesised by replacing individual n-terminal residues with alanine. alanine scan studies of these n-terminal residues showed that the gip( - )ala was the only analogue to show insulin secreting activity similar to that of the native gip. however, when compared with glucose its insulinotropic ability was reduced. for the first time, these nmr and modelling results contribute to the understanding of the structural requirements for the biological activity of gip. a knowledge of the solution structure of gip and of the role of its individual residues will be essential in the understanding of how they interact with the gip receptor. efrapeptins are pentadecapeptides produced as a mixture of six closely related analogues (efrapeptin c-g) by the fungus tolypocladium niveum and other members of this species. they consist predominantly of the nonproteinogenic amino acids -aminoisobutyric acid (aib), isovaline (iva), -alanine ( ala) and pipecolic acid (pip), have an acetylated n-terminus and bear an unusual cationic c-terminal headgroup derived from leucinol and , -diazabicyclo[ . . ]non- -ene. efrapeptin c is a competitive inhibitor of the f -atpase and active against the malaria pathogen plasmodium falciparum. an anti-proliferative effect was also reported. conformational analysis of efrapeptin c in trifluoroethanol and dimethylsulfoxide was conducted to obtain structure-affinity relationships. the absence of amide-and -protons resulted in an imperfect assignment and unsatisfying conformational study. specific deuteration of methyl groups in aib did not simplify the assignment. cd and ft/ir spectra hint to helical or beta-turn secondary structures as main structure elements. residual dipolar couplings (rdc) were measured in a stretched cross-linked poly(dimethylsiloxane) gel in dichloromethane. the impact of the rdc on the conformational analysis led to an improved high resolution structure from simulated annealing protocols and consolidated the formation of a helical structure of efrapeptin c in nonpolar solution which is comparable with the binding pocket of the f -atpase. finally, the dynamics of the resulting structures was studied using the gromos force field in explicit solvent. serotonin selective reuptake inhibitors (ssris) are currently among the most frequently prescribed therapeutic agents of depression. their therapeutic use includes also obsessive-compulsive disorder, panic disorder, bulimia. the serotonin transporter (sert) is the target of serotonin selective reuptake inhibitors (ssris). altough the inhibition is the proximal event in antidepressant action, the clinical benefit of antidepressant medications requires weeks of continuous dosing, indicating that their mechanism of action involves events downstream from acute transporter blockade. long-term effects of ssri treatment may be due to changes in intrinsic properties of sert structure, function, or regulation. thus, understanding the mechanism of action of sert remains a primary goal in the search for developing novel treatments for diseases associated with serotonergic dysfunction. in the present study experimentally determined ligand selectivity of the buspirone analogues toward the serotonin transporter was theoretically investigated on the molecular level. the model of serotonin transporter based on the crystal structure of bacterial homologue from aquifex aeolicus (leutaa) was constructed using the traditional homology modelling approach. a series of docking experiments with ssri's were conducted, using interactive molecular graphics techniques combined with energy calculations and analysis of the transporter-ligand complexes. structural information about the serotonin transporter and its molecular interactions with ssri's is important for understanding the mechanism of action of these drugs and for development of drugs with improved potency and selectivity. the protein kinase c (prkc) is a member of a super-family of the eukaryotic receptor protein kinases. it forms dimers and is anchored in the membrane, with a cytoplasmic kinase domain and an external domain, presumably acting as a sensor. prkc enables formation of biofilms of bacillus subtilis which show a high degree of spatial organization. they colonize various surfaces and produce complex antibiotic resistant communities. prkc acts as a ser/thr kinase with features of the receptor kinase family of eukaryotic hanks kinases. our current study involved theoretical modeling of the protein kinaze prkc complexes with the modified atp. the ligands were selected from a set of molecular probes developed by k. shah and coworkers [ ] . each modified atp molecule was docked to the active site of the kinase molecule using autodock genetic algorithm procedure. the optimized structures of the complexes were submitted to the molecular dynamics simulations in the amber force field. we obtained four optimized structures of prkcc complexes in water. the results suggest the great similarity of our complexes with human cyclin-dependent kinase [ ] complexes. background and aims. indolicidin is a -residue antimicrobial peptide, which was isolated from bovine neutrophils. this molecule possesses a wide spectrum of antibacterial, antifungal and antiviral activity, furthermore it has also haemolytic effect. data derived from structural investigations led to considerably diverse conclusions regarding the secondary structure of this peptide, therefore the aim of this study was to examine the effect of cis-trans isomerization on the conformational properties of this antimicrobial peptide. methods. the conformational analysis of indolicidin containing cis or trans xxx-pro peptide bonds was performed by simulated annealing calculations with the use of amber force field. results. for the conformers of indolicidin with cis or trans xxx-pro peptide bonds, the evolving secondary structural elements were examined and poly-proline ii helix and type vi beta-turn were identified. in the case of this peptide, various intramolecular interactions may play an important role in stabilizing the structure of conformers. therefore the presence of the h-bonds between backbone atoms, the aromatic-aromatic interactions between the side-chains of trp amino acids and the proline-aromatic interactions between the side-chains of trp and the pyrrolidine rings of pro amino acids was investigated. conclusions. the conformational comparison of the peptides possessing cis or trans xxx-pro peptide bonds resulted in different secondary structural elements for both isomers, which are the poly-proline ii helix and type vi beta-turn for the trans and cis isomers of indolicidin, respectively. the occurrences of various intramolecular interactions are in agreement with the observed secondary structures. we have shown the monte carlo conformational search using macromodel is useful for conformational study of oligopeptides prepared from alpha, alphadisubstituted alpha-amino acids. moreover, we have studied conformational analysis of oligopeptides containing chiral alpha, alpha-disubstituted alphaamino acids to predict the helical screw sense of helical structures. here we report computational study on conformation of oligopeptides containing cyclic alpha, alpha-disubstituted alpha-amino acids with side-chain chiral centers. background and aims. the homopolymeric amino acids (hpaas) are polypeptides consisting of the same amino acids. some of them play a relevant role in the formation of several neurodegenerative diseases. most probably the poly-(ala) and poly-(gln) are the best representatives of these peptides because of their important biological effects. our aim was to perform conformational analysis and structural investigation of these two hpaas. methods. to explore the conformational spaces of the peptides, simulated annealing (sa) and random search (rs) calculations were carried out using amber force field. two different forms of the hpaas were modelled: either with charged n-terminal amino group and c-terminal carboxyl group, or with the n-and c-termini blocked by acetyl and n-methyl amide groups, respectively. results. for the conformers obtained by sa and rs calculations, the occurrences of various secondary structural elements like different types of beta-turns, gammaand inverse gamma-turns, alpha-helix, -helix, poly-proline ii helix and beta-strand were investigated. in the cases of various helices and beta-strand, segments with different lengths characterized by these secondary structures were determined along the entire sequence of peptides. for the conformers of the hpaas, the intramolecular h-bonds formed between the backbone atoms as well as between the backbone and side-chain atoms were identified. the vasopressin and oxytocin receptors (v ar, v r and otr) are membrane-embedded proteins belonging to the large family a g protein-coupled receptors (gpcrs). they are involved in crucial physiological functions as the regulation of water metabolism, control of blood pressure and stimulation of labor and lactation, mediated via v r, v ar and otr, respectively. as such, they are involved in a number of pathological conditions and are important drug targets. understanding their inhibition and activation mechanisms may improve design of ligands capable of selective stimulation or blockade of the respective receptors presenting the therapeutic targets. to investigate the otr, v ar and v r interactions with agonists and antagonists thirty computer models of receptor-ligand complexes have been modeled via docking and molecular dynamics (md) and analyzed in details. the receptor models were built on rd crystal structure template or using the coordinates of mii-gtα( - ), for non-active and activated models, respectively. the ligands (arginine vasopressin, oxytocin, desmopressin, atosiban ([mpa ,d-tyr(et) ,thr ,orn ]ot) and barusiban (mpa ,d-trp ,ile ,allo-ile ,asn ,abu ,mol ) were docked into the receptors. the complexes have been embedded into the hydrated popc bilayer and submitted to ns unconstrained md in the amber force field. the relaxed systems have been obtained and analyzed in details. the receptor residues responsible for agonists/antagonists binding have been identified and mechanism of binding involving the highly conserved residues has been proposed. a three-dimensional models of the neurohypophyseal hormone receptors were constructed using a multiple sequence alignment and either the crystal structure of bovine rhodopsin or the complex of activated rhodopsin with gta c-terminal peptide of transducin rd*-gt( - ) prototype to obtain nonactive or activated receptor models, respectively. analogs were docked to v ar, v r and otr, both non-active and activated models. the low-energy receptor-ligand complexes, with properly docked analogs were submitted to the constrained simulated annealing (csa), in vacuo. the relaxed receptoranalog models were obtained. the residues responsible for analogs binding to v ar, v r and otr have been identified and presumable biological activity of these compounds was determined. n-methyldehydroamino acids belong to non-standard amino acids found in nature. n-methyl-(z)-dehydrophenylalanine was found in tentoxin, a selective weed killer, having been produced by several phytopathogenic fungi of the alternaria genus. n-methyl-(z/e)-dehydrobutyrine and n-methyldehydroalanine are components of nodularins and microcystins, families of hepatoxins produced by species of freshwater cyanobacteria, primarily nodularia spumingena and microcystis aeruginosa. the simplest n-methyl dehydropeptides, ac-delta(me)xaa-nhme (where xaa = ala, (z/e)-abu, (z/e)-phe, and val) and, for comparison, the saturated ac-l-(me)ala-nhme analogue were investigated using computational methods. cis-trans b lyp/ - +g**//hf/ - g ramachandran potential energy surfaces were created. the conformers found were optimised at the b lyp/ - +g** level. the effect of the electrostatic solute/solvent (water) interaction on the solute energies was investigated within the scrf method using the polarisable continuum model (pcm) on the geometries of solutes in vacuo. it was found that for all the studied dehydropeptide molecules the lowest conformer (phi, psi = ~ - °, °) has the cis n-methyl amide bond. this feature seems to be independent of the dehydroamino acid moieties, the c-beta substituent and the z/e configuration. the pi-electron conjugation as well as the n-h···n hydrogen bond play the dominant role in the stability of this conformer (see figure) . the preliminary nmr investigations into the conformational preferences of the studied molecules in solution confirm the theoretical results obtained. the strong tendency of the n-methyl amide bond to adopt the cis configuration seems to be the reason why n-methyldehydroamino acids are found in small natural cyclic peptides, where they ensure the conformational flexibility necessary for biological action. the purpose of this study was to determine the potentials of mean force (pmf) of the interactions between models of nonpolar amino acid side chains in water. the potentials of mean force (pmf's) dependent on orientation were determined for systems forming hydrophobic and diagonal complexes composed of side-chain models of alanine, valine, leucine, proline and iso-leucine, respectively, in water. for each hydrophobic pair in water a series of umbrellasampling molecular dynamics simulations with the amber force field and explicit solvent (tip p water model) were carried out and the pmfs were calculated by using the weighted histogram analysis method (wham). in all cases a characteristic shape of pmf plots for hydrophobic association were found, which was manifested as the presence of contact minima and solvent separated minima. depths of contact minima for all systems studied were about kcal/mol. in this work we compared the ability of two theoretical methods of ph-dependent conformational calculations to reproduce experimental potentiometric-titration curves of two models of peptides: ac-k -nhme in % methanol (meoh)/ % water (h o) mixture and ac-xx(a) oo-nh (xao) (where x is diaminobutyric acid, a is alanine, and o is ornithine) in water, methanol (meoh) and dimethylsulfoxide (dmso), respectively. in theory, in all three solvents, the first pka of xao is strongly downshifted compared to the value for the reference compounds, the water and methanol curves have one, and the dmso curve has two jumps characteristic of remarkable differences in the dissociation constants of acidic groups. the predicted titration curves of ac-k -nhme are in good agreement with the experimental ones; better agreement is achieved with the md-based method. the titration curves of xao in methanol and dmso, calculated using the md-based approach, trace the shape of the experimental curves, reproducing the ph jump, while those calculated with the edmc-based approach, and the titration curve in water calculated using the md-based approach, have smooth shapes characteristic of the titration of weak multifunctional acids with small differences between the dissociation constants. quantitative agreement between theoretically predicted and experimental titration curves is not achieved in all three solvents. the poorer agreement obtained for water than for the nonaqueous solvents suggests a significant role of specific solvation in water, which cannot be accounted for by the mean-field solvation models m a. papakyriakou , g.f. vlachopoulos , g.a. spyroulias , e. manessi-zoupa , p. cordopatis angiotensin-i converting enzyme (ace) belongs to the m family of the ma clan of zinc metallopeptidases and can act either as a dipeptidyl carboxypeptidase, which catalyses the proteolytic cleavage of dipeptides from the carboxy terminus of a wide variety of peptides, or as an endopeptidase, which hydrolyses peptides bearing amidated c-termini. among the former category of ace peptide substrates, the most distinguished are those involved in blood pressure regulation, such as angiotensin i (angi) and bradykinin (bk). in the latter category falls the gonadotropin-releasing hormone (gnrh) in an attempt to analyze molecular interactions at atomic level we simulated the ace-substrate complexes, using the recently determined d crystal structure of ace testis isoform and a knowledge-based docking method in order to insert the peptide substrate (angi, bk and gnrh) of ace into its catalytic cleft. in order to introduce the effect of protein mobility and gain information about enzyme-substrate recognition and interaction we have sampled the conformational space of these complexes via molecular dynamics simulations with explicit solvent representation. we have also performed molecular dynamics calculations with tace-inhibitor complexes, such as lisinopril, as well as with tace mutated at specific sites, such as the ligands of the two buried chloride ions that have been shown to affect substrate activity. our results provide new insights into the role of specific domains of tace and their implication in the enzyme activity, which is not readily apparent from the available crystal structures. two main mechanisms for the propagation of action potential in myocytes are: ) the free flow of local circuit current through gap junctions and ) the effect of electrical field. here we study effect of each mechanism and their importance during action potential propagation. method: we simulated the cardiac myocyte by the orcad software, then used the model of sinoatrial node to stimulate the myocytes model and studied the propagation of action potential with and without gap junction. result: our results show that, although gap junction solely is not able to mimic physiological condition, but it is necessary for normal cardiac functioning. on the other hand, electric field is not sufficient for successful propagation of action potential and the existence of gap junction is necessary. anthrax is a disease of animals and humans, caused by the bacterium bacillus anthracis. anthrax toxin (at) consists of three proteins, one of which is the anthrax lethal factor (alf). alf is a gluzincin zn-dependent highly specific metalloprotease (~ . kda), which belongs to the m family of the ma clan of zinc metalloproteases. alf cleaves most isoforms of mitogen-activated protein kinase (mapk)-kinases (meks) close to their amino termini, leading to the inhibition of one or more signaling pathways. no data are available on the enzyme-substrate interaction at the molecular level. therefore, we performed classical molecular dynamics simulations on the alf-mkk/mek complexes in order to probe protein-substrate interactions. the simulations pinpointed specific hydrophobic as well as electrostatic alf-peptide substrate interactions and these data were exploited in the building of virtual combinatorial libraries of di-and tri-peptides using the twenty native aminoacids. by applying docking simulations to anthrax zn-metalloprotease around . peptide substrates were virtually screened according to their binding affinity. data suggest that complexes of alf with peptides substrates bearing arg, trp, lys and phe aminoacids, exhibit the highest binding affinity providing evidence for electrostatic interactions between negatively charged residues of alf's active site and positively charged side-chains of di/tri-peptides. new libraries of substrates were built incorporating non-protein residues, organic moieties and chelating groups. alf-substrate complexes with the best score (in terms of binding energy) are further analysed. in the present studies we designed and synthesised seven new bradykinin (bk) analogues and evaluated them in the in vivo rat uterotonic assay using a modified holton method in munsick solution on a strip of rat uterus and in blood pressure test. we used [arg , hyp , thi , , d-phe ]bk, the b antagonist of vavrek and stewart as a model, when designing our analogues. in all cases, the n-terminus of our peptides is acylated with bulky substituent. we previously reported that acylation of the n-terminus of several known b antagonists with various kinds of bulky acyl groups has consistently improved their antagonistic potency in rat blood pressure assay. on the other hand, our earlier results seem to suggest that effects of acylation on the contractility of isolated rat uterus depend substantially on the chemical character and size of the acyl group, as we observed that this modification may either change the range of antagonism or even transform it into agonism. the peptides were synthesized by the solid-phase method using the fmoc-strategy the modifications proposed either preserved or increased the antagonistic potency in the rat blood pressure test. on the other hand, the seven substituents, differently influencend the interaction with the rat uterine receptors and except one led to decrease of antiuterotonic activity. in both cases acylation of the n-terminus led to enhancement of antagonistic potencies. our results may be of value in the design of new b agonists and antagonists. the formations of amyloid fibrils have been reported as for various amyloidosis. several structural models of fibrils are proposed for respective proteins so far. however, their common basic structures and universal features to induce amyloid fibril formations are not known in detail. previously, we examined intermolecular interactions among the several amino acid residues in barnase, which is known to form amyloid-like fibril. based on the experimental results using a series of mutant barnase, we discovered that the interactions between hydrophobic side-chains are the most essential driving force to form the fibrils and that both intermolecular and inter-sheet interactions in the fibril maintain highly ordered molecular packing. in the present paper, we describe a novel prediction method for core regions of various fibril-forming proteins and show the verification of the above possible structural principle. at first, we calculated the interaction's score between side-chains in the antiparallel orientation of beta-strands. next, the peptides with predicted sequences of fibril cores, a couple of high-scored regions with a designed turn moiety to induce a hairpin-like form, were chemically synthesized by spps. as a result, the formation of amyloid fibrils was confirmed for most of high-scored sequences. in addition, we also applied this method to prion protein, we could predict possible beta-strands with hetero-paired orientation. some synthetic peptides involving these strands were proved to have fibril-forming ability. thus, we have developed the novel method to predict the core regions that induce amyloid fibrils. a principal factor analysis (pfa) is a very efficient way of identifying patterns in the data sets even if the patterns are hard to find (e.g. in the high dimensional data sets). this is the reason why the pfa method can be powerful tool for analyzing molecular dynamics (md) trajectories. it is possible to reduce dramatically the trajectory size without loosing significant structural information by applying the pfa procedure. we used this tool for interpretation of results from the molecular dynamics simulations of the model of the transcription factor nf-kb. nf-kb is a protein involved in the numerous biological processes such as regulation of immune response, inflammation, various autoimmune diseases and is used by many viruses, including human immunodeficiency virus (hiv), to activate transcription of their own genes. only the trajectory of the backbone atoms of the nf-kb were subjected to the further analysis. peptides contain many basic sites such as side chains of basic amino acid residues, oxygen and nitrogen atoms of amide groups, and terminal amino groups. these parts can interact with protons. this interaction can change conformational behaviour of peptides and, consequently, their biological functions. the interaction becomes even stronger in the gas phase. in that case, the stability of the peptide chain is influenced, which may have impact on peptide fragmentation during mass spectroscopy analysis of peptide structures. in this study, we will present the interaction of proton with carbonyl oxygens in the model of alanine tripeptide. quantum chemical calculations employing density functional theory using hybrid b lyp functional and - ++g** basis set were used to describe this interaction and also to find possible pathways of proton transfer among interaction sites. two different mechanisms of proton transfer were found. the first mechanism is represented by an isomerization of the proton around the double bond of the carbonyl group. the second mechanism is based on the large conformational flexibility of the tripeptide model where all carbonyl oxygens cooperate. the later mechanism exhibits nearly half energy barrier of the rate-determining step compared to the first one. we focus our attention on situation, in which methyl groups attached to alpha atoms in tripepetide model influence the conformational behavior. results will be presented for all four possible stereochemical configurations. a. papakyriakou , p. galanakis , p. gazonis , g.a. spyroulias p protein is one of the most effective defensive weapons of human body against carcinogenesis, due to its tumor suppression properties. it has been noticed, in many types of cancer, that the functions of p are being downgraded or even suppressed and this fact is ought to the presence of mutated forms of p or to the complete absence of the protein. the suppression of p levels is being indirectly regulated by the protein itself, which activates the expression of a gene, the oncogene mdm (murine double minute ), which expresses the mdm protein, known as human-mdm or just hdm . hdmx protein is a homologue protein to hdm and is being implicated, through various biological processes, in the suppression of p . however, recent experimental evidence suggests that hdm and hdmx proteins are not the only ubiquitin ligases that negatively regulate p through ubiquitin pathway. two recently discovered e ligases, cop and pirh , are also proposed to promote p for degradation. all these proteins function as e ligases bearing a ring finger domain. these domains are characterized by their high content in cysteines and the binding of two zn(ii) ions while they catalyze the latter stage of protein signaling for proteolysis by the s proteasome, through the ubiquitin pathway. the structure variation and the stability of these ring fingers is studied through molecular dynamics simulations of - ns and structure variations are analyzed in a structure-function correlation basis. semax is a synthetic analogue of adrenocorticotropic hormone acth - . it is a nootropic agent containing seven amino acids met-glu -his-phe-pro-gly-pro without hormonal (adrenocorticotrophic) activity. semax is neuroprotective via a mechanism involving the regulation nitric oxide (no) and lipid peroxidation. semax proved to be highly effective in abating the rise in no and restoring neurologic functioning [ ] . it was found to improve intellect and memory in healthy human. it is effective in rehabilitation of people with memory and motor disorders, parkinson's and hantington's diseases, after cerebral stroke and head trauma [ ] . to study conformation dynamics in connection with in vivo activity of semax the molecular dynamics method of standard protocol was applied [ ] . semax and about twenty its analogs were studied. using cluster analysis method semax was found to be more labile among various synthesized analogous (met-gln -his-phe-pro-gly-pro; gly-glu-his-phe-pro-gly-pro; lys-glu-his-phe-pro-gly-pro; glu-his-phe-pro-gly-pro; his-phe-pro-gly-pro). because of collective degree of freedom it has one more stable configuration that is unreachable in analogs. singularities of semax and analogous were studied using -d, -d poincare maps, auto and crosscorrelation functions of special type in terms of topological structure of energy hypersurface. this work was supported by rfbr (pr. - - ), russian ministry of education and science, moscow government and crdf. rhodopsin (rd) is the only representative of g-protein coupled receptors (gpcrs) whose structure has been described with high resolution. thus, it has become the structural prototype for other gpcr. these receptors are involved in transduction of various signals into the cell and actions of many hormones and neurotransmitters. about % of all drugs act through gpcr. growing evidence that rd and related gpcrs form functional dimers/oligomers, followed by direct proof (using atomic force microscopy -afm) that in the retina rd associates into a paracristalline network of rows of dimers, need models of rd-transducin (g t -heterotrimeric protein) complex that would envision an optimal rd dimer/oligomer amenable to satisfy all well documented interactions with gt. current model includes tetramer built of two activated (metaii) and two inactive rd molecules, ligands stabilising metaii: gtα(ile -phe ) and gtγ(asp -cys )farnezy, lipid bilayer built of pc (phosphatidylocholin head groups) , ps (phosphatidyloserine) and pe (phosphatidyloetanolamine) (all three types of phospholipids contain the polyunsaturated docosahexaenoyl chain -dha) and water. experimental data concerning shape of oligomer, conformational changes in metaii, proper interactions and distances among residues have been looked upon. the poster shows results of the molecular dynamic carried in amber force field for ~ ps in the periodic box. conformational changes which took place during simulation caused proper adaptation one another monomers in tetramer and ligands to activated receptors. the human cystatin c (hcc) is a one of known domain swapping proteins. during this process, one of the hcc β-hairpins (β -l -β ) changes its conformation forming long β-strand. this conformational transition destabilizes the monomer structure and leads to domain-swapped dimer. the causative force for changing the βhairpin conformation is assumed to be the alleviation of distortions of the l -loop val amino acid residue's backbone. following the above assumption and our previous conformational studies of the hcc β-hairpin peptide we investigated the influence of the point mutations, v d, v p and v n of the val residue, on the β-hairpin peptide structure. the conformational studies by means of cd spectroscopy and molecular dynamics studies were performed. the study revealed that the hcc peptide with the wild-type sequence has the strongest tendency from all studied peptides to form a β-hairpin structure. on the basis of these results we conclude that the presence of distortions in the val residue of l -loop is unlikely to cause the d domain swapping of the human cystatin c. acknowledgments: this work was financially supported by the ministry of scientific research and information technology of poland under grant t a . temporin a (ta) (flpligrvlsgil-nh ) and temporin l (tl) (fvqwfskflgril-nh ) are small, basic, hydrophobic, linear antimicrobial peptides amide found in the skin of the european red frog, rana temporaria. these peptides have variable antibiotic activities against a broad spectrum of microorganisms, including clinically important methicillin-sensitive and resistant staphylococcus aureus as well as vancomycin-resistant enterococcus faecium strains. to gain further insight into the mechanism of action of these small antimicrobial peptides, we have investigated their conformational behaviour in different environmental conditions. more specifically, we deeply investigated by solution nmr spectroscopy in water and water/dmso ( : ) solutions as isotropic solutions and mm aqueous solution of dpc (dodecylphosphocholine) was used as membrane mimetic environment. understanding the basis of the interactions of temporins with membranes could be crucial for the design and synthesis of potent antimicrobial agents. cripto is the founding member of a family of soluble and cell bound growth factors known as egf-cfc [ ] distinguished by the presence of an n-terminal signal peptide, two distinct cysteine-rich domains (crd) and a c-terminal hydrophobic region involved in cell surface attachment by a post-translational gpi modification. the characteristic crds, known as egf-like and cfc domains (from the first members cripto, frl and cryptic), both span about residues with disulfide bridges [ ] each, which, presumably, beside a possible functional modularity, confer them also a structural independence. in this work we have focused our attention on the cfc domain of mouse cripto. the domain has been produced by ssps, along with variants bearing mutation on h and w , that have been described as crucial for alk receptor recognition. the two variants have been purified and refolded, achieving the correct disulfide bridges, and then comparatively analyzed by cd spectroscopy under different ph conditions; thus obtaining experimental insights on the structural arrangements of this new class of protein domains. furthermore, the binding properties of wild type and mutants cfc domains to alk receptor have been determined by using an elisa-based assay. our results demonstrated that the cfc domain alone can directly bind alk in the absence of additional ligands and, furthermore, confirmed a role of h /w in cripto/alk interaction. there is considerable interest in the pharmacology of the two cholecystokinin (cck) receptors ccka-r (or cck- ) and cckb-r (or cck- ) that mediate the biological action of the cck hormone. they are membrane receptors belonging to the superfamily of g-protein coupled receptors (gcpr) and are predominantly located in the gastrointestinal tract and in the central nervous system, respectively. a library of cyclic peptide analogues derived from the octapeptide c-terminus sequence of the human cholecystokinin hormone [cck( - ), or cck ] has been designed, synthesised and characterised. the peptide analogues have been rationally designed to specifically interact with the cck type b receptor (cckb-r) on the basis of the structure [ ] of the bimolecular complex between cck and the third extracellular loop of cckb-r [namely, cckb-r( - )]. the new ligands showed binding affinities generally lower than that of parent cck . anyway, structure activity relationship data underline that preservation of the trp -met motif is essential, and that the phe side chain and a carboxylic group close to the c-terminal end must both be present. the nmr conformational study in dpc micelles of the compound endowed with maximal binding affinity (cyclo-b , ic = m) shows that this compound presents the turn-like conformation, centred at the trp -met segment, as planned by rational design, and that such conformation is stabilised both by the cyclic constrain and interaction with the micelle. cripto is the founding member of a family of extracellular growth factors called egf-cfc found in mouse, human, chicken, xenopous and zebrafish [ ] . these proteins are characterized by the presence of an n-terminal signal peptide, a c-terminal hydrophobic region and two highly conserved cysteine-rich domains, the egf-like (epidermal growth factor) and the cfc (cripto/frl /cryptic). cripto is strictly required in the early embryonic development and contributes to deregulated growth of cancer cells in adults, since it is highly over-expressed in many solid carcinomas. it has been proposed that each single domain of cripto could bind different protein partners, playing different functional roles [ ] . on this grounds, investigation of the single domains d-structures can have also strong functional implications. we present here an extensive conformational analysis of the mouse cfc domain ( - sequence) and of the w a mutant based on nmr data. sequences have been synthesized by spps and refolded reconstituting the correct disulfide bridges [ ] . the molecular models have been built by computational methods using the nmr data collected under both acidic (ph ) and nearly physiological (ph ) conditions. both domains show a globally extended folding with three strands linked by the three disulfide bridges and two connecting loops, in which h and w , key residues in receptor binding, are exposed to the solvent urantide, a selective antagonist. thus, we carried out a study aiming at the characterization of conformational arrangement and affinity properties of ut extracellular segments.we measured by surface plasmon resonance (spr) technology the binding affinities of the three ligands, u-ii, urp and urantide towards the three extracellular loops of ut. furthermore, the secondary structures of the synthetic receptor fragments in presence of dodecylphosphocholine micelles and interaction with ut ligands were analysed using nmr spectroscopy. spr data showed that the ec loop ii was able to recognize the ligands u-ii, urp and urantide with similar affinities while none of these two ligands were able to interact with the extracellular loop i. furthermore, the absence of binding of urantide, a peptide antagonist, suggested strongly that loop iii would be involved in the signal transduction process and implies that u-ii and urp, but not urantide, would bind to ut according to a common pattern. moreover, the results indicate that potent ut antagonists could be designed by producing highaffinity ligand targeting the extracellular loop ii. also, the spr and nmr studies revealed that the synthetic structural ut domains contained some of the conformational and chemical features essential for the binding of hu-ii, urp and urantide to hut. synthetic cysteine-rich replicates of naturally occurring peptides such as hormones, neurotransmitters, enzyme inhibitors, defensins and toxins often can be oxidatively folded in high yields to their native structure. the presence of identical cysteine patterns in the sequence were found to lead to identical disulfide connectivities and homologous spatial structures despite significant variability in the non-cysteine positions. therefore, it is generally accepted to attribute the disulfide connectivities based on the homology of their cysteine pattern. minicollagen- from the nematocysts of hydra is a trimeric protein containing n-and c-terminal cysteine-rich domains involved in the assembly of an intermolecular disulfide network. examination of three-dimensional structures of peptides corresponding to these folded domains by nmr spectroscopy revealed a remarkable exception from the general admitted rule [ ] . despite an identical cysteine pattern, they form different disulfide bridges and exhibit distinctly different folds. additionally, comparative analysis of the oxidative folding revealed for the c-terminal domain a fast and highly cooperative formation of a single disulfide isomer, the n-terminal domain proceeding mainly via an intermediate that results from the fast quasi-stochastic disulfide formation according to the proximity rule. to our knowledge, this is the first case where two short peptides with identical cysteine pattern fold uniquely and with high yields into defined, but differing, structures. therefore, these cysteine-rich domains may well represent ideal targets for structure calculations to learn more about the elementary information encoded in such primordial molecules. the conformational change of the cellular prion protein, prpc, to its virulent "scrapie" form, prpsc, is believed to be responsible for prion infectivity. and several studies suggest that the prion disulfide bond is important for the stability, structure, and propagation of prion oligomers. to test this hypothesis, we selected two conserved peptides flanking the disulfide bond in the sheep prion protein, and measured the secondary structure of these peptides with circular dichroism, hydrogen/deuterium exchange, and molecular dynamics simulations. our preliminary data suggests that the two peptides do not adopt stable secondary structure, native or otherwise. thus, the folding intermediate of a prion protein seems unlikely to comprise local structure around the disulfide bond. the conformationally labile cα-tetrasubstituted α-amino acid residue bip possesses non isolable (r) and (s) atropoisomers. we have previously reported that in the linear dipeptides boc-bip-α-xaa*-ome with α-xaa* = ala, val, leu, phe, (αme)val and (αme)leu residues at the c-terminal position of bip, the onset of an equilibrium between diastereomeric conformers with unequal populations could be observed by cd and h nmr. the phenomenon of induced circular dichroism (icd) represents the basis for the "bip method", an easy and fast configurational assignment for chiral α-amino acids. in search for an extension of the bip method, we investigated the boc-bip-β-xaa*-ome dipeptide series with β-xaa* = β -hala, β -hval, β -hleu, β -hpro, β -hphe, or the cyclic β , -amino acids ( s, s)/( r, r)-achc and ( s, s)/( r, r)-acpc. low-temperature ( k) h nmr spectra in cd od revealed the presence of two conformers. significant d.r. (diastereomeric ratio) values were observed for all combinations of bip with both β -and cyclic β , -amino acids. cd analysis in meoh solution of the boc-bip-β-xaa*-ome dipeptides allowed us to conclude that the cd resulting from the induced axial chirality in the biphenyl core of the bip residue gives clear information on the β-xaa* configuration for both β -and cyclic β , -amino acids (except the aromatic β -hphe), with a p torsion of the biphenyl axial bond of bip being preferentially induced by (l)-β -xaa* as well as cyclic ( s, s)-β , -xaa* c-terminal residues. we have recently reported that the induced circular dichroism (icd) of the biphenyl core of boc-bip-xaa*-ome dipeptides based on the conformationally labile cαtetrasubstituted α-amino acid residue bip could allow an easy and fast configurational assignment for both α-and β-xaa* amino acid residues. in search for other biphenyl/xaa* architectures in which a transfer of central to axial chirality could result in a potentially useful icd, we considered n-substituted , -dihydro- hdibenz[c,e]azepine (daz) derivatives from α-and β-amino acids as interesting candidates. in the present communication, we report the syntheses, and the h nmr and cd analyses of a series of (daz)xaa*-ome amino esters derived from α-, β -, and cyclic β , -xaa* residues, namely d- β-peptide molecules possess interesting conformational characteristics and biological properties. they may represent a new class of rigid foldamers potentially useful as templates or spacers. d-structures of β-peptides have been experimentally investigated using x-ray diffraction and various spectroscopic techniques, but they have never been doubly spin labelled and studied by epr. a terminally protected β-hexapeptide, based on trans-( r, s)-β-toac and trans-( s, s)-achc, synthesized using classical solution methods, was found by ft-ir absorption and cd techniques to adopt the - -helical conformation. a set of four, terminally blocked, hexapeptide sequences, each characterized by four strongly helicogenic aib residues and all combinations of the two isomeric ile/allo-ile residues at positions and was synthesized by solution methods and fully characterized. a detailed solution (by ft-ir absorption, nmr, and cd) and solid (crystalline)-state (by cd and x-ray diffraction) conformational investigation allowed us to validate our assumption that all four peptides are folded in well developed - -helical structures. however, the most relevant conformational conclusion extracted from this d-analysis is that the handedness of the - -helical structures formed does not seem to be sensitive to the configurational change at the β-carbon atom of the constituent ile versus the diastereomeric allo-ile residues (in other words, the dominant control on this important structural parameter appears to be exerted by the chirality of the amino acid α-carbon atom). these results complement published findings on the diverging relative stabilities of the intermolecularly h-bonded β-sheet structures generated by ile versus allo-ile homo-oligopeptides. taken together, these data represent an experimental proof for the intuitive view that potentially different conformational properties are magnified in a strongly self-aggregated homo-peptide system (as compared to weakly self-aggregated, helical, host-guest peptides such as those investigated in this work). in a first approach to β-sandwich proteins the hydrophobic core between two symmetrical sheets each with four antiparallel β-strands was computationally designed by packing of amino acid side chain conformations (rotamers) in an initially given backbone structure. the proteins were synthesized by coupling four peptides with β-hairpin structure to a cyclic decapeptide template (tasp). an aggregation observed by equilibrium ultracentrifugation with the first designed proteins was decreased to a dimer by increasing the surface charge in two further variants of this protein from - to + and + . replacement of l-pro by d-pro in the loops and the template proved to stabilize the β-structure. these results led us to an improved design of an asymmetric core with algorithms for selection of proteins with a minimal number of atom clashes and cavities in the core, and a maximum number of hydrogen bonds after energy minimization. this protein termed beta-mop (modular organized protein) was synthesized in amounts to allow a characterization by cd, ftir, tryptophan fluorescence during reversible unfolding, and by high resolution nmr. nmr measurements of diffusion indicate a dimeric structure. the β-structure is stable up to °c ( k) as determined by d h nmr showing sharp resonance lines. the d h, h dqf-cosy spectrum at mhz shows a typical βsheet distribution extending well into the characteristic regions > . ppm (for amide protons) and > . ppm (for hα signals). all data indicate a well folded protein with β-structure. a.s. galanis , z. spyranti , n. tsami , g.a. spyroulias , e. manessi-zoupa , g. pairas , i.p. gerothanassis , p. cordopatis angiotensin-i converting enzyme (ace) belongs to the m family of the ma clan of zinc metallopeptidases and can act either as a dipeptidyl carboxypeptidase, or as an endopeptidase. among the ace peptide substrates, the most distinguished are angiotensin i (angi) and bradykinin (bk) due to their role in blood pressure regulation. despite the fact that biological data strongly suggest that the two active sites exhibit different selectivity and activity towards physiological and exogeneous substrates none experimental evidence for the interaction of angi and bk with ace catalytic sites, is available so far. a dual approach for studying the structure and physicochemical determinants of ace-angi/bk interaction has been performed. the first involves the application of molecular dynamics simulations (presented elsewhere in this book) and the second is making use of the solid-phase synthesized - aa ace catalytic site maquettes (csm) bearing the native sequence and the application of the nmr spectroscopy, and presented herein. therefore, high-resolution multinuclear nmr spectroscopy was applied to analyze the conformational features of ace substrates angi and bk in dmso or aqueous mixtures. then titration experiments were conducted and ace csms were titrated by angi/bk peptides, while monitored by nmr. d h- h tocsy and noesy experiments were used in order to map the interaction site of both substrates and csm through chemical shift perturbation and comparison of noe signal differentiation. competitive binding studies were also carried out through titration studies of csm-angi/bk and known ace inhibitors. a. carotenuto , p. grieco , l. auriemma , e. novellino , v.j. hruby the melanocortine receptors are involved in many physiological functions, including pigmentation, sexual function, feeding behavior, and energy homeostasis, making them potential targets to treat obesity, sexual dysfunction, etc. understanding the conformational basis of the receptor-ligand interactions is crucial for the design of potent and selective ligands for these receptors. the conformational preferences of the cyclic melanocortin agonists and antagonists mtii, shu , [pro ]mtii, and pg (table ) when two chromophores are chirally oriented and close enough to one another in space, their excited states couple and become non-degenerate. this phenomenon, termed exciton coupling, produces a typical bisignate cd curve. the intensity of the cd couplet is dependent on the molar extinction coefficient and the distance between the interacting chromophoric moieties, while the sign is governed by the angle between the effective electron transition moments. in particular, exciton coupling over a long distance can be observed only with strongly absorbing chromophores, e. g. porphyrin derivatives, characterized by their extremely intense and sharp soret band near nm. in this work we examined by the exciton coupled cd method the combined distance and angular dependencies, generated by the seven conformationally restricted β-turn and - -helical spacer peptides -l-ala-[l-(αme)val]n-(n = - ) on a system formed by two intramolecularly interacting -carbamido- , , , -tetraphenylporphyrin chromophores. these porphyrin derivatives are confirmed to be excellent reporter groups. we find that not only the centerto-center separation (from to Å) between the two chromophores, but the orientation (roughly parallel or perpendicular) between the directions of their effective transition moments as well, are responsible for the onset or even for the modulation of the intensity of the exciton coupling phenomenon. in particular, the porphyrin…porphyrin interaction is still clearly detectable over the long distance of ca. Å when the two chromophores are about perpendicularly oriented. a. hetényi , g.k. tóth , c. somlai , t.a. martinek , f. fülöp β-peptides are probably the most thoroughly investigated peptidomimetic oligomers. to extend the field of β-peptides towards the construction of possible new secondary structures, the replacement of the cα and cβ atoms of the β-amino acid with heteroatoms could be an attractive modification, for example cβ-atom of β-peptides by an nr moiety, leading to hydrazine peptides. in the literature, there are only a few studies [ ] [ ] [ ] about hydrazine peptides, and hydrazine peptides with cyclic side-chain have not been studied yet. in order to determine the secondary structure preference of -amino-pyrrolidine- s-carboxylic acid homo-oligomers (figure ), their potential energy hypersurface were probed at the ab initio b lyp/ - g** level. the calculations predicted the -strand to be the most stable structure. the hydrazino-peptides in question were synthetized on solid support, and their structures were characterized by nmr and cd methods. the results were found to be in good accordance with the -strand structure. cathepsin c [ec . . . ]( ), which belongs to family of cysteine proteases, catalyzes hydrolysis of n-terminal peptide, preferential glyphe. this enzyme may play a part in chronic airway diseases ( ) . also increaser level of enzyme was found in case of cancer, rheumatism and muscle's distrophy ( ) ( ) ( ) . for this reason we have undertook investigations of peptides containing two dehydroamino acid residues, which could act as alkylating inhibitors of this enzyme. to define structure and conformation of investigated peptides we were used different methods of nmr spectroscopy, including standard d experiments, protonproton correlations, proton-carbon correlations, and d noe experiments. to complete structural research computational chemistry methods had been used. in order to predict the biological activity of investigated peptides, the simulation of docking process of these peptides to enzyme active site had been made and after that correlated with results of enzymatic test.. the obtained results suggest, that investigated peptides containing two ∆phe residues (z and e isomers respectively) in solution have bent conformation, which is stabilized by intermolecular hydrogen bonds. these results are confirmed by the results of theoretical calculations. also simulation of docking process have showed two possible peptide's orientation in active site of cathepsin c and allowed the rational interpretation of biological test's results. turns are important elements of secondary structure in peptides and proteins. different types of turns are distinguished according to the number of residues involved. the most abundant is the β-turn, which involves four consecutive amino acids with the co at position i hydrogen-bonded to the i+ nh. the γ-turn is centred at a single residue and is generally stabilized by a hydrogen bond between the i co and the i+ nh. model dipeptides rco-l-pro-xaa-nhr' are the smallest systems able to adopt the β-turn conformation, which is favoured by the presence of proline at i+ . a peptide of this series, incorporating a cyclopropane amino acid (xaa), has been shown to accommodate two consecutive γ-turns in the solid state [ ] , instead of the expected β-turn conformation. the double γ-turn encountered is unique among crystalline short linear peptides. in fact, the γ-turn is observed almost exclusively in low-polarity solvents, and only a few oligopeptides of cyclic structure exhibit a γ-turn in the crystal. this is the first time that the strong tendency of pro-xaa dipeptides to adopt a β-turn in the solid state has been switched to the γ-turn. theoretical calculations [ ] also show the high preference of this cyclopropane amino acid for the γ-turn conformation. [ oxidative stress plays an important part in the development of cardiovascular disease (cvd). haptoglobin is a hemoglobin-binding protein that has a major role in providing protection against heme-driven oxidative stress. there are two common alleles for haptoglobin ( and ), and the three phenotypes, haptoglobin - , haptoglobin - , and haptoglobin - , differ in their ability to function as antioxidants. we determined whether there was a relation between the haptoglobin phenotype and the development of coronary artery diseases. haptoglobin (hp) phenotypes were determined in iranian patients with coronary artery diseases. we performed haptoglobin (hp) genotyping by polymerase chain reaction (pcr) using allele-specific primer-pairs. in multivariate analyses controlling for conventional cvd risk factors, haptoglobin phenotype was a highly statistically significant, independent predictor of cvd. the odds ratio of having cvd in patients with the haptoglobin - phenotype was . times greater than in patients with the haptoglobin - phenotype. an intermediate risk of cvd was associated with the haptoglobin - phenotype. these results suggest that haptoglobin phenotype is an important risk factor in determining susceptibility to cardiovascular disease which may be mediated by the decreased antioxidant and antiinflammatory actions of the haptoglobin allelic protein product. the special feature of proteins involved in alzheimer's or prion diseases is their ability to adopt at least two different stable conformations. the conformational transition that shifts the equilibrium from the functional to the pathological isoform can happen sporadically. it can also be triggered by mutations in the primary structure, changes of different environmental conditions, or the action of chaperones. elucidation of the molecular interactions that occur during the transformation from α-helix to β-sheet and the consecutive formation of amyloids on a molecular level is still a challenge. therefore, the development of small peptide models that can serve as tools for such studies is of paramount importance. we succeeded in generating model peptides that, without changes in their primary structure, predictably react on changes of diverse environmental parameters by adopting different defined secondary structures. these de novo designed peptides strictly follow the characteristic heptad repeat of the α-helical coiled coil structural motif. furthermore, domains that favour β-sheet formation and aggregation can be generated. alternatively, those peptides can be equipped with functionalities that allow either the binding of metal ions or the interaction with membranes. as proof of our concept we showed that the resulting secondary structure of such peptides will strongly depend on environmental parameters. thus, this system allows to systematically study the interplay between peptide / protein primary structure and environmental factors for peptide and protein folding on a molecular level. the pathogenesis of alzheimer's disease (ad) is strongly linked to neurotoxic assemblies of the amyloid β protein (aβ). aβ is a soluble component of human plasma which by an unknown mechanism becomes aggregated and neurotoxic. some genetic mutations within the aβ sequence cause very early onset of ad-like diseases, probably by facilitation of aβ assembly into neurotoxic species. recently, it was found that not amyloid fibrils, but smaller aβ assemblies initiate a pathogenic cascade resulting in ad. therefore, preventing the folding of nascent aβ monomers would have therapeutic benefit. to uncover details of structural changes accompanying the aggregation process, especially its initial stage, we have decided to study the aβ( - ) fragment and its mutation-related variants. our recent studies on this aβ fragment using the cd method and the aggregation test have proved it a good model for structural studies. the obtained results confirmed that the aggregation process follows the scheme with an α-helical intermediate and pointed out differences in the behaviour of aβ variants. to further confirm the scheme of the structural changes accompanying aggregation we have applied ftir spectroscopy and analysed aggregation-induced changes of the amide i band which is directly related to peptide backbone conformations. the ftir spectra analysis indicate that water addition provoked conformational changes are strongly dependent on the aβ( - ) variant and in some cases the formation of α-helical intermediate seems to be preceded by helix formation. to verify this hypothesis the temperature dependent atr ftir spectra will be analysed. supported by ug bw grant. amino acid octarepeats present in the prion protein bind to cu + and are considered as a potential periplasmic copper ion transporters. this octarepeat is located in the unstructured region of the prion protein, which is supposedly not intricately involved in prion aggregation. our group is involved in exploring the function of octarepeats with a special emphasis on their possible role in amyloid fibril formation and aggregation. in this context, we have prepared truncated peptide constructs derived from the prion protein octarepeat phgggwgq and have reported their fibrillation activity. we will present aggregative behavior of a truncated bis-pentapeptide, containing gggwg segment, when tethered with a flexible linker diaminobutane. fibrillar architectures were observed by this bis conjugate after incubation in water which was probed by different microscopic and steady state fluorescence techniques. further investigations with ki revealed a homogeneous environment of the two tryptophan moieties in the conjugate. in the absence of other side-chains, it is likely that fibril formation involves hydrophobic interaction between tryptophan indole moieties and main chain backbone interactions. interestingly, a facilitator role for aromatic-glycine motifs for amyloid aggregation has been proposed based on bioinformatics search of the swiss-prot and trembl databases. collagens are known to fold into a highly ordered rode-shaped triple helix with stretches of lower and higher suprastructural stability and even disruptions to modulate recognition by other proteins that interact with the extracellular matrix [ ] . to increase understanding of folding and stability of the collagen triple helix, we have adressed the design of photocontrolled collagenous peptides. our aim was to crosslink two side chains of the repetitive (xaa-yaa-gly) sequence motifs of collagen model compounds via an azobenzene chromophore in analogy to our previous studies on photomodulation of the conformational preferences of cyclic peptides and more recently of hairpin-peptide model systems [ ] . molecular modeling experiments suggested appropriate sequence positions that could result in triple-helical peptides with conformational stabilities that can be modulated by cis/trans isomerization of the azobenzene moieties. as light switchable crosslinker azobenzene- , '-n-( -iodo- -butynenyl)carboxyamide was synthesized for reaction with two ( s)-mercaptoproline residues placed in suitable xaa and yaa positions, respectively. by this approach a fully folded triple helix was obtained upon thermal relaxation, and unfolding was induced by irradiation at nm. the favorable optical properties of the azobenzene derivative together with the regular suprastructure resulted in a valuable model system that allows for ultrafast time-resolved studies of collagen folding and unfolding. amyloid formation is connected with alzheimer's disease, parkinson's disease, finnish familial amyloidosis. after protein misfolding short peptide sequences act as "hot spots" providing the driving force for protein aggregation in amyloid fibrils. we have identified one of these sequence stretches in the abl-sh domain of drosophila (dlsfmkge) whereas the human homologous region (dlsfkkge) is predicted to be less amyloidogenic. the possible reason for the difference of amyloid formation propensities of the two peptides was investigated by molecular dynamics (md) of β-sheet structures. the antiparallel alanine β-sheets consisting of two and ten strands were constructed, minimized, and mutated to the sequences dlsfmkge and dlsfkkge. all four systems: ) dlsfmkge -two strands, ) dlsfkkge -two strands, ) dlsfmkge -ten strands, ) dlsfkkge -ten strands, were surrounded by Å layer of water molecules over the solute and subjected to md, amber . force field, ntp protocol. the md runs were started at the temperature of k and the temperature was elevated stepwise by degrees till k. the results show considerably higher hydrogen bond percentage for dlsfmkge than that one for dlsfkkge during the course of the simulation, thus suggesting that dlsfmkge is a potential fibril-maker, but dlsfkkge is not. two strand β-sheet systems were stable until k. the ten strand β-sheets are more stable. angiotensin-i converting enzyme (ace) has a critical role in cardiovascular function, which consists of cleaving the carboxy terminal his-leu dipeptide from angiotensin-i producing a potent vasopressor octapeptide, angiotensin-ii. there are two isoforms of ace. the somatic isoform is present in all human cells except the testis cells, where the testicular isoform is produced. the major difference between these two types is that, the somatic form has two active sites, at the n-and c-end respectively while the testicular has only one, which is almost identical to the somatic c-terminal active site. here we report the structural study of a aa peptide (previously expressed in bacteria), which corresponds to an extended domain of the human somatic n-terminal active site of ace (ala -gly ) by circular dichroism experiments, and the overexpression in bacteria, purification and structural study, using circular dichroism techniques, of a aa peptide which corresponds to an extended domain of the human somatic c-terminal active site of ace (ala -gly ). following the subclonning into an appropriate expression vector and the expression, the peptide was isolated from the inclusion bodies using chromatography techniques. the recombinant protein fragment had a molecular weight, measured by esi-ms, of kda which was in consistence with the theoretical calculation based on the dna sequence. the recombinant peptides acquired their theoretically calculated secondary structure only when , , -trifluoroethanol is present at a concentration of ~ %. in order to elucidate their structures, solutions of these peptides, labeled with n and/or c, will be studied by nmr spectroscopy. aggregation of peptides is believed to trigger various degenerative diseases but it also plays an important role for the preparation of peptide fibres and peptide-based biomaterials. it is therefore extremely important to understand the mechanisms involved in peptide aggregation and be able to control them. studies were performed on a library of amphiphilic peptides, designed around the sequence of a model antimicrobial peptide rich in leucine and lysine. the library also included peptide hybrids in which natural amino acids were replaced by non-proteinogenic omega-amino acids, such as -aminohexanoic acid and -aminononanoic acid. the aim was to estimate the aggregation and its correlation with the biological activity by using a fluorescence technique commonly employed to calculate the cmc (critical micelle concentration) of surfactants. peptides and peptide hybrids were synthesized on solid support using the fmoc polyamide protocol. they were purified by semi-preparative rp-hplc and characterized by esi-ms and analytical rp-hplc. the aggregation behaviour of the synthesized molecules was investigated in water by steady-state fluorescence measurements using pyrene as fluorescent probe. peptides were dissolved in water/pyrene or water/pyrene/ . fluorescence spectroscopy has become an extremely valuable technique for conformational studies of biopolymers, the development of peptide-based chemosensors, and biochemical research in general. in this connection, synthetic amino acids as fluorescent probes to be incorporated into a peptide chain may exhibit significant advantages over the related protein (trp and tyr) residues in terms of potentially different and ameliorated properties. we recently designed and prepared a new fluorescent amino acid, antaib, based on a planar anthracene core and belonging to the class of achiral, ciα↔ciα cyclized, cα-tetrasubstituted α-amino acids (strong β-turn and helix inducers in peptides). peptides based on antaib combined with (l)-ala residues were synthesized and subjected to a conformational analysis. more specifically, the protected derivatives boc-antaib-oet (oh) and fmoc-antaib-otbu (oh) were prepared in seven steps from , , conformational transitions in peptides and proteins emerge to play the major role in the genesis and evolution of prion related diseases and alzheimer's disease (ad). in this context, conditions influencing this transition and the following aggregation process are of paramount interest. peptides and proteins that are involved in aggregation processes contain potential metal binding sites. the concentration of metal ions in the brain tissue is naturally high and zn in the mm range has been found in ad amyloid plaques. thus, it is widely accepted that metal complexation is one of the key incidents that lead to conformational transitions and aggregation. we present here a coiled coil based model peptide system with an intrinsic amyloid forming tendency which can be used to study the impact of different metal ions on secondary structure and aggregation. metal complexing histidine residues were incorporated to create potential binding sites which, depending on their position and the nature of the metal ion, dictate folding and aggregation. the time dependent conformational transition was monitored by cd-spectroscopy. aggregates were characterized by cryo tem. high resolution fticr-ms experiments revealed information on the stoichiometry of the peptide-metal complexes. in the absence of metal ions the presented peptides formed amyloids in a time range of weeks. depending on the his positions and milieu conditions, the nature of the metal ion determines folding and aggregate morphology. furthermore, metal binding was shown to inhibit the amyloid formation. a challenge to our understanding of protein folding is the design of a protein from first principle, i.e. starting from geometric restraints and applying properties of amino acids expected to be essential for folding to a defined structure. we developed a program to calculate the backbone coordinates of antiparallel strands to match the surface of an elliptical cylinder. various parameters like the number and shearing of the strands and the ellipticity of the structure can be varied. the relative orientation of the β-strands and the geometrical features of the hydrogen-bonds were derived from statistical analyzes of natural β-sheet structures. iterative cycles of core-packing with amino acid rotamers, molecular dynamics simulation and energy minimization with the charmm forcefield are used to include backbone movements and to minimize the risk of trapping an energetically unfavorable structure. the quality of residue packing is assessed with the help of criteria which proved to be successful in our design of β-sandwiches. at the protein surface, a network of salt-bridges with an excess of positive charges has been designed to increase the stability and the solubility of the protein. the final sequence is synthesized by standard solid phase fmoc-chemistry. insights gained from the analysis of the synthesized structure with ftir and cd spectrometry should help us to refine the parameters for subsequent designs. with this strategy, we hope to contribute to a better understanding of protein folding. the immunoglobulin binding protein g ( igd) from streptococcus species consists of amino acids residues, which form two antiparalell-packed beta-hairpins and an alpha-helix in the middle of the sequence packed to the beta-sheet. the second hairpin was found to be stable in isolation. this fragment is therefore likely to be the first folding initiation site of the protein which could provide an adequate nucleation center on which the rest of the polypeptide chain would find a favorable environment to fold. thus, among the two beta-hairpins, the - fragment of igd corresponding to the c-terminal beta-hairpin was synthesized. in our studies, we investigated different environmental and temperature conditions for formation of the - beta-hairpin structure. its structure was examined by means of cd spectroscopy in water, buffer solutions (ph = to ) and in aqueous solutions of trifluoroethanol. additionally, its structure was investigated in the solid state by ftir spectroscopy. the cd studies revealed that the - fragment of igd in water forms mainly a statistical-coil structure, whereas the ftir technique shows formation of a regular beta-sheet structure. nmr spectroscopy and calorimetric measurements were carried out at various temperatures. our studies show that the - fragment at low temperature exists in an equilibrium between two conformations -a regular beta-hairpin and a statistical-coil. although increasing temperature resulted in shifting the equilibrium in the direction of the statistical-coil structure, the overall beta-hairpin shape of the - fragment was maintained. prion diseases are characterized by the conversion of the physiological cellular form of the prion protein (prpc) into an insoluble, protease-resistant abnormal scrapie form (prpsc) with an highly beta-sheet content [ ] . the analyses of intrinsic structural propensities of the prp c-terminal domain showed an high conformational flexibility for the αhelix fragment which indicates that this region may be particularly important in the prpc→prpsc transition [ ] . therefore conformation-based approaches focalized on helix region appear to be the most promising for the study of prion protein misfolding. recent studies on tetracycline properties showed that this molecule binds and disrupts prp peptide aggregates and inactivates the pathogenic forms of prp [ , ] . a fluorometric titration of the fluoresceinated peptide corresponding to prion protein helix- with tetracycline has been carried out to determine the value of the apparent dissociation constant of this interaction (estimated to be ± nm). remarkably, the fluoresceinated peptide exhibits in water a canonical α-helical cd spectrum, that is maintained even in presence of tetracycline. accordingly, docking calculations and molecular dynamics simulations suggest that tetracycline interacts preferentially with the c-terminal end (residues - ) of helix- with a significant involvement of the treonine rich region. in the last decades, a series of discoveries have shed light on the role played by the carbohydrate moiety in glycoproteins. it has been shown that covalently linked sugar moieties influence peptide/protein properties such as hydrophobicity, conformation, biostability and bioactivity. the design of carbohydrate-peptide analogs with increased, retained or modified biological activity requires an understanding of their conformational preferences both in solution and in the receptor-bound state. in our recent work we have created two classes of well-structured linear and cyclic carbohydrate-modified analogs of opioid peptides, leu-enkephalin and leuenkephalin amide. the first class represents a group of compounds in which the linear peptide is alkylated at the n-terminal position by -deoxy-d-fructose unit, while its cyclic analog possesses an ester bond between the c- hydroxy group of the sugar moiety and the c-terminal carboxy group of peptide, -deoxy-dfructofuranose acting as a bridge between the leu-enkephalin terminal parts. the rigid -membered imidazolidinone ring is characteristic for the second class of compounds. in these adducts an imidazolidinone moiety connects the acyclic sugar residue with the linear peptide chain. in the corresponding bicyclic imidazolidinone analogs -membered ring is formed through an ester bond between the primary hydroxyl group of the d-gluco-pentitolyl residue and the cterminus of peptide. this work reports the comparative cd and ftir spectroscopic properties of the prepared glycopeptides in comparison with data on the non-modified flexible parent peptides performed in different solvents in order to expose the structural and conformational differences caused by a keto-sugar, rigid membered imidazolidinone ring and/or cyclization. the α-helical coiled coil structural motif consists of two to five α-helices which are wrapped around each other with a slight superhelical twist. the simplicity and regularity of this motif have made it an attractive system to study the role of complementary interactions for protein folding. here we present a systematic study showing that intermolecular electrostatic interactions between positions e and g of the helices are in competition with the intramolecular interactions between positions e/b and g/c. those competitive interactions affect folding and stability of the motif which were monitored by temperature dependent cd-spectroscopy. incorporation of oppositely charged amino acids in positions e/b and g/c reduced considerably electrostatic repulsion between equally charged amino acids in positions e and g. in addition coiled coil stability can be increased by the alkyl part of the amino acid side chains in positions e and g. studies with natural and unnatural amino acids showed that the longer this alkyl part the better is the hydrophobic core protected from solvent. therefore the repulsion of equally charged amino acids in positions e and g can be overruled by involving them either into attractive intrahelical electrostatic interactions or into hydrophobic core formation. human cystatin c (hcc) is a amino acid residues protein that reversibly inhibits papain-like cysteine proteases. this inhibitor belongs to the amyloidogenic proteins shown to oligomerize through d domain swapping mechanism. the crystal structure of hcc reveals the way the protein refolds to produce symmetric dimer while retaining the secondary structure of the monomer. the monomeric form of hcc consists of a core with a five-stranded antiparallel β-sheet wrapped around a central α-helix. the hcc dimerization is preceded by an opening movement of l loop from β -l -β hairpin and separation of the β -helix-β fragment from the remaining part of the molecule. the amino acid sequence of β -helix region suggests additional possible partial unfolding in the n-terminal part of helix. in order to investigate the structural stability of β -helix region the peptide corresponding to the helix and its n-terminal truncated analogs was synthesized along with the peptide analogs of helix containing point mutations that could stabilize helical structure of the n-terminus. the peptides were synthesized by the solid-phase method using fmoc/tbu tactics. purified products were identified by maldi-tof. the secondary structure content was calculated from the cd spectra using selcon . the random coil was the predominant structure of the peptide corresponding to the α-helical fragment of hcc and its n-truncated analogs. however, an increase of αhelix content was observed in some of the peptides corresponding to the helix containing point mutations. we expect that these mutations could stabilize the hcc monomer and suppress dimerization. the tumor suppressor protein p is a trarnscription factor that triggers cell-cycle arrest and apoptosis in response to genotoxic stress signals. the tetramereric structure of the p , which is essential for its activity as a transcription factor, is formed as a dimer of dimers. while the primary dimer is constructed from inter molecular formation of a two-stranded anti-parallel b-sheet and a two anti-parallel a-helix bundle, the secondary dimer is stabilized through interactions between residues on the surface of the primary dimer. from various substitution experiments on p , it has been shown that hydrophobicity of phe is critical for the tetramer formation of p . also we have substituted three phenyl groups of p with cyclohexylalanine (cha) and showed that phe cha is dramatically stabilized against temperature, chemical denaturant, and organic solvent by cd measurements. here, to clarify the mechanism of the stabilization of phe cha, we analyzed its three dimensional structure using x-ray crystallography. we obtained two kinds of crystals, one is a hexagonal bipyramid crystal in the space group of p<i> <sub> </sub> </i> with <i>a</i>= . Å, <i>b</i>= . Å, <i>c</i>= . Å diffracted to about . Å, and another is tabular crystal in the space group c<i> </i> with <i>a</i>= . Å, <i>b</i>= . Å, <i>c</i>= . Å diffracted to about . Å. in these crystals, the peptides formed tetramers which are very similar to those observed in the wild-type. the structure of the pocket where the side chain of cha is incorporated was defined to elucidate the hydrophobic interaction to determine the stability. helices shown in proteins, as a secondary structure, almost always form right-handed screw sense. this right-handedness is believed to result from the chiral center at the αposition of proteinogenic l-α-amino acids. among proteinogenic amino acids, l-isoleucine and l-threonine possess an additional chiral center at the side-chain β-carbon besides the α-carbon. however, no attention has be paid how the side-chain chiral centers affect the secondary structures of their peptides. recently, we have reported that sidechain chiral centers of chiral cyclic α,α-disubstituted amino acid (s,s)-ac( )c(dom) affected the helical secondary structure of its peptides, and the helical-screw direction could be controlled without a chiral center at the α-carbon atom. herein, we synthesized a chiral bicyclic α,α-disubstituted amino acid (r,r)-ab( , )c and its homopeptides, and studied the relationship between the chiral centers and the helical-screw handedness of peptides. contrary to the left-handed helices of (s,s)-ac ( ) four threefinger-toxins (tfs) have been purified from the pooled venom of golden krait (bungarus fasciatus, i.e. bf) from thailand and studied previously. these peptide toxins contain - residues and or pairs of disulfide bonds, and are rich in β-structure. we herein analyzed the tf-isoforms in bf venoms from kolkata (eastern india), hunan province (eastern china) and indonesia to study the geographic variations and structure-function relationships of the venom polypeptide family. a total of five or six tfs of low lethality were purified from each of the geographic venom samples, the n-terminal sequences and accurate masses of the peptides were determined. the cdnas encoding some of these tfs were also cloned and sequenced. full peptide sequences were deduced and match with those of the tfs purified from crude venom. intra-species variations of the venom tfs were found to be surprisingly high since sequence-identities between the majorities of orthologous toxins in different geographic samples are only - %. most of the bf proteins were not neurotoxic by electrophysiological assays using chick biventer-cervicis and mouse diaphragm neuromuscular tissues. the toxins appear to be associated with weak toxins or non-conventional snake venom tfs as analyzed by a phylogenetic tree. the reason behind their lack of neurotoxicity would be discussed. v. moussis, e. panou-pomonis, c. sakarellos, v. tsikaris peptides involved in neurodegenerative diseases can adopt at least two different stable secondary structures. amyloid-forming proteins can experience a conformational transition from the native, mostly α-helical structure, into a ß-sheet rich isoform. the latter conformation is probably present in intermediates for the formation of amyloids. the conformational change can be triggered by protein concentration or environmental changes. therefore, our aim was to generate a de novo designed peptide that contains structural elements for both, stable α-helical as well as ß-sheet formation. this model peptide can be used to elucidate the conformational changes dependent on concentration and ph. the design is based on the well studied α-helical coiled coil folding motif. the conformation and structure of the resulting aggregates were characterized by cd-spectroscopy and cryo transmission electron microscopy. as a result, three distinct secondary structures can be induced at will by adjustment of ph or peptide concentration. low concentrations at ph . yield globular particles of the unfolded peptide whereas, at the same ph but higher concentration, defined ß-sheet ribbons are formed. in contrast, at high concentrations and ph . , the peptide prefers highly ordered α-helical fibers. in conclusion, we successfully generated a model peptide that, without changes in its primary structure, predictably reacts on environmental changes by adopting different defined secondary structures. thus, this system allows to systematically study now the consequences of the interplay between peptide primary structure and environmental factors for conformation on a molecular level. cells respond simultaneously to a multitude of different signals. inside a cell signals from activated receptors are integrated by networks of enzymatic reactions and molecular interactions, leading to a spectrum of cellular responses. in order to understand the relationship between a specific cellular stimulus and a cellular response, methods are required to detect in parallel the pattern of molecular interactions. a large number of molecular interactions is mediated by protein domains, binding to linear peptide motifs. lysates of activated and resting cells were incubated on peptide microarrays carrying peptides corresponding to such binding motifs of signalling domains. binding of proteins to a spot of the array was probed by immunofluorescence. jurkat t cells were stimulated either with the phosphatase inhibitor pervanadate or with antibodies directed against cell surface receptors. upon activation of t cells, numerous changes in the pattern of molecular interactions were detected for a total of proteins and peptides. these changes were caused either (i) by masking or unmasking of a binding domain which resulted in a reduced or increased binding of a protein to the microarray or (ii) by recruitment of a protein into a complex that in turn bound to the microarray. the changes were dependent on the nature of the stimulus. the human melanocortin receptor (hmc r) was constructed to contain a flag epitope and a hexahistidine tag at the amino-terminus as well as at the carboxyl terminus to facilitate purification. stably transfected hmc r in human embryonic kidney (hek ) cell lines that expressed the receptor resulted in a kd value of . and . nm respectively in each case when the super potent agonist mtii was competed with [ i]ndp-α-msh. treatment of the tagged receptors in the hek cells with agonist resulted in down-regulation which indicates that these tagged receptors retain their biological functions. the hmc r was solubilized from cell membranes with n-dodecyl-β-d-maltoside and purified at a nickel chelating resin and a newly constructed affinity column. the purified hmc r was a glycoprotein that migrated on sds/page with a molecular mass of kda. the results from matrix-assisted laser desorption ionization-time of flight (maldi-tof) mass spectrometry was used to identify and characterize peptides derived from the hmc r following in-gel digestion with chymotrypsin. the phosphorylation sites were identified on the purified human melanocortin receptor with agonists (peptide vs small molecule) treatment. the discovery of antibiotics in the s has been one of the most revolutionary events in the history of medicine. however, during last decades, the increase of antibiotic resistance has significantly hampered the application of antibiotics. therefore, further scientific effort to find new antibiotics with novel mechanisms of action is of high importance. insects are the largest and the most diverse group of living animals on earth. they have potentially been confronting high variety of microorganisms. as a result, they have evolved powerful defense system, thus representing vast source of novel potential therapeutics. we chose larvae of fleshfly neobellieria bullata for identification and characterization of new promising molecules, peptides or proteins, which participate in immunity response against microbial infections. the hemolymph of the third-instar larvae of neobellieria bullata was used for isolation. the larvae were injected with bacterial suspension of escherichia coli or staphylococcus aureus to induce antimicrobial response. the hemolymph was separated into crude fractions, which were subdivided by rp-hplc. isolated fractions were characterized by uv-vis spectroscopy, amino-acid analysis, mass spectroscopy, -d and -d sds electrophoresis, capillary zone electrophoresis, ion-exchange hplc, tryptic digests and n-terminal sequencing. we found out significant antimicrobial activities against escherichia coli, staphylococus aureus or pseudomonas aeruginosa in several fractions. using real-time pcr, we followed and compared levels of mrna of different proteins and peptides in induced and non-induced larvae. despite the fact that many technological advances are currently involved in proteome analysis, like two-dimensional gel electrophoresis and mass spectrometry, there is still a great need for the development of novel engineered chemical probes for proteomics and interactomics. here, we describe our approach concerning the study of proteome and interactome of proteins involved in cell-matrix interactions. it relies on the use of a small synthetic inhibitor chemically modified to allow for its immobilisation to magnetic beads or affinity chromatography materials. proteins will be detected together with their native interaction partners because of nondenaturing conditions. this general procedure is applied for the enrichment of metalloproteinases, especially matrix metalloproteinases, which are potential target in tumour therapy. hydroxamic acids are known to be potent inhibitors of metalloproteinases. marimastat is a reversible inhibitor with a good potency and shows activity towards a wide range of metalloproteinases. the synthesis of new marimastat derivatives will be reported. the parent compound is modified with a linker to allow immobilisation on a solid surface. binding studies were performed using surface plasmon resonance. this approach is not only appropriate for the generation of metalloproteinase proteome subsets by affinity column or using magnetic beads, but also to enrich and isolate interaction partners of the target proteins. the present report summarizes the latest data devoted to theoretical and experimental investigation of the high temperature solid state catalytic isotope exchange reaction (hscie) that takes place in peptides and proteins by the action of deuterium and tritium [ ] . the available ms-procedures, designed to estimate the amount of protein, are aimed at derivatization at different stages of sample preparation, and as the best result, it is only possible to achieve quality comparison of the objects involved. the hscie reaction allows the production of evenly deuterium labelled proteins and peptides, and their application makes it possible to create a qualitative mass spectrometry method for protein analysis. introduction of definite amounts of these deuterium-labeled proteins into biological objects, prior to isolation, separation and trypsinolysis, will generate quantitative information concerning the composition of the proteins under study. tritium labelled proteins produced at a temperature of - oc carry the isotopic label in all the peptide fragments and completely retain their enzymatic activity. the proteins' reactivity is dependent on their three-dimensional structure. the hscie reaction has been shown to be used both in the production of tritium labelled proteins and in the investigation of spatial interactions in protein complexes. in addition, evenly deuterium and tritium labelled peptides can be used in studies of the kinetics and transformation paths of peptides in the organism's tissues. immunization of mice with type ii collagen (cii) from rat leads to development of collagen-induced arthritis (cia). susceptibility to cia is associated with the major histacompatibility class ii protein h- aq that binds the glycopeptide epitope cii - ( ) and presents it to helper t cells. [ ] to explore the interactions in the system and to stabilize towards in vivo degradation, amide bond isosteres have been introduced in its backbone.glycopeptide was virtually docked into the binding site of a comparative model of h- aq. based on the hydrogen bonding network between the peptide backbone and h- aq, the amide bond between ala -gly was chosen for isosteric replacement. to vary the geometric and hydrogen bonding properties, mimetics of the dipeptide were synthesized with the amide bond replaced by ψ[ch nh], ψ[coch ] and ψ[(e)-ch=ch], respectively. these were introduced in using solid-phase synthesis to give glycopeptidomimetics that were biologically tested for their ability to bind to the h- aq protein and for recognition by t cells. shown to be a potent neuroprotective factor in various pathophysiological models. despite its therapeutic potential in diverse neurodegenerative diseases, its short in vivo half-life limits its utility as a useful clinical agent. moreover, the development of a peptidomimetic that reproduces the pharmacological activity of pacap is unlikely since the pharmacophores are spreaded throughout the entire peptide chain. therefore, the development of pacap analogues with lower susceptibility to proteolysis represents a first step toward clinical applications. in the present study, derivatives of both pacap and pacap with particular chemical modifications were developed targeting specific peptidase sites of action. results indicate that the incorporation of an acetyl or a hexanoyl group at the n-terminus and modifications at the ser residue contributed to increase stability against dipeptidyl peptidase iv, the major enzyme involved in pacap degradation. moreover, after determination of pacap metabolites in human plasma, the amide bond between residues and was substituted by a ch nh surrogate and this derivative showed increased plasma stability. all modified peptides were tested for their ability to induce pc differentiation. the effects of pacap analogs on pc cells are mediated through the pac receptor which is the major receptor involved in the neuroprotective effects of pacap. this study exposes interesting data concerning pacap metabolism in isolated human plasma and demonstrates the possibility of increasing the metabolic stability of pacap without significantly reducing its biological activity. species of the fungal genus trichoderma are commercially used as bioprotective agents against fungal plant diseases. more than strains were collected from their natural habitats and evaluated for biocontrol properties. seven of the most active isolates exhibiting strong biological activity towards eutypa dieback and esca diesease of grapevine were classified as trichoderma brevicompactum, or shown to be closely related to that species. these strains were screened for production of peptaibiotics. the formation and synergistic action of hydrolytic enzymes and peptaibiotics were to play an important role in mycoparasitism. after background and aims: conotoxins are short, disulfide-rich neurotoxins that target various ion channels and receptors. these peptides have desirable pharmacological properties to become therapeutics for neurological disorders; several conotoxins have already reached clinical development stage. our long-term goal is to improve bioavailability, metabolic stability and pharmacokinetics of conotoxins using a variety of chemical modifications. methods: we designed and chemically synthesized conotoxin analogs containing two distinct types of backbone modifications: ( ) peptide-peptoid chimeras (conopeptoids) of alpha-conotoxin imi and ( ) peptide chimeras of mu-conotoxin kiiia containing non-peptidic "backbone spacers". results: conopeptoid-imi, containing ala replaced by n-methyl glycine potently blocked activity of nicotinic acetylcholine receptors. in mu-conotoxin kiiia, aminohexanoic acid or amino- -oxapemtanoic acid were inserted to be a part of the peptide backbone. the two oxidized analogs containing "backbone prosthesis" differed in their hydrophibicity profile, but they both potently inhibited neuronal sodium channels. conclusion: our results suggest that backbone engineering may become an effective method of producing conotoxin analogs with modified bioavailability. to increase the stability and the therapeutic efficacy of peptide sequences from myelin oligodendrocyte protein (mog) that act as multiple sclerosis (ms) antigens, we grafted them onto a framework of a particularly stable class of peptides, the cyclotides. they are a recently discovered family of cyclic plant peptides with superb intrinsic stability. the limitations of linear peptides as drugs due to their instability and poor bio-availability can be overcome by using the cyclotide scaffold as a framework for novel drug design. peptide epitopes from mog protein were incorporated onto the framework of the model cyclotide kalata b by means of boc-spps approach. after successful backbone cyclisation and oxidation of the cysteine residues, the peptides were purified to high purity with rp-hplc. nmr chemical shift analysis was used to assess whether the grafted analogues have a stable scaffold, similar to that of kalata b . a structure of a representative peptide was determined and it shows remarkable resemblance to the native scaffold of kalata b . the activity of the bioengineered peptides has been tested in vivo. a group of mice injected with one of the peptides have shown a depression in the clinical score and have not fallen ill. this is an exciting result that shows the first active bioengineered cyclotide in an animal model of disease. the structural information from nmr studies will be used in conjunction with the results from the activity studies in a feedback loop to design second-generation lead molecules. a.d. de araujo, p.f. alewood conotoxins are small, disulfide-rich peptide neurotoxins produced in the venom of marine cone snails that enable these molluscs to capture their prey. these compounds exhibit a high degree of selectivity and potency for different ion channels and their respective subtypes, making them useful tools in the investigation of the nervous system. due to the key role of ion channels in many physiological processes, conotoxins are also excellent drug lead candidates in drug discovery, with some examples currently undergoing clinical trials and one recently approved drug (prialt®). like other peptide drugs, the use of conotoxins in drug development is limited by the low oral bioavailability of these compounds due to pre-systemic enzymatic degradation and poor penetration through the intestinal mucosa. peptide analogs that mimic the native structure and incorporate rationally designed non-natural modifications in the disulfide framework or peptide backbone may exhibit increased resistance to proteolytic degradation. in biological environments, disulfide linkages are susceptible to attack by enzymes and reducing agents (such as glutathione). therefore we carried out the synthesis of redox-stable conotoxin analogs in which intrinsic disulfide bonds were replaced by thioether linkages. in this work, wehave explored two solid-phase synthetic routes in the preparation of thioether conotoxin mimetics: the first route based on peptide assembly using a tetra-orthogonally protected lanthionine building block, and the second route based on intramolecular on-bead cyclisation between cysteine and betabromoalanine residues. thyrotropin releasing hormone (trh, l-pglu-l-his-l-pro-nh ), a tripeptide synthesized in the hypothalamus, operates in the anterior pituitary to control levels of tsh (thyroid-stimulating hormone) and prolactin. the thyrotropin-releasing hormone (trh) receptor (trh-r) belongs to the rhodopsin/β-adrenergic receptor subfamily of seven transmembrane (tm)-spanning, g protein-coupled receptors (gpcrs). the two g-protein-coupled receptors for trh, trh receptor type (trh-r ) and trh receptor type (trh-r ), have been cloned from mammals and are distributed differently in the brain and peripheral tissues. the trh receptor subtype- appears to mediate the hormonal and visceral effects, whereas trh receptor subtype- has been implicated in its central stimulatory actions. identification of critical features of the trh, separation of its multiple activities through design of selective analogues and affinity labels have been elusive and unfulfilled goals for more then years. this presentation will highlight our studies on effect of the biological activity of trh with the introduction of alkyl groups of varying sizes at the n- and c- position of the centrally placed histidine residue of trh peptide. the requisite n--boc-dialkyl-l-histidines as building scaffolds have been synthesized in six steps from l-histidine methyl ester dihydrochloride and used for the synthesis of various trh analogues. ghrelin, the natural ligand for the growth hormone secretagogue receptor (ghsr- a), has received a great deal of attention due to its ability to stimulate growth hormone secretion and to control food intake. during these last years, ghrelin analogues or mimetics gained interest for their implication in food intake regulation. in the course of our studies concerning ghrelin analogs, we developed new ligands of the ghsr- a based on heterocyclic structures. interestingly, one heterocyclic family presented high affinity for the ghrelin receptor. a structure activity study was performed within this family and led to potent ghsr- a agonists and antagonists. the binding affinities were determined by displacement of radiolabelled ghrelin and the agonist or antagonist character was measured by intracellular calcium mobilisation. the first in vivo results revealed that in vitro activities and in vivo responses were not correlated. particularly, binding to ghsr- a and in vivo gh release and food intake control were not fully correlated. these results strongly suggest the presence of receptor subtypes that modulate ghrelin actions. some examples will be reported. further investigations are going on in the laboratory. background and aims: alzheimer's disease (ad) related beta-amyloid (aβ) peptides possess high propensity towards aggregation. nowadays one of the major directions of the drug-design against ad is the synthesis of putative amyloid aggregation inhibitor molecules (aai) which are able to hinder the formation of these toxic amyloid aggregates. in the present work we report the design, synthesis and investigation of putative aais derived form the peptide sequence aiigl identical to aβ( - ). methods: aβ( - ) peptide and putative aggregation inhibitors were synthesized manually using fmoc-chemistry and dcc/hobt activation. studies on both the aβ aggregation and the effect of the aais were performed with several instrumental techniques. the total amount of the aggregates was determined by thioflavine-t binding assay, while their size distribution was characterized with dynamic light scattering (dls). aggregated aβ forms were visualized with transmission electron microscopy (tem). the binding affinity of the aais to aβ fibrils was studied in saturation transfer nmr experiments, while in vitro viability assays were performed on cultured human sh-sy y neuroblastoma cells to monitor the neuroprotective effect of the amyloid aggregation inhibitors. results and conclusions: derivatives of aβ( - ) were synthesized and tested concerning their neuroprotective effect against aβ-mediated toxicity. the most promising candidates were examined with physico-chemical methods to characterize their aggregation altering ability. the pentapeptide riigl-amide proved to be the most powerful neuroprotective compound, and it was also able to alter considerably the aggregation kinetics of aβ( - ) . this molecule might serve as a lead compound in the drug-design against ad in the future. m. mattiuzzo, a. bandiera, m. benincasa, i. samborska, m. scocchi, r. gennaro antimicrobial peptides (amps) are ancient molecules of the innate immune defence system. most of them kill bacteria by lysing their membranes. the proline-rich group of amps represents an exception, as they act via a permeabilization-independent mechanism that is likely based on recognition of molecular targets/transporters. however, specific internal targets have not yet been identified for most of these amps. bac is a pro-rich amp isolated from bovine neutrophils that is capable to translocate across membranes to target hypothetical intracellular proteins. in this study, we used a molecular approach to identify putative targets for bac by selection of peptide-resistant mutants followed by identification of the genes responsible of this resistance. to this aim, an e. coli strain susceptible to the fully active fragment bac ( - ) was subjected to chemical mutagenesis and a number of peptide-resistant mutants was obtained. a pool of genomic dna from these mutants was used to construct a plasmid library that was used to transform a susceptible strain. this approach allowed the identification of different clones that provided a high level of resistance to bac . sequence analysis revealed the presence of genes originating from five different regions of the e. coli chromosome. among them, one clone contained ptrb, a gene coding for a serine peptidase broadly distributed among gram -bacteria, which could be involved in resistance by degrading the peptide. other resistanceconferring clones, which encode for membrane proteins that may be involved in peptide translocation across the memmbrane, are currently under investigation. lycotoxins are peptides from the venom of the wolf spider that were predicted to have an amphipatic alpha-helical structure and confirmed to possess significant antimicrobial and pore-forming activities. [ ] we became interested in these peptides as potential leishmanicidal agents against leishmania donovani promastigotes and leishmania pifanoi amastigotes. thus, lycotoxin i (lyi) and lycotoxin ii (lyii), [ ] and shortened analogues lyi - , lyi - , lyii - and lyii - , were synthesized as c-terminal carboxamides. short-and long-term parasite proliferation measurements showed all peptides except lyi - to be active against both promastigotes and amastigotes at the micromolar range, and suggest peptide effects on parasites to be irreversible. lyii, that showed the lower activity, became more leishmanicidal upon residue trimming, whereas the most active lyi displayed the opposite behaviour. a set of complementary techniques showed that lycotoxin peptides are membrane-disruptive to promastigotes. electron microscopy showed that two populations of promastigotes, one with intact parasites and the other extensively damaged, are formed upon addition of the peptides at their ec . all peptides were non-hemolytic for sheep erythrocytes below micromolar. tissue transglutaminase (tg ) is an enzyme that plays a key role in the pathogenesis of the celiac disease. tg is the main autoantigen recognized by the antiendomysium antibodies and, furthermore, catalyzes the deamidation of strategic glutamine to glutamic acid within the sequence of immunodominant gliadin epitopes. recently, another unexpected role for surface tg , in the innate immune response in the celiac disease, has been suggested. it follows that tg inhibitors might represent a potential attractive pharmacological alternative to the gluten-free diet that, nowadays, is the only possible therapy for celiac patients. starting from the sequence of the heptapeptide pqpqlpy, known to be a high affinity substrate of human tg , we have synthesized new analogs replacing pro with different constrained amino acids (d-pro, pip, chg, ind, deg, inp, hyp, thz)) with the aim to develop specific inhibitors of tg . actually, proline residues present in the gluten epitopes are important in determining the immunogenicity of the epitopes and the specificity of tg . herein, we describe the preliminary conformational studies of the synthesized analogs by cd and nmr spectroscopy and molecular modeling methods. the structural features of the peptides have analyzed in different environment. given the role of the domain pqpqlpy in the gliadin proteins, structural analysis on its analogs are of considerable interest. the results of our studies might be useful to clarify the role of the proline residues in the interaction of the gluten epitopes with tg and, consequently, to gain new insight in the molecular mechanism of celiac disease. carnosine and related histidine-containing dipeptides are known to react with high efficiency with the products of lipid peroxidation, namely -hydroxy-trans- , nonenal (hne) and other alpha, beta-unsaturated aldehydes, preventing their reaction with nucleophilic residues in proteins and nucleic acids. histidyl hydrazide alone or conjugated with aminoacids, long chain fatty acids, cholesterol and alpha-tocopherol synthesized in our laboratories demonstrated higher aldehydesequestering efficiency than carnosine, and were also efficient in protecting sh-sy y neuroblastoma cells and rat hippocampal neurons from hne-mediated death. the cytoprotective efficacy of these compounds suggests their potential as therapeutic agents for disorders that involve excessive membrane lipid peroxidation. lantibiotics are antibiotic natural products that are synthesised ribosomally and undergo extensive post-translational modification, resulting in multiple thioether bridges and dehydro amino acids in the mature peptide. one such lantibiotic is mersacidin, which is produced by bacillus sp. and exhibits extremely promising in vitro and in vivo efficacy versus a number of clinically relevant pathogens, most notably methicillin-resistant staphylococcus aureus (mrsa). mersacidin is believed to function by binding to the bacterial cell wall precursor lipid ii, thereby preventing its incorporation into the growing peptidoglycan network. in an attempt to better understand this mode of action and develop more active analogues, we have embarked upon its chemical derivatisation. some of these modifications resulted in an altered antibacterial spectrum, permitting some insight into tentative structure-activity relationships. the characterisation of these structurally complex compounds via a combination of multidimensional nmr and tandem mass spectrometry will also be presented. conjugation of peptides with different moieties, such as peg, lipids, carrier proteins and toll-like receptor ligands is an established approach to improve their pharmacokinetic profile for drug use and/or to enhance their immunogenicity as subunit vaccines. the development of suitable conjugation strategies for peptides of any complexity is therefore fundamental for their effective use in human therapeutic applications. here we describe our strategy to engineer a trimeric coiled coil to obtain a covalently linked, structurally stable construct endowed of extra functionality for further derivatization. we previously showed that covalent stabilization of the designed trimeric coiled coil izn , by interchain disulfide bonds, yielded an extremely potent and broad inhibitor of viral infection, (ccizn ) , [ ] . this potent inhibitory activity makes (ccizn ) not only attractive as an antiviral compound, but also as an immunogen to elicit a neutralizing antibody response [ ] . we have now developed an alternative synthetic strategy to obtain the covalently-linked izn trimer, which allows the presence in the molecule of a free thiol for subsequent chemoselective reactions. first, we showed that stable interchain thioether bonds can be effectively substituted for the disulfides. second, we devised an orthogonal cysteine protection scheme which allows formation of the thioether bonds, while leaving an extra free cysteine on demand. this thiol group can be used for conjugation of the trimeric coiled coil to adjuvant/carrier proteins or, as reported more specifically here, to a peg kda moiety. human igg is a most abundant type of immunoglobulin in serum and most of antibody drugs applied for therapy of cancer and autoimmune diseases also belong to this group of human immunoglobulin. specifically binding peptide to human igg is very useful for detection and purification as an affinity ligand of human igg. although some previous reports described such peptides, we tried here to isolate high-specific and high affinity ligands by employing random peptide-displayed t phage library. our t random peptide phage library possesses a total diversity of powered th consisting of different sequence length constrained by disulfide bond. by biopanning against human igg, we isolated several igg specific clones from our library. the peptides displayed on these phages shared some common sequences in the limited region surrounded by cys residues, which suggests they are essential for binding. these clones bound only to fc region of igg and did neither other types of human immunoglobulin nor igg of other animals. a synthetic peptide derived from a phage clone showed a sub-nanomolar of kd value in binding to human igg fc on spr analysis. these results indicate that the peptide motifs obtained here are a strong candidate of human igg-specific affinity ligand for detection and purification of igg. therefore, we are now going on constructing detection and purification system using modified and improved peptide motifs. synthesis of glp- analogues as potential agents for blood glucose control p. kanda, r.p. sharma, c.p. hodgkinson in this study, we synthesised a panel of glp- analogues stabilised against dpp-iv degradation through either selective amino acid substitutions for ala , or introduction of amide bond surrogates into the peptide backbone between ala and glu . each was made by standard fmoc or boc chemistry, purified by hplc, and characterized by electrospray mass spectroscopy. all derivatives except one bearing a hydrazine modification were stable to dpp-iv proteolysis for up to hours at ph . , o. each was tested for its ability to augment insulin release from a glucose-sensitive, murine insulinoma-derived tc- cell line in culture. it was found that each compound acted as a glp- agonist to varying degrees, with some exhibiting higher activity than native glp- toward promoting insulin release. the most active analogues have been chosen as candidates for stabilisation against renal clearance in efforts to develop new glp- analogues with therapeutic potential. the high propensity of the glucose regulatory hormone human islet amyloid polypeptide (iapp) to misfold and aggregate into cytotoxic beta-sheets and fibrils is strongly associated with beta-cell degeneration in type ii diabetes (t d) and precludes its pharmacological use for the treatment of diabetes. iapp analogs that combine solubility, lack of cytotoxicity, and bioactivity with the ability to block iapp aggregation and cytotoxicity could thus be of high biomedical interest. here we apply a minimalistic conformational restriction strategy to redesign the extremely insoluble and amyloidogenic -residue iapp sequence into a soluble, nonamyloidogenic, noncytotoxic, and bioactive iapp mimic (yan et al., pnas ( ) ). the designed mimic has nearly the same sequence as iapp but is highly soluble, nonamyloidogenic, noncytotoxic and a full iapp receptor agonist. in addition, the mimic binds with high affinity iapp and blocks and reverses with nanomolar activity its cytotoxic self-assembly which makes it to the most potent known iapp cytotoxic self-assembly inhibitor. due to its bifunctional nature, the mimic might find therapeutic application for the treatment of diabetes both as an inhibitor of amyloid cytotoxicity and as a soluble iapp receptor agonist. our findings offer a proof-of-principle of a chemical design approach for generating a novel class of highly potent inhibitors of polypeptide cytotoxic selfassembly which are nonamyloidogenic mimics of the native amyloidogenic sequence as well. such reengineered biomolecules -the design of novel mimics is in progress-are of high biomedical significance for understanding the mechanism of protein aggregation diseases and for the development of prospective therapeutic treatments. peptides that are capable of targeting abnormal changes of living tissue can be very useful in early detection or diagnosis of, e.g., cancer. conjugating a functional agent, an effector unit, to such a peptide may provide the agent with improved pharmacodynamic properties.the specificity of a thiol group for reactive groups offer a unique way to attach effector units to cysteine-free linear or cyclic peptides. tumor targeting peptides were synthesized by fmoc-type solid phase methods. peptides cyclic by cystine were modified by lactam bridges. fluorescein, metal (e.g. lanthanide) chelates and cytotoxins were coupled to tumor targeting peptides via e.g. a peg-type spacer, or the conjugates were immobilized on plates for adhesion assays. the method was uncomplicated and gave stable conjugates with good solubility. the approach is useful in making stable peptide-effector conjugates and sets of them for e.g. detection assays such as delfia method and has prospective use in development of diagnosis and therapy. antibacterial proline-rich peptides -synthesis and antibacterial activity d. knappe, r. hoffmann the antibacterial proline-rich peptide family, originally isolated from insects, shows remarkable activity against diverse bacterial and fungal pathogens. while more and more bacterial pathogens become resistant to common drugs, this part of insect innate immunity provides a new promising approach to develop future peptide-drugs. proline-rich peptides possess a significant sequence homology and share a common mechanism of action. in addition oglycosylated threonine residues of drosocin and formaecin appear to be necessary for full antimicrobial activity, although the significance of the carbohydrate moiety in interaction with intracellular targets is still unknown. we synthesized analogues of different antibacterial peptides on solid phase by the fmoc-tbustrategy. the combination or insertion of sequence regions from different native antibacterial peptide sequences offers several advantageous effects, including further reduction of toxicity and broadening of the antimicrobial activity. furthermore, mimicking the o-glycosylation site and changing the carbohydrate moieties, may yield new synthetic approaches to increase both the activity and the selectivity of these oligopeptides. new immunotherapeutic approaches have been developed for treatment of neurodegenerative diseases of the alzheimer's dementia (ad) type. the identification of a ß-amyloid-plaque specific epitope, aß( - ) (frhdsgy) [ ] , recognised by therapeutically active antibodies from transgenic ad mice, provides the basis for development of new ad vaccines and for molecular ad diagnostics. in order to produce immunogenic conjugates, the aß - epitope was attached via thioether linkage to synthetic carriers of well-defined structures, such as tetratuftsin derivative (ac-[tkpk(clac)g] -nh ) and its elongated version by a helper t-cell epitope (ac-fflltriltipqsld-[tkpk(clac)g] -nh ); sequential oligopeptide carrier (ac-[lys(clac)-aib-gly] -oh) and multiple antigenic peptide (clac-lys(clac)-lys(clac-lys(clac))-arg-arg-ßala-nh ). the epitope conjugates containing a cysteine residue either at the c-or n-terminus, and the chloroacetylated carriers were prepared by spps according to boc/bzl strategy. conjugation reactions were performed in solution under slightly alkaline conditions, and monitored by hplc and high resolution-ms. structures and molecular homogeneities of all epitope peptides, carriers and conjugates were ascertained by hplc, maldi and esi-fticr-ms. conformational preferences of the synthesized compounds in water and in tfe were examined by cd spectroscopy. comparative binding studies of the conjugates with a mouse anti-amyloid protein beta-( - ) monoclonal antibody were performed by indirect elisa. experimental data showed that the chemical nature of the carrier, the epitope topology and the presence of a pentaglycine spacer between the epitope peptide and the carrier, had significant effects on the antibody recognition and on the secondary structures of the conjugates. the new cla analogue , containing ethylene bridge between phe nitrogen atoms, was found to exhibit unexpected stimulatory effect in the model of the in vitro humoral immune response in mice. to disclose the structure-activity relationship the nmr solution conformational analysis was carried out. the solid-state and solution conformational analysis of native cla indicated the existence of this cyclic system as a complex mixture of conformations [ ] . the nmr spectra recorded at mhz in chloroform at k showed the different conformational behaviour of both cyclopeptides: cla exists as one isomer [ ] , peptide is in an equilibrium among at least of three conformers. the picornaviruses are small nonenveloped rna viruses with a single positive strand rna. the virus replication cycle starts after the penetration of the virus in the cytoplasm of the host cell. there are several stages of the virus life cycle used for attack. one of the most useful strategies for attacking of the virus includes inhibition of important for the virus lifecycle enzymes. the key enzymes in the replication of the picornaviruses are c and a proteases. changes in the active center of these enzymes make them incapable to produce polyprotein in vitro, therefore the inhibition by low molecular weight molecules could stop the viral replication in vivo. c protease plays a major roll in the enzymatic proteolysis of the initial viral polyprotein. the target compounds were based on structural modifications in the known as crucial for the cp inhibition activity dipeptides phe-gln by incorporation of additional amino acid and pyrrole moiety. the synthesis was cared out as multiple-peptide synthesis in parallel using stepwise spps, fmoc-strategy. the obtained compounds were tested for antiviral activity by agar-diffusion plaque inhibition test against coxsackievirus b replication in fl cell and on this base some structure-activity interpretations were made. histone deacetylases (hdac) play important roles in various aspects of regulation such as proliferation, differentiation, and aging by counteracting with histone acetyl transferases. the hdacs categorized in class-i and ii have a metalloprotease-related mechanism in its catalytic activity. these enzymes could be inhibited by small molecules bearing various zinc ligands such as hydroxamic acid and mercaptan. based on the structure of chlamydocin, which has a cyclic tetrapeptide framework, cyclo(-l-aoe-aib-l-phe-d-pro-), where aoe is ( s, s)- -amino- -oxo- , -epoxydecanoyl, we have developed potent hdac inhibitors as shown in the figure. in the present study, we examined the effects of the chlamydocin hydroxamic acid ( ) and ss-hybrid ( ) on the diabetes model mice, kk-ay. the peptide ( ) exhibited satisfactory activity to reduce both the blood glucose and blood insulin levels comparable to or even superior to those by pioglitazone, a pparγ agonist. the ss-hybrid ( ) , which is expected to be reduced inside of cells to generate the corresponding thiol-containing cyclic tetrapeptide, also showed a significant effect but less than ( ). the effect was dose-dependent from mg/kg to mg/kg. the effects of hdac inhibitors were also confirmed by the observation of in vivo histone hyperacetylation induced in the lymphocyte cells. synthetic peptides have a number of advantages over current vaccines. however, exploitation of synthetic peptides as vaccines has been limited by the small size; copy number, inefficient delivery, poor peptide immunogenicity and mhc restriction. we have developed chemical methodologies that overcome these limitations by synthesising and polymerising vinyl-peptides. protective b-cell and t-cell peptide epitopes from the oral pathogen porphyromonas gingivalis were identified for three different mhc restricted mouse strains using pepscan techniques. fmoc chemistry for spps was used to synthesize these peptides and a vinyl moiety was incorporated at the n-terminus using acryloyl chloride. after rp-hplc purification free radical polymerisation using ammonium persulphate and temed was used to polymerise the vinyl-peptides in the presence of either acrylamide or other vinyl-monomers to produce single peptide or multi-peptide polymers. size exclusion chromatography indicated that the peptide polymers were > million da. the peptide polymers were used to immunise each mouse strain (balb/c, cba and c bl ) and the t-cell response induced was evaluated using proliferation and cytokine (elispot) assays. the peptide polymers were found to be highly immunogenic, the single peptide polymers were found to only induce a response in their respective mouse strain, however, the multi-peptide polymer containing all of the t-cell epitopes was found to induce a response in all three mouse strains. in conclusion, our data shows that the polymerisation method overcomes all of the limitations in developing a peptide vaccine and most importantly that of mhc restriction. cytotoxic substances are auspicious weapons in tumour therapy. this compounds inhibit cell division and proliferation, hence, they affect all cells that are able to divide. however, all these compounds act intracellularly, i.e. at first they have to enter the tumour cell efficiently. this is a serious obstacle when using highly effective cytostatica and the cause of severe adverse effects by using higher doses. our aim is to overcome this problem by using synthetic hybride molecules composed of the cytostatic agent, in our case derivatives of arglabin, covalently linked to shuttle peptides. in order to identify the most effective possibility we tested two different strategies. by using the peptide hormone npy, whose specific y receptors are often overexpressed by tumour tissues, we intended to address the chemotherapeutic selectively to y receptor expressing tumour cells via receptor mediated endocytosis. on the other hand, the cytostatic agent was covalently bound to a cell penetrating peptide derived from human calcitonin (hct). recently, a c-terminal fragment of human calcitonin was found to internalize into excised nasal epithelium while the receptor activating n-terminal part is lacking. for the class of hct derived carrier peptides previous studies suggested a receptor independent "lipid raft" mediated endocytotic mechanism of uptake. here we present comparative data investigating both strategies, the highly selective receptor mediated delivery and the highly efficient receptor independent delivery. we investigated the peptide uptake by various cell lines and examined the release of the cytostatic agents inside the cells and its toxic effects. background and aims: synthetic peptides provide a straight-forward access to rationally designed inhibitors of molecular interactions based on structural information of proteins. poor membrane permeability may be overcome via cell-penetrating peptides, but low stability remains a major drawback. an increase of stability and bioactivity of peptides by coupling to polymers is intended. methods: for the development of peptide-functionalized cell-penetrating polymer conjugates, peptides were coupled chemoselectivly to hpma (n-( hydroxypropyl)methacrylamide, average mw . kda), as an inert backbone polymer by native chemical ligation. hpma is water soluble and its capacity in drug delivery has been demonstrated. peptide-functionalized polymers and free peptides were incubated with proteases or cell lysates and proteolytic break-down was determined by fluorescence correlation spectroscopy (fcs) deriving information on the number and size of fluorescent particles based on temporal fluctuations of a fluorescence signal caused by diffusion of particles through a femtoliter-size confocal detection volume. the diffusion time depends on molecular size. therefore this technique is suited for the detection of proteolytic fragments. results: efficient chemoselective conjugation of unprotected fluorescein-labelled peptides was accomplished by means of native chemical ligation. our fcs investigations revealed that conjugation of peptides to hpma increased their biostability. this data also indicate that this effect is peptide-dependent. a proapoptotic peptide coupled to hpma and introduced into mammalian cells by electroporation retained its bioactivity. cofunctionalization of hpma with this peptide and nonaargine yielded an efficient cellular import. macrolides, a rather large group of natural or semi-synthetic antibiotics, are widely known translation inhibitors whose structure is based on - -member lactones with carbohydrate substitutes attached. macrolides bind to ribosomal tunnel (rt) in a way that their lactone ring is located orthogonally to the long axis of the rt, covering most of its cleft. carbohydrate residues of the macrolides are spread along the walls of the rt. hence, the mechanism of protein synthesis inhibition by macrolides relies on the mechanical obstruction they provide to the passage of nascent polypeptide chain through the rt. the goal of this study was to design and obtain peptide derivatives of macrolides interesting both as antibacterial agents and potential probes for investigation of nascent peptide chain topography in the rt. tylosin ( in order to reach target sites inside the cns, neurotherapeutic candidates must overcome the blood-brain barrier (bbb). while several transport mechanisms occur at the bbb, this work has focused on the passive diffusion mechanism. the prediction of a peptide's ability to cross the bbb is not a simple task; hence there exists the need for the rational study of the relevant factors that affect the movement across this physiological barrier. here we present two new approaches based on in vitro non cellular assays for this type of studies. firstly, the evaluation of compound mixtures on parallel artificial membrane permeability assays (pampa). this approach increases the throughput of the study and structure-activity relationships can be easily establish. secondly, the transport and biological activity evaluation in a single assay. this second approach has been applied to the search of inhibitors for cns proteases involved in different neurological diseases (such as prolyl oligopeptidase for schizophrenia) able to cross the bbb. these two new approaches allow assaying compound's permeability in the early stages of a drug development project, and then designing novel analogues with improved bbb transport properties or using blood-brain-barrier shuttles for their delivery. two newly synthesized compounds are derivatives of l-valin and are positional isomers of nicotinic (m- ) and isonicotinic acid (p- ). these functional groups, as well as established good lipid solubility suggest that the main target for their biological action probably will be central nervous system. the presence of aminoacid l -valin, supposes their low toxicity, confirmed by our earlier experiments. the aim of the present work is to study their pharmacological activity as putative drugs. methods: male albino mice ( - g, in groups) were used. next neuropharmacological parameters were studied: analgesic effect (acetic acid test), neuromuscular coordination (rota-rod test), orientation ("hole board" test) and learning and memory (passive avoidance-step down test). results: significant analgesic effect of both compounds was established (more pronounced by dose mg/kg i.p.). slight depressing effect on the orientation reactions in animals was registered, but the neuromuscular coordination and locomotor activity of treated animals were not changed. good dose-dependent effect on learning and memory was established and М- had stronger effect than Р- . the compounds modified the effects of some model substances with central nervous activity. hexobarbital sleeping time was significantly prolonged by p- , but was antagonized by m- . pentileneterazole threshold was increased significantly and suggests some anticonvulsant activity of both compounds. conclusion: as positional isomers m- and p- demonstrated some variations in their pharmacological activity probably due to the differences in their kinetics, metabolism and excretion. registered significant neuropharmacological activity, accompanied by low toxicity motivates the new synthesis and future experimental studies. the glyproline family of regulatory peptides includes pro-gly-pro, gly-pro, pro-gly and also the simplest peptides with hyp substituted for pro. a distinctive feature of these peptides is that they exhibit a broad spectrum of biological activities: the antiulcer activity, the inhibition of blood clotting and thrombosis, the reduction of degranulation activity of mast cells, and the normalization of stressogenic behavioral disorders. alzheimer's (ad) and parkinson's (pd) disease are progressive neurodegenerative disorders which are characterized by amyloid plaques. the main components of the plaques are β-amyloid peptides (aβ - and aβ - ) and α-synuclein. we have previously shown that small peptides structurally related to the sequence of aβ( - ) protect against the neurotoxicity of aβ peptides. recent studies by other groups have shown that β-synuclein can counteract the aggregation of α-synuclein in the neurodegenerative process of pd, hereby might protect the central nervous system from the neurotoxic effects of alpha-synuclein. it was found that a tetrapeptide (kegv) protected against the neurotoxicity of aβ peptides in vivo. based on the previous findings, the following sequences of β-synuclein have been synthesized: kegv-nh and kegv-oh. after comparing the sequences of α-and β-synuclein, we found common sequences which are keqv, regv, kqgv, keqa. we have synthesized these tetrapeptides in amide forms at their c-termini. the peptides have been synthesized on mbha resin using a manual solid phase peptide synthesis equipment and boc-chemistry. the neuroprotective effects of peptides have been investigated in vitro in mtt test in differentiated neuroblastoma cell culture (sh-sy y) and in electrophysiological test on rats using multibarrel electrodes in vivo. the neuroprotective peptides might stop neuronal death and can influence ad and pd progression. the present study was carried out to determine antinociceptive effect in vivo of plant peptide hormone phytosulfokine-alpha (h-tyr( -so h)-ile-tyr( -so h)-thr-gln-oh ) (i) and its selected analogues, such as h-phe( -no )-ile-tyr( -so h)-thr-gln-oh (ii), h-d-phe( -so h)-ile-tyr( -so h)-thr-gln-oh(iii), h-tyr-ile-tyr( -so h)-thr-gln-oh(iv), h-tyr( -so h)-ile-tyr-thr-gln-oh(v) and h-tyr-ile-tyr-thr-gln-oh (vi) in rats. peptides were injected into the lateral brain ventricle at the dose of nmol. in the preliminary investigation we found the psk-as well analogues ii and iii induced a significant antinociceptive effect determined in the test of hot plate. the probable mechanism of this effect was discussed. a.b. bozhilova-pastirova , b.a. landzhov , p.v. yotovski , e.b. dzambazova , a.i. bocheva the tyr-mif- family of peptides (tyr-mif- `s) includes mif- , tyr-mif- , tyr-w-mif- and tyr-k-mif- , which have been isolated from bovine hypothalamus and human brain cortex. all these peptides interact with opioid receptors and in addition bind to non-opiate sites specific for each of the peptides. data in the literature suggest that tyr-mif- `s have antiopioid and opioid -like effects. we used wistar rats to study distribution and density of the tyrozine hydroxylase (th) imunoreactive fibres and nadph-d reactive neurons in the rat ventral and dorsal striatum. our results showed that neuropeptides mif- and tyr-mif- may affect them. opioid peptides have been recognized as modulators of reactive oxygen species (ros) in mouse macrophages and human neutrophils. data in the literature suggest that peptides of the tyr-mif- family -mif- and tyr-mif- have antiopioid and opioid -like effects. these neuropeptides are isolated from bovine hypothalamus and human brain cortex. so far no data about direct scavenger properties of tyr-mifs peptides were available. in this study we tested the hypothesis that they may scavenge ros in vitro. the antioxidant activity of these two substances was studied in the concentration range of - - - mol/l. we investigated the luminol-dependent chemiluminescence to test their ability to scavenge the biologically relevant oxygen-derived species: hydroxyl radical, superoxide radical, hypochlorous acid in vitro. we found that tyr-mif- was a powerful scavenger in all tested systems. the effects were higher for hypochlorous anion and weaker for superoxide radical. mif- had no scavenge activity against the hydroxyl and superoxide radicals and showed a moderate scavenger effect on hypochlorous anion. we have compared different strategies to increase the immunogenicity of an antigenic hiv peptide as a vaccine candidate. our selected b-cell epitope comprises amino acids ( - ) of the v region of hiv- , jy isolate (subtype d), and is in tandem with a t-helper epitope corresponding to the - region of tetanus toxoid. several presentations, including oligomerization, map dendrimer, conjugation to dextran beads or to other macromolecular carriers, have been synthesized and evaluated. murine sera from the different presentations of the v epitope have been compared with regard to antibody titers and cross-reactivity with heterologous hiv subtypes. the map dendrimer version of the peptide, conjugated to recombinant hepatitis b surface antigen protein, was a better immunogen than the dendrimer alone, and showed higher immunogenicity than other multimeric presentations, or than the peptide alone conjugated to dextran beads. the map dendrimer version, either alone or conjugated to hbsag, enhanced cross-reactivity towards heterologous v sequences relative to monomeric peptide. group a streptococcus (gas) responsible for critical diseases (eg. acute rheumatic fever and rheumatic heart disease) are classified over serotypes according to their surface virulence m proteins. development of vaccine to prevent infection with gas is hampered by the widespread diversity of circulating gas strains and m protein serotypes, and multivalent vaccine strategy would contribute to prevention against various gas infections and provide better protective immunity. we have studied the efficacy of incorporating four different epitopes derived from gas m protein into a single synthetic lipid core peptide (lcp) construct, in inducing broadly protective immune responses against gas following parenteral delivery to mice. peptide vaccine was synthesized on mbha resin by manual spps in situ neutralization/hbtu activation in boc-chemistry. immunisation with the mono-or multi-epitopic lcp vaccine led to high titers of antigen-specific systemic igg responses, and the production of broad protective immune responses as demonstrated by the ability of immune sera to opsonise multiple gas strains. systemic challenge of mice after primary vaccination showed that mice were significantly protected against gas infection in comparison with control mice demonstrating that vaccination stimulated long-lasting protective immunity. these data support to the usefulness of lcp system in the development of synthetic multiepitope vaccine to prevent gas infection. glycoprotein d represents a major immunogenic component of the virion envelope of herpes simplex virus and able to induce high titres of neutralizing antibodies. one of its optimal epitopes is the - region ( lkmadpnrfrgkdl ). several cyclic peptides possessing thioether bond and different ring size have been already prepared and some of them were conjugated with tetratuftsin derivative (ac-[tkpkg] -nh ) by thioether bond formation using selectively removable cys protecting groups. antibody recognition results suggested that the size of the cycle has considerable influence on antibody recognition, however, the replacement of met in position by nle is permitted. conjugation of cyclic peptide might increase the antibody recognition, but the binding depends on the structure and/or conjugation site of the cyclic peptide. conjugate with the best binding capacity ( . pmol/ ul) as well as the conjugate containing the linear ( - c) epitope ( . pmol/ ul) were selected for immunization. in order to increase the production of antibodies a new group of conjugates was prepared. in these constructs promiscouos t cell epitope peptide derived from tetanus toxoid (ysyfpsv) was attached to both amino groups of lysine residue coupled to the n-terminus of the carrier (ac-ysyfpsv-k(ac-ysyfpsv)-[tkpk(clac)g] -nh ). the cys containing linear and cyclic epitope peptides were conjugated to the carrier in solution ( . m tris buffer, ph ). this work was supported by grants of the spanish-hungarian intergovernmental program and cost chemistry action. background. primary hyperparathyroidism (pht) is characterized with increased parathyroid hormone (pth) secretion and in % of pht patients with hypertension. it was previously shown that pro-analogue of pth with a reversed sequence (which include strong alkali sequence -arg-lys-lys-) induced significant hypertensive response at dose - m/kg b.w. one of the hypothesis attributed hypertension in pht patients to the presence of fragments of degraded pth possessing -arg-lys-lys-sequence. aim. to compare influence on mean arterial blood pressure (map) of analogue of - pth fragment (amide) and - fragment of pth, with -arg-lys-lys-sequence and also responsible for binding to pth receptor. methods. chosen peptides were synthesized manually by a solid phase peptide synthesis method. the purity of the products was tested by reversed-phase high performance liquid chromatography. the synthesized peptides showed the right molecular mass. the influence on map of synthesized peptides was tested in wistar rats. sequential increasing boluses of each peptide: - , - and - m/kg.b.w. in the same animal were given i.v. blood pressure was measured continuously in carotid artery. results. injection of synthesized analogue of - fragment of pth does not show influence on map vs. control group. synthesized - fragment of pth increased map in min. of experiment for mmhg ± mmhg vs. time of administration of first dose and for mmhg vs. control group. conclusion. it seems to be possible, that in case of alternate degradation of pth, accumulation of - fragment of pth may partially play role in the mechanism inducing hypertension in pht patients. biologically active domains of a high affinity receptor for ig Е (fcεri) were determined, the fragments - and - of the receptor. the program of research of biological properties of synthesized tetrapeptide rnwd and heptapeptide rnwdvyk included the study of their binding with ige, which was contained in standard solutions and in patients' blood serum. the binding of peptides with ige was explored by the ifa method using ige antibodies labeled with horse-radish peroxidase (hrpo). peptides in the concentration of mkg/ml were used for sorption on immunological plotting boards. higher correlation between the ige concentration and the optical density of the solution after introducing monoclonal antibodies labeled with hrpo and substrate chromogenic mixture (r= . ) was found in the diagnostic system with sorption rnwdvyk-peptide than in the diagnostic system with sorption rnwd-peptide (r= . ). similar investigations were conducted with the diagnostic systems with sorption rnwd and rnwdvyk peptides, but rwnd peptides conjugated with hrpo were used as antibodies against immunoglobulin e, instead of hrpo-labeled monoclonal antibodies. almost equal correlation was found between the concentration of ige in standard serum and serum of allergy patients with the known concentration of ige and the optical density of the solutions after introducing the rnwd peptide, conjugated with hrpo. after introducing allergy patients' blood serum in the holes on the plotting board, the heptapeptide bound . % more ige than the tetrapeptide. our experiments demonstrated a high ige binding activity of synthetic rnwd-and rnwdvyk-peptides. in this study, the information spectrum method (ism) of the ha subunit of the h hemagglutinin protein of the influenza virus, h n , of different reference isolates was performed in order to identify possible antigenic determinants resistant to virus mutations. results of this analysis demonstrated that the primary structures of ha subunit of h hemagglutinins encode a common information corresponding to one characteristic frequency in their iss, which is probably important for the biological function of these proteins, including their possible recognition by the immune proteins targeting this molecule. besides, comparison of the iss of ha proteins of h "spanish flu" and h n isolates demonstrated an informational similarity between them. based on these results, a segment of the n-terminus of ha h n was identified to play a crucial role in the inhibitory and immunological properties of all possible h n variants. the identified core segment, being highly conserved in all h strains, was selected as an antigenic determinant and coupled to the sequential oligopeptide carrier (socn), (lys-aib-gly)n, to the lys-nεh groups, in order to develop a diagnostic immunoassay and formulate a vaccine candidate for the highly pathogenic h n influenza virus. background and aims: thrombin plays a key role in various disorders such as arterial thrombosis, atherosclerosis, restenosis, inflammation and myocardial infarction. insights into the way in which thrombin interacts with its many substrates and cofactors have been clarified by crystal structure and site-directed mutagenesis analyses, but until recently there has been little consideration of how its non-proteolytic functions are performed. we investigated cardiovascular effects of seven modified proline-and rgd-containing peptides designed from three surface-exposed sites of prothrombin, corresponding to residues - , - , and - . methods: cardioprotective effects of synthetic peptides were tested on the two rat models of heart failure produced by coronary artery occlusion ( -or -min) and reperfusion ( -or -min). arterial blood pressures from left carotid artery, heart rate and ecg ii standard lead were measured throughout experiment. at the end of second experiment hearts were morphologically investigated by light microscopy and electron microscopy methods. results: on animal model with short-term ischemia investigated peptides did not protected from myocardium ischemia during occlusion, however, tp-l , bk-mc and tp-h protected rat hearts from ventricular fibrillation, contributed more significant functional recovery during reperfusion and raising survival rate. on the model with prolong ischemia, acceptable cardioprotective effect revealed tp-h and bk-mc. these peptides significantly diminished necrotic zone of left ventricle, protected hearts from ischemia-reperfusion induced functional and morphological changes. conclusions: investigated proline-containing peptides revealed activity on cardiovascular system -decreasing of blood pressure, cardioprotective properties and improved recovery from ischemia. r. mansi, d. tesauro, c. pedone, e. benedetti, g. morelli the widespread use of compounds containing the gamma-emitting radionuclide mtc in nuclear medicine for the scintigraphic imaging, as well as the recent introduction of the beta-emitting radionuclides re and re in radiotherapy, have led to a rapid development of their chemistries, in order to produce novel radiopharmaceuticals. we have developed new peptide based radiopharmaceuticals based on a scaffold in which the radioactive metal ion is complexed by two different peptides that are able to bind two target receptors (see figure) . the + mixed-ligand approach has been used for the preparation of neutral oxotechnetium(v) and oxorhenium(v) peptide complexes. the complex preparation requires the simultaneous action of a dianionic tridentate ligand and a monoanionic monodentate thiolato on a suitable metal precursor. the dianionic tridentate ligand is based on the snn donor set able to stabilize the metal complex. the chelating agent (hsc(ch ) conhch ch(co-r)nh ) was coupled step by step to a bioactive peptide synthesized on solid phase. the second ligand, based on monodentate thiolato moiety, was coupled on n-terminus of the second peptide. labelling procedures and biological tests on tumour cells overexpressing receptors are described for mtc(o) complexed by cck and octreotide peptide derivatives. background: endostatin inhibits the proliferation of endothelial cells and induces their apoptosis. the measurement of serum endostatin can predict tumor vascularity. tumor angiogenesis is a strong prognostic factor in patients with hepatocellular carcinoma(hcc). significantly high levels of endostatin were noted in patients with trabecular-type tumors , and with hepatitis infection. methods : patients with hcc, patients with git malignancies, patients with liver metastasis and without metastasis, and normal persons . all subjects were tested for alfa-feto protein (afp) , ca . , carcinoemberyonic antigen (cea), and endostatin by elisa results : endostatin in normal control persons was . ± . ng/ml with a significant elevation (p< . ) between the hcc group and all the other tested groups .afp was . ± . ng/ml in normal persons with a significant elevation between hcc and all the other tested groups ( p< . ). ca . was . ± . u/ml in normal persons with a significance elevation ( p< . ) relative to hcc., and a significance of ( p< . ) relative to git cancers with metastases. cea was tested to be . ± . µg/l in normal persons , and had a significance of ( p< . ) relative to git metastases. endostatin was elevated in of patients with git cancers not proved to have metastasis. conclusion : endostatin can be used to denote metaplasia and can also detect possibilities of metastasis or liver cell affection even before the frank development of metaplasia affibody® molecules are a novel class of affinity proteins which are generated by combinatorial engineering of the aa three-helix bundle scaffold, originating from the b domain of staphylococcal protein a. we have used fmoc/tbu chemistry for total chemical synthesis of the affibody zher : , binding with picomolar affinity to the cell surface receptor her . the synthetic protein was investigated for molecular imaging of her -overexpressing tumours. in vivo detection of her in malignant tumours provides important diagnostic information which may influence patient management. to enable gamma camera imaging of the tumours, a panel of potential mtc-chelating sequences was designed and introduced into the affibody. the well-studied tc-chelating sequence mercaptoacetyltriglycyl (mag ) was compared to serine-containing sequences with increased hydrophilicity, such as mercaptoacetyltriserinyl (mas ). the total synthetic yield was - % and the her -binding affinity of the affibody conjugates were all in the range - pm. binding specificity of tc-labelled affibody molecules was determined on her -expressing skov- ovarian carcinoma cells. all variants showed receptor-specific binding. the tumour-targeting properties were studied in skov- tumour-bearing nude mice. all conjugates demonstrated high tumour uptake, quick blood clearance and low uptake in most other organs. the biodistribution results further showed that the more hydrophilic, serine-containing chelators resulted in a reduced hepatobiliary excretion, which significantly decrease the background in the abdomen area and provide for more sensitive detection. gamma camera images of mice with grafted tumours showed clear visualization of her -expressing tumours using the mtclabelled mas -affibody conjugate, suggesting a potential future application of this agent for diagnostic imaging. antimicrobial peptides are molecules with a unique mechanism of action. they are widespread in nature and play the role of an effective weapon of innate immune system against bacteria, fungi and viruses. the purpose of this study was to investigate the in vitro activity of natural antimicrobial peptides: citropin, piscidin, protegrin, temporin, uperin and the analogues of antimicrobial peptides: iseganan, pexiganan and omiganan. the peptides were synthesized using the solid-phase method and purified by high-performance liquid chromatography. the peptides were subjected to microbiological tests [mic (minimal inhibitory concentration) and mbc (minimal bactericidal concentration)] on reference strains of bacteria, according to the procedures outlined by the national committee for clinical laboratory standards (nccls). for comparison, conventional antibiotics (vancomycin, rifampycin, piperacillin, chloramphenicol) were included in this research. both the natural antimicrobial peptides and the analogues inhibited the growth of bacteria, but at higher concentrations than did conventional antibiotics. nevertheless, both natural origin of antimicrobial peptides and their low toxicity constitute a considerable advantage and this is an argument for considering the antimicrobial peptides as good candidates for medicines. the linear hexapeptide cypate-grdspk (compound ; the cypate moiety is a near-infrared fluorescent label) whose rgd sequence was rearranged to grd showed high uptake in the αvβ integrin-positive tumor tissues in vitro and in vivo. despite low affinity of to the integrin in the binding assays, the uptake was inhibited by equimolar amounts of the cyclic peptide c(rgdfv), which possesses high affinity to αvβ integrin. these observations led to hypothesis that cell internalization of compound may be mediated mostly by only one of the integrin subunits, as the β one. indeed, blocking of αv integrin by the specific antibody did not inhibit the internalization of in tumor cells, which was in the contrast with successful blocking the cell internalization by the anti-β integrin antibody. similar results were obtained in immunocytochemical assays employing the anti-αv and anti-β integrin antibodies. also, studies utilizing the β -knockout and wild-type mouse cell lines demonstrated that deletion of the β subunit markedly decreased internalization of compound in the β knockout cells. the preferential interaction of compound with the β subunit of integrins relative to the αv subunit was supported also by molecular modeling studies. summarizing, the bulk of our experimental and modeling data emphasizes interaction with the β integrin as the primary mechanism of the uptake of cypate-grdspk by tumors. since this compound showed the superior biodistribution profile in vivo, our results may provide a strategy to image and monitor the functional status of the β integrin in cells and live animals. background and aim: a growing tumor is accompanied by tumor intoxication development. intoxication independs on tumor size and intensity of its break-up. tumor intoxication is one of variant of endogenous intoxication. concentration of tyrosine-contained peptides (tcp) in blood plasma have been proposed as biochemical marker of endogenous intoxication at different organs cancers. our aim was to determine the tcp concentration in blood plasma patients with ovary tumor and its association with the severity of tumor. materials and methods: patients with ovary tumor, mean age is years, were studied. the control group consisted of healthy women without tumor. patients were divided into groups: people with non-malignant and people with malignant ovary tumor. tcp content in blood plasma was estimated by our technique. results: tcp concentration in the control group were , ± , mmol. the tested marker was present in increased concentration in blood plasma of the patients with ovary tumor. the mean concentration tcp in patients with non-malignant tumor was , ± , mmol. the content of this marker in blood plasma of patients from second group was increased , ± , mmol compared with healthy control group. after treatment a significant decrease in tcp content was observed. conclusions: the result indicate that content tcp in blood plasma depends of the type of tumor. it could be suggested that determination of tcp concentration in blood plasma could be useful for improve the diagnostic of ovary tumor and monitoring of its progression. c. strongylis , ch. papadopoulos , k. naka , l. michalis , k. soteriadou , v. tsikaris troponin is a structural protein complex, which is responsible for the regulation of skeletal and cardiac muscle contraction. it consists of three components: troponin i ( kda), troponin c ( kda), and troponin t ( kda), each of which carries out different functions in the striated muscles. cardiac troponins are released into the bloodstream of patients after the onset of a cardiovascular damage. even minimal elevations over the normal values, of serum troponin t and i are being used to diagnose acute myocardial infarction and also to rule out the patients' condition. the development and commercialization of highly specific biological assays for the detection of cardiac troponins is based on the production of specific antibodies against the whole complex or individual subunits. however, the specificity and sensitivity of these assays vary due to problems mainly originated from the fact that cardiac troponins have a high homology with the skeletal isoforms. the aim of this work is to select and synthesize appropriate regions of the cardiac isoforms of troponin i, c and t, suitable for the production of more sensitive and specific cardiac troponin detecting reagents. in order to construct the immunogenic complexes, the selected sequences were conjugated to the tetrameric sequential oligopeptide carrier (soc ), either by the classic solid phase step-by-step methodology or by chemoselective ligation reactions. using the carrier conjugated troponin sequences, anti cardiac troponin complex specific antibodies in high titers were produced. the increasing problems with the reproductive systems of man and animals are recently linked to the presence of polluting chemicals with endocrine activity, the so called endocrine disrupting chemicals or edc's . the family of edc's is a heterogeneous one and consists of natural and synthetic hormones (like estradiol, ethynylestradiol and diethylstilbesterol), phyto-estrogens (like genistein and coumestrol) and industrial chemicals (like bisfenol-a, ftalates and various pesticides). because of the complexity of the environmental matrices and the low physiologically active concentrations of the edc's there is still a need for an efficient routine analysis protocol. we want to develop a solid-phase bound receptor that possesses a high selectivity for edc's and thus can be used in a simple solid-phase extraction protocol. this receptor must have the right functional groups that bind the edc's with a strong affinity and must be able to create a cavity in which the edc's can fit. by looking at nature's own estrogenic receptor for humans we have found the different amino acids responsible for the specific interactions . in order to create the cavity which mimics the behaviour of the hormone-binding domain of the human estrogenic receptor we have made a tripodal scaffold. this tripodal scaffold has three orthogonal protected amino groups that will allow the generation of three independent peptide chains. milk proteins are a source of opioid peptides. these peptides are liberated from milk proteins during enzymatic hydrolysis. some of these peptides are characterized with agonistic (β-casomorphins) and some with antagonistic (casoxins) properties. the aim of the investigations was to determine the presence of opioid peptides with antagonistic properties in milk products. the experimental material included cheeses, yogurts and kefirs. peptides were extracted with a methanol-chloroform mixture ( : v/v). the peptide extracts were purified by spe method on c or stratax columns and characterized by sds-page electrophoresis. the agonist opioid peptides (casoxins) were identified by hplc using standard agonist peptides. the opioid activity was measured by examining the effects of peptide extracts on the motor activity of isolated rabbit intestine. the results of sds-page electrophoresis indicated the presence of to fractions in peptide extract derived from cheeses and yogurts and to ones from kefirs. the presence of casoxin a ( . - . µg/mg of extract) was proved in all examined the milk products. lactoferroxin a ( . - . µg/mg of extract) was identified only in kefirs and yogurts. those products were also found to contain trace amounts of casoxin c. all peptide extracts showed the antagonistic activity in the relation to motor activity of isolated rabbit intestine. the highest antagonistic activity was reported of peptide extract from kefirs ( . - . %) and gouda cheese ( . - . %), as compared to morphine. the physiological and nutritional function of these antagonist peptides requires elucidation. a. péter , r. berkecz , f. fülöp the past decade has seen a growing interest in β-amino acids, which are important intermediates for the synthesis of compounds of pharmaceutical interest and can be used as building blocks for peptidomimetics. oligomers of β-amino acids (β-peptides) fold into compact helices in solution. recently, a novel class of β-peptide analogues adopting predictable and reproducible folding patterns (foldamers) was evaluated as a potential source of new drugs and catalysts. studies on synthetic β-amino acids can be facilitated by versatile and robust methods for determining the enantiomeric purity of starting materials and products. highperformance liquid chromatography (hplc) is one of the most useful techniques for the recognition and/or separation of stereoisomers including enantiomers. the aim of the present work was to evaluate hplc methods for the separation of enantiomers of eighteen -amino- -aryl-substituted propanoic acids (β-amino acids). direct separations were carried out on different macrocyclic glycopeptide based stationary phases, such as ristocetin a containing chirobiotic r, teicoplanin containing chirobiotic t, teicoplanin aglycon containing chirobiotic tag, vancomycin containing chirobiotic v columns and on a chiral crown ether based column. the effects of different parameters on selectivity, such as the nature of the organic modifier, the mobile phase composition, the flow rate and the structure of the analytes are examined and discussed. the separation of the stereoisomers was optimized by variation of the chromatographic parameters. the efficiency of the different methods and the role of molecular structure in the enantioseparation were noted. the elution sequence of the enantiomers was determined in most cases. a rational approach to evaluating peptide purity a. swietlow , r. lax amgen, inc., pharmaceutics, thousand oaks, ca polypeptide laboratories, inc., torrance, ca, usa recent years have seen an enormous increase in interest in peptide therapeutics. new peptide leads are often chosen by screening procedures using microgram to milligram quantities of peptides, frequently provided by specialized manufacturers utilizing automatic synthesizers to maximize output. the purity of the resulting compounds is often not very high. the use of spps synthetic procedures predetermines that most impurities are closely related and difficult to resolve by reversephase purification. these factors, combined with the use of generic analytical methods not specifically optimized for the peptide in question (e.g. the ubiquitous . % tfa/water/acetonitrile system), lead to erroneous results that frequently severely overestimate the purity of the peptide. the use of poorly characterized materials in pharmaceutical development leads to significant risks of obtaining false negative or false positive results that may cause potential leads to be overlooked or misinterpretation of their pharmacological profiles. we describe a rapid, systematic and reliable hplc procedure for evaluation of peptide purity. utilizing the increased separation efficiency by increasing the column temperature and adjusting the gradient in two steps in reverse-phase buffers containing tfa, naclo , or ion-exchange buffers containing kcl, we demonstrate that methods -suitable for preclinical research -can be developed rapidly. the proposed approach will be illustrated with examples of peptides ranging between and amino acids and a model peptide vypnga. it will be demonstrated that peptides showing an hplc purity close to % are often - % less pure. to approach a high-throughput cell assay format using peptides, we attempt to design and construct a peptide microarray for examination of cell activities of peptides including apoptotic cell death. peptides were immobilized onto solid surfaces via a novel multi-functional linker. the linker enable us to examine various types of peptide cell assays in an array format. we also designed and synthesized peptidyl capture agents on the basis of the cell-active sequences suitable for the peptide microarray. the utility of targeted nanoparticles as fluorescent probes for tissue imaging has recently been subject to widespread interest. one exciting prospect is the further development of nanoparticles conjugated to both targeting peptides and cytotoxic cargoes. these nanodevices could preferentially bind to specific cells and/or tissues to provide effective tools for drug delivery. hence, such multifunctional nanoparticles could provide both diagnostic and therapeutic functions by acting as fluorescent probes that offer targeted delivery of therapeutic agents. we have coated the surface of quantum dots (qdots) with cell-penetrating peptides (cpp) to target and label u m cells for fluorescence imaging. qdots were initially coupled to polyethylene glycol linkers via carboxyl functionalities on their surface. a heterogeneous mixture of poly-arginine peptides of varying lengths (arg( )-arg( )) were covalently coupled via amide bonds to the polyethylene glycol linkers, conferring a cell penetrating capacity to the modified qdots. fluorescence imaging of u m cells, after incubation with the conjugated qdots at concentrations of nm, gave clear signals indicating cell binding and internalisation of the modified qdots across the plasma membrane. we aim to further expand this work by employing racemic mixtures of cpp and cytotoxic agents to engineer conjugates that will facilitate both imaging and the therapeutic delivery of cytotoxic moieties. the ability of cell penetrating peptides (cpp) to deliver biologically active cargoes into different cell types has been successfully applied in several experimental systems. despite the progress and growing number of described cpps, reports about the internalization mechanisms and the intracellular routes of cpps still remain controversial. we have characterized the membrane interaction and cellular localization of proteins delivered into hela cells by cell penetrating peptide transportan (tp) and its shorter analogue tp on ultrastructural level. our previous results obtained by transmission electron microscopy showed that complexes of transportans with gold-labeled streptavidin translocated into cells inducing large invagination of plasmamembrane, suggesting the uptake by macropinocytosis. the complexes of transportan with gold-labeled neutravidin, in contrary, were taken into cells mostly via caveosomes and clathrin-coated vesicles. the cell-transduced transportanprotein complexes localized mainly in the vesicular structures of different size and morphology. most of the complexes-containing vesicles in the perinuclear area contained also lamp protein, marker of late endosomes and lysosomes. still, the transportan-protein complexes were not confined in the membrane-surrounded vesicles, but spread in the cytosol suggesting the escape of transportan-protein complexes from endosomes. our findings show the involvement of different endocytic pathways in the transportan-mediated uptake process of proteins. the concentration of a cpp and the properties of cargo protein seem to determine the pathway for the cellular uptake of a particular construct. rgd peptides (r = arginine; g = glycine; d = aspartic acid) have been found to promote cell adhesion upon interaction with alphav-beta receptors, which are strongly overexpressed during neoangiogenesis by solid tumor associated cells compared to healthy cells. in this study we designed new targeting motifs aimed to deliver various antitumoral drugs specifically to cells involved in tumor vascularization. we inserted this short rgd sequence in tetracyclopeptides closed with various means. we expect these new cyclotetrapeptides to be more specific for the targeted receptor. moreover, these new type of cyclic peptides were multimerized on different scaffold to further improve the receptor avidity. our purpose is first to scrutinize and to quantify the efficient cellular uptake of these molecules and second, to address the specific cell targeting of a fluorescent cargo by differents tools such as fluorescence activated cells sorting (facs) analysis or fluorescence microscopy. these new targeting units were evaluated on two different cell lines: human umbilical vein endothelial cells (huvec) with an over expression of the alphav-beta integrin receptor and a cells expressing a much lower level of this receptor. preliminary results about the selectivity and the efficacy of these new targeting units will be presented. we have recently developed new approaches for obtaining highly immunogenic peptide conjugates: synthetic polyelectrolytes (pe) were used for the conjugation with peptide molecules in which pe carry out the carrier and adjuvant roles simultaneously. in this study, epitopes of antigenic parts of surface antigen of hepatitis b virus ( - , - , - and - of the s gene.) had been synthesized.the synthesis of peptides was performed by explorer pls ® automated microwave synthesis workstation (cem). peptide conjugates of synthetic anionic polyelectrolytes (copolymers of acrylic asid and n-vinylpyrrolidone) were synthesized by carbodiimide condensation following the modification procedures described early. composition and structure of bioconjugates were characterized by hplc (shimadzu), nanospr- , zetasizer nano zs, steady state fluorescence spectrometer qm- and viscotek tda size exclusion chromatography. it was obtained that a single immunization of mice with pe-peptide conjugates without classical adjuvant increased the primary and secondary peptide-specific immune response to hbsag. moreover, these conjugates possess own selectivity for recognizing the antibody in blood sera of hepatitis virus injected people. tissue engineering requires delivery of transplanted cells to organ sites needing repair/regeneration. we have demonstrated that several active laminin peptide-conjugated chitosan membranes enhanced the biological activity and promoted cell adhesion in a cell-type specific manner. the most active laminin peptide (ag : rkrlqvqlsirt)-conjugated chitosan membrane could deliver keratinocytes to a wound bed. when human keratinocytes were seeded onto the ag -chitosan membranes under serum-free condition, more than % of the cells attached within hrs. the membranes carrying keratinocytes were stable enough for handling with forceps and were inverted onto the muscle fascia exposed on the trunk of nude mice. keratinocyte sheets were observed after days and colonies appeared after days on the fascia of host mice. these cells were multilayered on day- and expressed various keratinocyte markers, including cytokeratin- , involculin, and laminin ? -chain. these results suggest that the ag -conjugated chitosan membrane is useful as a therapeutic formulation and is applicable as a cell delivery system, such as delivering keratinocytes to the wound bed. the peptide-chitosan approach may be a powerful cell transplantation tool for various tissues and organs. the fluorescent semi-conductive (cdse/zns) nanocristals possess very attractive optical properties that could be used for tracking individually biological receptors in vivo. our aim is to design functionalized water-soluble semi-conductive nanocristals (or quantum dots) that interact selectively with lipidic or biological membranes. to valid our approach, the interaction between the decorated qd and giant vesicles were observed by optical fluorescent and dark field microscopies. in view to solubilize and selectively bind fluorescent nanocristals to a lipid membrane, heterobifunctionalized peptidic ligands (liipe) that presented an adhesion domain for the nanocristal surface, an hydrophilic spacer and a terminal recognition function, were synthetized. the colloïdal stability of the water-soluble nanocristals (nc-lipe) was checked by dynamic light scattering, optical and electron microscopies the interaction of grafted nanocristals (nc-lipe) with positive or neutral giant vesicles was observed by optical fluorescence and dark field microscopies. as shown in figure, negatively charged nanocristals (nc-lipe) selectively adsorbed onto the surface of positively charged giant vesicles without altering the morphology of the vesicle. the nanocristals appeared as fluorescent patches growing on the surface of the vesicle until completely recovering. therefore these ligands (lipe) permitted to chemically functionalize the nanocristals by keeping their colloïdal stability and their fluorescence in water. furthermore it was possible to selectively label vesicle membrane. creatine analogues for treatment of obesity: us patent bioactive peptides containing pairs of basic amino acids are rapidly metabolized as a result of cleavage by trypsin-like enzymes. to increase the metabolic stability of opioid peptides containing arg-arg and arg-lys pairs, the arg residues were replaced by homoarginine (har) kenes international leprince , f. cavelier , p. gandolfo , m. diallo , h. castel , l. desrues m new bradykinin analogues modified with -aminocyclopentane- -carboxylic acid effect on rat blood pressure and rat uterus o. labudda-dawidowska , d. sobolewski , m Śleszyńska , i derdowska synthesis and heparin binding sites identification f. baleux , f. arenzana-seisdedos chemokines are small proteins involved in numerous biological processes (inflammation, immunity, morphogenesis, tissue repair, and tumour development) the general goal of our project is to elucidate the role that hs plays in vivo in the physiologic and pathologic activities of the chemokine cxcl /stromal derived factor- , and to characterise the molecular and structural determinants accounting for the interaction. three sdf isoforms, alpha ( aa), beta ( aa) and gamma ( aa) have been identified. we previously identified the major heparin binding site on sdf alpha and demonstrated the importance of hs/sdf interaction in hiv entry cell inhibition ( , ). sdf gamma amino acids sequence corresponds to the sdf alpha sequence extended by a c-ter amino acids sequence containing putative heparin binding sites. in order to determine sdf gamma heparin binding sites, wild type and mutants proteins were synthesised by stepwise solid-phase peptide synthesis using fmoc chemistry prusiner proc. natl. acad. sci here we describe xenome's drug development process for the chi family, conopeptide -mria[ ] of the predatory marine snail conus marmoreus, leading to a suitable drug candidate (xen ). xen is highly selective for the norepinephrine transporter (net) compared to other transporters, such as dopamine and serotonin, and inhibits net via an allosteric mechanism. xen is currently in a phase i/iia clinical trial for the treatment of severe pain. an intensive synthetic analogue and screening program around -mria, incorporating early stage animal data xen isomers were synthesized via selective disulfide bond formation to identify the active connectivity. data from alanine-scans, single amino acid mutations and probing of backbone interactions combined with the full d nmr structure, led to the development of a pharmacophore for xen . this model is refined from further studies where structure-activity relationships were developed utilising binding and functional assay data for a range of peptides anti-cancer drug design anti-cancer drug design published structural data for hdac-like protein, a bacterial enzyme sharing high homology to the hdacs in its active site, confirmed that this protein contains a zinc in the active site. for the discovery of specific hdac inhibitors, a number of hydroxamic acis and related compounds have been designed based on the ligating function to the zinc atom. the mechanism also involves an appropriate nucleophile in the active site. chlamydocin is a cyclic tetrapeptide on the other hand, we have been focusing the cyclic tetrapeptide to develop the potent and specific inhibitors of hdacs. in the present report, we employed the chlamydocin scaffold and successfully introduced the series of thioether as the functional group to the cyclic tetrapeptide. it is well argued that the strong inhibition of hdac requires the best combination of zinc ligand, capping group, and appropriate spacer between them jerusalem department of biological regulation department of organic chemistry department of biological chemistry, weizmann institute of science, rehovot, israel estrogen has a key role in the regulation of skeletal growth and maintenance of bone mass. the use of estrogen and selective estrogen receptor (er) modulators in treatment of osteoporosis is limited due to substantial risks for breast cancer. recently, we developed peptides having estrogen-like activity peptide emp- had no effect on bone growth in normal mice, and did not influence the ovx-induced bone-loss. we then developed a new µct methodology to evaluate uncalcified and calcified growth-plate parameters. in the ovx mice, peptide emp- reduced volume and thickness of the uncalcified growth-plate, a possible cause for the inhibition of bone longitudinal growth. based on a reported enhancement of er-in female mice during protein biosynthesis, after the release of the nascent polypeptide chain, ribosome recycling factor (rrf) disassembles the post-translational complex. rrf has been shown to be essential for bacterial growth. thus, we are attempting to design suitable compounds to inhibit the rrf function as candidates for new-type antibiotics. we have determined the structure of rrf with amino acid residues by nmr and x-ray analyses and shown that rrf has two domains domain i with three stranded helical bundles and domain ii with &beta;/&alpha;/&beta furthermore, we recently determined the structures of the s ribosome-rrf complex by cryo-electron microscopy and the s ribosome-rrf domain i complex by x-ray analysis using the results of these experiments, we elucidated the interaction profiles between rrf and ribosome and found that the cationic center consisting of three arginine residues on the surface of the helical bundle, which we have shown to be essential for the activity of rrf, is bound to helix - of s ribosomal rna. we synthesized the rna and peptide fragments around this interacting site and characterized them by physico-chemical analyses. the results of cd and biacore experiments to investigate the details of the interactions between them showed that a mer of rna fragment is bound to rrf <i>biochemistry</i> <b> </b> causes an increase of pain by inhibiting the opioid response [ ]. recent research has shown further that melancortin receptors, mainly subtype mc r, produce an increase in response to pain stimuli [ ]. based on this previous work, we are developing chimeric ligands which will be of benefit to therapeutic pain treatment with enhanced opioid efficacy by acting as agonists at opioid receptors and antagonists at both cck and melanocortin receptors cck (i) and melanocortin (ii) pharmacophores were overlapped by trp, and different profiles of opioid pharmacophores (iii) were linked to the n-terminal of the melanocortin pharmacophore (figure). the designed ligands showed moderate to high biological activity at all three receptors depending on their respective structures. design considerations and structure-activity relationships will be discussed in detail along with in-vivo assay results china synthetic exendin- is a -amino acid peptide that exhibits potent anti-diabetic and dose-dependent glucose-regulatory activity. exendin- is susceptible to degradation in plasma, so its activity is limited. our aim is to find sites in exendin- that are susceptible to cleavage and provide information for designing new exendin- analogues. in this study the stability of exendin- in human plasma was evaluated in vitro. exendin- was incubated in plasma at ℃, extracted with sep-pak octadecyl columns and subsequently analyzed using high performance liquid chromatography (hplc). the results showed that exendin- was slowly broken down in plasma with a half-life of . h. the degradation products were identified by quadrupole time of flight mass spectrometry (q-tof-ms) with electrospray ionization pharmacology of exenatide(synthetic exendin- ): a potential therapeutic for improved glycemic control of type diabetes one of the proposed solutions for the pharmacotherapy of obesity, a major health problem in the western world, is to regulate the biochemical pathways that control the metabolic balance in the body. the melanocortin pathway regulates energy balance by binding of the catabolic endogenic neuropeptide αmsh to its mc receptor and thus causes a decrease in food intake. we have synthesized a backbone cyclic peptide library, based on the minimal αmsh sequence phe -d-phe-arg-trp [ ], that activates the mc receptor. all the members of the library shared the same sequence analysis using colorimetric liposomes confirmed that bbc- penetrate the intestinal cells by the transcellular mechanism. moreover, bbc- have high metabolic stability to intestinal enzymes ( % recovery after hr). ec analysis showed that bbc- selectively binds and activate the mc and mc receptors (ec . ± . and . ± . respectively). oral administration of bbc- in mice showed reduces food intake melanocortin tetrapeptides modified at the n-terminus, his, phe,arg and trp positions department of experimental and health sciences graduate school of pharmaceutical sciences graduate school of agriculture graduate school of medicine consisting of amino acids, is a product of the metastasis suppressor gene kiss- . this c-terminally amidated peptide was identified as the endogenous ligand of an orphan g-protein coupled receptor metastin-gpr signaling may regulate gonadotropin secretion and negatively regulate cancer metastasis. it is interesting that activation of gpr signaling negatively regulates the function of sdf- -cxcr axis in cho and hela cell transfectants we conducted the structure-activity relationship (sar) study on kisspeptin- using the neuropeptide-derived rw-amide scaffold to identify five-residue peptide amides as novel gpr agonists equipotent to kisspeptin pro-), where aoe is ( s, s)- -amino- -oxo- , -epoxydecanoyl. in continuation of our study to design and synthesize analogues bearing a zinc ligand to develop potent inhibitors of histone deacetylase (hdac) inhibitors, we shifted the aromatic ring of phenylalanine at the aminoisobutric acid (aib) position and also at the imino acid position. the aim is to explore the interaction of cyclic scaffold with the rim of hdac paralogs. we replaced the epoxyketone moiety of aoe with sulfhydryl group, which is protected as disulfide hybrid, as zinc ligand. benzene ring was introduced to aib structure to design amino- -indane carboxylic acid and amino- -indane carboxylic acid. aromatic ring-containing imino acids, such as d-tic were also employed to replace d-pro. the cyclic tetrapeptides were profiled by the inhibition of hdac , hdac , and hdac in adult goats, however, the infection remains unapparent and the virus may cause abortion, vulvovaginitis or balanoposthitis. the use of a vaccine could provide a powerful tool for the control of cphv- infection. synthetic peptide-based vaccines have advantages of being selective, chemically defined and safe. in order to further localize immunogenic epitopes, glycoproteins b, c and d of cphv- were analyzed with several prediction programs. peptide conjugates incorporating t and b cell determinants in multiple copies in branched architecture are better immunogens for the preparation of goat vaccines, we synthesized peptide conjugates bearing t cell epitope on the n-terminal of the core. b cell epitopes were conjugated via a thioether bond on the ne-amino group of four choroacetylated lysine residues of the core. elisas confirm that the b cell epitopes and the conjugates t celltetratuftsin induce epitope-specific and antibody responses two recorded electrodes implanted bilaterally. eeg recorded after the mirror focus was arises. trh applicated intranasal in ultra low doses ( - m and - m) or intravenously in high doses ( mg, mg, mg). for eeg registration and analysis the computer system conan was used and new modification of fractal analyze of quantized eeg. results: the synchronization of epileptic activity between primary and mirror focuses observed on the third day after operation. intranasal application of trh induced reduction of spontaneous focal epileptic activity as in primary cobalt damage focus as in the mirror focus more than h. the inhibition of mirror focus was more expressed. quite the contrary intravenous trh administration provocated the epileptic discharges in both local focus. the intense synchronized generalized activity was record during - min deraos , t.v. tselios , i. mylonas , g.n. deraos it is known that cyclic peptides are more stable in enzymatic degradation and conformationally restricted compared to linear. the cyclization was achieved using o-benzotriazol- -yl-n,n,n',n'-tetramethyluronium tetrafluoroborate (tbtu) and -hydroxy- -azabenzotriazole, , , collidine allowing fast reaction and high yield final product. the purification was achieved using reversed phase high performance liquid chromatography (rp-hplc) and the peptide purity was assessed by analytical hplc and mass spectrometry (esi-ms). the synthesized cyclic plp analogue was found to exhibit lower agonist eae activity compared to linear plp - epitope in sjl/j mice. this implies that the conformation of cyclic analogue does not trigger autoimmune reaction in the central nervous system and therefore encephalomyelitis the netherlands department of experimental and health sciences on the other hand, in both central and peripheral nervous system, cck acts as neurotransmitter. recently, cck is focused at modulation effects on feeding especially. in this study, we tried to establish a sensitive and specific enzyme immunoassay (eia) for detecting cck and to investigate the effect of some dopamine receptor antagonists using this eia, we measured plasma cck-like immunoreactive substance (is) levels in five healthy human subjects after single oral administration of some prokinetics. the minimum amount of cck detectable by our eia system was . pg/ml, and the ic of the calibration curve was pg/ml. we revealed that domperidone and itopride caused significant decreases in plasma cck-is levels but metoclopramide and sulpiride did not. we established a sensitive and specific eia for cck. furthermore pro-pro-phe-phe-), isolated from linseed oil [ ], possesses a strong immunosuppressive activity comparable at low doses with that of cyclosporin a [ ]. it has been postulated that the tetrapeptide sequence pro-pro-phe-phe is important for biological activity of cla on the basis of this information we have synthesized a series of cla analogues in which the alpha-proline residue was replaced by beta -iso-proline and beta -homo-proline residues, respectively (fig. ). the synthesis of titled beta-amino acids has been achieved according to the literature procedure italy synthetic peptides are largely used as antigens in solid-phase immunoenzymatic assays (elisa) for recognition of antibodies (abs) in biomedical research and, most importantly, in the set up of diagnostic methods. it is well known that the method of peptide immobilization on the solid support is very important for a correct ab recognition atherosclerosis in patients infected with helicobacter pylori (hp) we synthesized appropriate oligopeptides immobilized on cellulose via n-or c-termini, using standard -alanine linkers as well as a new linker, developed for this particular studies glc)ghsvflapygwmvk) we found the strongest recognition when the peptide was linked to the cellulose support via the c-terminus. however, in the case of ureb f hp urease smallest epitope (sikedvqf), and epitope ub- ( - hp fragment: chhldksikedvqfadsri) the strongest reactions with sera of atherosclerosis suffering patients were obtained for n-terminally anchored peptides the synthetic dipeptide gamma-d-glutamyl-l-tryptophan (scv- ) has been shown to stimulate t-lymphocyte differentiation and specific immune responses, and enhance il- and inf-gamma production. due to this preferential activation of th cytokine production, scv- may show utility in treatment of infectious diseases cba mice at a dose of , µg/kg. the same animals were used for all three methods of administration with a dosing interval of weeks. blood samples were taken from the right retro-orbital sinus. for determination of the scv- concentration in blood samples, an "eia-scv- " competitive solid-phase immuno-enzyme assay was developed (loq ng/ml) with a mean residence time of minutes. minutes postdose, indicating very rapid absorption. mean concentrations then declined and were measurable through . and hours postdose (mrt and minutes, for i/p and p/o, respectively). the estimated bioavailability of scv- after i/p and p/o administration was almost equivalent gastrin- (g ) is a peptide which promotes gastric acid secretion, cell proliferation, and occasionally gastrointestinal cancer in the gastric antrum. g also promotes the growth of cancerous colonic epithelial cells, but the cck /gastrin receptor, which mediates its activity, is largely not expressed on such cells. instead, our previous studies have shown that some other receptor mediates stimulation of proliferation of dld- and ht human colonic carcinoma cells by ncarboxymethyl gastrin (g gly) at namomolar concentrations and inhibition at micromolar concentrations, indication separate binding sites. we have shown previously that g ( - )-oh stimulates cell proliferation of ht- cells - )-nh , in order to determine their selectivity for and activation of the putative proliferation-stimulatory receptor. the results revealed that g ( - )-oh is not selective for a single receptor, but binds both sites as do g and g gly. g ( - )-nh promotes dose-dependent and non-biphasic proliferation of dld- cells and binds a single receptor with low affinity. m comparative study of ige-binding activity of synthetic peptides rnwd and rnwdvyk v th involvement of l-name in the antinociceptive effects of newly synthesized analogues of tyr-mif- peptide in rats tyr-mif- is able to interact with opioid receptors with a higher potency at m sites as well as to its specific non-opiate receptors in the brain. nitric oxide and tyr-mif- `s are potent modulators of opioid activities. involvement of no in nociceptive effects is well documented in various physiological and pathological processes in the cns. l-name when administrated i.c.v. or systemically exhibit antinociceptive activity in rats as evaluated by the pp test. in the present study, we investigated the involvement of l-name in the antinociceptive action of newly synthesized analogues of tyr-mif- peptide: nα-(me)tyr-mif- , d-tyr-(me)-mif- , tyr(cl )-mif- and tyr(br )-mif- . the experiments were carried out on male wistar rats ( - g). the changes in the mechanical nociceptive greece paclitaxel is one of the most important anticancer drugs used mainly in treatment of breast, lung, and ovarian cancer and is being investigated for use as a single agent for treatment of lung cancer, advanced head and neck cancers, and adenocarcinomas of the upper gastrointestinal tract. however, the development of resistance to paclitaxel, the side effects and low solubility of this drug remain major obstacles for its optimal use in the clinical practice. in this work, we present the synthesis of various analogues in which paclitaxel is covalently bound to peptides or as multiple copies to synthetic carriers. these peptide-paclitaxel derivatives possess greater solubility in water, could be suitable in producing anti-paclitaxel antibodies and inhibit the proliferation of human breast, prostate and cervical cancer cell lines. although, no major differences were found concerning the extent of the antiproliferative effect between various paclitaxel derivatives and paclitaxel, the analogue with four molecules of paclitaxel covalently bound to synthetic carrier [ac-(lys-aib-cys) -nh ] when used at low concentrations inhibited cell proliferation more potently than paclitaxel th involvement of the histaminergic system in the nociceptin-induced pain-related behaviors in the mouse spinal cord the purpose of the present study was to determine whether histamine-containing neurons in the spinal cord are involved in nociceptin-induced behaviors in mice. the i.t. injection procedure was adapted from the method of hylden and wilcox. immediately following the i.t. injection, the time spent for nociceptive behaviors including scratching, biting and licking were measured. the i.t. administration of nociceptin resulted in nociceptive behavioral responses, which were eliminated by the i.t. co-administration of opioid receptor like- (orl- ) receptor antagonists. the nociceptive behaviors were significantly attenuated by the i.t. co-administration of the h receptor antagonists, but not the h receptor antagonists. i.t. co-administration of the h receptor antagonist significantly increased the behavioral responses, whereas the behavioral responses were completely attenuated by the i.t. co-administration of the h receptor agonist. an antiserum against histamine injected i.t. reduced the nociceptin-induced behavioral responses. the same result was observed by i.p. pretreatment with histidine decarboxylase inhibitor. in conclusion, i.t.-administered nociceptin elicits the orl- receptor-mediated nociceptive behavioral responses. the activation of the orl- receptor by nociceptin may induce the release of histamine in previous studies, we demonstrated that highly constraint cyclic (s,s)-cxaac-containing peptides inhibit platelet aggregation and fg binding [ , ]. cyclization reduces the allowed conformations, of both the backbone and the side chains, and possibly induces a favourable for the biological activity orientation of the charged side chains. conformational studies revealed that orientation of the charged side chains toward the same side of the molecule increase the anti-aggregatory activity of the inhibitor. in this work we present the synthesis and the inhibitory activity of new cyclic compounds. for the design of the studied compounds we combined the available information from the -cdc-containing inhibitors and the gpiib - (ymesradr) sequence which has been shown to inhibit the adp induced human platelet activation however, its pharmacological effects and physiological functions are still unclear. the present study was designed to characterize the nociceptive behaviours induced by intrathecal (i.t.) administration of hk- in mice. the i.t. administration was made in conscious mice according to the method described by hylden and wilcox. immediately after the i.t. administration, the accumulated time for nociceptive behaviours was measured for min. the i.t. administration of hk- dose-dependently produced characteristic nociceptive behaviours consisting of scratching, biting and licking, which peaked at - min and almost disappeared by min after injection. the subcutaneous pretreatment with morphine dose-dependently attenuated the hk- -induced nociceptive behaviours. the nociceptive behaviours elicited by low-dose of hk- were significantly inhibited by i.t. co-administration with nk receptor antagonist, however, the nociceptive behaviours elicited by high-dose of hk- were not affected by i.t. coadministration with nk receptor antagonist. on the other hand, nmda receptor antagonists significantly suppressed both high-and low-doses of hk- -induced nociceptive behaviours in a dose-dependent manner. these results suggest that the nociceptive behaviours induced by low-dose of hk- may be mediated through both nk and nmda receptors, whereas high-dose of hk- may induce the nociceptive behaviours through nmda receptor the bacterial lp are strong modulators of the innate immune system. until recently, it was generally assumed that triacylated lp, like the synthetic pam cys-sk , are recognized by tlr /tlr heteromers, whereas diacylated lp, like fsl- , induce signalling through tlr /tlr heteromers. contrary to this model, we could show that depending on the peptide moiety, diacylated lp also signal in a tlr -independent and tlr -dependent manner. the aim of this study was to analyse more closely the structural basis of this heteromer usage. the synthesis of lp was carried out by fully automated solid phase peptide synthesis and fmoc/tbu chemistry on tcp or rink amide resin. information on the structural factors determining the tlr /tlr versus tlr /tlr heteromer usage was obtained by testing of ligands with cells obtained from tlr , tlr , and tlr -deficient mice. when stimulating b-lymphocytes of wild-type mice we found that ester-bound long-chain fatty acids are necessary to induce considerable responses. for triacylated lp with long chain length ester-bound acyl residues (like pam c-ssnask ) the response in tlr -deficient cells was only slightly decreased, whereas for lp with short length ester-bound fatty acids (like pamoct c-ssnask ) the response was completely abolished. in summary, a tri-acylation pattern is necessary but not sufficient to render an lp tlr -dependent and a di naples department of organic chemistry "ugo schiff sera from patients suffering from autoimmune disorders often contain multiple types of autoantibodies, some of which can be exclusive of a disease and thus used as biomarkers for diagnosis. identification of these autoantibodies, as disease biomarkers, should be achieved using native antigens in simple biological assays. however, post-translational modifications, such as glycosylation, may play a fundamental role for specific autoantibody recognition. in line with these observations, we previously described synthetic glycopeptides able to detect high autoantibody titers in sera of patients affected by multiple sclerosis, an inflammatory, demyelinating disease of the central nervous system. we also demonstrated that glycopeptides able to reveal high antibody titers in multiple sclerosis sera are characterized by a type i we describe here the result of a conformationally driven rational design exercise, which led to the preparation of new, optimized glycopeptides endowed with enhanced antigenic properties. most importantly, the same approach, based on structure alignment, was used to shed light on the native antigen(s), target of pathogenetic autoantibodies involved in demyelination processes vitro and in vivo evaluation for cholecystokinin-b receptor imaging istituto nazionale per lo studio e la cura dei tumori ome (f )<br> (aib, α-aminoisobutyric acid) to investigate its binding properties to tb(iii) ions. according to our published spectroscopic results f populate a set of ordered conformations involving /α-helical segments and compact structures generated by the formation of a turn around the flexible gly -gly central motif. cd experiments showed that the binding of tb(iii) to f gives rise to a structural transition of the peptide chain from a helical to a folded conformation. peptide binding is also responsible for the dramatic increase in the tb(iii) fluorescence intensity, suggesting that the tb(iii)/f complex may represent an interesting system for imaging applications or bioanalytical sensing the kda protein of mycobacterium tuberculosis provokes specific immune response, therefore related epitope peptides and peptide-conjugates can be considered as potential diagnostics. in our previous study we have determined the functional human t-cell epitope within the - region. based on this we synthesised two groups of peptides: a) nand c-terminal alanine and beta-alanine elongated variants of the - epitope and b) - peptides with alanine substitution at different position according to the hla dr and tcr binding sites. peptides were prepared by solid phase synthesis using boc/bzl or fmoc/tbu strategy. the homogeneity and the primary structure of peptides were checked by analytical rp-hplc, amino acid analysis and esi-ms. the t-cell stimulatory activity of the compounds was investigated using in vitro assays (proliferation and ifn-gamma production) on the - epitope specific human t-cell clones and pbmc (peripherial blood mononuclear cells) from patients and healthy (ppd+, ppd-) subjects. the effective peptides were conjugated to branched polypeptides with polylysine backbone (sak, eak), tetratuftsin derivative (h-[thr-lys-pro-lys-gly] -nh ) and lysine dendrimer (h-lys-lys(h-lys)-arg-arg-beta-ala-nh ) (map) carrier via thioether bond formation. the subtitution degree of the conjugates was determined by amino acid analysis. pbmc and human t-cell clones were stimulated with the free peptide alone or with peptide-conjugates containing an equimolar amount of peptide or with a mixture of free peptide and carrier italy we demonstrated, for the first time, that an aberrant post-translational modification (ptm, n-glucosylation) is possibly triggering autoantibody response in multiple sclerosis. this was possible because of a "reverse approach", which led to csf (glc), a structure based designed glycopeptide, as the first multiple sclerosis antigenic probe accurately measuring high affinity autoantibodies (biomarkers of disease activity) in sera of a statistically significant patients' population universal peptide scaffold" to be modified with a series of glycosyl amino acids (different in sugars and linkages), in the aim of developing personalized diagnostic/prognostic tests. the csf beta-turn structure, exposing at the best the aberrant ptm specific for antibody-mediated forms of other autoimmune diseases, will lead to a family of antigenic probes to be used in diagnostic this information is encoded by the distribution of the electron-ion potential (eiip) of amino acids along the sequence and is represented by the frequency components in is. proteins with the same biological functions or interacting proteins (e.g. antibody/antigen) share the information corresponding to the common frequency components in their iss. investigation of the hiv- envelope glycoprotein gp , as a model system for hypervariable proteins, revealed that this information is strongly conserved and is not significantly affected by natural mutations. the c-terminus of the second conserved region (c ) of gp , encompassing ntm peptide is important for infectivity and neutralization of hiv- , while human natural anti-vasoactive intestinal peptide (vip) antibodies reactive with gp play an important role in control of hiv disease progression. ntm/vip multiple copies were coupled to an artificial sequential oligopeptide carrier for developing an immunoassay (elisa) as a reproducible, reliable and sensitive tool for the detection of anti-ntm/vip derived antibodies these peptides have been utilized in an immunopeptidometric assay for specific measurement of active, noncomplexed psa. however, this assay has not been sensitive enough for the measurement of active psa in clinical samples. therefore, we aimed to develop an improved assay utilizing the same principle as previously, but using a more sensitive detection method based on proximity ligation assay. methods: in the assay, psa is first captured on a solid phase by a psa antibody czech republic rapidly increasing knowledge of new gonadotropin-releasing hormones (gnrh)of different species of the animal kingdom induces the need to prepare new synthetic derivatives and fragments of these peptides with higher potency and metabolic stability and suitable for the formulation of new immunogens. the species related differences in the sequence of the native mammalian gnrh pglu-his-trp-ser-tyr-gly-leu-arg-pro-glynh concern predominantly the positions , and , particularly tyr in position is replaced for his or leu, leu in position by val or trp, and arg in position is substituted by lys, ser, asn or gln. several gnrh derivatives with with the above substitution and gnrh fragments were prepared by solid phase peptide synthesis and purified by rp-hplc. purity of the synthetic peptides was checked by capillary zone electrophoresis (cze); peptides were analysed as cations in acidic backround electrolytes (ph . - .quantitative analyses for determination of their effective electrophoretic mobilities and the estimation of their effective charges.supported by grant of ministry of agriculture of cr-nazv qf by rants of ga cr nos we use peptaibiomics for the structural determination of peptaibiotics from fungi grown on single agar plates thus avoiding time-consuming isolation and purification procedures. the method comprises fast and effective solid-phase extraction followed by on-line rp-hplc coupled to tandem esi-ms. here we present a survey of the peptaibiome of hypocrea species. in extracts of hypocrea semiorbis, h. vinosa, h. dichromospora, h. gelatinosa, h. nigricans, h. muroiana and h. lactea a multitude of short and long-chain peptides containing aib could be characterized. the formation of new and known peptaibiotics could be established by comparison with sequences stored in data bases japan process scale rp hplc purification of peptides and proteins is increasingly important in bio-pharmaceutical production. besides selectivity, other crucial factors are loadability, recovery loadability is believed to depend on the surface area of the packing material. consequently, smaller pores providing larger surface area should lead to increased loadability. this principle is misleading in the case of large molecules, because they cannot penetrate smaller pores. therefore the chromatographically accessible surface area has to be taken into account. recovery problems like irreversible adsorption or aggregation are frequently caused by hydrophobic surface properties of ods phases. the less hydrophobic c is a substituent to avoid considerably these problems. however c is less durable than ods under extreme acidic conditions. our new proprietary c modification technology combined with a perfect end-capping minimizes the presence of residual silanol groups and protects the silica surface sp- -c -bio demonstrates high mechanical stability by no obvious alteration of back pressure and particle size after repeated packing cycles in dac columns. by overcoming the common weaknesses of the conventional c rp silica phases, daisogel sp- -c -bio opens new avenues for process scale separation of peptides and proteins. m determination of peptide: protein molecular ratio in conjugates by seldi-ms method synthetic peptides are widely used as antigens in various research and practical areas of biology and medicine. peptides with molecular masses < kda should be conjugated with carrier proteins in order to ensure their immunogenicity and protect from proteolysis. in these cases the comparison of peptide immunogenicities and immunotest system development should be performed having in mind exact peptide-to-protein ratios. conjugates of peptide fragments of hepatitis c virus envelope protein e with ovalbumin, bovine serum albumin, and myoglobin were prepared using glutaraldehyde (ga), m-maleimidobenzoyl n-hydroxysuccinimide ester, dimethyl suberimidate (dms), -ethyl- -( -dimethylaminopropyl)-carbodiimide as conjugating reagents. the rough evidence of the peptide-protein conjugate formation was obtained by page. the exact peptide:protein molar ratio was estimated in all conjugates by seldi-ms. almost all conjugates had oligomeric structures due to the formation of intermolecular linkages between proteins. the peptide : protein molar ratio in conjugates varied from : to , : . conjugates obtained with the ga were more diversified in the number of peptide molecules linked to carrier proteins (peptide:protein ratios ranged from : to : ) than other conjugation reagents mazur-marzec poland posttranslational modifications (ptms) like phosphorylation, acetylation, or methylation have been shown to play a significant role in directing the function of various proteins [ ]. in eukaryotes, most of proteins have been shown to be posttranslationally regulated by a variety of different modifications. many effects of ptms include a change of enzymatic activity capillary electrophoresis (ce) has been used to study electrophoretic behavior of ptm-peptides gal-nh , gal( - )-nh by capillary electrophoresis. using a phosphate buffer most of ptm-peptides were poorly separated at acidic or neutral ph. the best results were obtained using trifluoroethanol containing separation buffers. optimization of ce separation of maps of peptides containing ptms should allow to detect ptm-proteins and characterize their role in the living cell. comparison of modification events occurring in diseased and healthy cell may iran the purpose of this study was to use the application of multiplex reverse transcription polymerase chain reaction(rt-pcr) assay for detecting the two most common leukemia translocations t( ; ) and t( ; ) in childhood acute lymphoblastic leukemia in iranian children. cases of leukemia patients were screened with the rt-pcr assay. this assay will identify the all type bcr-abl transcripts encoded by the t( ; ) and all described variants of the e a-pbx transcript encoded by the t( ; ). rna was isolated from leukocyte cells of patient's samples. through the construction and optimization of specific primers for each translocation,we have been able to set up multiplex rt-pcr reactions.then pcr products was electrophoresed on agaros gel and were compared with size markers and expected fragtments key words: acute lymphoblastic leukemia -multiplex rt-pcr tu study on the syntheses and lc/esi-ms analyses of the glutathione conjugates of bile acids t. wakamiya , m. sogabe a carboxylic acid-containing drug, is metabolized to a glutathione (gsh) conjugate in vivo, and the conjugate is excreted in human urine [ ]. although bile acids, compounds with carboxylic acid in molecules, are also expected to form gsh conjugates in liver, no evidence is so far obtained to confirm such metabolism, since there are no suitable standard samples for the research. in the present paper we report the syntheses of the gsh conjugates of main bile acids in human, i.e., cholic acid (ca), chenodeoxycholic acid (cdca), deoxycholic acid (dca), ursodeoxycholic acid (udca) and lithocholic acid (lca) as shown below, and the detailed analyses of these synthetic conjugates by means of linear ion trap lc/esi-ms. furthermore, the evidence for conversion of cholyl adenylate [ ] and ca-coa thioester into ca-gsh conjugate will be presented these peptides do no exhibit such strong side effects as csa, but their practical application is hindered because of their poor solubility in water. the - fragment of tat protein and its analogs, including oligoarginine sequences, are known for their unusual ability to cross cell membranes, skin, and blood-brain barriers. moreover, these peptides are able to transport other substances into cells. this strategy was successfully applied in cases of csa, taxol, and other drugs to improve their bioavailability. now we have synthesized a series of analogues of cyclolinopeptide a, clx, and the immunosuppressive fragment of ubiquitin, covalently bound to the cell-penetrating fragment of tat and its analogues. the ability to cross the biological membranes and the immunosuppressive activities of these conjugates were tested. the conformation of the peptides was determined by circular dichroism methods: we used fluorescein-labelled cationic cell-penetrating peptides and analyzed the uptake efficiency (flow cytometry) as well as the intracellular distribution (confocal laser scanning microscopy). the bioactivity of a proapoptotic cargo-peptide, delivered into the cells either via electroporation or via cpps was quantified using a caspase- activity assay and cellular assays. to address the integrity of cpps during their trafficking, a fluorescent double-labelled antp peptide was designed and used as an intracellular fret-sensor. results: endocytosis-mediated uptake of the cpp-cargo conjugate led to a significant reduction of cargo bioactivity compared to its direct transfer via transient membrane permeation. this finding was related to the sequestration of peptides within endocytic vesicles but also, in the case of the tnf response, to the induction of receptor internalization during cell entry. moreover, during endolysosomal passage peptides undergo significant proteolytical degradation. conclusions: the endocytosis-dependent uptake mechanism of cholesterol pullulan (cp), in which maltose moieties are partially modified by cholesterol, is unique in forming self-assembled nanoparticles ( - nm) in water. combination of these characteristics is considered to be promising for development of effective non-viral vectors without toxicity. a conjugate of hiv-tat and cp was synthesized and its gene expression efficiency was evaluated. fully protected hiv-tat-( - )-cys(snm)-gly-nh-r was obtained by conversion of the corresponding cys(acm) peptide which was synthesized by the solid-phase method [snm: (n-methyl-n-phenylcarbamoyl)-sulfenyl] [ ]. the sulfhydryl function was introduced to the hydroxyl groups of cp by acylation with trt- -mercaptopropionic acid followed by acid treatment. resulting -mercaptopropionyl-cp was coupled with cys(snm) peptide to form disulfide bridge and the protecting groups of the peptide were removed to give the cp-tat conjugate. cp-tat and pcmv-luc complex was transfected into cos cells and luciferase activity was analyzed after h. cp-tat elicited remarkable cytoplasmic luciferase activity and low toxicity this finding provides a possibility to use gnrh-iii as a targeting moiety for intracellular drug delivery. therefore we have prepared methotrexate and doxorubicine conjugates of gnrh-iii. the drug molecules were attached to the lys side chain in position of gnrh-iii by thioether bond formation through the gflg spacer elongated with cys either at the c-or n-terminus. since we found earlier that the dimer derivative of gnrh-iii was more effective, new dimer derivatives with a combination of antitumour agents were also prepared. branched gnrh-iii derivative (pyrhwshdwk(clac-glfgc(acm))pg-nh ]) was synthesised by spps. the drug molecules were attached to this compound by thioether bond and finally disulfide bridge was formed between two peptide chains. the cytotoxicity of new derivatives was characterised by mtt test th oligopeptide antifungals are exceptionally active against multidrug-resistant yeast previous studies have demonstrated that longer sirnas that are processed by dicer can result in more potent knockdown than the corresponding standard -mer sirnas. dicer-substrate - -mer sirnas were conjugated with different structural classes of peptides and their cell uptake properties evaluated. peptides were conjugated to the ' end of the sirna sense or antisense strand via a thioether bond under denaturing conditions to prevent aggregation and precipitation. the ability of conjugates to translocate fluorescently-labeled sirna across the plasma membrane was evaluated by flow cytometry. the results indicate that some peptides can mediate higher efficiency uptake of sirna into cells compared with lipofectamine or cholesterol-conjugated sirna. the peptide-sirna formulation with -mer sirna conjugates showed higher knockdown of tnf-alpha mrna and protein levels in activated human monocytes in vitro compared to the conjugated -mer sirna species. the products resulting from in vitro digestion of peptide-conjugated rna duplexes with recombinant human dicer were identified using esi-ms and consisted predominantly of the desired -mer sirna several peptides containing the sequence arg-gly-asp (rgd) were studied and developed for their nanomolar affinity to the membrane receptor alfa v beta and alfa v beta integrins, which are over-expressed by endothelial cells during proliferation and by tumor cells. to improve the pharmacological profile of some camptothecin derivatives (cpts), five conjugates were designed, where the cytotoxic drugs were covalently attached to the rgd peptide analogues for preferential uptake into tumor cells. the peptides to be used have been selected among a series of new pentacyclic peptides bearing at -position a trifunctional pseudoamino acid with a carboxy-terminal side-chain. peptide analogues showing the highest affinity to alfa v beta and alfa v beta integrins were coupled with cpts at different positions. the conjugates have been optimized for binding to the receptors, proteolytic stability and an overall improvement in tumor selectivity. the nature of the linkage between rgds and cpts has a major impact on stability and biological activity of the conjugates. the conjugates with amide bond, but not those with ester bond, are sufficiently stable and show in vitro antitumor activity against a and a cell lines combination of amaranth protein with other plant proteins (cereals) enables to formulate the composite protein (near to milk or beef protein, but exclusively on vegetal basis). it is shown on graphs. the aim of the projects' proposals is a development and realization of the technology for fractionation of amaranths defatted flour product is a top protein obtained by removing starches and next polysaccharides decomposed on soluble monosaccharide by specific enzymes. there are shown the chromatograph measuring. we can see complete disintegration of starch and the unchanged proteins. the separate solution monosaccharide is usable for others fermentative processes or as a nutrient solution for yeasts there are description methods for isolation amaranth protein -extraction processes, enzymatic removal starch. the product is a isolate protein rich in essential amino acids. the waste monosaccharide solution was used to production yeasts biomass rich in proteins vitamins amaranth protein isolate have high nutritional value and can be used as food ingredients, for functional, probiotic formulation to begin with, we made epitope mapping with the highly sensitive spot array method ( ) in order to study antigenic regions of parvovirus b vp and vp capsid proteins. epitope mapping identified highly reactive, immunodominant early epitope on parvovirus surface that centered to kyvtgin residues of vp . in the subsequent phases we developed the kyvtgin epitope type specific (ets) igg serodiagnostics. a correlation between enhancing igg avidity to b capsid and a transient reactivity with the point-of-care kyvtgin peptide was clear. together the two assays enhanced the value of early diagnosis of b infections ( ) acute phase-specific heptapeptide epitope for parvovirus b diagnosis synthetic peptide arrays on membrane supports-principles and applications human parvovirus b infection during pregnancy--value of modern molecular and serological diagnostics laboratory of peptide & protein chemistry & biology department of organic chemistry "ugo schiff" and cnr-iccom department of chemistry department of pharmaceutical sciences glc) able to recognize specific autoantibodies in multiple sclerosis (ms) patients' sera has been developed by the laboratory of peptide & protein chemistry and biology [ and bio-plex suspension array system, biorad. the biosensor technology and bio-plex suspension array system will offer advantages such as rapid analysis, and high sensitivity for a high throughput screening. immobilisation will be based on different strategies that are anchoring the synthetic antigen on different solid supports such as polystyrene well plates (elisa) optimisation of the different techniques was performed with anti-csf (glc) autoantibodies isolated using affinity chromatography from ms patients' sera. the analytical parameters such as specificity, sensitivity, and matrix effect were evaluated. the different technologies have been used for a high throughput screening of ms sera which control specific cell adhesion. here we discuss the route for preparation of amphiphilic block copolymers composed of hydrophobic polylactide and hydrophilic polyethylene oxide (peo) blocks, carrying various cell-adhesion oligopeptide sequences at the end of peo block. fully protected peptide fragments were prepared by solid-phase peptide synthesis by using fmoc strategy and chlortrityl resin. the side-chain protected peptides were cleaved from resin by % hfip solution in dcm. the copolymers peptide-polytehyleneoxide were prepared by coupling of the activated peptide fragments with α-amino-ω-hydroxy-peo in dmf using pypop as an activation reagent. subsequently, the polylactide block was grafted to the ω-hydroxy end group of the peptide-peo copolymer via a controlled rop polymerisation of lactide r)- -aminocyclopentanecarboxylic acid (acpc) and beta-methylphenylalanine (beta-mephe) were designed and synthesized to obtain more potent and selective mu-opioid receptor ligands with higher stability against proteolytic enzymes. we have prepared the peptides by spps methods using racemic amino acids. the diastereomeric peptides were separated by hplc. the configuration of the unnatural amino acids in the peptides was determined by chiral tlc using enantiomeric standards. radioligand binding assays and in vitro gpi and mvd assays indicated that several analogues showed high, subnanomol affinity and high selectivity for mu-opioid receptors having agonist or antagonist properties. the incorporation of alicyclic amino acids into the endomorphins resulted in enzyme resistant peptides. the most promishing analogues (dmt-pro-phe-phe-nh and tyr-( s, r)acpc-phe-phe-nh ) were labeled with tritium using precursor peptides containing dehydroproline or dehydro-( s, r)acpc amino acids and tritium gas and pd/baso catalyst. the novel peptides and their radiolabelled analogues with high specific radioactivity ( . - . tbq/mmmol) have become useful pharmacological and biochemical tools for the opioid research iran background and aims: injectable drug delivery based on polymer solution platforms has gained in resent years, particulary for protein-based therapies. the influence of polymer molecular weight (rg h, rg h) on the morphology, erosion of matrices and also on their in-vitro drug release behavior over a period of days was assessed for leuprolide acetate in this study. methods: each formulation was composed of % (w/w) polymer and % (w/w) leuprolide acetate dissolved in nmp. release studies were performed in a home-made diffusion cell at °c. the polymer erosion was studied using two different methods as follows. (a): l-lactic acid detection (b): ph change study. the morphology of the matrices was then analyzed by scanning electron microscope as is shown, the lower molecular weight polymer formulation shows higher porosity and pore diameter due to a rapid phase inversion phase i) can be divided into three more phases with different release rates. results showed that burst effect for rg h, %, was significantly (p< . ) higher than rg h ( %) italy fabrication of photocurrent-generating systems based on bioinspired organic-inorganic hybrid materials is currently of great interest. more specifically, the photoelectronic properties of nanometric films formed by peptide self-assembled monolayers have been actively investigated. in this work interdigitated gold microelectrodes were modified by covalently linking a hexapeptide ester functionalized by a lipoic acid (lipo) at the n-terminus. the peptide chain [lipo-(aib) -trp-aib-otbu] comprises five α-aminoisobutyric acid (aib) residues and one trp, a fluorescent amino acid with strong absorptions in the uv region. due to the very high percentage of conformationally constrained aib residues in the chain, the peptide adopts a rigid - -helical structure. cyclic voltammetry measurements indicate that the peptide forms a homogeneously and densely packed monolayer on the gold surface, while current/voltage curves exhibit interesting rectifying properties of the peptide sam. photocurrent generation experiments, performed on the peptide-layered microelectrode, show peculiar modifications of the spectrum. at nm a notably higher photocurrent/voltage response was observed for the peptide-modified electrode, suggesting that a photoinduced electron transfer process from trp to gold does take place with high efficiency this may lead to randomly orientated enzymes and subsequently limited activity. the aim of this work is to selectively activate enzymes at their c-terminal position in order to allow specific immobilisation. we chose akr a , an enzyme of the aldo/keto reductase superfamily, for the synthesis of an artificially monolabeled redoxprotein. akr a is a monomeric enzyme and catalyzes the nadph dependent reduction of aliphatic/aromatic ketones and aldehydes. to produce monofunctionalized enzymes we applied the strategy of expressed protein ligation (epl). accordingly, we used the impact®-system and cloned the aldo/keto-reductase as fusion protein with an additional intein/chitin binding domain. through intein mediated splicing we could produce c-terminal thioester of the akr a . in the next step, the thioester was coupled to a biotin containing peptide by native chemical ligation. this specifically modified enzyme was immobilised on avidin coated surfaces. the attachment on the surface was tested by tryptic digestion, followed by maldi-tof-ms analysis since safe organic solvent waste disposal is an important environmental problem, we aimed to perform peptide synthesis in water. we have reported solid phase peptide synthesis in water using water-soluble n-protected amino acids, such as -[phenyl(methyl)sulfonio]ethoxycarbonyl and -( -sulfophenylsulfonyl)-ethoxycarbonyl amino acids. following to study on water-soluble n-protected amino acids, we developed a new technology based on nanochemistry for solid phase peptide synthesis in water. the new technology is based on coupling reaction of suspended nanoparticle reactants in water. fmoc-amino acids are used widely in peptide synthesis, but most of them show poor water-solubility. we prepared well-dispersible fmoc-amino acid nanoparticles in water by pulverization using a planetary ball mill in the presence of poly(ethylene glycol) (peg). the size of fmoc-amino acid particles was - nm. to evaluate the utility of this technique, leu-enkephalin was prepared using the nanoparticulate fmoc-amino acids on a peg-grafted rink amide resin in water supramolecular structures formed from n-lipidated oligopeptides immobilized in the regular pattern on the cellulose surface are able to bind ligand molecule, thus acting like artificial receptors. due to the conformational flexibility of lipidated oligopeptide chains, the supramolecular structure is highly flexible, forming the cavities with the shape and prosperities adjusted most effectively to requirements of the guest molecule. structural requirements for a peptide providing the most efficient fit the guest molecules are not known, therefore an array of the artificial receptors have been synthesized and used in the studies. thus, even in the case, when the single receptor in an array does not necessarily have selectivity for a particular analyte, the combined fingerprint response can be extracted as a diagnostic pattern visually, or using chemometric tools in order to improve the sensitivity of the competitive binding and to study the mechanism of molecular recognition, experiments involving fluoresceine and fluoresceine marked acp fragment were performed. we found that λmax and intensivity of fluorescence depends on the structure of the peptide motif and lipidic fragment of receptor this mts was linked with dhhp- by disulfide bond, and the new molecular was named mts~dhhp- . the peroxidase activity of mts~dhhp- ( . x u•µmol- ) was tested and similar to that of mp- ( . x u•µmol- ). mts~dhhp- coated with quantum dots (qds) [ ] were observed to accumulate into neonatal rat cardiomyocytes (nrcms) of wistar rats and co-localized with mitotracker red in mt. these results suggest that mts~dhhp- is an excellent apx mimics and may have potential proceedings of the twenty-eighth european peptide symposium, kenes international, israel, , . references: elastin-based polypeptide, poly(val-pro-gly-val-gly), undergoes self-assembly called coacervation, in which microcoacervate droplets with approximately nm diameters are formed [ ]. nanoparticles cross-linked by cobalt- γ-irradiation of these microcoacervate droplets are useful as drug release devices. to investigate the size optimization of nanoparticles, the stability of nanoparticles in the treatment of enzyme, and the drug release profiles from nanoparticles, the three copolymers; poly[ (val-pro-gly-val-gly), (val-ala-pro-gly-val-gly)], poly[ (val-pro-gly-val-gly) application of polyelectrolytes and theoretical models the synthetic heptapeptide rnwdvyk is a fragment of a high affinity receptor (fcεri) for immunoglobulin e (fragment - ). it is the active domain for binding with ige. the program of studies of biological properties of the heptapeptide included the investigation of its binding to ige contained in standard solutions and in patients' blood serum. the binding of rnwdvyk with ige was investigated by the ifa method using the ige antibodies labelled with horse-radish peroxidase (hrpo). we determined the optimum sorption concentration of the peptide in this experimental immunoenzyme system to be mkg/ml. the ability of synthetic rnwdvyk peptides to bind with ige was studied as a function of ige concentration in standard serum ( . to ng/ml ige). a high correlation was found between the ige concentration and the optical density of the solution after introducing monoclonal antibodies labeled with hrpo and the substrate chromogenic mixture (r= . ). similar investigations were conducted using the allergy patients' blood serum. the serum with a known concentration of ige was added to immunological plotting boards with sorbed synthetic rnwdvyk peptides. a high correlation was also found between the concentration of ige in the patients' blood serum and the optical density of the solution after introducing monoclonal antibodies labelled with hrpo and substrate chromogenic mixture (r= . ). our experiments showed the high ige binding activity of synthetic rnwdvyk peptides. we demonstrated the possibility of construction of diagnostic systems for the quantitative determination of ige and ige-antibodies. the fusion protein nucleocapsid-dutpase is present in virions of mason-pfizer monkey betaretrovirus and in virus-infected cells where it potentially contributes to rna/dna folding and reverse transcription (barabas, et al., ; bergman et al., ; berkowitz, et al., ) . in addition to trimeric dutpase core, the protein possesses flexible n-and c-termini consisting of the nucleocapsid segment, and a peptide motif conserved in dutpases. to analyze the function of the flexible cterminal peptide segment, reconstitution experiments were designed with truncated enzyme lacking the c-terminal mer oligopeptide and the synthetic oligopeptide prepared on rink-amid resin by solid-phase peptide synthesis, using fmoc strategy. the truncated enzyme proved to be practically inactive. addition of the synthetic mer (pyrgqgsfgssdiy) at fold molar excess resulted in partial complamentation of the catalytic activity (to % of original). a mixture of the truncated enzyme and the mer oligopeptide (this latter at fold excess) was put to crystallization trials. we conclude that the c-terminal mer is essential for catalytic activity. antifolate drugs are inhibitors directed to interfere with folate metabolic pathway. methotrexate (mtx) and pemetrexed (alimta®) are known folic acid analogues used in cancer treatment. different peptide conjugates of mtx have been prepared for intracellular delivery. ( ) in octaarginine conjugates one of the carboxylic groups of mtx was attached to the n-terminal of the peptides. ( ) however, as results showed, that both carboxylic groups are required to the biological effect of mtx. therefore we decided to synthesize peptide conjugates of folic acid analogues in which the carboxylic groups are untouched. octaarginine, penetratin and a cyclic peptide cgnkrtrgc, which can deliver a cargo molecule in the lymphoid system, were used as delivery peptides. we introduced squaric acid or aminoxy acetic acid as linker moiety between the peptides and cargo molecules. the conjugation was monitored by rp-hplc, the crude products were purified by rp-hplc and were identified by mass spectrometry. the biological activity of the conjugates was evaluated in vitro on sensitive and resistant human leukemia (hl- ) cell lines. besides its endocrine activity, trh (the tripeptide pglu-his-pro-nh<sub> </sub>) has also been long recognized as a modulatory neuropeptide with broad range of physiological and pharmacological activities in the central nervous system (cns). although numerous centrally active and metabolically stable analogues and peptidomimetics have been synthesized using trh as a template, selectivity of their cns action has remained an issue to be addressed. we aimed at discovering novel analogues with enhanced cns-selectivity by incorporating pyridinium building blocks. the design also allowed for enhancing transport across the blood-brain-barrier and increasing residence time in the cns through prodrug strategy. solid-phase chemistry was used to prepare the analogues and novel methods previously not used to incorporate pyridinium moieties into resin-bound peptides such as the zincke reaction were also introduced. comprehensive evaluation included measurement of affinity to trh-receptor, acetylcholine-releasing, analeptic and antidepressant-like activity in animal models, as well as prediction of membrane affinity, determination of in vitro metabolic stability, and pharmacokinetics and brain uptake/retention studies that employed in vivo microdialysis sampling. a strong connection between acetylcholine-releasing potency and analeptic effect in animals was obtained for close analogues of trh, while pyridinium compounds designed from the structurally related pglu-glu-pro-nh<sub> </sub> maintained the antidepressant-like effect of the parent peptide, while showing significant decrease in analeptic action.in conclusion, an increase in the selectivity of cns-activity profile was obtained by the incorporation of pyridinium moieties. we have also demonstrated the benefits of the prodrug approach on the pharmacokinetics, brain uptake and retention of the analogues upon systemic administration. the use of small radiolabelled compounds such as peptides is a very attractive tool for the diagnosis of several different pathologies, specially cancer, through the use of nuclear medicine techniques. among the various membrane receptors, the two cholecystokinin receptors ccka-r and cckb-r are very promising biological targets for radiolabelled compounds due to their overexpression in many tumours . in order to develop radiolabelled peptide derivatives able to target these receptors, the binding mode of the c-terminal cholecystokinin octapeptide (cck ), toward the two cholecystokinin receptors ccka-r and cckb-r has been, recently, structurally characterized. the structural data suggest that modifications on the n-terminal end of cck obtained by introducing chelating agents and their metal complexes should not affect the interaction of the derivatized cck peptides with both ccka-r and cckb-r. here we report the labelling procedures and the in vitro and in vivo characterization of new mtc cck derivatives. a stable mtc-nitrido complex is obtained by using the coordinative set formed by: ) the n-terminal amino group and the sh cystein of the cck derivative cys-gly-cck peptide; and ) a pnp aminodiphosphine used as coligand. several phosphines are used in order to define the best labelling procedures and to optimize the in vivo biodistribution properties of the mtc labelled peptides.references various combinations of pore size and chemistry of silica-based materials were investigated for high performance liquid chromatography (hplc) of peptide separation. incorrect pore size and ligands have been suggested to cause peak broadening, poor resolution and poor recovery. our study suggests that an appropriate combination of pore sizes and ligands is necessary to obtain the most efficient usage of reversed-phase hplc columns according to the molecular weights of peptides and proteins. we will also show the possibilities of an improved method development for the separation of complex peptide mixture by ph or additives.the development of new biopolymer materials as drug delivery systems is of enormous interest on biomedicine. dendrimers are polymers with particular properties; they are highly branched polymers with well-defined chemical composition, and show compact globular shape, monodisperse size and controllable surface functionalities. peptide dendrimers incorporates amino acids in their structures and have additional features such as biocompatibility and biodegradability.in previous studies we described the solid-phase synthesis of a new class of polyproline-based dendrimers. these biopolymers have the capacity to cross the mammalian cell membrane and moderate toxicity. these promising results open up the possibility to explore these dendrimers as delivery systems.to design more versatile polyproline dendrimers, we have developed a methodology that involves the combination of solid-phase and solution strategies. diverse multivalent peg-and proline-based cores were synthesized to attain dendrimers with distinct topologies. dendrimers were synthesized by iterative building block addition [(glypro ) imdoh], around an inner core, using a peptide solution convergent approach. a variety of coupling methodologies and protecting groups for the n-terminal function were explored. the novel high throughput protein detection system using designed peptide arrays has been successfully indicated on their capabilities as the "protein-chip" [ ] [ ] [ ] [ ] . our concept has many advantages especially for high-quality industrial production and practical applications compared to arrays with antibodies or recombinant proteins. the deposited peptide solution can be dried without covalent immobilization, although, when the resulted arrays are exposed in protein-solution they showed planned conformation [ ] . based on these basic results, several hundreds of α-helical, β-loop and β-sheet peptides, which involved a cysteinyl residue for covalent immobilization and tamra as a fluorescent label, were successfully synthesized, characterized and used as capture molecules. a novel material for chips made from amorphous carbon suitable for our concept has been developed, which has significant advantages over conventional glass or polymer plates, such as no selffluorescence, mechanically more stable, easy manufacturing in the aspects of precised and high throughput processing. thus, chip-plate with nano-l wells could also be easily manufactured. peptides were deposited on these chips surface covalently as well as non-covalently in picol/spot (diameter: ca µm). the resulted chips were used for protein detection. a part of this work was funded by the okinawa-bio-project and nedo-grant. key: cord- -ad i wgj authors: nan title: th international congress on amino acids and proteins : vienna, austria, august – , date: journal: amino acids doi: . /s sha: doc_id: cord_uid: ad i wgj nan the raised concentration of protein bound homocysteine in homocystinuric (hcu) patients displaces protein bound cysteine and increases the free/bound cysteine ratio in plasma. this ratio is independent of albumin concentration. results from hcu patients were compared to controls. free cystine concentrations in hcu were poorly discriminated from the control range but the total cysteine results were almost invariably lower than control data. this appears to result from an increased free/bound cysteine ratio in hcu [mean (range) for control . ( . - . ) and for hcu . ( . - . ); p ϭ . ]. ex vivo protein binding experiments in albumin solution revealed the free/bound cysteine ratio to be linearly related to the amount of homocysteine bound (r ϭ . , p Ͻ . ). we conclude that measurement of total cysteine is essential for assessment of the true cysteine status in hcu. however, any cysteine deficit, or alteration to free/bound cysteine ratios, does not obviously effect glutathione synthesis as assessed by measurement of plasma total glutathione. ( nmol/g; . %); sprague-dawley rat (rattus norvegicus, rodentia) (n ϭ ; - nmol/g; . - . %); rabbit ( nmol/ g; . %); pig (sus scrofa f. domestica, artiodactyla) ( nmol/ g; . %); bovine (bos primigenius f. taurus, artiodactyla) ( nmol/g; . %); seal (phoca vitulina, carnivora) ( nmol/g; . %), and rob (halichoerus grypus, carnivora) ( nmol/g; . %). from the date it is concluded that d-aas are common in body fluids and certain tissues of vertebrates. in order to determine the quantity of cyst(e)ine and methionine, the oxidation of cyst(e)ine and methionine (Ϫ) refers to not detected or not determinable; asx ϭ asp ϩ asn; glx ϭ glu ϩ gln; his, arg, trp, cys not determined; a) feed fortified with dl-met; b) nmol/g lyophilized serum. mentation and subsequent purification. the separation of the enantiomers of cysteic acid, methionine sulphone, aspartic acid and glutamic acid is displayed on the chromatogram. into cysteic acid and methionine sulphone with performic acid is often applied before hydrolysis of protein. the authors examined the applicability of this process in case of quantification of cyst(e)ine and methionine enantiomers. the rp-hplc analytical method was developed for the determination of the amount of cysteic acid and methionine sulphone enantiomers. the rate of conversion during oxidation from cyst(e)ine into cystic acid and from methionine into methionine sulphone was determined. the racemisation of l-cyst(e)ine and l-methionine was negligible during oxidation with performic acid, therefore this process can be applied before hydrolysis during quantification of cyst(e)ine and methionine enantiomers. after the performic acid oxidation and the m hcl hydrolysis of the protein, opa/tatg (o-phthaldialdehyde/tetra-o-acetyl- -thio--d-glucopiranoside) precolumn derivatisation method was used, and the enantiomers of sulphur containing amino acids were separated by rp-hplc (lichrosphere rp- e, ϫ mm, µm column, merck-hitachi lachrom hplc). the resolution of the peak of cysteic acid and methionine sulphone enantiomers was better than , . the method was used to determine the amount of l-and d-cyst(e)ine and land d-methionine containing preparations prepared by fer- d/l rate of aspartic acid and the individual age of specimens. a method for age determination based on d-aspartic acid content and on the racemisation of l-aspartic acid of teeth was developed. d-glutamic acid, beside d-aspartic acid, was found to be eminently suitable for the estimation of individual age, as it showed a sufficiently high sensitivity. calibration curves based on these investigations were used for the age estimation of adults ( males and females) of unknown individual age from the avar period series of kereki-homokbánya (hungary). the age distribution of the sample was the following: individuals ( %) belonged to the adult age group, persons ( %) to the mature and ( %) to the senile one. the correlation between our results and those obtained using standard paleoanthropological methods was over . . quantitative determination of free and bound -nitrotyrosine in rat plasma and tissues using isotope dilution liquid chromatography-electrospray tandem mass spectrometry t. delatour, p. a. guy, j. richoz, j. vuichoud, and nestlé research centre, nestec ltd, vers-chez-les-blanc, lausanne, switzerland since -nitrotyrosine was reported to be readily formed in proteins by reactions with nitrite or nitrogen dioxide, it has been postulated to be a possible marker for investigating peroxynitrite-mediated nitration of proteins. thus, several methods were developed to assess nitration of tyrosine in proteins and determine -nitrotyrosine in physiological fluids. methods based on hplc or gc/ms techniques were described to quantify -nitrotyrosine within tissues or biological fluids. unfortunately, it has been demonstrated that an artifactual nitration of tyrosine occurs with gc/ms assays leading to an overestimation of the response. in the present work, lc-esi-ms/ms methods for quantification of free -nitrotyrosine in rat plasma as well as bound -nitrotyrosine in tissue samples are reported. plasma samples were spiked with , , -d - -nitrotyrosine and the following steps were applied prior to injection into the lc-esi-ms/ms system used in selected reaction monitoring (srm) mode (m/z ae for the analyte and m/z ae for the internal standard): protein precipitation, solid phase extraction on aminopropyl cartridge and derivatization in nbutanol in hcl n. -nitrotyrosine butyl ester has lead to a dramatic increase of the sensitivity (ca. -fold) by comparison with -nitrotyrosine. under such conditions, calibration curves exhibited excellent linearity (r Ͼ . ) within concentration range . to . nm (equivalent to . - , fmol on column) and recoveries above %. inter-and intra-assay precision was determined below % over the concentration range . to . nm. no artifactual nitration of tyrosine occurring during sample clean-up was observed. this was unambiguously established by plotting experimental ratio of analyte response/ internal standard response versus expected within the range . - . nm. this curve strongly correlated with a linear model (r Ͼ . ) and slope was . Ϯ . (mean Ϯ sd). basal level of -nitrotyrosine in rat plasma was measured to be within concentration range Ͻ lod to . nm. -nitrotyrosine basal level in rat plasma, kidney and liver proteins was established by performing enzymatic hydrolysis in order to avoid artifactual nitration of tyrosine which may occur under strong acidic conditions (hcl n at °c). resulting hydrolysates were analysed by lc-esi-ms/ms and nitrotyrosine was monitored in srm mode (m/z ae for the analyte and m/z ae for the internal standard). t. guszczynski , r. b. kapust , d. s. waugh , and t. d. copeland basic research laboratory, and macromolecular crystallography laboratory, national cancer institute at frederick, maryland, u.s.a. the set-can fusion gene was first detected as associated with acute undifferentiated leukemia. set (also called phap ii) is a nuclear phosphoprotein with a long acidic tail. set has been shown to inhibit phosphatase pp a and is a substrate of human granzyme a. in order to determine any zn(ii) binding properties of set, we utilized affinity capillary electrophoresis (ace) to detect shifts in mobility as zn(ii) ions bind to the protein. we have earlier employed ace to measure the binding constants of zn(ii) to the nucleocapsid protein of hiv- . with a constant concentration of recombinant set as a receptor and varying concentrations of zn(ii) as ligand in the sample buffer, we observed changes in electrophoretic mobilities of set when complexes were formed with zn(ii). scatchard analysis of the mobility provided the stoichiometry and binding constant of zn(ii) to set. interdisciplinary research center, institute of nutritional science, department of food sciences, university of giessen, germany peptaibols are defined as fungal polypeptides containing a high proportion of aib (α-aminoisobutyric acid) and a cterminal bound amino acohol. the mold trichoderma aureoviride (strain imi ; commonwealth mycological institute, kew, uk) was cultured in complex medium consisting of casein peptone, g; soy peptone, g; yeast extract, g; dglucose, . g; nacl, g; dipotassium hydrogen phosphate, . g in l demineralized water adjusted to ph . . fermentation was conducted in nineteen -l shake flasks, each containing ml medium, for d at °c. mycelia were obtained by filtration and extracted with meoh and meoh/chloroform. extracts were evaporated to dryness and subjected to sephadex and silica gel chromatography (eluent chloroform/meoh/acoh/water : : : ) yielding . g and . g, respectively, crude peptaibol mixture named trichoaureocins. the peptide mixture was uniform on tlc but could be separated by analytical (fig. ) and semipreparative hplc (nucleosil c- ; ϫ mm id; µm). six peptides could be isolated each of which was subjected to sequencing using on-line hplc (fluorocarbon stationary phase) esi-ms/ ms (lcq, thermoquest, finnigan mat) as described for peptaibols trichovirins and antiamoebins. sequences are presented in fig. . the -residue peptaibols represent a natural peptide library and cause hemolysis of sheep erythrocytes and exert antibiotic activity against bacillus subtilis and staphylococcus aureus. national institute of chemistry, ljubljana, slovenia wine consists of several hundred components present at different concentrations. the dominant ones are water, ethanol, glycerol, sugars, organic acids, and various ions, while amino acids are present at much lower concentration. the composition of amino acids is of great importance in wine production. they act as a source of nitrogen for yeast during fermentation, they influence the aromatic composition of wine and their composition can be used to differentiate wines according to vine variety, geographical origin, and year of production. among already established analytical methods high-field nmr has been shown to be a promising method for the nondestructive analysis of low-molecular mass compounds in complex mixtures like wine due to its selectivity and capability of simultaneously detecting a great number of compounds. h and c one-dimensional nmr spectra of wine are very crowded and many signals are overlapped. due to a great difference in concentration levels the signal intensities of particular compounds may vary for the factor of . the tails of the dominant frequencies of water, ethanol and glycerol obscure weak signals of minor compounds like amino acids in the near surroundings. the use of d homo-and heteronuclear experiments and the suppression of strong signals are a prerequisite for a successful h and c signal assignment. a complete assignment of h and c nmr resonances of seventeen amino acids commonly present in wine and of γ-aminobutyric acid at ph was accomplished using gradient-selected cosy, tocsy, gradientselected hsqc and hmqc experiments with incorporated wet pulse sequence for the supression of large signals. unambiguous assignment of h and c nmr resonances of amino acids is necessary for the selection of appropriate signals in fast and simple one-dimensional nmr that can serve as parameters in the chemometric classification of wines according to the provenance, vine variety, and year of production. institute of medical biochemistry, jagiellonian university collegium medicum, kraków, poland highly sensitive colorimetric method for determination of aldehydes in the reaction with n-methyl benzothiazolone hydrazone (mbth) turned out to be not very specific for such carbonyl compounds. namely, it has been found that tryptophan and to higher degree its n-derivatives (n-acetyl-trp, ala-trp, gly-trp) and also tripeptides (gly-trp-gly and leu-trp-leu) in the reaction with mbth and fe ϩ are converted to coloured products, with maximum wavelength at nm. the properties of the products and the kinetics of the reaction under defined conditions are described in the spectrophotometric procedure. proteins containing tryptophan are also substrates in the reaction with mbth. comparison of molar extinction coefficients of mbth-fe ϩ -treated various proteins with those of simple n-derivatives of tryptophan shows, that not all molecules of tryptophan in proteins are accessible to the reagents, and in order to determine all tryptophan moieties partial unfolding of protein has to be performed. it should be emphasized that aldehydes cannot be detected and accurately determined in the presence of tryptophan derivatives and protein, and also aldehydes interfere with determination of tryptophan derivatives. natural product laboratory, department of chemistry, the university of burdwan, w. bengal, india detection of protein amino is of utmost importance for the evaluation of protein structure and also their presence in numerous natural products. several specific and non-specific reagents have been used for their detection using thin-layer chromatography, an important tool for such purpose. of the reagents in general use, ninhydrin is the most popular for its high sensibility, however, nihydrin produces same purple color with most of the amino acids (only proline and hydroxproline produce yellow color). an endeavour has been made to resolve this color problem with a reagent which is capable of developing various distinguishable colors with many of the protein amino acids and also shows its high sensitivity comparable to ninhydrin. a probable mechanism for such color formation has also been proposed. measuring enrichments below the sensitivity range of conventional gc-ms. the gc-c-irms technique combines the resolution capabilities of gc with the accuracy and precision of irms. at low abundance gc-c-irms analysis it is superior in terms of time, labor, and sample requirement as compared to the conventional off-line analysis. we discuss some latest advancements and applications of gc-c-irms amino acid analysis related to nutrition research. plasma amino acids in omnivorous human subjects show a characteristic n-isotopic pattern with phenylalanine and threonine showing the lowest abundance, whereas e.g. alanine and leucine are higher by ‰ δ n. in rats fed diets containing intrinsically labeled c casein or the corresponding amino acid mixture labeled with c leucine and n lysine whole-body protein homeostasis is better supported by casein-bound than free amino acids. there is no adaptation to a low lysine diet by an enhanced bioavailability of intestinal microbial lysine to extra-splanchnic tissues in minipigs. highly selective hplc determination of tyrosine, tryptophan and their related compounds based on precolumn derivatization followed by intramolecular fluorescence resonance energy transfer detection h. nohta , m. yoshitake , h. yoshida , t. yoshitake , and m. yamaguchi faculty of pharmaceutical sciences, fukuoka university, nanakuma, johnan-ku, fukuoka, and chemical evaluation and research institute, ishii machi, hita, oita, japan we have developed highly selective hplc method for the determination of tyrosine, tryptophan and their related compounds (l-dopa, catecholamines, -hydroxytryptamine, etc.). the compounds were precolumn-derivatized with a commercially available fluorogenic reagent for amines by usual manner. each derivative afforded intramolecular fluorescence resonance energy transfer (fret) from the tyrosyl or tryptophoryl moiety (donor) to the labeled fluorophore (acceptor); the acceptor fluorescence was observed with the excitation of the donor at nm. the derivatives were separated on a reversedphase column and then effectively detected by monitoring their fret. through the screening study of fluorogenic reagents, o-phthalaldehyde (with -mercaptoethanol) and dansyl chloride gave the best results for the purpose. the fret detection method was highly selective and sensitive by comparison with the previous methods detecting native fluorescence of the compounds or typical fluorescence of the acceptor. the presented study was devoted to determination of the energetic effect of interactions in aqueous solutions between urea and neutral amino acid derivatives. the principal reason for studying of interactions of peptides with urea is the hope that such investigations will give insight into the factors affecting protein denaturation in aqueous solutions. the enthalpies of solution of n-acetylglycinamide, n-acetyl-l-alaninamide and n-acetyl-l-leucinamide were measured in water and in aqueous solutions of urea of molality . to . mol·kg Ϫ using the "isoperibol" type calorimeter at . k. from the obtained standard dissolution enthalpies ∆ sol h ϱ m the enthalpic pair interaction coefficients h xy for urea-nacetylamino acid amide pairs in water were calculated. these parameters derived from mcmillan-mayer theory are regarded as a measure of effect of interactions between solute molecules in solution. the h xy values for the systems investigated suggest that the interactions between urea and amide molecules dominate the effects of dehydration of nonelectrolyte and of peptides. the replacement of the hydrogen atom in the hydrocarbon chain with a methyl group causes a positive change in the value of the enthalpic pair interaction coefficient. the obtained results were compared with those of earlier studies of interactions between electrolytes, namely sodium chloride, potassium chloride and sodium iodide and the same n-acetylamino acid amides. the effect of the solute type on the magnitude of the interaction parameter was also analysed. the side chains of amino acids in solution react in various ways with the water molecules which surround them as well as with other components of solution depending on the fact whether they possess non-polar, polar or ionic groups. many research laboratories carry out studies intended to describe precisely the intermolecular interactions with the participation of amino acid side chains. such a description may allow one to describe better the spatial structures of protein and the mechanisms of folding its surface area. the present work reports the results of calorimetric measurements of the dilution enthalpies of l-α-amino acids in water. using modified mcmillan-mayer's theory, these results served to calculate the enthalpic homogeneous interaction coefficients which characterise interactions between the amino acid zwitterions with the competitive participation of water molecules. thus, these coefficients illustrate the differences in amino acid molecules interactions both with the homogeneous amino acid molecules and water molecules around them, and consequently they may play the part of a parameter which differentiates the hydrophobic/hydrophilic properties of amino acid side chains. the enthalpic interaction coefficients of the homogeneous pairs of l-α-amino acids were compared also with the hydrophobicity parameters obtained by fauchere et al., which describe the side substituents of natural amino acids as well as aminobutiric acid (aba). based on the above statement, one may conclude that the obtained enthalpic homogeneous pair interaction coefficients of l-α-amino acids in water make it possible to systematise amino acid side chains according to their affinity to water or their hydrophobic-hydrophilic properties. thus the enthalpic homogeneous pair interaction coefficients may play the role of parameter describing the lipophilicity (hydrophobicity) of amino acid side chains. compounds (iii) with amino acid ligands. in this work we present results of x-ray investigation of fourth amino acid complexes of rhenium (iii), which have different coordination of amino acids around binuclear complexforming center -re ϩ . substances (glyh) . h o -in inner, but gaba has cisposition according to re -re bond. influences of fatty radical length in the amino acid ligand on week interaction between binuclear anion [re cl ] Ϫ and protonized amino acid are discussed. role of hydrogen bonds in formation of crystal unit cell of investigated substances is shown. these two factors are the reason of formation of staggered conformation of an anion [re cl ] Ϫ in the substance (glyh) [re cl ]cl together with existence of quadruple re -re bond that is described first. in the substance [re (gaba) cl (h o)]cl . h o axial position of re ϩ fragment are substituted by ligands of different kind: h o and cl Ϫ -that says about possibilities to coordinate a substrate of biological nature exactly to these position. a precise, sensitive and reliable rp-hplc/uv method was developed to enable determination of α, and k caseins in cow's milk. the optimised method using a chrompack p- -rp column allowed separation of caseins in min. this column differs from conventional alkyl-bonded silica rp matrices in that it is an underivatised polystyrene-divinylbenzene matrix, a material which proved excellent chemical and ph stability. gradient elution was carried out at a flow rate of ml/min and a temperature of °c, using a mixture of two solvents. solvent a . % trifluoroacetic acid in water and solvent b was % acetonitrile- % water- . % trifluoroacetic acid. the effluent was monitored by a uv detector at nm. the determinations were performed in the linear range of . - . mg/ml for k-casein, . - . mg/ml for α-casein and . - . mg/ml for -casein. the detection limits were . , . and . mg/ml, respectively. the validity of the method was verified. the recoveries ranged from to % for cow's milk. the precision of the method was also evaluated, the % cv being less then . %. the developed methodology was also applied with success to the separation of caseins in ewe and goat milks. different chromatographic profiles were obtained for the three kinds of milk. department of aquatic biosciences, the university of tokyo, bunkyo-ku, tokyo, japan several aquatic crustaceans and bivalve molluscs accumulate a large amount of free d-alanine ( - µmol/g wet wt.) in their muscle tissues. during seawater acclimation from freshwater to % seawater, red swamp crayfish procambarus clarkii largely accumulated d-and l-alanine by . -and . fold, respectively, together with l-glutamine, l-proline, and glycine. the percentage of d-alanine to total alanine increased from % in freshwater to % in % seawater. these data indicate that d-and l-alanine are the major compatible osmolytes responsible for the intracellular isosmotic regulation of this species as well as other crustaceans. under anoxia stress for h in freshwater, and % seawater, crayfish increased d-and l-alanine in muscle and hepatopancreas in addition to the increase of lactate. the increase was much higher in seawater than in freshwater. thus, d-and l-alanine may be anaerobic end products during prolonged anoxia of this species. alanine racemase [ec . . . ] has been proved to catalyze the interconversion of d-and l-alanine in crustaceans and bivalve molluscs. this enzyme was isolated to homogeneity from the muscle of black tiger prawn penaeus monodon. the purification was , -fold with % yield. the molecular weight of the enzyme was estimated to be kda on sds-page and kda on gel filtration, suggesting the dimeric nature of this enzyme. the amino acid sequences of the peptide fragments obtained from the isolated enzyme showed low homology below % with those of microbial enzymes. syntheses and immunological effect of thymic humoral factor-γ analogues research laboratory, global shinwa pharmaceutical co. ltd., yoriki, matsuomura, iwate-gun, iwate-ken, japan nine analogues of thymic humoral factor (thf)-γ , were prepared by the solid-phase method and their in vitro restoring effect on the impaired blastogenic response of phytohemagglutinin (pha)-stimulated t-lymphocytes of uremic patients with infectious diseases were examined. the results were as follows: [arg ]-thf-γ exhibited higher restoring activity than that of our synthetic thf-γ . phylos has developed a powerful combinatorial biology platform for peptide and protein selections. phylos' proprietary profusion tm technology enables the selection of peptides and proteins with desired properties. the fundamental advance represented in this unique platform is the in vitro covalent linkage of a peptide or protein (phenotype) to the encoding messenger rna (genotype). this linkage permits the selection of a protein based on its characteristics and allows the recovery and amplification of that protein through pcr, an efficient means of bring the desired proteins to easily detectable levels. profusion tm technology has routinely selected peptide and protein binders with affinity constants in the nanomolar to picomolar range. the starting library size of randomized peptide or protein profusion tm constructs is typically . linear and constrained loop peptide libraries, for ligand generation, enzyme: substrate interaction, peptidomimetic design, and epitope mapping have been successfully used. randomized constrained loops have also been incorporated in a betasandwich scaffold, resulting in the successful selection of binders against targets of therapeutic interest. antigenic properties of three biological active de novo proteins were investigated by peptide scanning approach, using noncleavable multipin technology. a de novo protein albebetin (pid caa ) was engineered to attain a pre-designed d structure and later modified by grafting short peptide fragments from human α interferon (aag ), and insulin molecules (aag ). such protein constructs carrying important biological activities may be used in future as potential protein pharmaceuticals. despite artificial proteins are investigated for more than years, immunological properties of these substances are not known. in our experiments we applied an innovative approach of raising antibodies in yolks of egg-laying hens. three continuous antigenic determinants with different immunogenic potentials have been revealed in two proteins with partially overlapping sequences. it was shown that the octapeptide interferon fragment is the immunodominant site in albeferon and albeferon-insulin molecules. on the contrary, the hexapeptides, corresponding to the insulin fragment displayed low immunogenic activity. thus we recognise that the fragments attached to the de novo frame could essentially govern immunological properties of resulting construct. no preference of any type of secondary structure was observed in antigenic determinants. nevertheless, all of them are located at the boundaries of the secondary structure elements and on the predicted surface-located sites of albebetin molecule. peptide fragments from human α -interferon and insulin corresponding to the functionally important sites of their molecules were grafted into de novo protein albebetin (pid caa ) engineered to attain a pre-designed tertiary structure with a unique topology that has not been observed in natural proteins. by means of genetic engineering the dna fragments corresponding to these peptides were inserted into the albebetin gene to obtain two variants of albebetin with antiviral fragment of human α -interferon and two variants of albebetin with insulin-like peptide. the chimerical genes were expressed in escherichia coli in a fusion expression system with thioredoxin based on the plasmid pet- (novagen). the fusion proteins were digested by highly specific protease factor xa and the target chimerical proteins were purified and tested for their structure and biological activity. according to the cd spectroscopy study the chimerical proteins maintained the pre-designed structural properties of albebetin. toxicological testing of the proteins in the mtt-test did not reveal their cytotoxicity. antiviral activity of de novo proteins with human α -interferon fragments was studied in vitro using human fibroblasts cell line l- and simian cell line vero. treatment of these cell lines with the proteins revealed the dose-dependent stimulated antiviral activity on fibroblasts and direct dose-dependent antiviral activity on the vero cells. one of two de novo proteins including insulin-like fragment (pid aag ) acquired ability to stimulate glucose uptake by l- cells although the efficacy of stimulation was lower than that for the synthetic peptide and insulin. these results demonstrated that albebetin can be used as a scaffold for constructing of the functionally active de novo proteins possessing the pre-designed tertiary fold of albebetin and various biological activities. the identification of genes encoding unique tumor associated antigens (taas) has facilitated the development of novel immunotherapeutic strategies in cancer patients. clinical investigations have focused on targeting these cancer antigens for the generation of anti-tumor t-cell responses. taa epitopes come from differentiation antigens, from embryonal reexpressed or overexpressed proteins, from mutated proteins and from viral proteins in viraly associated tumors. we have recently developed a novel screening system for identification of immunogenic and antigenic ctl peptide epitopes using d b-/-x m -/double knockout mice, transgenic for a single-chain hla-a - m molecule (hhd mice). specific ctl were derived by immunization of hhd mice with tumor peptide extracts loaded on antigen presenting cells and with hhd transfected human tumor cell lines ctl induced against peptides from various tumors recognized tumor peptides more effectively than peptides extracted from normal tissues and also reacted with a serie of peptides derived from overexpressed candidate proteins, identified by differential display methods (sage, microarrays) comparison of ctl derived from hhd mice to ctl induced from patient's pbmc showed overlapping recognition of many candidate peptides. using these hhd mouse derived ctl we identified novel peptide sequences from prostate, bladder, breast and colon carcinomas, antigens pap and steap, from breast carcinoma antigens muc and ba - . analysis of tumor differentially expressed genes by the sage method in colon, followed by screening for hla-a binding peptides resulted in candidate peptides for immunogenicity screening. we have identified antigenic peptides of which peptides were found to be immunogenic in hhd mice. interestingly of these peptides are derived from the same protein. differential expression studies, using "dna chips" were performed on prostate and bladder tumors versus normal tissues. ten new candidate genes from tcc were analysed for expression and potential immunogenic peptides. novel peptides from uroplakins and from mage- were identified. surface plasmon resonance biosensing in the study of viral antigenic sites mimicked by synthetic peptides p. gomes , e. giralt , and d. andreu centro de investigação em química da universidade do porto, portugal department de química orgànica, universitat de barcelona, spain antigen-antibody binding has been regarded as one of the most representative examples of specific molecular recognition in nature. the simplistic view of antigenic recognition in terms of a lock-and-key mechanism is superseded, since it is now evident that both antigens and antibodies are flexible and can undergo substantial mutual adaptation. this flexibility is the source of complexities such as degeneracy and non-additivity in antigenic recognition. we have used surface plasmon resonance to study the effects of combining multiple amino acid replacements within the sequence of the antigenic gh loop of foot and mouth disease virus. our aim was two-fold: to explore to what extent can antigenic degeneracy be extended in this particular case, and to search for potential non-additive effects in introducing multiple amino acid replacements. combined analysis of one such multiply substituted peptide by spr, solution nmr and x-ray diffraction shows that antigenic degeneracy can be expected as long as residues directly interacting with the paratope are conserved and the peptide bioactive folding is unaltered. structural properties of creatine kinase from amphioxus, branchiostoma belcheri gray f. inoue, s. obase, t. suzuki, and t. imai department of physiological chemistry, faculty of sciences, toho university, funabashi, japan to further our knowledge of creatine kinase (ck) in the fields of molecular evolution and comparative enzymology, we analyzed the ck gene of the protochordate amphioxus. amphioxus is thought to be the phylogenetic predecessor of vertebrates and thus possesses characteristics, such as enzymological properties, that are associated with ancestral vertebrates. the results clarified the sequence of bases including the active site. the homology of the active site and the surrounding bases for the amphioxus ck gene to that of the human and electric ray ck-m gene was . % and . %, respectively. the amino acid sequence of this region of amphioxus ck was also identical to that of human and electric ray ck-m. in addition, the estimated secondary structure of amphioxus ck was compared to that of human and electric ray. there were no marked differences in the relative ratio of the α helix, sheet and turn structures for the peptide structure of ck consisting of amino acid residues. there was a high degree of homology in the sequence of amino acid residues (met ϳhis ) near the active site of ck between amphioxus and other organisms, suggesting that this region of ck is functionally essential for transphosphorylation. gelsolin is a ca ϩ -activated and phosphoinsitide-regulated cytoskeletal actin-binding-and-severing protein, its fragments - : ksglkykk (g - ) and - khvvpnev vvqrlfqvkgrr (g - ), are responsible for the binding of this protein to actin and the cellular messenger phosphatidylinositol , -bisphosphate (pip ). the binding of peptides g - and g - to a cluster of four pip molecules in a dimyristoyl-phosphatidylcholine lipid was in vestigated by means of molecular-dynamics (md) simulations of , ps. the binding of the pip molecules to the peptides g - , g - showed both electrostatic and hydrophobic nature: lysine residues of the peptides formed salt bridges with the phosphate groups of the pip molecules, while hydrophobic interactions occurred between the nonpolar residues of the peptides and the fatty-acid tails of pip . during the binding some of the pip molecules were dragged out of the lipid, thus disrupting the bilayer. after the binding dissociated a draggen-out pip molecule tend to incooporate back to the lipid. division of applied physiology, institute of veterinary physiology, university of zürich, switzerland chemical modification of the proteins: bovine serum albumin, α-lactalbumin, -lactoglobulin and chicken egg white lysozyme by -hydroxyphthalic anhydride ( hp) yielded compounds which exerted antiviral activity in vitro as compared with the native unmodified proteins. of the three enveloped viruses tested: human herpes simplex virus (hsv- ), bovine parainfluenza virus (pi- ) and porcine respiratory corona virus (prcv), the hp proteins were shown to be active against human herpes simplex virus only indicating that a perturbation of the viral envelope is unlikely. pre-incubation of vero cells with hp-albumin, hp--lactoglobulin and hplysozyme resulted in protection against hsv- infection whereas pre-incubation with hp-α-lactalbumin had no antiviral effect. however, all hp modified proteins showed a more significant inhibition when present during or after the viral infection step. thus multiple mechanisms appear to be involved in the inhibition of hsv- infection. the blocking of cell receptors may contribute to the antiviral activity as shown by the preincubation data. however, a direct interaction between the modified proteins and the hsv- glycoproteins responsible for viral entry and spread, seems to play a more important role, as indicated by the smaller ec values obtained during and after the infection. a dummy or a protagonist on the stage of inflammation? r&d department, zambon group, bresso, milan, italy amino acids are usually present in large excess in healthy and the excess is used as source of calories. however, metabolic alterations are observed in ill patients and preferential retention of sulphur amino acids (saa) occurs during the inflammatory response. the metabolism of cysteine is modified during the acute phase of sepsis in rats. sulphate production is lower, whereas the higher liver production of taurine seems to play a protective role; glutathione concentration is greater in liver, kidney and other organs and cysteine incorporation into proteins was higher in spleen, lung and plasma (acute phase proteins) while albumin level decreases. another important phenomenon is the impairment of methionine conversion to cysteine during stressed condition. premature infants or hiv patients synthesise cysteine from methionine at a much lower rate. thus, the metabolic flow through the trans-sulphuration pathway may be insufficient to meet the glutathione and cysteine requirement in critical conditions. the pro-inflammatory cytokines, interleukin- , interleukin- and tnf-α are the main initiates that alter protein and amino acid metabolism. in this complex picture, saa supply may contribute to the immune system regulation. our previous investigations showed some biological activity of newly synthesized cluster rhenium compoundtetrachlorodi-µ-(γ-aminobutirato)dirhenium(iii) chloride -i such as antitumour activity, cell-stabilizing activity against osmotic hemolysis, changing of morphology of cells, and other. there exists some information about stabilizing effects of some metal-organic substances with antitumour properties on the isolated ishaemic-reperfused rat heart (leperre a. ) throughout decrease of malonaldehyde (mda) production. some new investigations showed the influence of metal-organic substances on apoptotic processes (winter b. , syrkin a. , that are considered now as the main mechanism of such tissue damages as ishaemia, myocardial infarct, etc. thus we tried to analyze such activity of i. two models of hemolytic anemia was used: a -on rabbits by introducing of pbac -solutions; this model permits to investigate dynamics of anemia in one experimental animal; bon rats by introducing of phenylhydrazine chloride. i was administrated as in solution as in lyposomic (lyp) forms. all measurements and models were accomplished according to described procedures. administration of i led to: increase of hemoglobin and resistance of erythrocytes and to prolonging of life for hemolytic animals; significant decrease in quantities of mda and increase in quantities of reduced glutathion (gsh), glutathionreductase (gsr) and glutathionperoxidase (gsp) in myocardium, blood, brain, liver, splenic and entherocites of anemic animals. the most effective was i in lyposomic form. mechanism of antioxidant action of rhenium cluster compound is speculated and experiments with some well-known antioxidants to compare with i are working out. at present problem of finding remedies against the mostly dangerous human disease -aids is one of higher interest. the aim of this work was the investigation of inhibiting effect of high-pure l-lysine-α-oxidase (lo) e.c. . . . , extracted from trichoderma sp., on hiv-virus reproduction, comparatively to azidotymidin (azt), being now in use for treatment of aids-patients. for studying of inhibiting effect of lo, the mt- cells, sensitive to citopathical action of virus, were used. the experimental studying has shown, that the enzyme at concentration - ng/ml suppresses hiv reproduction and synthesis of virus' proteins, not exerting toxical effect on mt- cells. toxical dose of lo has been determined preliminary. a comparison with standard preparation -azidotymidin, which causes suppression of virus reproduction at concentration mkm ( , mg/ml) not exerting toxical effect on mt- cells. the same effect is attained having used lo in doses - ng/ml. using lower concentrations of enzyme leads to partial increasing of virus' titre comparatively to control cultures. obtained data allow to conclude that lo from trichoderma sp. is more high specific agent than azidotymidin, because it needs times lower concentration for the same action. comparison of azt and lo action on synthesis of virus' antigens presenting in cultural media of mt- cells infected with virus, leads to conclusion, that lo has inhibitory action both on virus' reproduction and virus' protein synthesis. department of microbiology, dokkyo university school of medicine, mibu, tochigi, japan our previous studies showed that the cellular amino acid composition obtained by amino acid analysis of whole cells, differs in various organisms. these results suggest that the difference in the cellular amino acid composition reflects biological evolution. however, the basic pattern of cellular amino acid composition is relatively constant in all organisms, and the cellular amino acid compositions of the archaeobacteria are quite similar to those determined from codon usage data, based on the complete genomes. in the present study, the free amino acid compositions in archaeobacteria, eubacteria, protozoa, blue-green alga, green alga, slime mold, plants and mammalian cells were analyzed, to investigate whether changes in their free amino acid compositions reflected biological evolution. cell homogenates were treated with - % ethanol to separate cellular proteins and free amino acids contained in the cells. rat hepatoma cells (r-y b) were cultured in eagle's minimum essential medium (mem) containing % serum or in a modified mem lacking arginine, tyrosine and glutamine. no significant difference in the free amino acid composition was observed between the two cell groups cultured under two different conditions. the patterns of the free amino acid compositions differed completely from those of the cellular amino acid compositions, and from each other in various organisms. characteristic differences were observed between plant and mammalian cells, and between archaeobacteria and eubacteria. the patterns of the free amino acid composition in blue-green alga, green alga, protozoa and slime mold differed from each other and from those of eubacteria and archaea cells. it has been suggested that the free amino acid composition reflects apparent biological changes as the result of evolution. ) catalyzes the hydrolysis of gamma-glutamyl compounds such as glutathione, and the transfer of their gamma-glutamyl moieties to amino acids and peptides. we previously developed enzymatic methods for the synthesis of various gammaglutamylamino acids using the transfer reaction of ggt from e. coli k- as a catalyst. it has been reported that gamma-lglutamyltaurine has a potent and long-lasting antiepileptic action, and its chemical synthesis has also been reported, but it required protecting and deblocking of reactive groups. thus, the purpose of this study was to develop an enzymatic method for the synthesis of gamma-l-glutamyltaurine using ggt. the optimum reaction condition was mm l-glutamine, mm taurine and . unit/ml ggt, ph , and -hr incubation of °c. forty-three mm gamma-glutamyltaurine was obtained and the yield was .%. gamma-glutamyltaurine was purified by dowex ϫ column and c column, and then identified with gamma-l-glutamyltaurine by nmr and polarimeter. in this study the yield of gamma-l-glutamyltaurine was comparatively low because synthesized gamma-lglutamyltaurine was promptly converted into the by-product, gamma-l-glutamyl-gamma-l-glutamyltaurine. the production of antimicrobial peptides is an important aspect of host defense in animals ranging from insects to mammals. they do not target specific molecular receptors on the microbial surface, but rather assume amphipathic structures that allow them to interact directly with microbial membranes, which they can rapidly permeabilize. they are thus perceived to be one promising solution to the growing problem of microbial resistance to conventional antibiotics. insects express a battery of potent antimicrobial proteins in response to injury and infec-tion. until now, approximately immune peptides have been characterized from insects and other invertebrates. an antimicrobial gene (md-cecropin) belonging to cecropin family was cloned from the bacteria-charged adult house fly, musca domestica. expressed in the vector pgex- t . mrna was isolated and degenerated primers were designed according to the conserved sequences of cecropins. the full-length cdna encoding md-cecropin was cloned by rt-pcr and Ј, Ј-race and sequenced. the deduced amino acid sequence indicated that a prepeptide with amino acid residues is first translated and then processed to a mature peptide with amino acids. the dna encoding the mature peptide was subcloned into expression vector pgex- t , and expressed efficiently in e. coli bl as a fusion protein. the fusion protein was purified and specifically digested and the md-cecropin was further purified to homogeneity and the activity spectrum was investigated. escherichia coli with metabolic engineering methods l. yun, x. zhang, s. wang, q. xu, and l. ma biotechnology laboratory, institute of beijing radiation medicine, beijing, p.r. china a bioengineering escherichia coli strain was obtained by metabolic engineering method. three genes related to the biosynthesis of phenylalanine, arog, phea, and tyrb encoded key enzymes: -deoxy-d-arabino-heptulonate- -phosphate synthetase (ds), a bifunctional protein-chorismate mutase (cm)/prephenate dehydratase (pd) and aminotransferase (at), respectively. in this work, the feedback inhibition of ds and cm/pd were relieved by site-directed mutagenesis on bases of homology comparison of related sequences of the key enzyme. the feedback inhibition resistant genes encoding ratelimiting enzymes in the main and terminal pathways were amplified by co-expressed in order of arog-phea-tyrb on the plasmid by their own operator plpr, pl, and pr. in the recombinant strain showed great resistant to the l-phenylalanine analogues, the specific activities of ds, cm, pd and at were increased by . , . , . and . folds, respectively. as the result, the amount of phenyalalnine biosynthesis of the bioengineered strain was increased greatly compared with that of the host strain. an enzymatic approach for the mapping of phosphoproteins resolved on two-dimensional polyacrylamide gels hiroshima proteome laboratory, regional science promoter program, kagamiyama higashihiroshima, japan an enzymatic approach for high-throughput mapping of phosphorylated proteins resolved on two-dimensional ( -d) polyacrylamide gels is presented. proteins of cultured rat skin fibroblasts were divided into two aliquots, one of which was dephosphorylated using recombinant protein phosphatase. the two aliquots were then subjected to -d electrophoresis. the phosphoproteins could be mapped on the -d gel by com-paring the gels of the phosphatase-and non-treated samples, because the dephosphorylated proteins shifted to more basic positions on the gel. this technique revealed that approximately % of the detectable proteins were phosphorylated. fifteen phosphoproteins were identified by mass spectrometry, including proteasome component c and small glutamine-rich tetratricopeptide repeat-containing protein. furthermore, the extent of phosphorylation of two actin-modulating proteins, destrin and cofilin, was found to be significantly reduced when the cells were chemically or enzymatically detached from the culture dishes. the presented technique can be applied to all biological materials because it requires no protein-labeling step, and is therefore useful for high-throughput mapping of phosphoproteins in proteome research. with the completion of the human genome sequence maldi-tof-ms is increasingly becoming an established method for identification of proteins separated by d gel electrophoresis. mono-isotopic peptide mass fingerprinting (pmf) has been previously shown to be amenable to full automation encompassing the process of acquisition, data processing and databank searching under full software control. until now the throughput of maldi-tof-ms for proteomics has been limited to several hundred samples in a working day and this represents approximately - % of the total proteins resolved by a large format d gel. to reduce the number of proteins to be identified the d gels are imaged and analysed to determine differences in expression levels within a set of gels. although much of the image processing is semiautomated the comparison is labour intensive as manual pattern matching has a role in the gel alignments (land marking). increased ms sample throughput allows the possibility of identifying every protein spot in a d gel within a day. this could eliminate the potentially erroneous step of human gel image alignment, whereby land marking could be achieved using the ms data. increased sample throughput requires greater capacity and robust unattended instrument operation. in this poster we describe an integrated robotic multiple plate loader that allows overnight unattended ms operation. other improvements include an increased laser repetition rate that allows the data capture rate to increase four fold. sample tracking, data archiving and data reporting are essential attributes of this new technology and these aspects are outlined in the presentation. the proteinchip tm biology system for ciphergen biosystems: a novel proteomics platform for rapid biomarker discovery, validation and identification ciphergen biosystems ltd., surrey technology centre, the surrey research park, guildford, surrey, u.k. the proteinchip system uses seldi (surface enhanced laser desorption/ionization) proteinchip technology to perform the separation, mass detection and analysis of proteins at the femtomole level directly from biological samples. surfaces are based on either chromatographic based chemistry (ion exchange, reverse phase, imac etc.) that bind large classes of proteins or biologically defined surfaces (antibodies, dna, receptors, etc.) that are used to investigate specific proteininteraction events. as with conventional elution chromatography each type of surface is designed to bind a different subset of proteins from a crude mixture. sample complexity is reduced on the surface by washing with standard biological buffers compatible with the chosen proteinchip array. unlike elution chromatography, proteins are detected directly from the stationary phase using laser based mass spectrometry greatly increasing throughput whilst reducing sample loss and improving reproducibility. multiple proteinchip surface and wash conditions are explored with a small sample set to resolve hundreds of proteins and establish assay conditions that reveal candidate biomarkers or diagnostic protein profiles. the resulting custom built assay is then used to monitor disease processes or drug toxicity profiles by screening large banks of samples such as tissue extracts or physiological fluids (serum, urine, csf, etc.). pharmaceutical research, genomics technologies, f. hoffmann-la roche ltd., basel, switzerland to the present, samples representing the total protein mixture have been usually analyzed by proteomics technologies mainly only the abundant, hydrophilic components have been visualized. these proteins could be solubilized with reagents compatible with isoelectric focusing, for example urea and chaps. such an analysis provides us with a limited image of the proteome, which is insufficient for the detection of the majority of the proteins. in a -d gel, where about mg of protein amount has been resolved, , - , protein spots can be detected, using coomassie blue staining. the spots represent the products of only - different genes. other gene products, not visualized, are most likely expressed at too low levels for detection or they can not be identified because of limitations of the current technology, they are too small, too large, basic or hydrophobic. here we will discuss protein enrichment approaches prior to the analysis, which we have applied for the enrichment of bacterial and eukaryotic proteins. proteomic analysis of the rat liver mitochondrial proteins m. fountoulakis , j.-f. juranville , and l. suter genomics technologies, and drug safety, f. hoffmann-la roche ltd., pharmaceutical research, basel, switzerland subcellular fractionation increases the probability of detection of low-abundance proteins. we prepared mitochondrial, microsomal and cytosolic protein fractions from total liver of male rats. the proteins of the three fractions were analyzed by two-dimensional electrophoresis using broad and narrow ph range immobilized ph gradient strips. the proteins were identified by martix-assisted laser desorption ionization mass spectrometry. in the mitochondrial fraction, different gene products were detected. approximately % of the identified mitochondrial proteins are enzymes with a broad spectrum of catalytic activities. most of the identified proteins had been detected before in other samples as well, analyzed in our laboratory. eight gene products were detected for the first time. these were represented by one spot each, whereas most of the frequently detected proteins were represented by multiple spots. in average, approximately spots corresponded to one gene product. centre for molecular medicine, university college london, u.k. three kinds of experiments have been carried out successfully in our labs. ( ) identification of post-translational modifications of the endothelin a and b receptors (etar and etbr) including both phosphorylation and acylation. we have developed new, very efficient methods for single step isolation of highly pure etar and etbr from cells. this has allowed us to obtain evidence that the post-translational modifications are very complex and result in multiple phenotypes showing different forms of modification for receptor. as with other systems, e.g. insulin-like growth factors, it is probable that these multiple phenotypes of the et receptors correspond to different forms of signalling dependent on cellular state, e.g. the cell cycle. it is, for example, already clear from the phosphorylation of the receptor that a series of different kinases must be involved. ( ) following stimulation of fibroblasts with endothelin, phosphorylation/dephosphorylation signalling cascades involving several hundred proteins have been observed by use of high resolution d electrophoresis and detection of phosphorylated proteins labeled with p by autoradiography or immunological methods. the large number of proteins involved are being identified by mass spectrometric methods such as mass fingerprinting or sequencing by mass spectropmetry. ( ) differential gene expression has been followed by using s met pulse chase labelling concurrently with endothelin stimulation. at least proteins showed significant changes in expression of d gels and these proteins are also being identified. these experiments demonstrate that it is now possible to use proteomics methods to investigate the integration of response to an extracellular signal at the levels of the receptor itself, the subsequent signalling cascades and the ensuing gene expression. the proteomics technology permits concurrent monitoring of large numbers of protein phenotypes (the forms and amounts of individual proteins and is therefore able to provide a global overview of signalling processes which greatly augments more traditional investigations of individual proteins or pathways. furthermore, these new methods will allow quantitative determination of the changes in protein phenotypes, which is very important in view of the highly non-linear amplification properties of such signalling processes. an integrated approach to automated high throughput protein identification by d gel electrophoresis and mass spectrometry d. gostick , s. cohen , p. young , b. karol , j. langridge , j. randell , t. slyker , and a. jacobson micromass, manchester, u.k. waters corporation, milford, massachusetts, and bio-rad laboratories, hercules, california, u.s.a. establishing the function of gene products is the major challenge of the post genomic era. the rate-limiting step in this endeavour is the speed with which proteins can be isolated and identified. separation of proteins from cell lysates or sub-cellular domains by d gel electrophoresis is an established method of visualising these complex systems. recently mass spectrometry has proved to be a powerful method of further characterising these proteins. from the mass spectrum of the enzyme digest of a d gel spot, the resulting digest map is compared with the theoretical maps from the databases and the protein identified when these correlate. maldi-tof is of great benefit in these studies since it requires a minimal amount of sample, is relatively tolerant to salts and other contaminants arising from the gel and may be configured for automated sample analysis. high sample throughput with automated analyses including data processing and client-server database searching are already available. our system automatically acquires the data and processes the maldi mass spectrum into a monoisotopic peak list. this peak list is then automatically sent to a networked database for protein identification. when proteins are not identified from the maldi analysis or an ambiguous result is obtained, then further analysis of the sample by electrospray caplc-ms-ms is required. the development of a hybrid quadrupole orthogonal acceleration timeof-flight mass spectrometer (micromass, q-tof) has facilitated the generation of unambiguous amino acid sequences from the ms-ms analyses of tryptic peptides. these ms-ms spectra can be automatically searched against protein, nucleotide or est databases. thus enabling protein identification from gel spots, despite non-specific enzymatic cleavage, protein co-migration and post transitional modifications. for organisms who's genome sequences are poorly represented in the data bases de novo amino acid sequencing may be required. inferring de novo peptide sequences from ms-ms data is complex and is often the rate-determining step in this method. however, it is now possible to interpret the ms-ms spectrum automatically. in our approach the raw ms-ms spectrum is reduced to the plausible single-charge, monoisotopic mass spectrum. sequence interpretation is achieved by generating "trial sequences" consistent with the experimentally determined molecular weight. a probabilistic fragmentation model is used to transform the trial sequences to predicted spectra for comparison to the single-charge, monoisotopic spectrum and to calculate the likelihood that the trial sequence would account for the observed data. the possible number of trial sequences for any peptide is large, for example there are possible sequences for a peptide containing any of the naturally occurring amino acids and having residues. to reduce the scale of the problem a terminated markov chain monte carlo algorithm is used to produce sequences. this bayesian method simulates an exhaustive search of all sequences having the correct mass. the huge increase in genomic sequence information available, combined with the increased sensitivity and selectivity provided by mass spectrometry, has allowed large-scale protein identification. however the analysis of the post translational modifications present on the identified proteins is a more challenging problem. currently the approach that offers the most expedient and specific solution, to determine modified peptides, is precursor ion scanning. this approach has primarily been performed on a triple quadrupole mass spectrometer where the rear quadrupole, (ms ) is set to transmit only the fragment ion of interest. the ms quadrupole is then scanned across the appropriate mass to charge range. in this paper we describe a method that allows specific post translationally modified peptides to be identified and sequenced during the course of an hplc experiment on the q-tof mass spectrometer. during the hplc run the instrument is switched alternately at one-second intervals between low and high collision energy with argon in the collision cell. the quadrupole, ms is not mass selective, operating in the rf only mode. the first data set at low energy ( ev) shows only the normal pseudo molecular ions. the second at higher energy shows their fragments. wherever a product ion of interest occurs in the high-energy data all its possible precursors are revealed by the corresponding ev data. since the two data sets contain the entire set of precursor and product ions that can be formed it is clearly possible to generate the equivalent of a constant neutral loss scan. this is invaluable in the case of phosphorylated peptides where the neutral loss of da (h po ) occurs via -elimination from the phosphoserine and phosphothreonine residues. this allows the q-tof mass spectrometer to switch from the ms mode to the ms/ms mode of operation when a potential pseudo molecular ion exhibits a neutral loss of da between the high energy and low energy data sets. the product ion ms/ ms spectrum can then be acquired on the phosphorylated precursor ion. in the case of phosphotyrosine, neutral loss of the h po moiety is not observed, however a low mass immonium ion at m/z can be detected. this characteristic ion (from the high energy data) is used to direct the mass spectrometer to fragment potential phosphopeptide precursor ions, which are selected from the low energy data. in this case several precursor ions may require ms/ms interrogation at one decision making time-point. with the first draft of the human genome completed largescale protein identification by mass spectrometry, even for samples originating from higher organisms has become relatively straightforward. this requires a high throughput facility to identify proteins that have usually been separated by d page. the approach providing the highest level of automated sample throughput, in terms of samples per hour, is currently maldi-tof-ms. this technique provides a peptide mass fingerprint of the protein digests and allows the rapid and accurate identification of the parent protein by comparison to a databank. however, under some circumstances, for example if the number of peptides detected is small or if the sequence coverage is poor, it is advantageous to be able to include even a short piece of sequence information to provide added specificity. in a conventional maldi-tof-ms instrument post source decay (psd) can be used to try and generate sequence information, however this approach is notoriously unreliable in producing good quality ms/ms data. one reason for this is that the peptide ions do not undergo fragmentation in a controlled environment such as a gas cell with selected collision gas and collision energy. an alternative approach is to use the predictable fragmentation obtained from a hybrid quadrupole ortho maldi source has been fitted to a hybrid quadrupole orthogonal acceleration time-of-flight (q-tof) mass spectrometer. in contrast to a conventional maldi-tof-ms instrument the resolution and mass measurement accuracy of the data is comparable between the ms and ms/ms modes. this allows superior data acquisition in the ms-ms mode compared to conventional maldi-tof-ms. a number of modifications have been made to optimise the system for high throughput proteomics. the maldi source has been configured with a high-density target plate, compatible with a well microtiter plate. the acquisition software has been modified for automated data acquisition in both the ms mode and the ms to ms/ms switching mode. dedicated processing software has been developed to fully automate the post acquisition and databank searching. this software has been optimised to consider the unique nature of the data acquired from this configuration of instrument. in this paper we demonstrate the how an maldi-q-tof instrument can be used for high throughput proteomics. we also compare and contrast is functionality in comparison with alternative strategies for high throughput proteomics, namely conventional maldi-tof-ms and electrospray lc-ms/ms. pseudomonas putida is an ubiquitous, metabolically and physiologically extremely variable soil bacterium. it is kown to be a good colonizer of plant roots and a plant growth promoter. now, after the sequencing of the total genomic dna has been finished we have focused on the functional analysis of this strain. plant growth promotion is achieved in different ways. one is the inhibition of fungal and bacterial phytopathogens, which is known to be a multifactorial mechanism. an important factor of this mechanism is the production of siderophores (iron-transport-agents), small linear or cyclic peptides, which are synthesized in a ribosomal-independent manner by special synthetases. the siderophore production is induced by iron limitation. the regulation of this process was investigated by pulselabelling with [ p] inorganic phosphate. d-protein patterns generated from cells grown with and without fesupplementation were compared. proteins which were phosphorylated under iron limitating conditions were analysed by maldi-tof peptide mass fingerprint. for the identification of the proteins we used an in-house peptide mass database which has been built based on the genomic sequence data. bio-rad laboratories, inc., hercules, california, u.s.a. worksbase software for proteomics is a platform independent information management system encompassing laboratory experimental workflow and bioinformatics for protein and biochemical research. the worksbase system is designed to allow direct internal integration between laboratory experimental data and background biological knowledge found in reference and in-house data, such as gene, protein and functional annotation databases. worksbase provides a crossdisciplinary research infrastructure for drawing together multiple lines of evidence for characterization of proteins, and integration of this data with domains such as gene expression, pharmacological screening, structure and related areas. while the focus is on the biology underpinning the experimental work, the system is also designed with the capability of providing a sample and workflow tracking system for use in the wet lab, effectively a proteome lims (laboratory information management system). as experimentation proceeds in the laboratory, worksbase software can be used for development of hypotheses on protein, biochemical pathway, and post-translational processing involvement in biological systems and disease processes. as such, identifications that are derived from lab work and user observation can be used to augment the reference data repository. however, unlike databases ands systems where the methods and reasoning for assignment of annotations are obscure, by maintaining the link between the source data and the biological roles derived from them, the accuracy and integrity of any information stored in the worksbase system can be directly ascertained. changes in the brain protein levels following administration of kainic acid k. krapfenbauer , , m. berger , g. lubec , and m. fountoulakis f. hoffmann-la roche ltd., pharmaceutical research, genomics technologies, basel, switzerland institute of cancer research, and department of pediatrics, university of vienna, austria kainic acid (ka), a potent neurotoxin and excitatory amino acid, leads to derangements and modulation of brain proteins. no global brain protein expression pattern induced by ka-treatment has been reported yet. we studied the effect of systemic ka administration on the levels of brain proteins. rats were injected placebo or ka intraperitoneally and brain was taken after one week. the mitochondrial and cytosolic fractions of the brain proteins were analyzed by proteomics technologies. heat shock protein hsp was exclusively detected in brains of animals treated with ka. the levels of neurofilaments and alpha-internexin were significantly decreased and a fragment of tubulin alpha- chain was manifold increased in ka-brains. the mitochondrial enzymes dihydrolipoamide dehydrogenase, atp synthase beta chain and isocitrate dehydrogenase were reduced and pyruvate kinase m was increased following ka treatment. the results indicate altered regulation of heat shock proteins, neuronal death, cytoskeletal disruption and mitochondrial derangement by systemic ka administration. this report confirms and extends previous studies on the effect of ka on the expression of brain proteins and suggests that our analytical system can serve as a model for neurotoxicological, neurobiological and neuropathological proteome studies. the rat brain mitochondrial proteins genomics technologies, f. hoffmann-la roche ltd., pharmaceutical research, basel, switzerland we constructed a two-dimensional database for rat brain mitochondrial proteins. rat is a useful model of human diseases of the central nervous system. in order to detect alterations in the levels of the low abundance brain proteins, the mitochondrial, microsomal and cytosolic fractions were prepared. the proteins of each fraction were analyzed by two-dimensional electrophoresis, followed by martix-assisted laser desorption ionization mass spectrometry. approximately proteins were identified in the mitochondrial fraction, which were the products of different genes. about % of the identified proteins were detected in the mitochondrial fraction only and the rest were detected in the cytosolic and about % were found in the microsomal fraction as well. of the proteins had not been detected before in our laboratory. the identified proteins were in the majority enzymes or enzyme subunits with a broad spectrum of catalytic activities and heat shock proteins. whilst lc-ms/ms has been utilised for the identification of proteins from complexes and cell lysates (qualitative proteomics), the quantitative study of gene expression using differential display has until recently been the preserve of a d gel based proteomic experiment. however, recently a great deal of interest has been generated on the use of isotope coded affinity tags (icat) for the quantitative study of gene expression at the proteome level. the technique is based upon chemically modifying the cysteine residues of proteins isolated from cells in two different states with light and heavy isotopically labeled reagents. the two cell states are then combined, digested with trypsin and the cysteine containing peptides preferentially selected by binding to an avidin column, prior to analysis by mass spectrometry. the eluent from this column is then analysed by capillary lc esi-ms/ms. interrogation of the eluting peptides by tandem mass spectrometry and databank searching results in the identification of the associated protein. we describe how icat data analysis has been automated within a software environment. the ms and msms data acquired using the qtof instrument are processed and analysed using a new algorithm which recognises related isotope clusters and quantifies their relative intensities. based on a user defined ratio threshold the software will automatically carry out an lc-ms/ms experiment and databank search in a client-server mode and provide a report of the identified proteins and their expression ratio in the two cell states. deterioration of the transcriptional, splicing and elongation machinery in brain of fetal down syndrome b. lubec and m. fountoulakis department of neonatology, university of vienna, austria gene technologies, cns research, f. hoffmann la roche, basle, switzerland perturbation of brain development i.e. regulation of gene expression, differentiation, growth and migration in down syndrom (ds) has been reported to occur early in life pointing to impairment of the complex system of transcription and or translation and indeed, altered expression of transcription factors has been reported in adult ds brain. we therefore decided to compare the transcriptional and translational machinery in cortex of brains of controls and fetuses with down syndrome in the second trimenon of gestation. we determined a series of transcription/translation factors by d-electrophoresis followed by maldi -identification and quantification with specific software. the protooncogene c-crk, crk-like protein, elongation factor -alpha , elongation factor , elongation factor tu and two out of four spots representing ptb-associated splicing factor psf were significantly downregulated in brain of fetal ds fetuses as compared to controls. the finding of reduced transcription and translation factors may indicate deranged protein synthesis. the underlying cause for individual reduced transcription, splicing and translation factors may be explained by chromosomal imbalance or by posttranslational modifications as e.g. phosphorylation, known to be aberrant in ds. reduced expression of transcription factors in fetal ds during early life may be responsible or reflecting impaired brain development and deficient wiring of the brain in ds. r. mazzoli , m. g. giuffrida , e. pessione , g. dellavalle , c. barello , e. griva , and c. giunta dipartimento di biologia animale e dell'uomo, università di torino, and csaapz-cnr. c/o bioindustry park canavese colleretto giacosa (to), italy a fast phenol degrading acinetobacter radioresistens strain was isolated in our laboratories and selected for bioremediation applications. this bacterium is also able to grow on benzoate and catechol as sole carbon-energy sources, metabolizing them via the ortho route. in previous researches we detected, by means of proteome analysis, some marker enzymes of the phenol and benzoate degradative pathways. in the present work we extend the identification of the proteins involved in the aromatic-ring opening (the different components of the phenol hydroxylase and benzoate dioxygenase, the catechol dioxygenase isozymes) together with other satellite proteins specifically induced by the aromatic growth substrate. of these last proteins some are probably related to the cellular uptake of benzoate and phenol while others are ascribed to the groel family of heat-shock chaperonines, involved in proteins processing and folding. aromatic substrates may thus act as stress-agents like heat or cold. proteomic studies on rat body fluids i. miller , r. wait , l. sironi , i. eberini , m. gemeiner , e. tremoli , and e. gianazza veterinärmedizinische universität, wien, austria imperial college school of medicine, hammersmith, london, u.k. universita' degli studi, milano, italy previously, we have characterized rat serum proteins, both under "normal conditions" and during experimental inflammation, using two-dimensional electrophoretic separation, densitometric quantitation and identification by mass spectrometry and immunological procedures (http://linux.farma.unimi.it/ homeframed.html). we have now extended these studies to the protein composition of cerebrospinal fluid (csf) and urine, and have identified several proteins specific to these fluids, including major urinary protein, uromodulin, and prostaglandin d synthase. these baseline data provide a useful comparison to the biological fluids of stroke-prone spontaneously hypertensive rats, an inbred strain, which develops cerebrovascular abnormalities following high blood pressure. our studies have detected signs of an inflammatory condition several weeks prior to stroke. we have confirmed the sharp rise in proteinuria preceding stoke onset, and have identified the excreted proteins. following stroke we observe a massive increase in csf protein concentration as serum proteins, even those of large molecular size, cross an impaired blood-brain barrier. as a first step to discover useful disease markers from the urinary proteome, we have developed a unique and systematic approach for detection of low molecular weight urinary proteins by using high resolution two-dimensional ( d) electrophoresis and mass spectrometric methods. unlike previous studies on urinary proteins, and most importantly as observed in present study, our results show that a large number of low molecular weight protein spots can be visualized in the d electrophoresis pattern. it was observed that protein concentration and fractionation methods were critical for our ability to detect many proteins in the gel pattern. therefore, several approaches were carefully considered to concentrate and fractionate proteins in urine samples. initially, urine specimens from normal individuals were concentrated by using centrifugation and ultrafiltration methods. the concentrated samples of urine proteins were then fractionated by size exclusion and immunoaffinity chromatography. the size exclusion method was used to generate two fractions of proteins based on their native molecular weights. further, this method allowed us to enrich concentrations of less abundant proteins for each fraction. the immunoaffinity method was used to specifically remove well-known abundant urinary proteins (such as albumin) from the above mentioned two fractions. that the d pattern includes many native low molecular weight proteins was confirmed by analyzing both protein fractions from size exclusion chromatography. a detailed mass spectrometric analysis of the protein spots is carried out to identify the proteins observed in d pattern. since urine is an ultrafiltrate of plasma, many factors in urine are present in proportion to their rate of synthesis in the body. these factors include many low molecular weight proteins that remain undiscovered due to their low abundance. therefore, the present analysis of urinary proteins would serve as the most useful guide for the discovery of novel diagnostic markers in urinary proteins. i. pucci minafra , , s. fontana , p. cancemi , g. alaimo , and s. minafra , centre of experimental oncobiology, department of cell biology and development, and institute of histology and embryology university of palermo, italy breast cancer is one of the leading causes of death for cancer among women. there are different types of breast cancers, grouped as invasive and non-invasive types. among the invasive types "infiltrating ductal carcinoma" (idc) accounts for about % of all breast cancers. in order to study some biological properties related to this type of cancer, we have developed and well characterized an "in vitro" system, consisting of an idc-derived cell line, -bc (minafra et al., br. j. cancer, , - , ) and some of its cloned cell lines, selected for their high and low invasive activity in matrigel. using this model we are producing proteomic maps to compare with that of non-tumoral breast epithelial cells and with breast tissue fragments, existing in our collection or available at the expasy proteomics server. protein identification is currently done by means of gel matching, edman-microsequencing and immuno-detection. to rationalize data we grouped proteins into functional categories: a) cytoskeletal proteins, b) metabolic enzymes, c) chaperonins and other functionally related proteins, d) peptides and enzymes with regulatory functions. a fifth group consists of peptides with unknown identity. among these sets of proteins we found that glycolitic enzymes and some chaperonins are overexpressed in cancer cells. in addition, new isoforms of potential interest as biomarkers for breast cancer, were identified by means of microsequencing. a. santucci , l. trabalzini , d. soldateschi , e. ferro , a. paffetti , and p. martelli dipartimento di biologia molecolare, sezione di chimica biologica, universita' degli studi di siena, and diesse diagnostica senese srl, siena, italy human cytomegalovirus (hcmv) is an ubiquitous virus, belonging to the herpesviridae family, betaherpesvirinae subfamily, able to induce morbidity in immunocompromised patients and congenitally infected new-borns. hcmv has the largest genome among the herpes-viruses ( kbp): ad strain genome was completely sequenced, containing about open reading frames encoding polypeptides, most of which are not characterized. the viral genes are activated in a cascade fashion: ) alpha, immediate-early genes, coding for regulatory proteins necessary for the activation of ) beta, early genes, needed for dna replication, and, finally ) gamma, late genes, coding for structural proteins of the mature virions. this latter category includes the virus surface antigenic proteins responsible for the main immune response during hcmv infections. although the sequencing of hcmv genome has been completed, very little is known about the actual nature of the viral proteins. the most appropriate approach to characterize hcmv phenotype is to study its protein expression as it is carried out within the host cell. for this purpose, we analyzed by two-dimensional electrophoresis ( d-page) the protein phenotipic repertoire of human fibroblasts and compared it with that of the same cell type following infection with hcmv strain ad . the phenotypic d map of human fibroblasts dramatically changes following infection with hcmv. a relevant amount of newly appeared spots is attributable to hcmv proteins, mainly of the structural category, since we analyzed host cells at the - th day of infection, when the late, gamma genes are supposed to be the only to be activated. on the other hand, a marked decrease of protein synthesis can be easily evidentiated in the infected fibroblasts respecting to uninfected cells. a temptative mapping of the main structural viral proteins (those against which patients sera are directed) was carried out by immunoblotting, microsequencing and mass spectrometry. comparative proteomics of cultured cells: identification of genetic defects and molecular mechanism of apoptosis regulation v. seyrantepe , k. landry , s. taurin , s. n. orlov , and a. v. pshezhetsky sainte-justine hospital research centre, and research centre, chum, university of montreal, montreal, pq, canada we employed a comparative proteomics of cultured cells to study mechanism of genetic disorders and for identification of key proteins involved in cell proliferation, differentiation, and death. in particular, this technology proved to be very useful to understand molecular basis of severe inherited diseases resulting from deficiency of lysosomal membrane transporters, and a role of programmed cell death (pcd) of vascular smooth muscle cells (vsmc) in cardiovascular disorders. to increase sensitivity of the identification of cellular proteins we have either have isolated cellular organelles such as lysosomal membranes or performed the differential extraction of soluble, membrane and cytoskeletal proteins. by comparison of pro-teomic cell maps from normal controls and individuals affected with lysosomal transport disorders we have selected and identified several candidate disease-causing proteins, which have to be further studied by mutation analysis and functional expression. for the second group of disorders we identified proteins, which de-novo synthesis could result in survival of vsmc including a two members of hsp family, a molecular chaperone grp , and so-called mortalin (grp ) highly expressed in non proliferative tissues and associated with mortal cell phenotype. two-dimensional polyacrylamide gel electrophoresis ( d-page) is the established technology employed for the separation of proteins from a cell lysate, sub-cellular organelle or tissue sample prior to identification of the excised protein spots by mass spectrometry. in the order of several hundred to several thousand proteins, can be separated and visualised on a d gel by conventional staining or utilising fluorescent labelling techniques. the advantage of performing a two dimensional gel based separation is the ability to obtain quantitative information by comparing and contrasting two samples in a differential display experiment, for example, between a healthy and diseased state. the last stage however stipulates that the gels are reproducible which can be both difficult and time consuming to achieve. the relativity poor dynamic range that the gels exhibit also limits quantification. other restrictions include the under representation of certain classes of proteins, such as membrane proteins, large or small proteins and very acidic/basic proteins. for these reasons, amongst others, alternatives to d-page are being investigated. advances in both lc and mass spectrometry instrumentation have allowed the analysis of protein complexes, which have not been separated on a d gel. in this case protein identification is achieved via database searching of esi-ms/ms data. this provides qualitative information on the proteins that are present and has recently been coupled with isotope dilution experiments to provide relative quantiative information. these experiments normally involve separation of the complex digest mixture by microcapillary liquid chromatography connected to an instrument capable of data dependant switching between the ms and ms/ms modes. using this approach it has been demonstrated that hundreds of ms/ms spectra can be acquired in a fully automated fashion, resulting in the identification of significant numbers of proteins, including low copy number proteins, from a single lc-ms/ms experiment. if, however, a complex protein mixture is to be investigated then a fractionation step prior to separation of the peptides on the basis of their hydrophobicity would be advantageous. we have, therefore, adopted a d lc-ms/ms approach using a capillary lc system (caplc) operating at nanoliter per min flow rates coupled to a q-tof mass spectrometer. by replacing the standard sample loop within this system with a strong cation exchange (scx) cartridge followed by a c trap cartridge it is possible to pre-fractionate the peptides before separation on a c column. after loading the sample, discreet fractions are sequentially eluted from the cation exchange cartridge using a salt step gradient; the eluted peptides are then retained on the trapping c cartridge whilst they are desalted. finally the peptides are eluted from the c pre-column, at nl/min, onto a um id ϫ cm waters symmetry analytical column for separation and elution into the mass spectrometer. this analytical approach will be discussed with examples where this methodology has been used for the analysis of standard protein mixtures and also for the analysis of cell lysates and sub-cellular fractions. monoclonal igg are commonly observed in various b cell disorders, the most clinically relevant being multiple myeloma. in a series of serum samples, immunofixation identified igg , igg , igg , and igg in , , , and cases, respectively. their light-chains were k in cases and λ in cases. these monoclonal igg were further characterized by high resolution two-dimensional polyacrylamide gel electrophoresis ( -de) with various isoelectric focusing conditions as well as by -de ( -de of the proteins extracted from agarose after serum protein agarose electrophoresis). after -de or -de, the monoclonal γ-chains were not visualized in out cases, whatever the isoelectric focusing conditions that were tested. in cases, γ-chains were only detected using alkaline ph - gradients. monoclonal γ-chains and light chains were highly heterogeneous in terms of pi and mr. however, a good correlation (p Ͻ . ) was observed between the index of migration of the monoclonal igg in agarose gels and the pi of their γand of their light-chains (r ϭ . , multiple linear regression). because of the extreme diversity of the different γ-chains as well as of the k-and γ-chains, it appears that a classification of monoclonal igg based only on their electrophoretic properties is not possible. alzheimer's disease (ad) is one of disorders caused by protein conformational changes and recent studies have shown that several chaperone proteins are involved in this process. as information of chaperone expression in ad brain is limited, we aimed to study the expressional pattern of chaperones in several brain regions as this may be essential to understand how folding defects can lead to disease. we studied the concomitant expressional patterns of molecular chaperones in seven brain regions of adults with ad using two-dimensional polyacrylamide gel electrophoresis ( -de) and matrixassociated laser desorption ionization mass spectroscopy (maldi-ms). we unambiguously identified and quantified nine different chaperone proteins. six chaperone proteins, heat shock protein (hsp ), hsp ry, heat shock cognate (hsc) , alpha crystallin b chain, glucose regulated protein (grp) and grp showed aberrant expressional patterns depending on brain region. hsp . , grp and t-complex (tcp- ) epsilon subunit did not show any significant expressional change. these findings are compatible with neuropathological and biochemical abnormalities in ad brain and this report presents the first approach to quantify nine different chaperones simultaneously at the protein level in individual ad brain regions providing evidence for the relevance of aberrant chaperone expression to ad neuropathology. the mainstream approach to protein separation, visualisation and identification has been to use two-dimensional gel electrophoresis coupled to mass spectrometry for the identification of the separated proteins. however this approach is limited with the level of protein that may be loaded onto the d gel and the nature of the proteins that may be incorporated onto the first dimension (ipg strip). an alternative approach for the qualitative analysis of complex protein mixtures is the use of tryptic digestion followed by electrospray lc-ms/ms. this approach is dependent on a high degree of chromatographic separation prior to the mass spectrometer, such that ideally individual peptides are eluted into the source. if this is the case then the dynamic range of protein identification can be increased and low copy number proteins can be identified. often, however there is a large degree of redundant sequence information acquired, as in theory one peptide ms/ms spectrum is sufficient to identify a protein from a sequence databank. if a protein identification is obtained from a databank search of an ms/ms spectrum, it is potentially valuable to exclude the rest of the theoretical tryptic peptides to "mine" deeper into the protein complex being studied. we have introduced a new protein databank search engine capable of matching a tryptic peptide from the swissprot/ trembl databank to an ms/ms spectrum in one second. using this search engine we are able to generate dynamic tryptic peptide exclude and include lists, based upon the theoretical tryptic peptides from the identified protein, which can be passed to the acquisition software of our q-tof mass spectrometer in real time. thus, we are able to automatically steer the q-tof, during acquisition, to select and switch to the ms/ms mode only on those peaks that meet the modified selection criteria. experiments can be designed in which peaks that belong to a protein already identified during acquisition can be avoided. this exclusion list is based upon m/z, charge state and a user definable mass tolerance. the mass measurement of the data from the q-tof mass spectrometer is typically better than ppm and as a consequence of this a tight mass tolerance can be selected, thus making the exclude list extremely specific. alternatively, in the case of samples derived from d gel spots, the mass spectrometer may abandon the current sample, re-equilibrate the lc column and move on to the next sample. to illustrate this methodology we show examples, both on standard samples and complex protein mixtures where q-tof data acquisition has been directed based upon the results from a databank search. this data will be compared and contrasted to data acquired in the normal automated lc-ms/ms mode. the specific anti-cancer activity of green tea (؊)-epigallocatechin- -gallate (egcg) department of molecular biology, hebrew university-hadassah medical school, jerusalem, israel the effect of the green tea polyphenol-(Ϫ)epigallocatechin- -gallate (egcg) was tested in cultures of normal and transformed nih-patmras fibroblasts. in this system transformation can be induced at will by the addition of dexamethasone, which induces the expression of h-ras by activating the mammary tumor virus long terminal repeat (mmtv-ltr) promoter. this facilitates a reliable comparison of the susceptibility of normal and transformed cells to egcg. it has been shown that egcg inhibited the growth of transformed but not of the normal fibroblasts. in an attempt to elucidate the mode of the preferential inhibitory activity of egcg, its effect on growth promoting factors has been examined. the level of ornithine decarboxylase (odc, ec . . . ), which is a signal for cellular proliferation, was reduced by egcg in the transformed but not in the normal cells. egcg also showed strong inhibition of tyrosine kinase and mitogen-activated protein kinase (mapk) activities, without affecting the kinases in the normal cells. similarly, egcg also preferentially decreased the levels of the oncogenes ras and jun in transformed cell. egcg preferentially induced apoptosis in the transformed fibroblasts. in vitro chemosensitivity tests demonstrated that egcg inhibited the proliferation of leukemic cells. these findings suggest that egcg has a therapeutic potential in the combat against cancer. objectives: to develop a safo, affordable immune supportive therapy for hivϩ patients. design: a randomised, double blind, placebo-controlled study, testing an internationally patented l-methionine combination (lmc), in approximately hivϩ patients; not yet on anti-viral treatment (cd count to ). methods: parameters measured included: cd count, total lymphocyte court, viral load, several clinical, as well as mechanistic parameters. the difference in the change from the baseline (active -placebo) was determined for each parameter. the study is ongoing. results: within months, significant trends are noted. the cd count of the patients on the active therapy, presented with a slower rate of decrease, compared to the placebo group, mean difference (md) in this change from baseline; . /cmm and % confidence interval (c ), this was confirmed by the total lymphocyte court values. after months the placebo group was placed on active, causing the difference to disappear. conclusions: although further trials are needed, these results already indicate t-methionine as an important role player in the immune system of patients with impaired immune function. c. chiarla , i. giovannini , j. h. siegel , g. boldrini , and m. castagneto centro di studio per la fisiopatologia dello shock cnr, catholic university, rome, italy department of surgery, umdnj, newark, new jersey, u.s.a. in critical illness and sepsis, changes in amino acid plasma levels (aapl) have been assessed extensively, while little is known about the relationship with changes in other plasma components, such as those involved in fluid-electrolyte and osmotic balance; their investigation is also limited, in large clinical samples, by inter-patient variability. we analyzed the relationships between plasma sodium (na ϩ pl, meq/l) and aapl (µm/ l) in eighty consecutive measurements performed in one single patient with post-traumatic sepsis and severe, prolonged illness. unique feature of plasma taurine (tau) was maintenance of a highly significant inverse correlation with na ϩ pl (r ϭ . , p Ͻ . ). all other aapl were correlated directly, or unrelated, to na ϩ pl, the only exception being a weak inverse correlation between tryptophan and na ϩ pl. tau was correlated, strongly and directly, also to phosphoethanolamine (pea), glutamate (glu) and aspartate (asp): tau ϭ . ϩ . (pea) Ϫ . (na ϩ pl) r ϭ . , p Ͻ . tau ϭ . ϩ . (asp) Ϫ . (na ϩ pl) r ϭ . , p Ͻ . tau ϭ . ϩ . (logglu) Ϫ . (na ϩ pl) r ϭ . , p Ͻ . and unrelated, or weakly and inversely related, to other aapl (measurements of beta-alanine were not included). co-variation of na ϩ pl and these aapl (particularly tau and pea) was influenced by severity of illness, and more complex regressions were needed to quantify this effect. these results provide useful information on interdependency of tau, na ϩ pl and other aapl in critical illness. the central nervous system (cns) shows an exceptionally high degree of vulnerability to reactive oxygen species. considerable evidence suggests that free radical formation and oxidative stress might play an important role in the pathogenesis of parkinson's disease (pd). moreover, it has been reported that the levels of glutathione and vitamin e increase in the brain of patients with pd as a compensatory mechanism to deal with oxidative stress. since vitamin e is an effective free radical scavenger in the brain, its neuroprotective function is the issue of new therapeutic approaches in neurodegenerative diseases. to elucidate the possible role of vitamin e in the pathogenesis of pd, we assessed the plasma levels of vitamin e, measured by high-performance liquid chromatography, in patients with pd. vitamin e concentrations were also assessed in age and sex matched normal individuals. the mean plasma levels of vitamin e did not differ significantly between these two groups ( . Ϯ . mmoli/l for pd patients and . Ϯ . mmoli/l for controls). the results of our study suggest that plasma vitamin e concentrations do not play a major role in the pathogenesis of pd. vitamin e and cardiovascular disease: nutritional and intervention approaches f. galli , institute of biological chemistry, university of urbino, italy department of cardiovascular research, st thomas' hospital, london, u.k. vitamin e is represented by a family of eight natural vitamers ( tocopherols and tocotrienols) of which αtocopherol (α-t) form has the highest biological activity. this vitamin accounts for most of the lipid-soluble, chain-breaking antioxidant activity in mammalian tissues and plasma. in addition, it shows nonantioxidant properties through which it modulates cell signaling and the expression of specific enzyme in cell models playing a role in atherogenesis (e.g. endothelial and inflammatory cells). the preventive effect of vitamin e on acdv is still a matter of debate. the largest epidemiological investigations and out of main intervention studies at yet available have suggested a correlation between levels of vitamin e and incidence of atherosclerotic cardiovascular disease (acvd) and related mortality. an overall conclusion rising from these studies is that the major effect (if any) of vitamin e is to be found with intakes higher than iu ( mg all-rac α-tocopheryl acetate) per day. however, other investigations have failed to demonstrate a beneficial effect of vitamin e against acvd, suggesting the need for more studies on its metabolism and function. recently a family of tocopherol binding and transport proteins has been identified. they play a key role in the selective uptake and delivery of tocopherols to lipoproteins and tissues. genetic abnormalities of these proteins have been demonstrated to be responsible for conditions of vitamin e deficiency in humans. their tissue distribution and regulation are now under investigation. the information available on vitamin e metabolism and its response to supplements or diet changes are at yet poorly characterized. the synthesis of stable isotopes and the characterization of major metabolites of main vitamers provide important advances in this research. in the last years, both plasma levels and urinary excretion of relevant metabolites of α-t have been characterized. little information is available on metabolites formed by other vitamers. the emerging role of γ-t and its main catabolite , , -trimethyl- -(b-carboxyethyl)- hydroxychroman (γ-cehc) in the defense against nitrogen oxide species formed during the activation of inflammatory cells is now well established and suggests the need for further studies on the bioavailability and transformation of this homologue of vitamin e in humans. at the same time, an oxidation byproduct of α-t found in human plasma, namely α-tocopherylquinone, has been proposed to be also de novo synthesized from phenylalanine with a role in the genesis of a defective polyunsaturated fatty acid metabolism observed in phenylketonuric patients. this suggests a possible, and at yet unexplored relationship between vitamin e and phenylalanine/fatty acid metabolism which might have also a role in atherosclerotic process. r. gaspari , s. mensi , g. mercurio , c. callà , l. colacicco , e. sacco , and s. lippa department of anaesthesiology and intensive care medicine, and department of biochemistry and clinical biochemistry, catholic university of rome, italy four patients ( females, male; aged from to years) affected by severe liver failure, were treated by a new blood purification method, namely molecular adsorbent recycling system (mars). mars removes albumin-bound toxins using a specific membrane with a dialysate solution containing albumin. in the patients the plasma levels of methionine (meth), branched chain and aromatic amino-acids and liposoluble antioxidants were measured. the fischer's index did not show any significant variation, whereas the plasma levels of meth were well correlated with the levels of liposoluble antioxidants (vitamin e and coq ). in fact, in the patients receiving just branched chain amino-acids, the plasma levels of both meth and antioxidants progressively decreased. on the contrary, if meth and branched chain amino-acids were administered, the plasma levels of coq and vitamin e showed a positive correlation with the plasma meth levels (p Ͻ . ; r ϭ . and p Ͻ . ; r ϭ . , respectively). since vitamin e and coq are mutually dependent-molecules, the administration of meth, essential substance for coq synthesis, may be effective to maintain a good antioxidant status in patients with severe liver failure undergoing mars treatment. we obtained new synthetic peptide preparation epitalon to be widely applied as a pharmaceutical due to its properties important in medical care. epitalon was found to stimulate repair processes in retinal diseases via restoring the retinal functions, in particular its photoreceptors. this promising peptide drug is a linear tetrapeptide of formula h-ala-glu-asp-gly-oh (alanyl-glutamyl-aspartyl-glycine). the substance was obtained by classic peptide synthesis in a solution (scheme: ( ϩ ) ϩ ) with n-oxysuccinimide activated esters. coohgroups of lateral radicals of glutamic and aspartic acids were defended as benzyl esters, benzyloxycarbonyl (ala) and tert.butyloxycarbonyl (glu) n-defending groups were employed, deblockade conducted by trifluoroacetic acid and catalytic hydrogenolysis. preparative hplc on a reverse phase was applied for purification. the product was fully characterised by the data of analytical hplc (substance content - %), amino acid analysis, ir-and hmr-spectra. the ready drug form is ampoules containing µg of the substance in ml of isotonic solution. epitalon application in patients with pigmented retinal degeneration stopped the pathology development in % and increased visual functions in % of the cases. in % of the patients visual acuity raised by . - . . electroretinography confirmed the retinal functional activity increase. an increasing number of proteins are implicated in apoptosis and several of them have been shown to be altered in alzheimer's disease (ad) brain. because of this apoptosis is thought to be the underlying mechanism of neuronal cell loss in ad. to further substantiate this hypothesis we investigated the expression of a recently identified apoptosis related proteins and other apoptosis regulators in frontal cortex and cerebellum of ad by western blot and elisa techniques. quantitative analysis revealed unaltered levels of bax and raidd (receptor interacting protein associated ich- (caspase- )/ ced- (caenorhabditis elegans death protease- )-homologous protein with death domain) in both regions. zip (zipper interacting protein) kinase, bim/bod (bcl- interacting mediator of cell death/bcl- related ovarian death gene) and p were significantly increased only in ad frontal cortex (p Ͻ . , in all cases). cerebellar bcl- levels were significantly increased in ad (p Ͻ . ) while in ad frontal cortex, although the levels tended to increase did not reach significance level. the results indicate that apoptosis indeed account for the neuronal loss in ad. however, it does not seem to involve bax and raidd. a. magyar , m. brózik , r. tóbi , t. szabó , j. szakonyi , b. rojkovich , p. gergely , and f. hudecz research group of peptide chemistry hungarian academy of science, budapest, central laboratory of immunology, semmelweis university, budapest, and national institute of rheumatology, budapest, hungary rheumatoid arthritis (ra) is a systemic autoimmune disease of unknown etiology. it is the most common of the inflammatory joint diseases, affecting - % of the world population. anti-filaggrin antibodies (afa) directed against the epidermal protein, filaggrin, belongs to the most specific markers of ra. epitopes, containing citrulline within the sequence of filaggrin, have been recently identified as major antigenic sites recognised by afa. the aim of our study was to identify these epitopes of filaggrin derived-peptides targeted by ra specific antibodies to provide further information about the nature of the initial autoantigenic substance. the most immunogenic six sequences of filaggrin and further, on the n-and c-terminal, shortened version of the original peptide ( shqestrgrsrgrsgrsgs ) were synthesized. we used conventional solid-phase peptide synthesis (fmoc strategy) carried out on "multipin ncp" noncleavable kit. in elisa experiments the presence of afa was deter-mined using serum samples of ra patients and healthy blood donors. in conclusion our results provide further evidence that not simply the presence of citrulline but also the nature of its surrounding amino acids have important role in the creation of autoantigenic epitope reactive with anti-filaggrin antibodies. the autoimmune nature of multiple sclerosis (ms) has introduced cytokine genes as logical candidates for the loci determining susceptibility to the disease and/or influencing disease progression. interleukin (il)- alpha and beta are major proinflammatory cytokines that have been related with several chronic inflammatory diseases such as ms. the il -receptor antagonist (il- ra) is a protein structurally related to il- beta that effectively inhibits the proinflammatory effects of il- . a polymorphism in the Ј-flanking regulatory region at Ϫ of the il- alpha gene, which may cause an overexpression of il- alpha and a variable number tandem repeats (vntr) polymorphism in the il- ra gene have been also associated with several inflammatory diseases. two biallelic base change polymorphisms in the il- beta gene have been reported to influence the protein production: one is located in the promoter region at position Ϫ and the other is in exon at position ϩ . to analyze the contribution of il- alpha, il- beta and il- ra genes in the genetics predisposition to ms, we have examined four polymorphic genetic markers in italian patients with clinically definite ms and healthy controls. in summary, no significant differences in genotypes and allele frequencies were found between ms patients and healthy controls. fibronectin -the extracellular matrix protein is oxidatively modified with oxygen reactive species (ros) in inflammation site. activated neutrophiles release the hypochlorite acid (hocl) and chloramines as products of myeloperoxidase/ h o /cl Ϫ system. these reactive chlorine species chlorinate in turn matrix proteins. the resulting changes of tertiary protein structure could be evaluated by monitoring the antigen/antibody complex formation. the formation of the complexes between native/chlorinated fibronectin and igg class antibodies were examined by means of elisa with luminol chemiluminescence detection. the degree of fibronectin modification was monitored with spectroscopic methods. since the oxidation leads to the fibronectin aggregation -the tryptophane contents in resulting aggregates were evaluated with stern-volmer approach (acrylamide quenching). moreover, the aldehydes influence on the ag/ab complex formation was examined -since aldehydes are known products of amino acids n-chloramines deamination. also the native and modified fibronectin adherence to the matrix proteins was monitored with use of hrp labeled antifibronectin antibodies. the preliminary results suggest that chlorination impairs the ab/ag complex recognition but also prove that igg bounded chlorinated fibronectin promotes igg clusters formation. it was found also that mm concentration of the serine derived glycoaldehyde decreases the fibronectin/igg recognition and the effect could be attributed to the igg aggregates formation. we demonstrate also that hrp-labeled iggs detect the collagen and fibrynogen adherent fibronectin in a dose dependent manner-details of the elisa method are discussed. in subjects with rheumatoid arthritis (ra) oxidized low density lipoproteins (ldl) are supposed to serve as mediators for joint damage, further exacerbating the inflammatory process. to better understand mechanisms of ldl oxidation in ra a specific marker of oxidative modification of apolipoprotein (apo) b- proline and arginine residues, hydroxy- -aminovaleric acid (hava), had been measured in plasma and synovial fluid ldl subfractions (ldl , svedberg units (s f ) - and ldl , s f - ) by gc-ms. paired knee synovial fluid and plasma samples were collected from subjects with ra. additionally, plasma samples were collected from healthy controls. the ldl hava content in plasma was not different between the groups (ra, . Ϯ . vs controls, . Ϯ . mol/mol apob- , p ϭ . ). the ldl hava content in plasma was significantly higher in ra ( . Ϯ . vs . Ϯ . mol/mol apob- , p ϭ . ). furthermore, synovial fluid ldl and ldl in ra contained elevated hava levels when compared with plasma concentrations (ldl syn , . Ϯ . mol/mol apob- (p Ͻ . ) and ldl syn , . Ϯ . mol/mol apob- (p Ͻ . )). results suggest that proline and arginine residues of apob- are highly reactive toward oxygen radicals in both plasma and synovial fluid in ra. furthermore, susceptibility of apob- to oxidative modification increases along the lipoprotein metabolic cascade. particularly small dense ldl were prone to direct oxidation of apob- . correlation between hava content in plasma and synovial fluid ldl and ldl in ra may allow the use of hava as a clinical marker of antioxidant barrier impairment in ra. vascular collagen accumulation contributes to development of hypoxic pulmonary hypertension (ph). we have shown that injections of a polymer of the proline analogue cis- -hydroxy-l-proline (chyp) in liposomes attenuated acute ph in rats (ajrccm ; : ) . we now treated rats with established ph with a new polymer containing an increased "payload" of chyp. chyp was conjugated to a low mw poly(ethylene glycol)-lysine carrier {poly (peg )-lys-chyp} to increase the % by wt of the analogue. rats were exposed to % o for da to induce ph. on da , and after da of hypoxia, animals were injected iv with chyp polymer in liposomes (hc) or bioinactive trans-hyp polymer in liposomes (ht). air controls received thyp polymer in liposomes (at). at and da, we measured mean right ventricular pressure (rvp) and hydroxyproline (hyp) content in main pulmonary arteries. on da , rvp (mmhg) was Ϯ and hyp (µg/vessel) was Ϯ in at. rvp and hyp increased to Ϯ * and Ϯ *, respectively, in hypoxic animals (n ϭ ; *p Ͻ . vs. at). on da , rvps were at Ϯ , ht Ϯ *, hc Ϯ * †; hyps were at Ϯ , ht Ϯ *, hc Ϯ * † (n ϭ ; *p Ͻ . vs. at; †p Ͻ . vs. ht). from da to , rvp did not increase and hyp decreased in the hc group vs. ht. we conclude that weekly injections of polymeric chyp prevented progression of established hypoxic ph and reversed hyp accumulation. targeted delivery of antifibrotic polymers may prevent and reverse the progression of ph. (support: phs, barbara cornwall foundation). glucosinolates are amino acid-derived natural plant products found throughout the capparales order, which includes agriculturally important crops such as oilseed rape, brassica vegetables and the model plant arabidopsis. glucosinolates and their degradation products have a wide range of biological activities, e.g. in plant defense as deterrents against insect and fungi and as attractants to insects that are specialized feeders in brassicaceae. the conversion of amino acids to oximes is a key step in glucosinolate biosynthesis. we have recently shown that cytochromes p belonging to the cyp family catalyze the conversion of aliphatic, aromatic as well as indole amino acids to the corresponding oximes. cyp e catalyzes the oxime-metabolizing step in the biosynthesis of the cyanogenic glucoside dhurrin. we have recently shown that the oxime-metabolizing enzyme in the glucosinolate biosynthetic pathway is a cytochrome p homologous to cyp e . the post-oxime enzymes in the glucosinolate pathway have high substrate-specificity for the functional groups, and low substrate-specificity for the side chain. therefore, we have been able to metabolically engineer new glucosinolate profiles into arabidopsis by altering the level of endogenous cyp s and by introducing new cyp s. the approach has great potential for design of "biotech crops" with improved pest resistance and increased nutritional value. hypercalcemia as a potential threat in the dietary treatment of maternal phenylketonuria f. eyskens and s. beernaert pediatrician, metabolic diseases and dietitian, azm-koningin paola childrens hospital, metabolic lab pcma, antwerp, belgium over % of infants born to mothers with blood phenylalanine (phe) concentrations above µmol/l exhibit evi-dence of foetal dammage, low birth weight, microcephaly, dysmorphic facies, slow postnatal growth and development and long-term intellectual impairment. keeping maternal phe concentrations below µmol/l before conception and throughout pregnancy reduces significantly the risk of abnormalities in the offspring of women with phenylketonuria (pku). we describe a woman, years old, who showed phe blood levels of - µmol/l under a strict diet (total protein content of . g/kg body weight/day with . g/kg natural proteins and . g/kg proteins provided by the aminoacid mixture pku (milupa, germany); , cal/day) at the beginning of her first pregnancy. the first weeks she developed vomiting which gradually increased in severity. at weeks of pregnancy, she had diarrhea, severe bouts of vomiting and manifested a deficient nutritional status with intake of . g/kg bw proteins and , cal/day. she was hospitalized to start refeeding using continue drip feeding administered by nasogastric tube. after days on this regimen she developed vomiting, heart palpitations and mental confusion. her serum calcium level, that was normal at admission in the hospital, showed an elevation to . - meq/l (ref. value . - . meq/l). the feeding was stopped immediately and under an intravenous infusion and gradually introducing a feeding composed of pku , carbohydrates and mct fats the serum calcium and the blood phe levels dropped to normal values. and volatile components of caramel obtained by heating commercial maltose solution for different time intervals. one sample containing maltose only was used as control, the caramelization was conducted at c° for total time period minutes and subjected to sensory analysis and isolation of volatile components. the odour and colour sensory tests were evaluated according to the international standard methods (iso). the results showed that addition of lysine as a catalyst gave rise to a significant (p Ͻ . ) increase in intensity of the whole flavour in comparison with the control sample. the sweet and caramel notes, the most characteristic attributes of caramel, showed remarkable increase. on the other hand the increase in heating time in presence of lysine as a catalyst resulted in high significant increase in browning rate of caramel solution. the volatile components of each sample were isolated by using the new technique, solid phase microextraction (spme) and subjected to gc and gc-ms analysis. over volatile components were separated, however only the most important component for caramel flavour were reported. maltol and hydroxymethyl- -furfural (hmf) and . h-pyran- -one, , dihydro- , -dihydroxy- -methyl (dihydro dihydroxy maltol), the main characteristic caramelization products were present in high concentration in samples contaning lysine heated for minutes. in addition one pyrazine was only identified in the samples contaning lysine. a comparative study between the present results and those of our previous study concerning addition of alanine as a catalyst was carried out. short-term exposure of human umbilical vein endothelial cells (huvecs) to hyperglycemia increases l-arginine transport (system y ϩ /cats) and nitric oxide (no) production (via enos). it has been reported that enos could also be activated by a ca ϩ -independent mechanism involving phosphorylation of ser by a phosphatidylinositol -kinase (pi -kinase) dependent pathway. we investigated the involvement of pi kinase on the stimulatory effect of acute hyperglycemia on enos and l-arginine transport in huvecs. l-arginine transport, no synthesis and phosphorylation of ser in enos were increased by d-glucose ( mm, min). similar results were obtained in huvecs exposed to insulin. incubation of cells with wortmannin (pi -kinase inhibitor) prevented the effects of d-glucose and insulin. no changes in the intracellular ca ϩ and enos protein levels were detected. thus, acute hyperglycemia increases l-arginine transport and enos activity through a pi -kinase dependent, ca ϩ independent mechanism in huvecs. [ the hypercalcemia in this patient was due to a very high content in calcium of the feeding administered ( - times the adh value) associated with a high vitamine d concentration (see table) and a clinical state of dehydratation. the further pregnancy was uncomplicated and a healthy girl was born who developed normal. • the aminoacid mixtures used in the treatment of pku contain a high level of calcium, phosphate, magnesium and iron. they also contain a high concentration of vitamine d. • nutritional monitoring of pregnant pku patients should include the calcium, phosphate, iron, zinc and vitamins status. • vitamins a and d suppletion is contraindicated in these patients based on the high concentrations of these vitamins in the aminoacid mixtures used in the dietary treatment. flavour and aroma chemistry department, national research centre, dokki, cairo, egypt caramelization of various carbohydrates leads to product with a high tinctorial strength provided by different additives catalyzing the process. the present study was conducted to evaluate the catalytic effect of lysine on the sensory attributes administered pku ( g ϭ adh , g/kg/bw) lipoic acid is a prosthetic group of h-protein of the glycine cleavage system and e components of the pyruvate, oxoglutarate and branched-chain -oxoacid dehydrogenase complexes. in mammals, attachment of lipoic acid to these proteins requires two enzymes. lipoate-activating enzyme (lae) catalyzes the activation of lipoate to lipoyl-nucleoside monophosphate. then, lipoyltransferase transfers the lipoyl moiety to the specific lysine residue of the proteins. we purified lae from bovine liver mitochondria. lae activated lipoate with gtp at a -fold higher rate than with atp. the reaction absolutely required lipoate and mggtp, and the reaction product was lipoyl-gmp. lae activated both r-and senantiomers of lipoate to the respective lipoyl-gmp although preference for r-lipoate was observed. lipoyltransferase equally transferred both r-and s-lipoyl moiety from respective activated lipoate to apoh-protein. however, only h-protein carrying r-lipoate was active in the glycine cleavage reaction. cdna clones encoding a precursor lae with a mitochondrial presequence were isolated. amino acid sequence of lae was identical with that of xenobiotic-metabolizing/medium-chain fatty acid : coa ligase-iii, but an amino acid substitution due to snp was found. these results indicate that the medium-chain acyl-coa synthetase in mitochondria plays a novel function with gtp, the activation of lipoate. instituto di chimica biologica "g. fornaini", università di urbino, italy nitric oxide (no) can modulate red blood cells (rbc) glycolysis by translocation of the enzyme glyceraldehyde- phosphate dehydrogenase (gapd) [e.c. . . . ] from the cytosolic domain of the membrane protein band (cdb ) in the cytosol. in this study we have investigated which no-reactive thiols might be involved influencing gapd translocation, and which is the role of glutathione (gsh) in this context. two highly reactive cys residues (k ϭ . m Ϫ s Ϫ and . m Ϫ s Ϫ , respectively) were identified by transnitrosylation with nitrosoglutathione (gsno) of cdb and gapd. the cys in the catalytic site of gapd is exclusively involved in this gsno-induced nitrosylation. reassociation experiments carried out at equilibrium with preparations of rbc membranes and gapd revealed that different no-donors may form Ϫsno on, and decrease the affinity between, gapd and cdb . in intact rbc, both the no-donors -morpholino-sydnonimine (sin- ) and peroxynitrite (onoo Ϫ ) significantly increased gapd activity in the cytosol and glycolysis measured as lactate production and energy charge levels. however, we obtained data suggesting that onoo Ϫ is the main no-derivative able to cross the rbc membrane leading to gapd translocation and Ϫsno formation. both in cell-free experiments and intact rbc, diamide (a thiol oxidant able to inhibit gapd activity) was observed to reverse the effect of sin- on gapd translocation. the results demonstrate that cdb and gapd contain reactive thiols that can be transnitrosylation mainly by means of gsno, these can ultimately influence gapd translocation/ activity and the glycolytic flux. abteilung für allgemein-viszeral-und gefässchirurgie, kliniken dr. erler gmbh, nürnberg, germany new surgical procedures like minimal-invasive-surgery brought many advantages for the surgical patient: less pain and shorter hospitalization. regarding nutrition, patients gets normal food on the ward still on the operation-day and need only saline-infusions overnight for fluid and electrolyte substitution but no hypocaloric parenteral nutrition. hypocaloric parenteral nutrition had been developped as a peripheral intravenous nutritional concept for patients with a normal body mass index over a period not longer than - days. multiple clinical studies showed that bowel movements increase earlier after an early postoperative enteral feeding which allows an earlier discharge of the patient. the result is a remarkable decrease of costs and an increase in patient benefit. still some years before surgeons preferred in visceral surgery parenteral nutrition over a period of - days under the opinion not to stress an anastomosis. this opinion changed in the last years under the aspect that about , - , ml of bile fluid, , - , ml pancreatic juice and , - , ml gastric juice per day are passing a small intestine anastomosis without any complications. concerning colon-anastomoses, the colon is preoperatively washed out, so it lasts until days until defecation. multiple studies also showed a benefit for the patient regarding immunostimulation by early postoperative enteral feeding. conclusion: in our hospital with surgical patients we recommend postoperatively either early normal enteral feeding or a high caloric parenteral nutrition if parenteral nutrition is needed for longer than days. if artificial nutrition is necessary for more than days we recommend enteral nutrition given by a tube or peg (percutaneous endoscopic gastrotomy). department of food technology, national research centre, dokki, cairo, egypt in the near east, "frekeh" has been known for many centuries as a stable food made from wheat. it is generally claimed that "frekeh" is better than wheat regarding its storage stability. the protein quality of parched immature durum wheat (frekeh) produced from variety was evaluated. frekeh from four maturing levels during the dough stage of the seed development, were analyzed for approximate analysis. results showed that "frekeh" produced at the beginning of the dough stage was of better nutritional value than that produced at the following maturity levels, since the former was higher in protein, fat, minerals and crude fiber as well as in reducing sugar content. in addition, it was shown that these results confirm well with the sensory quality evaluation of the cooked product. further more, it was found that the cooking time was suitable to produce a "freqkeh" meal with high levels of acceptability. the observed decrease in protein content with increasing maturity level raised the question of how the protein quality of "frekeh" versus that of nature wheat grains varied. in this investigation, the amino acid of "frekeh" was determined. dietary treatment and carnitine supplementation has greatly improved long-term outcome of patients with ppa and (vitamin b unresponsive) mma. however, metabolic decompensation may be frequent and final outcome in most patients show various handicaps. to investigate the usefulness of measuring free carnitine and acylcarnitines in dried blood by tandem mass spectrometry, we investigated patients with ppa and with mma in a period of months by weekly capillary blood punctures performed by the parents. age of the patients were from . until years. clinical status at the time of blood drawing was evaluated by regular phone calls. free carnitine in all patients substituted by oral carnitine treatment ( - mg/kg/day bw) was normal. the parameter best reflecting clinical status was the c /c -acylcarnitine quotient. mean value in mma and ppa patients showed a range of . - . (normal . ϩ/Ϫ . , n ϭ ), there was no difference between ppa and mma patients. individual mean values of the patients significantly increased when the patient was ranked higher in the clinical score system or during decompensation. since measurement of acylcarnitines in dried blood by tandem mass spectrometry is easy to perform, this method may be used for home monitoring of patients with mma and ppa. influence of acute treatment with , , , tetrahydroisoquinoline on the levels of glutathione and reactive oxygen species, and on the enzymatic activity of γ-glutamyl transpeptidase in dopaminergic structures of rat brain e. lorenc-koci , m. sokoĺowska , m. zapaĺa , and l. wĺodek institute of pharmacology, polish academy of sciences, kraków, and institute of medical biochemistry, collegium medicum, jagiellonian university, kraków, poland , , , -tetrahydroisoquinoline (tiq) and its derivatives generated considerable interest as molecular species that may be implicated in the pathogenesis of parkinson's disease (pd). in pd, apart from the lack of dopamine in the striatum, a decreased concentration of glutathione (gsh) is found in the substantia nigra (sn). it is also known that gsh depletion potentiates the toxicity of mptp and -hydroxydopamine. however, there are no data available on the tiq influence on gsh metabolism. the aim of the present study was to exemine the effect of acute tiq administration on the levels of gsh and reactive oxygen species (ros), and on the enzymatic activity of γ-glutamyl transpeptidase (γ-gt) in dopaminergic structures of rat brain. the investigation was carried out h after a single dose of tiq ( mg/kg i.p.). at that time, a marked increase in the tissue gsh level and simultaneous significant inhibition of γ-gt were found in the structures studied. in tiq-treated rats, the production of ros was reduced in the sn, but it was markedly enhanced in the striatum. our results suggest that the increase in gsh level in dopaminergic structures stems from inhibition of γ-gt and refers to the extracellular pool of this peptide. apparently, the tiq-mediated alterations in the levels of gsh and ros may have some implications for the etiology of pd. tetrahydrobiopterin-responsive phenylalanine-hydroxylase deficiency with mutations distant from the tetrahydrobiopterin binding site z. lukacs , r. steinfeld , a. kohlschütter , j. zschocke , and k. ullrich department of pediatrics, university of hamburg, and university-children's hospital, heidelberg, germany phenylalanine hydroxylase (e.c. . . . ) catalyzes the hydroxylation of phenylalanine to tyrosine in the presence of oxygen and the cofactor ( r)-l-erythro- , , , tetrahydrobiopterin (bh ). mutations in the phenylalanine hydroxylase gene may cause phenylketonuria or hyperphenylalaninemia. alternatively, disorders in bh metabolism also result in an increase in phenylalanine concentrations but simultaneously affect other bh -dependent enzymes, consequently, causing a severe neurological disorder. recently, several patients with a phenylalanine-hydroxylase deficiency but with normal bh -metabolism were reported who showed a significant decrease in blood phenylalanine concentrations upon treatment with bh . indeed, two such patients in our hospital were also sensitive to daily oral doses of - mg bh /kg. the subsequent molecular genetic analysis revealed that patient was homozygous for the widespread mutation y c and patient was compound heterozygous for the mutations a d and k n. it is striking, that all mutations are located distant from the known bh -binding site and thus, should not be associated with bh -sensitivity. additionally, further patients who share the same genotype are not sensitive to bh . therefore, it must be concluded that factors independent of the phenylalanine hydroxylase gene, like e.g. individual chaperone proteins, influence the three-dimensional structure of the enzyme and thereby, enhance enzymatic activity in the presence of elevated concentrations of bh . retrotransposons are structurally similar to retroviral gag and pol which are required for their replication via reverse transcription, and seem to be an ancestral form of specialized retroviruses. reverse transcription of retrotransposons was assumed to occur in virus-like particles as well as in retroviruses. rna-packaging in this particles suggests a possibility of infection. presumably, the formation of functional virus-like particles requires the interaction of gypsy rna with a protein encoded by gypsy first open reading frame (orf ) or a product of its processing. the objective of this work was to study whether the protein by this frame can bind with nucleic acids similarly to retroviral gag-protein and how phosphorilation of that protein may influence to this interaction. then gypsy orf was cloned and expressed in escherichia coli, and its protein product was purified by ion-exchange chromatography on deae-cellulose and affinity chromatography on heparin-sepharose and tested electrophoretically. it was shown that recombinant protein bound with its own mrna and with dna. the affinity for ssdna bing higher than for dsdna. the binding constant was estimated with rna. the method utilizes the ability of nitrocellulose to bind proteins but not nucleic acids. binding of % gypsy rna was achieved with about ng of the protein in ml of the reaction mixture. the binding constant was &times; m- , which is consistent. the structure of the putative nucleic acid-binding domain suggests that the protein is more similar to the core proteins of spumaviruses of the family retroviridae that to those of other retroviruses. phosphorilation of gag-like protein encoded by first open reading frame of retrotrasposon gypsy (mdg ) affects to interaction with nucleic acid. tryptophan (trp) in humans is catabolized by several pathways leading to various metabolites of kynurenine and indolic compounds formation. a number of diseases are connected with abnormalities in its excretion, but relationship of cause and effect is usually unclear. we introduced a two-step procedure for the detection of defects in metabolism of trp: ) tlc is employed when starting the investigation, ) two hplc methods were proposed and used at the next step, when pathological findings are to be proved and the individual metabolites quantified. the first hplc procedure enables the assessment of tryptophan, indolylacry-loylglycine (iag) and other five indolic compounds. the second method is intended to the monitoring of kynurenine and seven of its catabolites. the same sep-pack pre-treated sample of plasma and urine is used for all methods. the reference values and the excretion pattern in some groups of patients ( in total) were assessed. hepathopathy, gastrointestinal defects, myopathy and seizures with other neurological symptoms were the conditions connected with changes in the excretion of some metabolites of trp. significant decrease of iag excretion was found in burn patients early after the injury. urine analyses were performed at patient with hartnup disease and benign xanthurenic aciduria, inherited metabolic defects of trp. in other experiments, trp effect on the decarboxylation of other aromatic amino acids in the liver was investigated; only weak inhibition under physiological conditions was recognised. ( two hypothetical proteins of escherichia coli, ybbq and yhae, show high sequence similarity to d-threonine dehydrogenase from pseudomonas cruciviae ifo . we cloned each gene encoding ybbq and yhae into e. coli jm . both ybbq and yhae showed no d-threonine dehydrogenase activity and showed significant activities for d-serine in the presence of nad. ybbq and yhae were purified to homogeneity from the e. coli clones. ybbq consisted of two identical subunits with a molecular mass of kda, whereas yhae was a tetramer (native molecular mass, kda). ybbq showed the maximum activity at ph . for the oxidation of d-serine. whereas optimum ph of yhae was ph . . they catalyzed oxidation of glycerate and -hydroxyisobutyrate. d-glycerate was the best substrate for both enzymes. both enzymes also catalyzed reduction of tartronate semialdehyde in the presence of nadh. at physiological ph, the rate of tartronate semialdehyde reduction was much higher than that of d-glycerate oxidation. the ybbq gene is in the operon of glyoxylate utilization and the yhae gene is in the operon for d-glucarate/galactarate utilization. these results suggest that both ybbq and yhae are dglycerate -dehydrogenases and function physiologically in conversion of tartronate semialdehyde into d-glycerate. a serine protease inhibitor model: synthesis and biology z. mucsi , Á. bódi , l. gráf , a. perczel , a. patthy , and g. orosz department of organic chemistry, biochemistry, eötvös university, budapest, agricultural biotechnology centre, gödölló´, and research group of peptide chemistry, hungarian academy of sciences, budapest, hungary sgci is structurally related to the pmpd- family of canonical serine protease inhibitors. in these peptides, there is a p -p Ј position which is responsible for reversible binding to chymotrypsin. their structure is characterized by structural compactness: the molecule contains three -sheets and three disulfide bonds. in the sgci molecule the p -p Ј corresponds to lys-leu bond, which is cleaved by chymotrypsin extremely slowly. the question arises why an excellent substrate behaves at the same time as inhibitor. it was assumed that the threedimensional structure of the molecule is responsible for the inhibitory activity. a model was designed to include all the known features of the inhibitor: the structurally necessary -sheet structure and the fragment containing the p -p Ј environment. three model peptide were synthesized. two model peptides had no inhibiting effect and were cleaved by chymotrypsin. one of the cleavage points is the expected p -p Ј position, while the other positions found to be chymotrypsin preferred positions after the first cleavage. the three-dimensional structures of the model peptides were mapped by nmr. on the basis of nmr structures obtained it has been shown that the cyclopeptide part is more flexible in the models than in sgci. the initial process in the reaction mechanism of a bisubstrate enzyme, rat mercaptopyruvate sulfurttransferase: inactivation study by using chloropyruvate n. nagahara , t. nakagawa , and m. minami department of hygiene and public health, nippon medical school, sendagi bunkyo-ku, tokyo, japan institute for organic chemistry, darmstadt university of technology, darmstadt, germany to investigate the reaction mechanism of a bisubstrate enzyme, rat mercaptopyruvate sulfurtransferase (ec . . . , mst), inactivation kinetics with -chloropyruvate (chloropyruvate) was studied; each inactivation reaction was completed in a preincubation procedure. chloropyruvate is an analog of -mercaptopyruvate (mercaptopyruvate) and irreversibly inhibits mst. the inactivation depended on incubation time and the concentration of chloropyruvate and showed saturation kinetics. the plot for the logarithm of % activity remaining versus preincubation time showed pseudo-first-order. the kinact is . ϫ Ϫ min Ϫ and k is . mm. these suggest that chloropyruvate serves as a mechanism-based inactivator. mercaptoethanol , so that chloropyruvate can approach cys via the donor substrate route and acceptor substrate one, and a ternary complex may be formed prior to the inactivation. these findings suggest that a donor substrate enters the catalytic cavity prior to an acceptor one in the initial process of the mst reaction: mst follows an ordered sequential mechanism. polyketides are natural products of bacteria, fungi, marine organisms and higher plants, many of which have clinical usage. actinorhodin ( ) is an antibiotic produced by streptomyces coelicolor via an iterative type ii polyketide synthase (pks) system. this consists of a multi-enzyme complex with a single catalytic function for each enzyme. departments of chemistry and pediatric surgery, school of medicine, fujita health university, toyoake, japan it has been reported that, in rats with a single intoxication of α-naphthylisothiocyanate (anit), acute liver injury develops with enhanced lipid peroxidation and neutrophil infiltration in the liver tissue. melatonin functions as an antioxidant. melatonin is known to inhibit neutrophil infiltration into damaged liver tissues. therefore, we examined whether melatonin exerts a protective or preventive effect on anit-induced acute liver injury. male wistar rats received a single i.p. injection of anit ( mg/kg) and oral administraton of melatonin ( mg/kg) at or h after anit injection. animals administered with melatonin at and h after anit injection were sacrificed and h, respectively, after the injection. liver injury appeared h after anit injection and developed at h. melatonin administered at h after anit injection prevented liver injury formation with attenuation of increases in hepatic lipid peroxide level and myeloperoxidase activity, an index of neutrophil infiltration. melatonin administered at h after anit injection prevented liver injury development with attenuation of further increase in hepatic lipid peroxide level. thus, melatonin protects against and prevents anit-induced acute liver injury in rats possibly through its antioxidant action and/or its inhibitory action against neutrophil infiltration in the liver tissue. k. okamura-ikeda, s. katayama, k. fujiwara, and y. motokawa t-protein is a component of the glycine cleavage system and catalyzes the tetrahydrofolate-dependent reaction. mutation in human t-protein (ht) gene results in clinical nonketotic hyperglycinemia (nkh). eight point mutations have been identified so far in nkh patients with t-protein deficiency. to understand the structure and function of ht, the wild-type (wtht) and three mutant t-proteins (g r, g d and r h) were expressed in escherichia coli with chaperons groel and groes which facilitated the recovery of the expressed proteins as a soluble form. levels of expression of these proteins were similar but the recovered soluble forms of mutants were about one-third of wtht. g r showed comparable specific activity to wtht, whereas g d and r h mutants exhibited remarkable reduction in specific activity. since homoallelism for g d mutation and heteroallelism for g r and r h mutation were identified in typical and atypical nkh, respectively, these results suggest that g and r h mutations are highly deleterious in the aspects of not only protein folding and/or stability but also catalytsis. on the other hand, g r mutation might affect mainly on the protein stability. detailed characterization of these mutants is now in progress. laboratory of animal nutrition and biochemistry, miyazaki university, miyazaki-shi, japan the minimal actinorhodin pks, shown in black below, consists of the ketosynthase (ks), chain length factor (clf) and acyl carrier protein (acp) and is the minimum set of enzymes required for polyketide production. we have investigated the stoichiometry of the ks-clf complex and the ks-clf:acp minimal system using three methods: . native gel electrophoresis. . cross-linking of proteins using dibromoacetone. . radical cross-linking of proteins. this new method has also been used with wild type s. coelicolor cell free extract with % ks-clf in order to elucidate which proteins are in close proximity to ks-clf during in vitro actinorhodin production. in ruminant animals, essential amino acids have never been completely established, because of the difficulty of its estimation due to the presence of microorganisms such as bacteria and protozoa in the first stomach called rumen. in our previous paper, histidine was shown to be the first limiting amino acid in the rumen contents when evaluated by chemical score. recently we have also reported that rumen microorganisms cannot synthesize histidine from histidinol. on the other hand, there have been some reports which showed that nitrogen balance of ruminants was not improved by supplementation of histidine to rumen microbial protein together with methionine, lysine and threonine which had been known to improve. based on these facts, we have a hypothesis that histidine may not be an essential amino acid for ruminants. in the present paper, we will report about the abilities of cattle liver and kidney to synthesize histidine from histidinol comparing with those of swine liver and kidney. the ability was demonstrated by examining the activities of histidinol dehydrogenase (crude enzyme) by means of direct measurement of an increase in histidine and decrease in histidinol. the amount of histidine produced from histidinol by the enzyme seemed sufficient for meeting the histidine requirement of cattle. the browning reaction is the sequence of events which begins with the reaction of amino group in amino acids, peptides or proteins with glycosidic hydroxyl group of sugars; the sequence terminates with the formation of brown nitrogenous compounds or melanoidines. this reaction gives rise to tremendous number of components such as volatile alcohols, ketones, aldehydes, esters, ethers and sulfur and nitrogen containing heterocycles in addition to nonvolatile amadori compounds and complex brown pigments of medium to high molecular weights. the present study was designed to choose a currently occurring system (aspartic acid -fructose) as a model system, since aspartic acid was found to be one of the most important amino acids in many kinds of food varieties. the reaction was done under controlled conditions of reactants ratios, temperature and time. the reaction mixtures were subjected to successive extractions with suitable solvents where the obtained corresponding flavour concentrates were thoroughly investigated. the results indicated different classes of compounds such as aldehydes, furans, alcohols and alkylated pyrazines varying in quantities depending on the reaction conditions. these products were also investigated concerning their toxicological effects. so, such products of nonenzymatic reactions showed different chemical and biological properties. purification and characterization p. piyarat , s. nagata , h. misono , and k. packdibamrung department of biochemistry, faculty of science, chulalongkorn university, bankok, thailand department of bioresources science, faculty of agriculture, kochi university, nankoku, kochi, japan nad ϩ dependent alanine dehydrogenase was purified fold to homogeneity from aeromonas hydrophila. molecular mass of , daltons was estimated for alanine dehydrogenase by sephadex g- chromatography. sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified en-zyme showed polypeptide band with molecular mass of , daltons, indicating that the enzyme is hexamer. the enzyme is highly specific for alanine and nad ϩ . sulfhydryl group of the enzyme plays an important role in the catalysis. the enzyme retained its activity on heating at °c for h. optimum ph for reductive amination and oxidative deamination were . and . , respectively. the steady state kinetic studies including product inhibition on the enzyme reaction indicated that the oxidative deamination proceeds through a sequential ordered binary-ternary mechanism in which nad ϩ binds first to the enzyme followed by l-alanine and products are released in the order of pyruvate, ammonia and nadh, respectively. the k m values for nad ϩ , l-alanine, pyruvate, ammonia and nadh were . , , . , and . mm, respectively. an elevation of apolipoprotein (apo) b- concentrations is a particular feature of several metabolic disorders, such as type diabetes (t d), impaired glucose tolerance (igt), and familial combined hyperlipidemia (fchl). to further understand the in vivo turnover of apolipoprotein b- of very low density lipoprotein subfractions (vldl , svedberg units (s f ) - and vldl , s f - ) kinetic studies were performed in subjects with t d, igt, fchl, and healthy controls using a tracer of either l-[ring- c ]-phenylalanine or l-[ , , - h ]leucine. these studies showed direct hepatic vldl apob- secretion to be increased in patients with t d and igt when compared with controls. in contrast, patients with fchl showed a discrete increase in hepatic vldl apob- secretion. in all patients vldl catabolism is not essentially impaired. vldl apob- secretion is associated with plasma insulin and free fatty acid (ffa) concentrations, resp., whereas vldl apob- secretion is correlated with plasma mevalonate and lathosterol levels. in conclusion, vldl overproduction is supposed to be completely responsible for higher triglyceride (tg) levels found in patients with t d, igt, and fchl. vldl overproduction seems to be regulated by tg and ffa substrate and appears to be an indicator of decreased insulin sensitivity. in contrast, vldl overproduction is more likely to be regulated by the availability of cholesterol substrate. these data give further in vivo evidence that vldl and vldl secretion is regulated independently. arabidopsis resulted in enhanced production of cysteine and glutathione graduate school of pharmaceutical sciences, chiba university, chiba, japan serine acetyltransferase (satase) catalyzes the formation of o-acetyl-l-serine (oas) which is the key intermediate of cysteine biosynthesis. oas is not only a dominant limiting factor but recently suggested as a possible signal molecule for gene expression in cysteine biosynthesis. in has been shown that the activity of cytosolic satase from watermelon was feedback inhibited by l-cysteine. to enhance the ability of cysteine biosynthesis in plants and to reveal the role of oas in the regulation of sulfur assimilation, we made the point-mutated watermelon satase gene (satg c) whose product was not inhibited by cysteine, and introduced satg c into arabidopsis. the contents of oas, cysteine, and glutathione in transgenic arabidopsis were increased significantly as compared to the wild-type arabidopsis. we are currently dealing with the expression analysis of sulfur-related genes in transgenic arabidopsis accumulating oas due to the overexpression of satase. certain amino acids as source of specific branched chain fatty acids in fish sauce manufacture n. g. sanceda , e. suzuki , and t. kurata institute of environmental science for human life, and department of human biological studies, ochanomizu university, tokyo, japan the source of some branched volatile fatty acids (vfa) during the fermentation process in the manufacture of fish sauce was investigated. we previously reported that straight chain volatile acids seemed to have been derived from fish fats but unlikely for branched fatty acids which was believed to be derived from other sources. to clarify the source of branched volatile acids, specific amino acids, alanine, leucine, iso-leucine and valine were used in this study. these amino acids were first mixed with salt and added to fish. the fish mixtures were then aerobically and anaerobically incubated for one and a half months. results showed that addition of valine significantly increased the production of iso-butyric and iso-hexanoic acids and leucine increased that of iso-valeric in the aerobically fermented fish mixtures. a similar tendency was observed in the anaerobically fermented fish mixture except that an increase in the amount of iso-hexanoic acid was observed in the leucine added mixture, which was not observed in the aerobically fermented one. it seemed that specific branched volatile fatty acids were derived from certain amino acids. glutathione (gsh) is an important component of the cellular defense mechanisms that protect cells from oxidative injury. in the retina, the glial (müller) cells have been shown to synthesize and transport gsh, and thus are likely to be involved in regulating gsh levels. in the present study, we have characterized gsh transport system in a müller cell line using s-gsh uptake. the results showed that gsh was taken up in a na ϩ -and concentration-dependent manner with a k m of . mm. moreover, cellular gsh had no effect on the rate of gsh uptake. in related studies, we found that oxidative stress induced the expression of γ-glutamylcysteine synthetase (gcs) subunits, and that gcs mrna levels were correlated with the degree of gsh depletion. because organic anion transporters (oatps) have been implicated in glutathione cotransport, we examined expression of oatp members using rt-pcr. we found that the müller cell line expressed transcripts for oatp , oatp and oatp . these studies indicate that the müller cell plays important role in gsh homeostasis in the retina. in the active site of human porphobilinogen synthase (ec . . . , pbgs), two zinc ions are coordinated by cys , cys and cys , and his and cys , respectively. the fomer zinc ion, closer to catalytic site lys , plays an important role in catalysis. on the other hand, a role of the latter (distal) one has not been clarified. interestingly, in human hep b cell, his was replaced with arg (h r). to elucidate the role of his in catalysis, the kinetic properties of wild type and h r mutant enzymes were studied. these cdnas were cloned by rt-pcr with total rna from human peripheral lymphocyte and hep b cell, respectively. each cdna encoding pbgs with Ј non-coding region was inserted into pet- b(ϩ) vector and then the constract was transformed into e. coli strain bl (de ). the cells were cultured in lb medium containing mg/ml ampicillin and µm zn ion for h at °c. after addition of mm isopropyl--d(Ϫ)-thiogalactopyranoside, cells were further cultured for h at °c. the highly purified pbgss were obtained by ultora centrifugion, fractionation with ammonium sulfate and column chromatographies with deaecellulose, hydroxylapatite and superdex , serially. we are now investigating molecular properties of these pbgss. agriculture and agri-food canada, lacombe research centre, lacombe, alberta, canada handling and management procedures such as capture and restraint can be significant stressors for recently domesticated animals such as elk (cervidae elaphus). the objective of the current study was to investigate the use of pre capture nutritional therapy in attenuating hpa response and improving animal welfare. fourty eight adult male elk stags ranging in age from - years and raised on pasture were used in the study with as control and as nutritionally treated. twenty four hours prior to capture the elk were offered either kg of a cereal grain based dietary supplement or kg of a cereal grain based nutritional therapy product containing specified amino acids (usa patent # ). the amino acid content of the nutritional therapy product was minimally . g per kg animal weight of ala, lys, phen, meth, thre, isoleu, val and tryp plus g per kg weight of leu and g/ kg weight of glut. the animals were subsequently captured and held in appropriate facilities designed to handle elk. saliva samples were collected on all animals immediately following capture and salivary cortisol was monitored by ria. animals offered the nutritional therapy product containing the amino acid mixture displayed lower cortisol levels ( . nmol/l) compared to the untreated controls ( . nmol/l; p Ͻ . ). the data suggest that amino acid therapy can be used to attenuate hpa response to a stressor in captured elk. department of bioengeneering and technology, delhi, new delhi, india resistance to analogues of methionine by corynebacterium lilium results in the partial de-repression of methionine biosynthetic enzymes. the levels of enzymes involved in methionine biosynthesis also increased step-wise by successive endowing the resistant markers, resulting in the overproduction of methionine. moreover, the repressibilities of the enzymes were also reduced by the addition of methionine analogue resistance. analogue resistant mutants were developed by uv induced mutagenesis of corynebacterium lilium (wild type) strain. the single analogue (norleucine) resistant mutant c. lilium nl- produced µg/ml methionine in shake flasks with methionine yield at . g methionine/g glucose and specific methionine production at . mg/g dcw, while double analogue (norleucine and triazole) resistant mutant c. lilium nt- produced µg/ml methionine. a triple analogue (norleucine, triazole and ethionine) resistant mutant c. lilium nte- produced . g/l methionine. the methionine yield was . g methionine/g glucose and its specific productivity was . g methionine/g dcw. clinical biochemistry, laboratory , luxemburg, grand duchy of luxemburg blood plasma glucose level was compared on fast and minutes after oral administration of mg of acetylcysteine. in the group of healthy persons the plasma glucose level feel by . % over the minute period. in the diabetics on the contrary, the plasma glucose level observed minutes after administration of acetylcysteine was . % higher than in blood plasma taken on fast. similar tests were carried out "in vitro" to interpret these different results. the control group consisted of ml of distilled water ϩ . ml % glucose ϩ . ml god pad (boringer mannheim gmbh). in the acetylcysteine group the distilled water was replaced by ml . % solution of acetylcysteine. in the glucagon group the distilled water was replaced by . % solution of glucagen hypokit novo nordisk. spectrometric determination was carried out after minutes of incubation. a % diminution of glucose was observed in the acetylcysteine group in comparison with the control group. a . % increase in glucose was observed in the glucagon group in relation to the controls. the results with healthy persons and the tests "in vitro" indicate that acetylcysteine lowers the level of glucose. but it elevates the level of glucose in the blood plasma of diabetics. it may be presumed that acetylcysteine modifies the insulinglucagon balance in favour of glucagon. the objective of this study was to fortify yogurt with three oilseed protein hydrolysates prepared from soybean (glycine max), sesame (sesanum indicum) and rice bran (oryza sativa) flours. hydrolysis was carried with two enzymes one of plant origin (papain) and the other of microbial origin (alcalase). a yogurt fortification experiment was then carried using the previous hydrolysates. the hydrolysates were added to yoghurt at , and % levels of fortification and the fortified yoghurt was analyzed fresh, and after and days of consuming period. fortified yogurt was chemically examined for fermentation activity (ph values, acidity and proteolysis) as well as its organoleptic properties. results of this experiment indicate that the addition of soybean hydrolysates with papain ( . units/g) for minutes (tb) and rice bran hydrolysates with alcalase ( . units/g) for minutes (te) to yoghurt can ex-ceed - %, while fortification with sesame hydrolysed with papain ( . units/g) for minutes (td) and soybean hydrolysed with papain ( . units/g) for minutes (tc) can not reach up to %. it is well known that dna is fragile to reactive oxygen intermediates (rois) damage. evidences that dna fragmentation and apoptosis occur in cardiomyopathies, in the failing heart and in cultured cells under hyperbaric oxygen (hbo) stress, demonstrated that oxygen free radicals also play a critical role in heart failure. as a consequence, myocardial cell survival depends on response to oxidative stress. experimental data obtained in vitro suggested that polyamines, by acting as rois scavengers, play a role in prevention of endonucleasemediated dna fragmentation and inhibition of alkylating agents-mediated damage, potentially exerting a protective role against rois damage. thus we studied polyamine metabolism and superoxide dismutase (sod) expression in an in vivo model of heart oxidative stress, such as rats subjected to hbo. four experimental groups were used: ) controls; ) rats subjected to hbo for min once and immediately sacrificed; ) rats treated as group but for consecutive days and immediately sacrificed; ) rats treated as group but sacrificed h later (recovery). northern blot analyses showed that odc mrna accumulation increased immediately (paralleled by activity) in groups - , while ssat mrna decreased remarkably, thus leading to higher polyamine concentration in rois-stressed hearts. contrariwise, sod mrna level decreased rapidly in groups - . this suggests that hbo-induced compensatory mechanism in rat heart is based on specific and rapid boosting of polyamine concentration, caused by coordinate induction of biosynthesis and inhibition of catabolism, and not of enzymes known to metabolise rois such as sod. amino acids oxidation was greater in tumor-bearing rats muscle. leucine is an important ketogenic amino acid that proves energy to the skeletal muscle. leucine supplemented diet was used to analyze the effects produced by walker growing in pregnant rats which were distributed into six groups. three groups received normal diet ( % protein): control (c), tumor-bearing (w), pair-fed rats (cp). three groups were fed with diet supplemented with % leucine ( % protein plus % leucine): pregnant fed with leucine (l), tumor-bearing with leucine (wl) and pair-fed with leucine (lp). after days, the animals were submitted to intestinal perfusion to measure leucine, methionine and glucose absorption. leucine absorption increased in w and wl groups. glucose absorption reduced in tumor-bearing. in pregnancy with cancer, metabolic changes provided both reduced fetal and tumor development. tumor-bearing rats showed increase in methionine and leucine absorption, probably diverting this nutrients to tumor cells. glucose absorption reduced in w and wl. leucine supplemented diet group promoted high leucine absorption which could be used by neoplasic cells, and mainly by fetus and host. probably, the transamination of the branch long chain amino acid provided energy substract for the skeletal muscle, keeping the nitrogen offered to host carcass. ( undernutrition cause several changes as body weight loss, in biochemical parameters, even microscopic alteration in absorptive epithelium. this means the nutrients absorption process has been harmfully and consequently increase the damages caused by malnourished. knowing leucine is used as a ketonic and oxidative amino acid our main propose was to recovery the malnourished young rats with normal (rc) and leucine supplemented diet (rl, % of leucine) for days. it was measured body, liver, and muscle weight, intestinal absorption of glucose, methionine and leucine, and body chemical composition. the body weight gain in rc and rl was higher than control group, suggesting that nutritional replacement for these groups could provided nutrients to support the body weight recovery, reaching as the same weight as the control. methionine and glucose absorption was reduced in malnourished group, but it was recovered (glucose, methionine and leucine) after nutritional replacement. leucine supplemented diet promoted a good recovery of carcass collagen nitrogen, keeping the carcass structural nitrogen. further studies are necessary to investigate this mechanism. [financial support: fapesp ( we diagnosed the very rare autosomal recessive disorder hyperprolinaemia type ii (deficiency of ∆ -pyrroline- carboxylate dehydrogenase, ec . . . ) in a girl aged months presenting with seizures and encephalopathy. l-∆ pyrroline- -carboxylic acid accumulates in this disorder and there is a - -fold increase in plasma proline. surprisingly, she also had vitamin b deficiency. this was an unrecognised association, which was not explained by her diet or medications. we hypothesised that pyridoxal phosphate (vitamin b coenzyme) was de-activated by l-∆ -pyrroline- -carboxylic acid. with high resolution h nuclear magnetic resonance spectroscopy and mass spectrometry, we have shown that these two compounds react at ph . and °c in vitro to form three novel adducts, which we characterised. they are products of a claisen condensation (or knoevenagel type of reaction) of the activated c carbon of the pyrroline ring with the aldehyde carbon of pyridoxal phosphate. if this previously unreported interaction occurs in vivo, pyrroline- -carboxylic acid is a unique endogenous vitamin antagonist. preliminary observations show that pyrroline- -carboxylic acid also condenses with other biologically important aldehydes and ketones. some of these reactions may contribute to the brain disturbances in hyperprolinaemia type ii. we have already identified adducts with acetoacetic acid in urine from our child, which is evidence that condensation can occur in vivo. the kidneys are characterized by a high activity of γglutamyl transpeptidase (γ-gt), as well as by a high cysteine level. the present paper was aimed to obtain information on how the activity of γ-gt and the levels of non-protein sulfhydryl compounds (npsh) changed with age in rat kidneys. simultaneously, protein-bound cysteine (pb-cys) and sulfane sulfur compounds were estimated. the kidneys were from following rats groups: young ( -month-old), middle-aged ( month-old) and old ( -month-old). the obtained results showed that the activity of γgt and npsh levels in the kidneys fell with age. at the same time, a significant increase in the level of protein-bound cysteine was observed. on the other hand, the content of sulfane sulfur compounds was elevated in the group of the oldest animals. these findings indicate that -due to disturbances in the γ-glutamyl cycle -the capacity for extracellular glutathione degradation and, in consequence, the availability of cysteine for intracellular gsh biosynthesis may be impaired. the increased pb-cys level indicates potentiation of the thiolation reaction, i.e. development of protein-mixed disulphides, cysteine, sulfane sulfur compounds, oxygen reactive species. national research centre, dokki, cairo, egypt in the past few years, many attempts have been made to prepare a synthetic insulin. the biological activity of insulin is known to be closely related to the c-terminal octapeptide fragment of its b-chain. this does not necessarily mean, however, that each of the amino acid residues of the octapeptide fragment is essential for its activity. it was found that b gly and b phe were present in all insulins so far obtained from various animal species indicating the significance of these two residues. it would therefore seem desirable to study the effect of each of these two amino acid residues or both on biological activity of the octapeptiede fragment of the b-chain. weitzel et al. found that the substitution of arginine b with another amino acid resulted in a very large decrease in biological activity, which indicates that it participates in the action of insulin. also it was found that the aromatic amino acid residues (b -b ) participate in the action of insulin. a heptapeptide arg-phe-tyr-thr-pro-lys-ala-och , corresponding to (b -b ) insulin des gly -phe , and an octapeptide arg-phe-phe-tyr-thr-pro-lys-ala-och , des gly were synthesized using the solid phase method. the c-termenal ends of both peptide were converted to methyl ester by transesterification cleavage from the resin. the side chain protecting groups were removed by hf. manual counter current distribution method was used for purification of the free peptides. the way to solve the evaluation of tyrosine containing peptide was studied. the free methyl ester peptides were administrated for insulin-like activity test by glucose metabolism in the rat fat cells technique in vitro. nitric oxide synthase inhibitors influence dynorphin immunoreactivity in the rat brain following hyperthermia p. alm and h. s. sharma department of pathology, university hospital, lund university, lund, and laboratory of neuroanatomy, department of medical cell biology, biomedical centre, uppsala university, uppsala, sweden nitric oxide (no) is a free radical gas that influences neuronal communication in the central nervous system (cns). recent reports suggest that no can influence dynorphin neurotransmission in the normal brain as well as in several pathological states. previous reports from our laboratory show that the enzyme nitric oxide synthase (nos) responsible for no formation is upregulated in several brain regions following hyperthermia. the present investigation was carried out to find, whether hyperthermia can influence dynorphin immunoreactivity in the brain, and if so, whether inhibition of nitric oxide synthesis will alter its distribution in heat stressed rats. rats subjected to hyperthermia at °c for h in a biological oxygen demand incubator (bod) resulted in marked redistribution of dynorphin immunoreactivity in several brain regions e.g., cerebral cortex, hippocampus, cerebellum and brain stem. pretreatment with two potent nos inhibitors, l-name ( mg/kg, i.p.) and l-nmma ( mg/kg, i.p.) min before heat stress significantly altered the dynorphin immunoreactivity in the brain. these drugs alone however, did not influence the peptide expression in normal rats. the results suggest that (i) hyperthermia has the capacity to influence dynorphin immunoreactivity in the brain, and (ii) inhibition of nitric oxide synthase considerably influences the dynorphin immunoreaction in hyperthermia, not reported earlier. the functional changes induced by uncompetitive and competitive nmda antagonists, memantine, amantadine and mk- , and cgp , respectively, were studied in both saline-pretreated and mptp-pretreated c bl/ mice. the nmda antagonists were administered acutely by themselves or in combinations of either: nmda antagonist plus subthreshold l-dopa dose or nmda antagonist plus suprathreshold l-dopa dose, to either the mptp-pretreated or the salinetreated mice. activity-enhancing or functional restorative effects of the nmda antagonists were variable with memantine and mk- distinguished from amantadine and cgp . in the study of long-term effect of nmda antagonists mk- was administered postnatally and spontaneous motor behaviour and motor activity in response to several pharmacological interventions was assessed. marked alterations associated possible with apoptotic penchance are discussed. t. archer and a. fredriksson department of psychology, university of göteborg, and department of psychiatry, university of uppsala, ulleråkers hospital, uppsala, sweden synergistic antiparkinsonian actions of different classes of putative therapeutic agents co-administered with a subthreshold dose of l-dopa ( mg/kg) in drug-naive mptp-treated mice as well as the restorative actions of those compounds in suprathreshold l-dopa-tolerant mptp-treated mice subjected to "wearing-off" of l-dopa efficacy were assessed in a series of experiments. the classes of compounds studied included the noncompetitive nmda antagonists, memantine, amantadine and mk- , the anticonvulsive and putative anticonvulsive agents, lamotrigine, fce , phenytoin, the monoamine oxidase inhibitors, l-deprenyl, amiflamine, α-ethyltryptamine, clorgyline and phenelzine, and the α -adrenoceptor agonists, clonidine and guanfacine. in this final case, the restorative effects of clonidine and guanfacine were antagonised by the α -adrenoceptor antagonist, yohimbine, but not the α adrenoceptor antagonist, prazosin. within each class of potentially therapeutic agents a differential restorative efficacy was obtained, but the combination of different doses of apomorphine with clondine failed to restore motor activity. in vivo proton mr-spectroscopy of the human brain: assessment of n-acetylaspartate (naa) reduction as a marker for neurodegeneration w. block , f. träber , s. flacke , f. jessen , ch. pohl , and h. h. schild department of radiology, department of psychiatry, and department of neurology, university of bonn, germany proton magnetic resonance spectroscopy ( h-mrs) is a well accepted non-invasive method to investigate changes in brain metabolite composition in different types of cerebral disease. we performed proton spectroscopy in patients with dementia of the alzheimer's type (ad) and in patients with motor neuron disease (mnd) with the aim to detect a specific metabolic pattern for each of these two neurodegenerative disorders. overall, more than spectroscopic data sets of patients with mnd and more than data sets of ad patients were acquired within the last years. in the mnd group we found a significant reduction of naa/tcr metabolite ratios in the central region, which correlates with the disease severity and the clinical lateralisation of neurological symptoms and increases in the time course of the disease. in ad patients a similar reduction in relative naa contents was observed in the medial temporal lobe. the observed regional metabolic alterations correlate well with the characteristic neurological symptoms in ad (dementia) and mnd (muscular palsy) and seem to follow the disease process over time. since naa is exclusively expressed in neurons as shown by immunohistochemical studies, reduced naa levels suggest neuronal loss or dysfunction in the observed regions. center for molecular imaging research, massachusetts general hospital, boston, massachusetts, u.s.a. non-invasive measurement of hemodynamic parameters and imaging neovasculature architecture during angiogenesis is highly important in determining tumor prognosis and in assessing treatment efficacy. we suggested a technique to map the tumor vascular (vvf) and interstitial volume fraction (ivf) noninvasively in vivo. a poly-l-lysine based macromolecular probe (mpeg-pl-gddtpa) with extended circulation in the bloodstream designed to shield chelated paramagnetic lantanide with poly(ethylene glycol) chains. we hypothesized that a magnetic resonance signal after intravenous administration of a vascular paramagnetic probe can be maximized so the signal change after administration of a second comound (gddtpa) reflects the ivf but not the vvf. the method and its assumptions were verified in animal models of cancer. tumoral vvf and ivf values were consistent with histology data and literature values. imaging showed heterogeneity of both parameters at submillimeter pixel resolution. this technique was used for characterizing differential angiogenesis in human mammary adenocarcinoma lines as well as for imaging anti-angiogenic drug effects. anti-angiogenesis was induced using synthetic d-reverse peptides derived from thrombospondin- . this study showed that peptide treatment results in slower brain tumor growth due to inhibition of de novo blood vessel formation and synergistic anti-proliferative effect on tumor cells. in conclusion, in vivo mr imaging can be used for non-invasive treatment assessment of novel antiangiogenic drugs. wallenberg neuroscience centre, lund university, lund, sweden we have recently found that -hydroxydopamine lesioned rats gradually develop dyskinetic-and dystonic-like movements upon repeated administration of a therapeutic dose of l-dopa. such movements simulate the time course of peak-dose dyskinesia in parkinson's disease. in this rat model, the severity of l-dopa-induced dyskinesia is strongly correlated with an upregulated expression of the prodynorphin gene in striatal neurons. using antisense technology and gel-shift assay analyses, we have addressed the role of transcription factors which may mediate this response. we have found that the camp response-element binding protein (creb) is essential in maintaining a basal expression of prodynorphin mrna in the intact striatum, but it is not required for l-dopa to induce the prodynorphin gene in dopamine-denervated striatal neurons. we have thus addressed the role of fos-and jun family tran-scription factors, and found very high levels of fosb-and jundlike proteins in the striata of dyskinetic animals. these proteins could bind to both ap and cre sites in the prodynorphin promoter. moreover, intrastriatal fosb knockdown could inhibit both the upregulation of prodynorphin gene expression and the development of dyskinesias under chronic l-dopa treatment. we propose that dimers of fosb-and jund-like proteins mediate abnormal changes in striatal gene expression which are linked to the development of l-dopa-induced dyskinesia. department of pharmacology, grünenthal gmbh r&d, aachen, germany glutamate plays important roles in both normal and pathophysiological nocieception. upon physiological conditions, glutamate release from primary afferents in the spinal cord activates largely ampa receptors. as those are ubiquitously involved in fast transmission in the cns, ampa antagonists have a broad side-effect profile. prolonged activation of nociceptors by tissue damage, inflammation or nerve injury evokes a long-lasting release of glutamate and neuropeptides, activating nmda receptors in the spinal cord. this mechanism appears to play a key role in pain chronification. the nmda receptor is, therefore, an important target for chronic pain treatment. both animal and human studies confirm the efficacy of nmda antagonists in chronic pain, however, clinically available compounds are weak or have unacceptable side-effects. glycine b antagonists and compounds selectively blocking nr b-containing receptors appear to be safer, the reasons for this remain unclear. central side-effects could potentially be avoided by using nmda antagonists with restricted central access. peripheral nmda receptors (as well as some other subtypes of glurs) could be activated by glutamate released from the site of injury, thus contributing to peripheral hyperexcitability. some other subtypes of glurs can also contribute to peripheral sensitisation. of ionotropic glurs, kainate receptors appear important in inflammatory and neuropathic pain. they can be activated by high intensity stimulation of nociceptive afferents, and may act as autoreceptors controlling release of glutamate. group i metabotropic glurs are also present on primary afferents and on second order neurones in the spinal cord, and may play a similar role. antagonists of these subtypes of glurs are active in some models of chronic pain. specific upregulation of group ii metabotropic glurs in some pain-relevant structures could reflect a possible adaptive role of these inhibitory receptors under chronic pain conditions; their selective agonists also have a potential for the treatment of chronic pain. we have performed a series of studies of the distribution and function of mglur subtypes in the basal ganglia that suggest that members of this receptor family could serve as targets for novel therapeutic agents that would be effective in treatment of pd. for instance, two group ii mglurs (mglur and mglur ) are localized on presynaptic terminals of striatal neurons in the globus pallidus where they could reduce gaba release. furthermore, activation of group i mglurs results in a depolarization and increased cell firing of neurons in the subthalamic nucleus (stn) and projection neurons of the substantia nigra pars reticulata (snpr). interestingly, this effect is mediated by mglur in snpr projection neurons and mglur in stn neurons. finally, activation of group ii mglurs results in inhibition of glutamate release from stn terminals in the snpr. furthermore, selective agonists of group ii mglurs inhibit haloperidol-induced catalepsy in rats, suggesting an antiparkinsonian effect of these compounds. the rich distribution and diverse physiological roles of mglurs in basal ganglia raises the possibility that these receptors may provide targets for novel therapeutic agents that could be used for treatment of pd and related disorders. a. cupello , m. parodi , and m. balestrino centro di neurofisiologia cerebrale, cnr, genova and dipartimento di scienze neurologiche, university of genova, italy in vitro rat hippocampal slices are commonly used to study the effects of hypoxia in the central nervous system, because they allow to differentiate the effects of hypoxia in the brain from that of systemic (e.g., respiratory and cardiac) failure that may accompany hypoxia. we used electrophysiology to monitor and evaluate the damage caused by transient hypoxia to the nervous tissue. a few minutes after oxygen deprivation brain tissue suddenly depolarizes. this event, which is termed ìanoxic depolarizationî is accompanied by dramatic metabolic changes: transmembrane ionic gradients disappear (na ϩ enters, k ϩ exits the neurons), neurons swell, there is intra-and extra-cellular acidosis. this event is caused by functional inactivation of (na ϩ / k ϩ )atpase caused by decreased atp content, as it is suggested by the fact that it is mimicked by ouabain treatment. one of us has contributed to show that if this event is not promptly reversed by reoxygenation it causes irreversible damage, mainly by determining a massive entry of ca ϩ into neurons. pretreatment of tissue with creatine ( mm or more) both increases neuronal energy store by increasing neuronal phosphocreatine and protects brain tissue from irreversible damage. in vivo increase in phosphocreatine has been shown using lower ( . mm) creatine concentration, injected directly into the lateral ventricle. a different type of hypoxia-induced damage is observed when hypoxia is of shorter duration. in this case upon reoxygenation one does not observe disappearance of evoked potentials but their increase. this phenomenon, originally described as ìpost-hypoxic hyperexcitabilityî has been later called ìanoxic long-term potentiation (ltp)î. we showed that this event can be prevented by inactivating the nuclear protein s- . while this damage is milder than that induced by anoxic depolarization, it may explain stroke-induced epileptic fits. we are currently investigating what role, if any, pretreatment with creatine may have in preventing also this type of damage. cytoskeleton is subject to continuous modification to yield changes in cell shape and function of plasmamembrane proteins linked to the cytoskeleton. gelsolin (gsn) depolymerizes filamentous actin and thus causes dynamic uncoupling of membrane ion channels. we have studied alteration of neuronal ca ϩ influx by the absence of gsn and its pathophysiological consequences during cerebral ischemia. cytosolic ca ϩ concentrations were determined ratiometrically in synaptosomes preloaded with fura- am. glutamate release from synaptosomes superfused with krebs' buffer was measured by hplc. transient focal cerebral ischemia was induced by h occlusion of the middle cerebral artery (mca). in gsn deficient mouse brain cortical synaptosomes [ca ϩ ] i increase in response to k ϩ ( mm) depolarization was % higher than in wild-type. ω-agatoxin iva . µm decreased ca ϩ -influx in neocortical wild-type synaptosomes by %, and abolished differences between gsnϩ/ϩ and Ϫ/Ϫ genotype. k ϩinduced release of glutamate in neocortical synaptosomes was % higher and lesion size after mca occlusion was % higher than in wild-type. it is concluded that presynaptic ca ϩ influx is increased in gsn deficient nerve terminals which, together with subsequently increased glutamate release, increases neuronal vulnerability. in vivo assessment of tissue alteration in cerebrovascular and neurodegenerative diseases s. flacke, w. block, f. träber, p. mürtz, h. urbach, and h. schild the combined used of perfusion imaging (pi) and molecular diffusion imaging (dwi) are opening a new window into the processes that occur during the first hours of ischemia. dwi detects early changes of proton diffusion associated with cytotoxic edema. pi has the potential to characterize the degree of regional hypoperfusion. mismatches between dwi and pi, i.e. hypoperfused areas with normal adc are considered potentially salvageable. we present results of patients with an angiographically defined thrombembolus in the middle cerebral artery and a spontaneous stroke evolution. whereas the infarct core was clearly visible on both dwi and pi, tissue at risk of infarction could only be detected by an increased blood volume and transit time. however only in a subgroup of patients (n ϭ ) these areas were incorporated into the final infarct. in these patients perfusion parameter of tissue at risk of infarction were more pronouncedly altered than in those where the tissue at risk was spared from infarction (ratios of tissue at risk vs normal (rcbv . Ϯ . , mtt . Ϯ . , ttp . Ϯ . , p Ͻ . ). these human data show that a detailed analysis of diffusion/ perfusion mismatches allow the identification of tissue at risk of damage. glucose deprivation enhances the sensitivity of cerebellar granule cells to die by excitotoxicity. we have previously reported that neither min of glucose deprivation, a treatment that depletes cell energy resources, nor exposure to µm glutamate (glu) for min, induce significant cell death in cerebellar granule cell cultures, h after treatment. in contrast, the combined treatment with µm glu and glucose deprivation induces both cell death and choline (cho) release. we investigated whether the neurotoxic effect of this treatment is related with inhibition of phosphatidylcholine (pc) synthesis. we found that exposure to µm glu alone for min, glucose deprivation for min, and the combination of both treatments inhibited pc synthesis when measured at the end of treatment by %, % and %, respectively. furthermore, we found that exposure to either µm glu, glucose deprivation or µm glu ϩ glucose deprivation decreased incorporation of [ h]cho into phosphocholine and increased the intracellular content of free [ h]cho, indicating that all these treatments inhibit the synthesis of pc by inhibiting choline kinase activity. since only the combined treatment with µm glu plus glucose deprivation evoked cho release and excitotoxic cell death, the present results indicate that other factors in addition to inhibition of pc synthesis are required to induce cho release and excitotoxic cell death in cerebellar granule cells. (supported by cicyt, saf - .) the role of striatal metabotropic glutamate receptors in degeneration of dopamine neurons k. goĺembiowska, j. konieczny, k. ossowska, and institute of pharmacology, polish academy of sciences, kraków, poland the present study was undertaken to characterize the effect of blockade of the mglu receptor subtype by -methyl- -phenylethynylpyridine (mpep), as well as the effect of stimulation of the mglu / receptor by (Ϫ)- -oxa- aminobicyclo[ . . ]hexane- , -dicarboxylic acid (ly ) on spontaneous and stimulated dopamine (da) release in rat striatum using an in vivo microdialysis. mpep ( - µm), perfused through a microdialysis probe affected neither the basal nor the veratridine ( µm)-stimulated striatal da release. however, mpep given intraperitoneally ( mg/kg) diminished either the basal or the veratridine-evoked da release. ly ( - µm) administered locally also inhibited the veratridine-evoked da release in rat striatum. antagonists of mglur-i and agonists of mglur-ii have been shown to have neuroprotective properties in several models of neurotoxicity in animals. we have approached this issue using a selective mglu antagonist in an animal model of neurotoxicity induced by methamphetamine. in our preliminary experiments, methamphetamine ( ϫ mg/kg sc every two hours) decreased the tissue content of striatal da and its metabolites dopac and hva.mpep ( ϫ mg/kg ip) given before every methamphetamine injection reversed its action. the effect exerted by the mglu antagonist mpep seem to be mediated by sites located outside the striatum due to relieving da neurons of the facilitatory influence of glutamate. in turn, the attenuation of da release from nigrostriatal terminals by ly may be a consequence of activation of striatal mglu / receptors. reversal of the methampetamine-induced da depletion suggests a potential for neuroprotective activity of mpep. o. golubnitschaja , h. h. schild , and j. flammer department of radiology, university of bonn, germany university eye clinic, basel, switzerland glaucoma remains a major eye illness with unknown etiology. although elevated intraocular pressure has been shown to be the major risk factor, there is a cohort of relatively young patients developing normal-tension glaucoma (ntg). assymptomatic ischemic events in brain have been shown to be often attributable to galucoma. perfusion of the retina and optic nerve head suffering from observed vasospastic dysfunction may be further reduced by changes in the intraocular pressure. ocular ischemia developed due to these blood flow deficits may play a major role in initiation of glaucoma. possibly secondary to ischemia the autoimmunogenic capacity is activated by ntg patients having an increased prevalence of systemic autoimmune diseases. therefore, the determination of potential molecular markers in blood lymphocytes could be useful for early diagnostics of ntg. our recent study using "gent hunting"-techniques showed indeed altered gene expression in lymphocytes of ntg patients. the demonstrated downregulation of xpgc gene expression which subsequently leads to the accumulation of damaged dna and an elevated p expression, together with the upregulation of a new abc-transporter seem to be specific for the pathogenesis of ntg. molecular imaging of ntg provides insights in mechanisms of disease initiation and allows the early diagnostics and preventive treatment. (supported by "bio-rad" and "amersham pharmacia aggregate cell cultures prepared from fetal rat telencephalon were used to study neuronal amino acid consumption during glucose restriction. to that end, both mixed (neuronglia) and neuron-enriched cultures were grown in chemically defined medium and tested at an advanced maturational stage. it was found that h of exposure to reduced glucose ( . mm instead of mm) caused significant increases in the consumption of several amino acids and the accumulation of ammonia. it also greatly changed the intracellular level of several amino acids in neurons, particularly of aspartate and glutamate. irreversible neuron-specific damage was observed one week after the insult. elevated glutamine media concentrations ( mm instead of . mm) during glucose restriction further increased ammonia production and neuronal damage, although the overall rate of glutamine metabolism remained practically unchanged. taken together, our findings suggest that glucose deficiency caused (i) the dysfunction of crucial transamination pathways; (ii) a shift towards the oxidative deamination of glutamine and several other amino acids used by neurons as alternative energy substrates; and (iii) the accumulation of neurotoxic ammonia levels. institute for brain research, university of vienna, austria the racemic (d,l) mixture of the naturally occurring neutral aromatic amino acid , -dihydroxy-l-phenylalanine (l-dopa) was first synthetized in . in , the natural levorotatory isomer was isolated from vicia faba beans and declared to be biologically inactive. however, in l-dopa was observed to lower the blood pressure in the rabbit, an effect opposite to the vasopressor effect of adrenaline. following the discovery, in , of the enzyme l-dopa decarboxylase, ldopa's conversion in tissues (by decarboxylation) to dopamine (da), the first biologically active substance in the biosynthetic pathway of catecholamines, was demonstrated. subsequent pharmacological studies, done between and , showed that the biological actions of l-dopa were, in principle, due to da formed from it in the body. in , the central antireserpine effect of d,l-dopa was described in mice and confirmed in with l-dopa in humans. following the demonstration of da's occurrence in the brain in the years / , d,l-dopa was found (in rabbits) to restore brain da levels, reduced by reserpine. in , the severe brain da deficit, confined to patients with parkinson's disease (pd) was reported and a year later l-dopa's superior anti-akinesia effect in patients with pd demonstrated. finally, in the high-dose oral l-dopa regimen was successfully introduced into clinical practise. in contrast to these supreme achievements, two related early studies remained, for different reasons, without consequence. despite some initial doubts about its mechanism of action, there is now convincing evidence for l-dopa therapy being a classic example of a central neurotransmitter replacement therapy, with the severe brain da deficit furnishing a rational basis for the amino acid's clinical use and high efficacy in patients suffering from pd. b. jakobsen , a. tasker , and j. zimmer anatomy and neurobiology, sdu-odense university, odense, denmark department of anatomy and physiology, university of prince edward island, canada the toxicity of domoic acid (dom) was studied in rat hippocampal slice cultures, prepared from -days old rats and grown on semipermeable membranes for - weeks before exposure. dom ( . - µm) was added to the medium, alone or together with the glutamate receptor antagonists ns- , nbqx or mk- , for hrs followed by hrs in normal medium. neuronal degeneration was monitored and ec values estimated by densitometric measurements of the cellular uptake of the propidium iodide (pi) at , , and hrs. the lowest ec values, obtained at hrs, were: ca ( µm), dentate granule cells (dg) and ca ab ( µm),ca c ( µm). protective effects of µm nbqx at hrs were seen against µm dom in dg, ca and ca c and against µm dom in ca and ca c. µm ns and mk only displayed protective effects together with nbqx. mk thus significantly increased the protective effect of µm nbqx in ca against µm dom in combination with µm nbqx and µm ns . we can confirm that dom neurotoxicity primarily involves ampa/kainate receptors, but also nmda receptors at high concentrations (glutamate release). department of physiology/neuroscience, medical university of south carolina, charleston, south carolina, u.s.a. although dopamine has been most clearly tied to the development of addiction to drugs of abuse, recent studies indicate that once addiction has been established the expression of addictive behaviors, such as drug craving, is mediated more by long-term neuroadaptaions in glutamate transmission. data will be presented which supports and extends this hypothesis. repeated cocaine injections were given for one week and three weeks after the last drug injection a number of molecular, neurochemical and behavioral neuroadaptations were measured. it was found that in the nucleus accumbens there is a increase in the expression of genes encoding mglur / and glur , and a decrease in the expression of mglur and its accompanying scaffolding proteins homer bc. this was accompanied by an increase in the capacity of mglur / receptors to regulate presynaptic glutamate release and a blunting in the effects of stimulating mglur / receptors. in addition, there is reduced activity in the cystine/glutamate exchanger wks after repeated cocaine. as a result of these changes there is a decrease in the basal release of glutamate, and a relative increase in the releasibility of glutamate upon stimulation. by using the reinstatement model of drug seeking behavior, it was shown that glutamate transmission in the projection from the prelimbic cortex to the core of the nucleus accumbens was particularly affected by the cocaine-induced changes in gene expression. taken together, these findings support the use of glutamate autoreceptor agonists as possible therapeutic adjuvants in treating the cravings associated with addiction. dopamine (da), a catechol that autoxidizes to an oquinone, is implicated as an endogenous pro-toxin, however, the following studies suggest that da has dual neurodegenerative and neuroprotective roles. in rats treated as neonates with -hydroxydopamine ( -ohda; :g icv), there was a % reduction in striatal tissue da content in adulthood, and a to fold increase in spontaneous hydroxyl radical (ho*) formation (indirect salicylate trapping method: dihydroxybenzoic acid analysis). additionally, systemic l-dopa ( mg/kg i.p.) suppressed ho* formation. however, when glutamate ( mm) was added to an in vivo microdialysate, ho* formation was increased substantially more in the microdialysate from dainnervated striatum. these findings indicate that da innervation is inherently neuroprotective, but in the presence of a high level of an excitatory amino acid, da innervation predisposes to formation of reactive oxygen species. ongoing neuronal activity is likely to interact with and to determine the role of da as a neurotoxic or neuroprotective substance. (supported by ns .) the glutamate hypothesis of schizophrenia along with the dopamine hypothesis was intensively discussed in the past. the last years however suggest more and more that neither a hypofunction of the glutamatergic system alone nor a hypofunction of the dopaminergic system alone is responsible for symptoms found in schizophrenia. the basal ganglia (bg) as the critical structures mediating symptoms of schizophrenia are innervated by dopaminergic fibers from the mesencephalon as well as by glutamatergic fibers from limbic structures; like prefrontal cortex, hippocampus, entorhinal cortex and amygdala. thus, limbic input is able to modulate information processing in each structure of the bg and by this way control dopaminergic functions through feedback mechanisms. dysfunction in limbic structrues may result in an imbalance of information processing via the bg and terminates in behavioral symptoms of schizophrenia. we showed in recent neurochemical studies in combination with behavioral analysis that a simple, generalized hypofunction of limbic glutamatergic input on bg nuclei is not the key mechanism inducing schizophrenic behavior. a dysfunction of a particular limbic structure or pathway seems to be responsible for an imbalanced information processing via the bg and imbalanced behavioral adaptation terminating in schizophrenic symptoms. [ there is a need to identify subtype-specific ligands for mglu receptors to elucidate the potential of these receptors for the treatment of nervous system disorders. to date, most mglur antagonists are amino acid-like compounds acting as competitive antagonists at the glutamate binding site located in the large extracellular n-terminal domain. we have investigated novel subtype-selective mglur antagonists which are structurally unrelated to competitive mglur ligands. using a series of chimeric receptors and point mutations we demonstrate that these antagonists interact with novel allosteric binding sites in the tm domain via a noncompetitive mechanism of action. recent studies in animal models implicate mglu receptors as a potentially important therapeutic target particularly for the treatment of pain and anxiety. vascular endothelial growth factor (vegf) is a major mediator in angiogenesis and vascular permeability. in central nervous system (cns) vegf plays pivotal roles such e.g., inductor of endothelial cell proliferation, migration and inhibition of apoptosis, as well as mediator of blood brain barrier (bbb) breakdown and subsequently of brain edema formation. these ubiquitous epiphenomena are major complications in several cns pathologies, including head trauma and stroke. reduced tissue oxygen tension (hypoxia) and hypoglycaemia triggers vegf expression that occurs in ischemic regions around postraumatic or postinfarct necrosis. after brain injury, the expression of vegf is increased contributing to disruption of the bbb. vegf increases the permeability of bbb via the synthesis/release of nitric oxide and subsequent activation of soluble guanylate cyclase. the immunohistochemistry shows an increase of stained astrocytes around a cortical micronecrosis. vegf participates in the response of the cns to injury in a dose dependent way. immunostaining correlates with infarct volume and clinical disability. vegf-antagonists reduce ischemic brain edema and injury, involving vegf in pathogenesis and eventually in treatment of stroke and related disorders. this cytokine also exerts a neuroprotective effect mediated by its receptor flk- . functions related to the inflammatory response, co-expression with proteins of the ecm and interaction with the two main receptors, flk- and flt- , will be discussed. n-methyl-d-aspartate (nmda) receptors can mediate excitotoxic or neuroprotective responses. one of the molecular mechanisms responsible for nmda neuroprotection involves the release of brain-derived neurotrophic factor (bdnf) which in turn binds to and activates its cognate receptor trkb. bdnf levels in the neuronal culture medium increased -fold when cells were preincubated for three hours with nmda. at three hours, the increase in bdnf protein levels in the medium was accompanied by a concomitant increase in bdnf mrna. thus, nmda elicited two temporally distinct responses: an early release of bdnf protein followed by a later transcriptional activation of dbnf mrna and protein release. these results suggest that nmda activates the trkb receptor via a bdnf autocrine loop resulting in neuronal survival. in addition, extracellular regulated kinases (erk / ) were rapidly activated, which peaked within six hours of nmda treatment. erk / activation is completely blocked by mk- and partially blocked by k a, suggesting the nmda and trkb receptors act in a coordinated fashion to activate erk / . as an extension of this work, we discovered a single nucleotide polymorphism in the human nr gene that, when transfected into hek cells, alters the electrophysiological properties of the nmda receptor complex. possible consequences of this nmda receptor variant in signaling will be discussed. this overview summarizes our recent knowledge of the role that tyrosyl radical (tyro • ) can play in neurochemical systems of brain and thereby lead to neural disorders (pd, ad, als). these could involve the interactions of tyrosine and tyro • with reactive oxygen species (ros) and reactive nitrogen species (rns), via radical mechanisms and chain processes in the presence of o and endogenous brain antioxidants. concentrations of tyro • , ros and rns can increase dramatically under conditions of generalized stress: oxidative, nitrative or reductive. this in turn can directly damage (by lipid peroxidation) or indirectly damage (by protein oxidation and/or nitration) cellular substructures which ultimately can lead to apoptotic neuronal cell death or autoschizis. enzymatically (classical peroxidase mechanisms) or non-enzymatically formed tyro • can react with no • and this reversible and intrinsic "combination" acts to "buffer' tyro • concentrations. the reaction of tyro • with superoxide (o •Ϫ ) is a scavenging reaction which proceeds rather by addition, not by electron transfer; and major resultant products are tyrosine hydroperoxides (tyrooh). however, the decay of tyro • can be also terminated by self-termination (dimerization) resulting in dityrosine (dt) formation. tyro • can catalyze ldl oxidation, although the precise mechanisms of this reaction in vivo remain unknown. nitration of tyrosine to -nitrotyrosine ( -nt) requires a one-electron oxidation as a primary step, with formation of tyro • , followed by addition of the nitrogen dioxide radical (no • ). the promoting effect of carbon dioxide on peroxynitrite-mediated tyrosine nitration (via radical mechanisms) (tyro • /no • /o •Ϫ /no • system) is due to the selective reactivity of the putative carbonate radical anion, as compared to that of the oxidizing hydroxyl radicals ( • oh). moreover, once formed, -nt may act to promote repetitive redox cycling; it may be reduced to the corresponding nitroanion radical, which is then oxidized by molecular o to o •Ϫ and parent -nt. one-electron oxidation of -nt can result in catalytically active imminoxyl radical. dt formation can outcompete tyrosine nitration at lowsteady state concentrations of peroxynitrite. it is unquestionable that very high fluxes of no • and o •Ϫ are requisite intermediates of peroxynitrite, a tyrosine nitration agent formed via tyro • . evidence for the existence of generalized stress within neurons includes the presence of protein peroxides (tyrooh), dt, and -nt. the nitration/denitration processes can be pathologic, but these also may play: ) a signal transduction role; ) a role of "blocker" for radical-radical reactions (scavenging of no • , no • and co •Ϫ by tyro • ); or ) a role of delimiting factors for peroxynitrite formation. it is still unknown whether oxidation/nitration of tyrosine (as dopamine precursor or protein residue) via tyro • formation, is a footprint of generalized stress and neuronal disorders, an important part of o •Ϫ and no • metabolism, or just a part of integral processes for maintaining neuronal homeostasis. the complete answer of these questions should be the first priority task of our recent search, wherein the problem of increased free radical formation in the brain and/or the imbalance of ratios: ros/rns/tyro • may be all important in determining neural cell and tissue injuries under pathological conditions resulted from generalized stress. [acknowledgements. this work was supported in part by kbn ( more than % of patients with type diabetes have coronary heart disease, related to silent ischemia, caused by an autonomic denervation of the heart in diabetic patients. oxidative damage to dna has been well documented in cardiac cells isolated from diabetic patients and rats with streptozotocin-induced diabetes mellitus (dm) . this dmmodel shows already seven days after onset of disease structural changes in vascular tissue typical for the development of atherosclerosis. this study evaluates possible molecular mechanisms for early events in the development of dm-induced cardiomyopathy. methods: using "expression array" we examined the activation of cardiac cell death in heart of dm-rats. ms-pcr was used to examine a differential dna methylation. results: an increased expression of genes encoding renin, angiotensinogen and p was detected in heart of dm-rats. substantial changes in the methylation status of the p dependent p waf /cip -gene and the cyclin d -gene were detected in dm-rats. conclusions: the renin-angiotensin system is upregulated with diabetes, and this may contribute to the development of cardiomyopathy via oxidative damage and p -dependent activation of cardiac cell death. this pathway includes de novo methylation of the p -inducible p waf /cip -gene encoding a protein which binds to and inhibits a broad range of cyclincyclin-dependent kinase complexes. (supported by "bio-rad" and "amersham pharmacia biotech") department of pharmaceutical biosciences, uppsala university, uppsala, sweden during the past decade studies have indicated that growth hormone (gh) may exert effects on the central nervous system (cns). for instance, gh replacement therapy was found to improve the psychological capabilities in adult gh deficient (ghd) patients. furthermore, beneficial effects of the hormone on certain functions, including memory, mental alertness, motivation and working capacity have been reported. likewise gh treatment of ghd children has been observed to produce significant improvement in many behavioural problems seen in these individuals. studies also indicated that gh therapy affects the cerebrospinal fluid (csf) levels of various hormones and neurotransmitters. further support that the cns is a target for gh emerges from observations indicating that the hormone may cross the blood-brain-barrier (bbb) and from studies confirming the presence of gh receptors in the brain. it was previously shown that specific binding sites for gh are present in discrete areas in the cns of both humans and rats. in peripheral tissues gh is shown to elicit its effects through a second mediator insulin-like growth factor (igf- ). igf- is well recognized as a protective agent against neural injury in the cns. the neuroprotective effect of this peptide has a broad spectrum affecting many brain regions and acts through its antiapoptopic effect. the production of igf- is upregulated in areas of brain damage and the igf- system may be an important part of an endogenous neuroprotective system. in spinal cord injuries, however, the content of igf- is reduced. we recently observed a neuroprotective effect of topical application of igf- in animals subjected to spinal cord trauma. the observed effect may be mediated via a mechanism involving nitric oxide. in the same animal model we have very recently observed a neuroprotective effect of gh. recent reports suggest that the level of gh is drastically reduced in patients with spinal cord injury. in victims of spinal cord injury the secretion of gh and igf- , as well, is known to be decreased. therefore, exogenous substitution of gh and igf- might be a promising approach in the future therapy of spinal cord injury victims. in fact, there is one report indicating that prolonged treatment with synthetic gh of spinal cord injured rats attenuates some of the neurological motor dysfunction seen in these animals weeks following trauma. in our animal model we observed that topical application of rgh significantly reduced traumainduced disturbances in the fluid micro-environment. we also noted that gh was capable of attenuating the trauma-induced depression of spinal cord evoked potentials. the mechanism by which gh exerts it neuroprotective effects will be discussed. chronically administered levodopa in parkinson's disease (pd) treatment is ultimately associated with alterations in motor response. in -hydroxydopamine lesioned hemiparkinsonian rats, chronic twice-daily administration of levodopa progressively shortens duration of contralateral turning and augments the period of turning at or below % of peak turning rate. the pathogenesis of the response alterations involves in part sensitization of the corticostriatal glutamatergic synaptic activity. characteristic changes involving interactions between striatal kinase and phosphatase signaling now appear to contribute to sensitization of spiny-neuron glutamatergic receptors. glutamate-mediated striatal dysregulation, subsequently, modifies basal ganglia output system in ways that favor the appearance of parkinsonian motor response complications. at a molecular level, transcriptional activation of striatal creb contributes to the persistent expression of the levodopa-induced motor response alterations. conceivably, a safer and more effective therapy for all stages of pd can be provided by drugs that target intracellularly on striatal kinases or phosphotases, or by agents that interact extracellularly on non-dopaminergic striatal receptors such as ampa and nmda, adenosine a , adrenergic a , opiod, and serotonergic b. the primary cause of parkinson's disease is a loss of dopamine in the corpus striatum. it has been postulated that this effect leads to disinhibition of the striopallidal pathway and, secondarily, to a functional shift towards glutamatergic stimulation. the aim of the present study was to find out whether inhibition of glutamatergic transmission at a level of metabotropic glutamate receptors (mglurs) in the striatum may alleviate parkinsonian-like symptoms in rats. the non-competitive antagonist of receptor subtype (mglur ), mpep ( . - mg/kg ip), or the agonist of group ii mglurs, ly ( - mg/kg ip), reduced the haloperidolinduced muscle rigidity and catalepsy. intrastriatal injections of the antagonist of mglur , (rs) aida ( . - µg/ . µl), but not of the agonist of group ii mglurs, r, r-apdc ( . - µg/ . µl), inhibited the muscle rigidity induced by haloperidol. in order to search for an influence of mglurs on the striopallidal pathway, the effect of mpep or of the agonist of group ii mglurs, dcg-iv, on the preproenkephalin mrna expression in the striatum was examined. the obtained results suggest that blockade of group i mglurs, or stimulation of group ii mglurs may be important to the amelioration of parkinsonian symptoms. striatal mglurs may contribute to at least some of these effects. several lines of evidence suggest an important role of glutamate in depression. the involvement of group i mglurs in depression has also been proposed. thus, we decided to evaluate whether group i mglurs antagonists have antidepressantlike effects. we also investigated if antidepressant treatment influences group i mglu receptors in the brain. the experiments were performed on male wistar rats ( - g) and male c bl/ mice ( - g). aida (group i mglurs antagonist) given i.v. in the dose of µg, decreased the immobility time in the despair test in rats. mpep (noncompetitive, systemically active mglur antagonist) given i.p., was not effective in the despair test in rats. however, in doses of . , and mg/kg, it significantly decreased the immobility time of mice in the tail suspension test. moreover, the deficit in passive-avoidance learning, which was observed in bulbectomized rats, was reversed by chronic, but not acute mpep ( mg/kg) treatment. prolonged imipramine treatment resulted in significant increase of the level of expression of mglu receptors in the ca field of the hippocampus, while prolonged electroconvulsive shock treatment (ect) enhanced significantly the chemiluminescence of mglu receptors in the ca field. the results indicate that group i mglu receptors are modified by chronic antidepressant treatment and that group i metabotropic glutamate receptors antagonists may play a role in the therapy of depression. (this study was supported by kbn grant no. .po a. . ) institute of pharmacology, polish academy of sciences, krakow, poland chronic exposure to nicotine, alcohol, opioids, sedatives, and cannabis results in development of drug dependence that becomes evident upon a cessation of drug administration and expresses itself as a withdrawal syndrome (with its physiological and motivational manifestations). adaptations at the nmethyl-d-aspartate receptor (nmda-r) complex have been observed in different brain areas during chronic exposure to, and upon withdrawal from, opioids, ethanol, benzodiazepines and barbiturates. behavioral studies employ the assessment of the effects of nmda-r antagonists on: a) the development of dependence (nmda-r antagonists are co-administered with the drug), b) the maintenance of dependence (nmda-r antagonists are administered to animals with pre-established dependence, and -most relevant to the clinical situation -c) on the expression of drug dependence (assessment of the withdrawal severity in subjects with nmda-r antagonists administered just before the expected emergence of withdrawal). the development of dependence to opioids and benzodiazepines is significantly retarded by nmda-r antagonists. studies from this laboratory demonstrate similar inhibition by nmda-r antagonists of the maintenance of opioid dependence. both in rodents and humans, the expression of opioid antagonist-precipitated as well as spontaneous (natural) withdrawal is inhibited by nmda-r antagonists, and animal data demonstrate similar inhibition of the expression of dependence produced by ethanol, barbiturates and benzodiazepines. the involvement of the excitatory amino acid, glutamate and the inhibitiory amino acid, gamma-amino butyric acid (gaba) in the pathophysiology of spinal cord trauma is not known in details. this investigation is focused on the involve-ment of glutamate and gaba in a rat model of spinal cord injury using immunohistochemistry. spinal cord injury induced by an incision into the right dorsal horn of the t - segments resulted in profound edema formation and cell damage in the adjacent t and t segments at h. pretreatment with h- / ( mg/kg, p.o.), a potent antioxidant compound, effectively reduced the edema formation and cell injury following trauma. at this time period, untreated traumatised rats exhibited a marked increase in glutamate immunoreactivity and a distinct decrease in gaba immunostaining in the t and t segments compared to the control group. the changes in glutamate and gaba immunoreactivity in traumatised rats were considerably attenuated by pretreatment with h- / . these results suggest that (i) oxidative stress contributes to alterations in glutamate and gaba in spinal cord injury, (ii) glutamate and gaba are contributing to edema formation and cell damage and (iii) the antioxidant compound h- / has a potential therapeutic value in the treatment of spinal cord injuries. dov pharmaceutical, inc., hackensack, new jersey, u.s.a. both preclinical (i.e., behavioral despair models) and clinical studies indicate that compounds reducing transmission at nmda receptors are antidepressant. conventional antidepressants may be viewed as "monoamine-based", increasing the synaptic availability of serotonin, norepinephrine, and/or dopamine. however, chronic administration of of conventional antidepressants alters both mrna levels encoding nmda receptor subunits and radioligand binding to this family of ligand-gated ion channels in circumscribed areas of the cns indicating that nmda receptors may be a downstream target of these monoamine-based agents. we have recently reported (li, et al., neuropharmacology, in press ) that a class of ampa receptor potentiators also exhibits antidepressant-like actions in preclinical models. in this presentation, i will describe how these two distinct, and (at a cellular level) seemingly diametric approaches employing glutamatergic mechanisms converge on intracellular targets that are also impacted by chronic treatment with biogenic amine-based agents. kainic acid is an essential pharmacological tool for many forms of neurobiological research. until several years ago, all commercially available kainic acid was derived from a single biological source (digenia simplex). commercial isolation of kainic acid in japan ceased in , creating a void in the marketplace. recently several different companies have become providers of kainic acid, but each uses a different source of the compound ( biological and synthetic) and different isolation procedures. our objective was to use three common assay systems to evaluate the comparative pharmacological and neurotoxicological properties of these three sources of kainic acid. dose response curves, both alone and in the presence of receptor selective antagonists, were constructed for each kainate formulation using (a) cerebellar granule neurons in culture, (b) isolated hippocampal slice preparations, and (c) whole animal behavioural toxicity studies. preliminary results reveal many similarities, but also distinct differences between the three formulations, especially when challenged with antagonists for different eaa receptors. full results will be presented and discussed with respect to their implications for both extending the known kainite literature and for future studies employing kainic acid as a ligand in both mechanistic investigations and in animal models of neurodegenerative disease. our results from in vitro studies further elucidate the role of cell-cycle related proteins in neuronal apoptosis induced by excitotoxins. exposure of primary cerebellar neurons to toxic concentrations of glutamate was found to produce a significant, short lasting increase in the expression of p and cdc . transcriptional activity of p was shown by increased p dna binding activity and by the concomitant induction of the cdk inhibitor p , the cell cycle regulator gadd and the apoptotic induced bax. cell-cycle proteins are also expressed concomitantly to dna damage in neurons undergoing excitotoxic degeneration. we found that excessive activation of glutamate receptor by nmda results in the formation of -oh-deoxyguanosine, which is a marker of oxidative dna damage. in addition, the expression of the dna repair factor msh increases in cultured cerebellar neurons or in ca pyramidal cells that have been challenged with excitotoxins. excitotoxicity may thus provide a further example of how re-expression of cell-cycle proteins might be tightly connected to dna damage and repair in neurons. rush-presbyterian-st. luke's medical center, chicago, u.s.a. patients with parkinson's disease by definition benefit from levodopa therapy. however, after years of therapy % of patients experience motor response complications (mrc's): the benefit from each dose becomes shorter (wearing-off), more unpredictable (on-off) and associated with involuntary movements (dyskinesias). when dyskinesias first arise, they are associated with high levodopa levels and may be prevented or minimized by lowering levodopa intake. later on, the therapeutic window of levodopa narrows progressively and dyskinesias occur at doses equal to those needed to induce an antiparkinson effect. while the pathogenesis of motor complications remains incompletely understood, recent clinical studies implicate mechanisms downstream from the degenerating nigrostriatal dopamine system, possibly involving glutamatergic projections to the basal ganglia. in a rat model of pd, blockade of striatal glutamate receptors of the n-methyl-d-aspartate (nmda) subtype reverses levodopa-induced motor fluctuations. similarly, in -methyl- -phenyl- , , , tetrahydropyridine (mptp) lesioned primates, several nmda-antagonists reduce levodopa-associated dyskinesias. in parkinsonian patients the nmda-antagonists dextromethorphan, dextrorphan and amantadine improve dyskinesias as well. these findings have lead to the suggestion that hyperfunction of nmda receptors on striatal efferent neurons, as a consequence of chronic non-physiologic dopaminergic stimulation, contributes to the pathogenesis of motor response complications. protein misfolding and aberrant polymerization are salient features of virtually all central neurodegenerative disorders, including alzheimer's disease (ad), parkinson's disease, polyglutamine diseases, tauopathies, and prion diseases. in many instances, a single amino acid change can predispose to disease by increasing the production and/or changing the biophysical properties of a specific protein. possible pathogenic similarities among the cerebral proteopathies suggest that therapeutic agents interfering with the proteopathic cascade might be effective against a wide spectrum of diseases. however, testing compounds preclinically will require diseaserelevant animal models. numerous transgenic mouse models of ad-like pathology have now been produced. our studies have found that tg mice overexpressing human -amyloid precursor protein (huapp k n/m l) produce copious deposits of diffuse and compact -amyloid as they age, and that females are more susceptible than are males (callahan et al., am. j. pathol. , - , . recently, we also found that the overexpression of p protein, an activator of the kinase cdk , results in tau hyperphosphorylation, axonopathy and severe motor deficits in transgenic mice, in the absence of neurofibrillary tangles. none of the existing transgenic models of -amyloidosis or tauopathy fully recapitulates the pathology of ad. in an attempt to more authentically model the human disease, we infused dilute ad-brain extracts into tg mice at -months of age (i.e. - months prior to the usual onset ofamyloid deposition). we found that infusion of ad brain extracts results in: ) earlier and more abundant deposition of -amyloid in app-transgenic mice (kane et al., j. neurosci. , - ); ) evidence for the spread of pathology to other brain areas, possibly by neuronal transport mechanisms; and ) tau hyperphosphorylation (but not neurofibrillary pathology) in axons passing through the injection site. the seeding ofamyloid by ad brain extracts suggests pathogenic similarities between -amyloidoses such as ad and other cerebral proteopathies, and could provide a new model for studying the proteopathic cascade and its neuronal consequences in neurodegenerative diseases. supported by warner-lambert/pfizer. purpose: the effects of essential amino acid deficiencies on function of cornea and lens were investigated. methods: dietary deficiencies of tryptophane and methionine were studied in young rats over months. transparency of cornea and lens were evaluated using slitlamp microscope and scheimpflug camera. after sacrifice, lens fresh weight and crystallin patterns were determined to evaluate effects on lens growth and protein synthesis. results: methionine deficiency had no effect on the parameters investigated. tryptophane deficiency caused severe loss of body weight in both rat strains (brown-norway, bn; sprague-dawley, sd), sd rats also lost their hair. they developed corneal neovascularisations and cortical cataracts. bn rats developed faint neovascularisations and a discontinuity zone in the lens. diet intermission arrested pathological processes restarting when feeding diet again. this observation is supported by lens fresh weight data. dna staining evidenced that tryptophane deficiency arrested lens fiber maturation. conclusion: a difference has been found for essential amino acids in their effects on transparency of cornea and lens. tryptophane deficiency stimulated corneal neovasculariseration, but arrested lens fiber cell maturation. the difference in reaction of cornea and lens to tryptophane deficiency between bn and sd rat eyes remains to be elucidated. dynorphin is a neuropeptide that is present in the dorsal horn of the spinal cord. the peptide is actively involved in pain processing pathways. however, its involvement in spinal cord injury is not well known. alteration in dynorphin immunoreactivity occurs following a focal trauma to the rat spinal cord. infusion of dynorphin into the intrathecal space of the cord results in ischemia, cell damage and abnormal motor function. antibodies to dynorphin when injected into the intrathecal space of the spinal cord following trauma improves motor recovery and reduces edema and cell changes. however, influence of dynorphin on trauma induced alteration in spinal cord bioelectrical activity is still not known. spinal cord evoked potentials (scep) are good indicator of spinal cord conduction that are altered following trauma. therefore, in present investigation, influence of dynorphin antibodies on trauma induced changes in scep was examined in our rat model. in addition, spinal cord edema formation and microvascular permeability disturbances were also investigated. our results show that intrathecal administration of dynorphin antiserum prior to injury has a beneficial effect on trauma induced electrical activity, microvascular permeability disturbances, and edema formation. these observations indicate that dynorphin is somehow involved in the altered bioelectrical activity of the spinal cord and participates in the pathophysiological processes leading to cell injury. fatty-acid binding proteins (fabps) are involved in the intracellular binding, targeting and transport of long-chain fatty acids (fas) to modulate cell growth and/or differentiation. fabp form a family of proteins displaying tissue-specific expression. the expression of brain type fabp (b-fabp) is spatially and temporally correlated with neuronal differentiation during brain development. heart type fabp (h-fabp) is widely distributed and present in skeletal muscles, kidney, lung, brain and endothelial cells. it is neuron-specific in postnatal brain and participates in neurite formation and synapse maturation. epidermal type fabp (e-fabp) is expressed at high levels during neurogenesis, neuronal migration, and terminal differentiation. although all three fabps could be involved in normal brain function in prenatal and postnatal life, a neurobiological role of fabps in neurodegenerative diseases has not been reported yet. these made us evaluate the protein levels of fabps in brains from patients with down syndrome (ds) and alzheimer's disease (ad) and fetal cerebral cortex with ds using two-dimensional ( -d) gel electrophoresis with subsequent matrix-assisted laser desorption ionization mass spectroscopy (maldi-ms) identification and specific software for quantification of proteins. in fetal brain, b-fabp and e-fabp levels were comparable between control and ds. in adult brain, b-fabp was significantly increased in occipital cortex of ds, and h-fabp was significantly decreased in ds (frontal, occipital, parietal cortices) and ad (frontal, temporal, occipital and parietal cortices). we conclude that aberrant expression of fabps, especially h-fabp in neurodegenerative diseases could be involved in impaired neurite outgrowth and synapse maturation. in our previous paper, it was shown that gaba-a receptor antagonist picrotoxin suppressed etoh (ethanol) selfadministration. recently, several authors indicated that systemic injection of dopamine or serotonine agonists reduced ethanol drinking in rats. therefore, in the present study we investigated the effects of thip ( , , , -tetrahydroizokasazolo, , -c pyridin- -ol) gaba-a receptor agonist in naive and long-term ethanol-experienced rats on etoh selfadministration and on cardiovascular system. adult - week-old male, normotensive wistar-kyoto (wky) and spontaneously hypertensive rats (shr) were used. naive rats were examined according to smith method. long-term ethanolexperienced rats were studied according to boyle method. thip was injected in naive rats at a dose of and mg/kg i.p. metabotropic glutamate receptors are coupled to phospholipase c stimulation and adenylyl cyclase inhibition through g-proteins. c glioma cells, that endogenously express the phospholipase c coupled metabotropic glutamate receptor type, were treated with different specific agonists of these receptors and the effect of these treatments on different components of metabotropic glutamate receptor pathway was studied by radioligand binding, phospholipase c activity and rt-pcr assays. agonists treatment caused a decrease in l-[ h]glutamate binding to intact cells and membranes in a time dependent manner being maximum at - hours and recovered at - hours. this decrease was associated with a significant increase in the mrna level coding mglurs. no changes on g q/ mrna level were detected in any case. however, a significant decrease in l-glutamate stimulated phospholipase c activity was detected after agonist treatments in both membranes and intact cells. this decrease was not associated to significant variations in mrna level coding phospholipase c isoform. all these results suggest that agonist exposure causes a desensitisation of glial metabotropic glutamate receptor decreasing not only receptors number but its functionality. in this study the interaction between these two nuclei were investigated by means of microinjection and microdialysis techniques in sprague-dawley rats. steroetaxic surgery was performed by placing intracerebral parenchymal microinjection cannula into the right dmh and microdialysis probe into the left pvn. iliac artery was also cannulated to monitor the pulsatile blood pressure and heart rate by means of pressure transducer connected to a polygraph microinjection of pmol nmda into the dmh was performed and microdialysis perfusates were collected simultaneously from the pvn in conscious rat model. γ-aminobutyric acid (gaba) and l-glutamic acid levels were analyzed by an isocratic hplc (high pressure liquid chromatography) method with the aid of a fluorescent detector. microinjection of pmol nmda into dmh produced significant increases in mean arterial pressure and heart rate. nmda microinjection into the dmh produced significant increase in l-glutamic acid release in the pvn, but no significant change in gaba release was observed. these results suggest that stimulation of dmh by nmda results in subsequent stimulation of the pvn. [this study was sponsored by marmara university research foundation (project no: /sag/ ).] and in long-term ethanol-experienced rats only at a dose mg/ kg i.p. control group (cg) received saline ml/kg i.p. as can be seen in fig. and table the lower consumption of ethanol in shr in comparison to wky rats was observed. systemic injection of thip decreased dosedependently etoh intake in naive rats of both strains. this effect was more pronounced in shr (fig. ) . similar phenomenon was observed after thip injection in long-term ethanolexperienced rats. there were no effect on systolic blood pressure and heart rate after thip treatment. born-bunge foundation, university of antwerp, and university of ghent, belgium increased neuronal excitability may underlie some of the neurological complications in uremic patients. in an effort to identify candidate neuroexcitatory compounds, different uremic retention solutes, including several amino acids and amino acid derivatives, were applied to mouse spinal cord neurons in primary dissociated cell cultures. using the tight-seal whole-cell technique, a few of the candidate toxins were shown to evoke whole-cell currents in cells clamped at Ϫ mv. in a first survey, each of the solutes was briefly applied in a concentration of mm. significant inward whole-cell currents were evoked by guanidinosuccinate, spermine, and -indoxyl sulfate, whereas phenol evoked an outward current. further experiments indicated that guanidinosuccinate-evoked whole-cell currents were due to activation of nmda-type glutamate receptors in concentrations similar to those found in uremic patients. high (mm) concentrations of spermine activated voltage-gated calcium channels, whereas low (µm) concentrations were found to potentiate guanidinosuccinate-evoked currents through its action on the nmda receptor-associated polyamine binding site. whole-cell currents evoked by indoxyl sulfate or phenol seemed to be due to complex interaction with several different ion channels. we conclude that guanidinosuccinate-evoked nmda receptor activation, possi-bly potentiated by the neuroexcitatory effects of polyamines and other putative uremic neurotoxins, could be an important mechanism underlying the increased neuroexcitability in uremic brain. glutamine (gln) is one of the key metabolites in the cns (energy metabolite, precursor of neurotransmitter amino acids, end product of ammonia detoxication, osmolyte), and as such is a routine supplement of cns cell culture media. c glioma cells relatively easily adapt to culturing in a gln-deprived medium. the present study investigated the effects of gln deprivation on the characteristics of the different systems that mediate gln cell membrane transport in the cells. in contrast to a variety of cns and non-cns cells, the absence of gln did not derepress the methyl-amino-isobutyric acid (meaib)-sensitive ("system adependent") uptake. system asc became relatively more-, and system n less active than in cells grown in the presence of gln, but the ion -and substrate specificity of the uptake remained unaltered. system asc in c cells grown in a glnsupplemented medium shows two features distinct from most other cell types: a) strong ph sensitivity and b) partial tolerance of lithium substitution, pointing to domination of system asct -an asc variant strongly expressed in cultured astrocytes. cells grown in gln-deprived medium lost lithium tolerance, but not ph-dependence of the uptake, their properties thus resembling system glnt (sat ), a neuron-specific variant of system a. by contrast, transport of threonine, a standard asc system substrate, was not affected by gln deprivation and showed neither ph dependence nor lithium tolerance, which is typical of an asc in all the non-cns tissues. (supported by scsr grant no. p a .) the classical the hypothalamic-neurohypophysial system (hns) is comprised of neurons originating within the supraoptic nucleus (son) which project to the neurohypophysis to release the nonapeptides oxytocin (oxt) and vasopressin into the blood after appropriate stimulation. previous experiments have shown that a single social defeat experience triggers the release of oxt from somata and dendrites into the extracellular fluid of the son, but not from axon terminals in the neurohypophysis. to further investigate the regulatory mechanisms underlying this dissociated release, we exposed male wistar rats to a -min social defeat experience and monitored the release of the inhibitory amino acids gamma amino butyric acid (gaba) and taurine into the son using microdialysis. social defeat caused a significant increase of the intra-son pre-stress basal release). to reveal the physiological significance of the intrahypothalamically released gababicuculline, a specific gaba a -receptor antagonist -was administered into the son by retrodialysis. this treatment increased significantly the release of oxt both within the son ( %; p Ͻ . vs. pre-stress basal release) and -as measured via chronically implanted jugular venous catheters -into blood under basal and stress conditions (up to %; p Ͻ . vs. prestress basal release). however, bicuculline did not affect plasma vasopressin. these data demonstrate that gaba is released within the son during social defeat to act as an inhibitor of both, central and peripheral oxt secretion during emotional stress. the mechanism described here contributes to the regulatory capacity of the hns to ensure the appropriate involvement of oxt in the stress response of the animal (supported by dfg, en - ). down syndrome (ds) is the most common human chromosomal abnormality caused by an extra copy of chromosome and characterized clinically by somatic anomalies, mental retardation and precocious dementia. the phenotype of ds is thought to result from overexpression of a gene or genes located on the triplicated chromosome or chromosome region. reports that challenge this notion, however, have been published. to add to this body of evidence, the expression ofamyloid precursor protein (app), ets- and collagen α (vi) chain precursor, encoded on chromosome , was investigated in fetal brain by western blot and two-dimensional electrophoresis ( -de). western blot detected app and ets- that migrated at ϳ and kda, respectively. subsequent densitometric analysis of app and ets- immunoreactivity did not produce any significant change between controls and ds. since the metabolic fate of app determines the propensity of amyloid production, the expression of the secreted forms of app (sapp) had been examined. neither the expression of sappα nor sapp showed any detectable changes among the two groups. collagen α (vi) chain precursor, a protein resolved as a single spot on d gel was identified by matrix associated laser desorption ionization mass spectroscopy. quantitative analysis of this spot using the d image master software revealed a significant decrease in fetal ds (p Ͻ . ) compared to controls. linear regression analysis did not show any correlation between protein levels and age. the current data suggest that overexpression per se can not fully explain the ds phenotype. apoptosis is the mechanism by which cells are programmed to die under a wide range of physiological and developmental stimuli. accumulating evidence indicates that enhanced apoptosis (programmed cell death) in down syndrome (ds) may play a role in mental retardation and precocious neurodegeneration of the alzheimer-type. in this regard, alteration of several apoptosis related proteins have been reported in adult ds brain. fetal ds neurons exhibited increased reactive oxygen species leading to early apoptosis, however, expression of apoptosis related proteins in fetal ds, has never been considered. to address this issue, we investigated the expression of proteins involved in apoptosis including fas (cd , apo- ), caspase- , bcl- and annexins in the cerebral cortex of control and ds fetal brain by western blot and two dimensional electrophoresis. here, we report that no detectable changes were obtained in fetal ds brain in the expression of fas, caspase- , bcl- and annexins (i, ii, v, and vi) compared to controls. in parallel experiment, we also examined the expression of neuron specific enolase (nse), a neuronal marker found to be decreased in adult ds brain, to see if there is any neuronal loss and no difference was observed between the two groups. protein expression did not correlate with age. the unchanged levels of fas, bcl- and annexins together with unaltered caspase- expression, a predominant caspase that executes apoptosis in the developing nervous system, suggest that enhanced apoptosis may not be apparent in fetal ds brain as demonstrated for adult ds brain. introduction. among the various metabolites indicating neuronal damage, amino acids are regarded particularly important. detection of amino acids by microdialysis is currently introduced as a neuromonitoring tool in patient care. here, we present changes in the extracellular concentrations of various amino acids in stroke patients and in experimental stroke in cats. method. cat focal ischemia was produced by occlusion of the middle cerebral artery (mca) for h followed by h reperfusion. glutamate, aspartate, gaba, taurine, glycine, serine, glutamine, methionine, threonine, tyrosine, asparagine, valine, phenylanaine, isoleucine and leucine were sampled by microdialysis in the ischemic core and subsequently analyzed by hplc. human microdialysis was performed in patients with large mca infarction. the microdialysis probes were inserted into primarily non-infarcted tissue in the border zone of the ischemic territory. results. transmitter amino acids rose immediately after occlusion in the cat model. correspondingly, these substances increased sharply in the human brain, when the tissue around the probes became infarcted, as shown by positron emission tomography (pet) and ct scan. in contrast, structural amino acids did not show marked increases or even decreased during severe ischemia in both, experimental ischemia and stroke patients. these substances did increase, however, when the brain tissue was only slightly ischemic, i.e. after reperfusion of the cat brain, when brain swelling occurred, or in human brain, when tissue did not show any infarction in the ct scan but hypoperfusion in the pet image. conclusion. extracellular amino acids detected by microdialysis can serve as markers for secondary ischemia. severe ischemia is reflected by rapid increases of transmitter amino acids, due to various mechanisms including synaptic release and reversal of reuptake systems. oligemia seems to be reflected by slow increases of structural amino acids, possibly due to a reduction in cerebral protein synthesis. apoptosis has been implicated in the selective neuronal loss of down syndrome (ds). apoptosis activates a family of cysteine proteases with specificity for aspartic acid residues referred to as, caspases that play a key role in dismantling a cell committed to die. caspases activity is regulated by a variety of proteins that possess a domain resembling the prodomains of caspases. little is known, however, about the changes of caspases and their regulatory proteins in ds. here, we investigated levels of nine such different proteins by western blot technique in frontal cortex and cerebellum of control and ds subjects. the protein levels of dff (dna fragmentation factor ), and flip (fadd like interleukin- -converting enzyme inhibitory proteins) were significantly decreased whereas that of rick (rip-like interacting clarp kinase) increased in both regions of ds. in contrast, cytochrome c, apaf- (apoptosis protease activating factor- ), procaspase- and arc (apoptosis repressor with caspase recruitment domain) were unchanged. procaspase- and - were significantly decreased in frontal cortex but no significant change was observed in cerebellum. regression analysis revealed no correlation between postmortem interval and levels of the investigated proteins. however, inconsistent correlation was found between age and levels of proteins as well as amongst the density of individual proteins. these findings demonstrate that dysregulation of apoptotic proteins does exist in ds brain and may underlie the neuropathology of ds. the study further suggests that apoptosis in ds may occur via the death receptor pathway independent of cytochrome c. hence, therapeutic strategies that target caspase activation may prove useful in combating neuronal loss in this disorder. in order to examine the differential roles of nitric oxide (no) induced by either endothelial no synthase (enos) or neuronal no synthase (nnos) after transient cerebral ischemia, we investigated the effects of the relatively selective cnos inhibitor, l-n -( -iminoethyl)ornithine (l-nio), the relatively selective nnos inhibitor, -nitroindazole ( -ni) and the no scavenger, -( -carboxyphenyl)- , , , tetramethylimidazole- -oxyl -oxide (ptio) on hippocampal dysfunction caused by cerebral ischemia. we measured no concentration, mean arterial blood pressure (mabp), hippocampal blood flow, direct current potential, ca population spike (ps) and release of amino acids from rat hippocampus after transient forebrain ischemia, which was induced by vessel occlusion for min. l-nio ( mg/kg), -ni ( mg/kg) and ptio ( mg/kg) were administered intraperitoncally min before ischemia. ptio, -ni and l-nio reduced ischemiainduced no production in the hippocampus during the early period of reperfusion. the rank order of inhibitory potency was ptio Ͼ -ni Ͼ l-nio. l-nio, but not -ni, reduced hippocampal blood flow during ischemia and increased mabp before, during and after ischemia, compared with the vehicle group. ptio increased mabp during and after ischemia. ptio and -ni, but not l-nio, reduced amplitude of anoxic depolarization induced by ischemia. -ni recovered in part ps amplitude min after ischemia. -ni, but not l-nio, reduced ischemiainduced release of aspartate and glutamate, but not taurine. the present study provides further evidence for the idea that in the early stages of transient forebrain ischemia, enos-derived no has a neuroprotective effect in the hippocampus, while nnos-derived no has a neurotoxic effect. the estrogen affects brain protein synthesis in ovariectomized female rats k. hayase , m. tanaka , k. tujioka , e. hirano , and h. yokogoshi department of home economics, aichi university of education, kariya, aichi, and laboratory of nutritional biochemistry, school of food and nutritional sciences, the university of shizuoka, japan the purpose of this study was to determine whether -estradiol affected the rate of brain protein synthesis in ovariectomized female rats. experiments were conducted on three groups of wk old female rats: group . ovariectomized to reduce the level of plasma estradiol; group . ovariectomized and treated with estradiol; and group . sham-operated control. the fractional rates of protein synthesis in brain of ovariectomized rats treated with estradiol were significantly greater than in ovariectomized rats without estradiol treatment. in brain, the rna activity [g protein synthesized/(g rnaᮀd)] significantly correlated with the fractional rate of protein synthesis. the rna concentration (mg rna/g protein) was not related to the fractional rate of protein synthesis in any organ. the results suggest that estrogen treatment of ovariectomized female rats is likely to increase the rate of protein synthesis in the brain, and that rna activity is at least partly related to the fractional rate of brain protein synthesis. we have synthesized a series of new peptides that have demonstrated potent antidepressant activity in animal models for depression and in phase iia and iib clinical trials. mif- (prolyl-leucyl-glycinamide) an endogenous brain peptide has been reported to have some clinical activity in patients with unipolar depression with few apparent side effects. we have undertaken a study to determine the effect of molecular structural changes on the antidepressant activity of this peptide. we evaluated our new derivatives in a stress-induced animal model for depression, i.e. porsolt test, we have found that -f-phe- -oh-pro-arg-gly-trp-nh (inn ) is superior in all the statistical parameters used. in comparative testing inn was more active than prozac (fluoxetine) and zoloft (sertraline) in our antidepressant model. a u.s. patent has been granted on these compounds. the clinical results of inn show that it is effective in over % of depressed subjects when blood levels exceeded therapeutic threshold with no significant side effects. inn has a rapid onset of action, - days (vs. - weeks for currently marketed products) with sustained effects for months following to doses over - weeks. h. iwama , , a. umino , a. hashimoto , k. takahashi , n. yamamoto , and t. nishikawa , section of psychiatry and behavioral science, tokyo medical and dental university graduate school, and department of mental disorder research, national institute of neuroscience, ncnp, tokyo, japan using an in vivo dialysis technique, we have studied the extracellular contents of endogenous free d-serine in comparison with that of l-serine, glycine and l-glutamate in the brain of the freely moving rat. a high amount of d-serine was detected in the dialysate obtained from the medial prefrontal cortex and striatum, whereas the cerebellar dialysate contained only a trace concentration of the d-amino acid. intra-medial prefrontal cortex perfusion of a sodium channel activator, veratrine, augmented the extracellular release of glycine and l-glutamate but a slight decrease in that of d-serine in a tetrodotoxin-sensitive manner in the prefrontal area. moreover, selective destruction of neuronal cell bodies in the medial frontal region by means of local infusion of an excitotoxin quinolinate resulted in a marked reduction of extracellular and tissue levels of d-serine in the infused prefrontal region. these findings suggest that endogenous d-serine might be liberated into the extracellular space from non-neuronal cells or certain exceptional neuronal cells probably by a carrier-mediated process in the mammalian prefrontal cortex. also, the endogenous d-amino acid has been indicated to be accumulated or synthesized, at least in part, in the neuronal cells. nucleoside diphosphate kinase (ndpk) catalyzes a transfer of the terminal phosphate from nucleoside triphosphates ((d)ntps) to nucleoside diphosphates ((d)ndps) and has been suggested to be involved in the regulation of wide variety of cellular functions. in addition, ndpk isoforms (a and b) are encoded by nm genes (h and h ) , which are related with the metastatic potential of some tumors. although ndpk/ nm has been also implicated to modulate neuronal cell proliferation, differentiation and neurite outgrowth, a neurobiological role of ndpk/nm in neurodegenerative diseases has not been reported yet. here we evaluated the protein levels of ndpk-a/nm -h in brains from patients with down syndrome (ds) and alzheimer's disease (ad) using twodimensional ( -d) gel electrophoresis with subsequent matrixassisted laser desorption ionization mass spectroscopy (maldi-ms) identification and specific software for quantification of proteins. ndpk-a/nm -h was significantly decreased in brain regions (frontal, occipital, parietal cortices) of both ds and ad compared to controls. we conclude that the down-regulated ndpk-a/nm -h upon neurodegeneration could play a pivotal role in a wide range of neurobiological functions such as neurite outgrowth and consequently these could result in functional disturbance of the nervous system in ds and ad. brain α-endosulfine is manifold decreased in brains from patients with alzheimer's disease: a tentative marker? and drug target? α-endosulfine has the sulfonylurea-like ability to block atp-sensitive potassium (k atp ) channels and is expressed in a wide range of tissues. although the blockade of k atp channels has been reported to be involved in the release of neurotransmitters, the neurobiological role of α-endosulfine has not been studied yet. we examined the levels of αendosulfine protein in frontal cortex and cerebellum from patients with alzheimer's disease (ad). α-endosulfine was extremely decreased in both regions of ad compared to controls. this could result in the continuous opening of k atp channels with subsequent decrease of neurotransmitters release and change of potassium fluxes. this study is of great significance for providing a neurobiological function of α-endosulfine in brain and furthermore, α-endosulfine could serve as a useful marker for the diagnosis of ad and a target for drug treatment. children's hospital heidelberg, and department of pharmacology and toxicology, university of marburg, germany d- -hydroxyglutaric aciduria is an inherited neurometabolic disorder of unknown etiology characterized by progressive neurodegeneration of vulnerable brain regions during infancy and early childhood, resulting in psychomotor retardation, hypotonia, seizures, macrocephaly, enlarged lateral ventricles, delayed cerebral maturation as frequent neurological presentation in affected children. the disease is biochemically characterized by the accumulation of d- hydroxyglutarate (d- ), which is structurally similar to lglutamate (ϭ -amino-glutarate). we therefore investigated in primary neuronal cultures from chick and rat, whether d- induces excitotoxic neuronal damage. here we report that d- decreased cell viability in a concentration-and time-dependent fashion by disturbance of intracellular calcium homeostasis as determined by fura- measurement. furthermore, fluorescence microscopy using dihydrorhodamine- revealed an increased generation of reactive oxygen species (ros) elicited by expo-sure to d- . n-methyl-d-aspartate (nmda) receptor blockade specifically prevented excitotoxic neuronal damage as well as increased calcium influx and ros production, suggesting that d- is an agonist at nmda receptors. patch-clamp investigation confirmed that d- activated recombinant nmda receptors in hek cells. furthermore, activity measurement of single respiratory chain complexes revealed a specific inhibition of complex v activity by d- . we conclude that excitoxic mechanisms contribute to the neuropathology of d- hydroxyglutaric aciduria, highlighting new neuroprotective strategies for this neurometabolic diseases. these studies were designed to determine the effects of aging and an aging intervention on nmda subunit expression. in situ hybridization and receptor autoradiography were performed on naïve or behaviorally-tested c bl/ mice of different ages ( , - , and - months old) and diet groups (ad lib-fed and diet restricted). there were age-related decreases in both e and z mrna density in naïve, ad lib-fed mice. correlations were found between changes in e subunit mrna and agonist binding and z mrna expression and antagonist binding. diet restriction significantly improved learning ability, slightly spared glutamate binding to nmda sites and improved z mrna expression in older mice. significant correlations were found between agonist binding and both learning ability and e and e mrna density. learning ability in the old mice also correlated with the ratios of mrna expression for e and e and/or z subunits. these results suggest that changes in nmda receptor binding and the relationship between subunit expression levels are important for maintaining memory functions in older animals. extracts of st john's wort (hypericum perforatum l.) are widely prescribed for the treatment of mild to moderate depression and the putative antidepressant constituent is probably hyperforin. in this study the effect of hyperforin was investigated on the release of neurotransmitter amino acids. coronal cortical slices ( mm) were cut and perfused with gassed ( % o , % co ) acsf at °c. two-minute samples of perfusate were collected and aspartate and glutamate were assayed by hplc. potassium-and veratridine-stimulated release was elicited by administering pulses of kϩ ( mm) or veratridine ( mm) minutes apart. in control experiments the second kϩ pulse elicited glutamate release which was % of the first pulse. hyperforin ( mm) perfused for minutes prior to, and during, the second kϩ pulse significantly increased glutamate release to % (p Ͻ . , n ϭ - ). release elicited by the second veratridine pulse was % of the first pulse for both glutamate and aspartate. hyperforin ( mm) increased this release to the second pulse to % and % respectively (p Ͻ . , n ϭ - ). when perfused on its own for minutes, hyperforin ( mm) increased the basal release of glutamate (p Ͻ . , n ϭ - ). in conclusion, the increase in the release of neurotransmitter amino acids observed following hyperforin is possibly mediated through a facilitatory action on voltage-operated ca ϩ or naϩ channels. glaxosmithkline group, glaxo wellcome s.p.a., medicines research centre, verona, italy n-methyl-d-aspartate (nmda) receptors are ligand gated ion channels widely distributed in mammalian brain, which play a crucial role in important physiological mechanisms, such as excitatory transmission, neuronal migration and memory formation. a peculiar feature of nmda receptors is the absolute requirement of l-glutamic acid and glycine for the opening of the channel. noteworthy, these two aminoacids reciprocally modulate binding at their respective recognition sites. aim of this work was to study nmda receptor glycine/glutamate interactions in rat and human brain. binding of the nmda antagonist [ h]cgp to rat cerebral cortical membranes was inhibited by glycine. the overall effect of glycine consisted in a decrease of [ h]cgp affinity, with a parallel increase of the receptor affinity for glutamate. the glycine antagonist gv a competitively reversed glycine inhibition, proving that the modulation was via the glycine binding site. [ h]cgp binding to rat brain sections revealed the existence of regionally distinct nmda receptor subtypes with difference glycine/glutamate interactions. in most regions of the human brain nmda receptors presented the same allosteric modulation of [ h]cgp binding, as revealed by large section autoradiography technique. nevertheless, detection of any regional variation was not possible, probably due to the high intersubject variability. the effect of long-term high k ϩ -treatment on neuronal survival, cellular maturation, nmda receptor (nr) splice variant expression, and receptor function was investigated in primary cultures of rat cortical neurones. long-term incubation (up to days) with mm k ϩ significantly increased neuronal survival and induced multiple morphological changes associated with promoted cellular maturation. cultures grown in medium containing mm k ϩ also exhibited multiple changes in nr splice variant expression according to rt-pcr studies performed with primer pairs flanking the alternatively spliced regions, in order to estimate the ratios of the corresponding Ј and Ј splice variants. nr - and nr - (each containing exon ) were decreased, whereas nr - and nr - (each lacking exon ) were increased, accordingly. the predominant expression of nr -b was further increased. after administration of ttx, each of the k ϩ -induced changes on mrna expression was virtually abolished. in voltage-clamp recordings (holding potential: Ϫ mv), nmda induced inward currents in a concentration-dependent manner with a maximum effect of Ϫ pa under control conditions. neurones treated with mm k ϩ showed a significantly diminished response to nmda (max. response: Ϫ pa). in conclusion, the present data indicate that a sustained increase in neuronal activity induces adaptive changes in nr splice variant expression and a decrease in receptor function. thus, alternative splicing associated with a diminished receptorcytoskeletal linkage may be important compensatory mechanism in preventing cellular damage due to long-term activation of excitatory nr. it seems conceivable that this mechanism contributes to the promoting effects of mm k ϩ on neuronal survival and maturation. (supported by bmbf grant gg / ) there is accumulating reports that kainate-induced seizures elicit expression of various heat-shock proteins (hsps) in the brain, such as hsp , hsp , and hsp . however, no investigation has been carried out on changes in level of apg- , a member of hsp family, after excitatory amino acid-induced seizures. by means of an immunoblot assay, we determined the levels of hsp and apg- in discrete brain structures of mice after a single intraperitoneal injection of kainate or nmda. apg- level was significantly decreased in the frontal cortex, hippocampus, and striatum days after kainate administration, while hsp level was increased in these regions following the administration. decreased level of apg- returned to the control levels in the three regions days after kainate administration. no significant changes were observed in levels of both hsp and apg- in hypothalamus, midbrain, medulla-pons, and cerebellum of kainate-treated mice. by contrast, nmda administration did not significantly affect both levels in any of the regions examined. these results suggest that, unlike the case of hsp , apg- expression could be temporarily down regulated by signals peculiar to kainate, but not by those peculiar to nmda, in murine telencephalon. high concentrations of glucagon-like peptide- ( - ) amide (glp- ) and its specific receptor (glp- r) have been found in the rat hypothalamus. in this study the actions of glp- and its related peptides, exendin- (glp- r agonist), exendin ( - ) (glp- r antagonist) and glp- ( - )amide (major glp- metabolite) on levels of amino acids (glu, asp, gln, gly, tyr, trp, gaba) in the hypothalamus were investigated. i-glp- binding in rat hypothalamic membranes was competed by the peptides in the following order of potency; glp- Ͼ exending- Ͼ exendin ( - ) Ͼ glp- ( - )amide. intracerebroventricular (icv) glp- ( nmoles) produced a statistically significant reduction in levels of all measured amino acids compared with saline injected controls, whereas nmoles of exendin ( - ) was ineffective. exendin- produced a statistically significant reduction in the levels of trp, glu, and tyr. glp- ( - )amide showed a statistically significant increase in the level all the amino acids tested in this study. prior administration of exendin ( - ) or glp- ( - )amide blocked the effects of glp- on the levels of the amino acids. these data are consistent with exendin- being a glp- r agonist and exendin ( - ) being a specific glp- r antagonist. glp- ( - )amide, a primary metabolite of glp- , appears to act as an endogenous antagonist at the glp- r. department of biophysics, instituto de fisiología celular, universidad nacional autónoma de méxico, méxico city, méxico cholecystokinin (cck), a family of neuropeptides, seems to be involved in anxiety. evidence from several laboratories indicates that the ansiogenic effects of cck are mediated by cck b receptors. however it has been reported that cck a receptors have been found in brain and cck a receptor antagonists have ansiolytic properties. the aim of this work was to study whether or not cck a receptors are also involved in the modulation of anxiety. male rats were cannulated in the lateral ventricle and cck ( fmol) and/or cck antagonists ( fmol) were injected days after surgery. anxiety was evaluated in the elevated plus-maze test and locomotion in an open-field test. ansiogenic effects were observed when cck b receptor agonists (cck ns; cck ) or a mixed cck b and cck a receptor agonist (cck s) were injected. in contrast, cck , a cck a receptor agonist or cck - and cck - were uneffective. furthermore, the ansiogenic effects of cck s were prevented by the previous ( min) administration of l , (cck b receptor antagonist) but not by devazepide (cck a receptor antagonist). no effects on locomotion were observed in any condition. these results indicate that cck a receptors are not involved in anxiety, as measured by the elevated plus-maze test. congenital conditions (i.e. neural tube defects: ntg) have a multifactorial aetiology. deficiencies in the folate and transsulfuration pathways have, in recent years, been positively linked to ntd and other dysmorhogenic syndromes. efficient one-carbon metabolism is crucial for the synthesis of dna precursors, the remethylation of homocysteine and biomethylation of dna. more than % of the one-carbon units that flow through the metabolic system in mammals and birds are derived from l-serine and glycine, the natural substrates for shmt. the mitochondrial glycine cleavage enzyme system (gces) can potentially compete with shmt for tetrahydrofolate (thf) in the generation of the methylenetetrahydrofolate pool. valproate (depakene, epilim), an anti-epileptic agent, appears to be strongly associated with hyperhomocysteinemia, several other induced metabolic conditions, the inhibition of the gces and an increased incidence of ntd in epileptic women of child-bearing age. the exact mechanisms of valproate-induced ntd are not yet clear. we investigated the association of the teratogenic properties of valproate with the inhibition of shmt and/or the gces in developing embryos. chicken embryos were treated with sodium valproate (vpa) and pregnant female mice (c bl) received intraperitoneal injections of vpa, during the critical period of embryonic neural tube development. control embryos were treated with sterilised saline solution. harvested embryos were subsequently investigated for congenital abnormalities and hepatic shmt and gces activities quantified with radiometric assays. the effect of vpa on hepatic dna synthesis was monitored ( h-thymidine incorporation into embryonic dna) and the dna-methylation status determined (dna n -methylcytosine levels). dose-responsive incidences of ntd were observed in vpa treated embryos. very few defects occurred in control embryos. shmt and gces appeared to be inhibited in liver extracts of vpa-treated embryos. hepatic dna synthesis was significantly compromised and -mc levels were altered in vpa-treated embryos. the inhibition of either shmt and/or gces activities appeared to be associated with valproateinduced ntd in the chicken and mouse embryo models. the primary mechanism of this effect can probably be ascribed to a restriction in the flow of one-carbon units through the metabolic system, decreased synthesis of dna precursors and alterations in the methylation status of dna. department of neuroscience, university of cagliari, italy ethanol is long known to cause dose-related biphasic effects and we recently found that ethanol bidirectionally affects also working memory. the euphoriant and excitatory effects produced at low doses are associated with the rewarding action of ethanol and are thought to be mediated by the activation of the mesolimbic dopamine (da) system. however, ethanol monophasically stimulates mesolimbic da release in the nucleus accumbens, even at doses that cause hypnosis and coma. in contrast, ethanol biphasically modulates mesocortical da release in the prefrontal cortex (pfc). the changes in da release induced by ethanol are time locked with corresponding changes in extracellular glutamate levels. these biphasic effects of ethanol on pfc da and glutamate are matched by biphasic changes in the performance in a spatial delayed alternation task -a working memory test that is sensitive to proper function of the pfc -suggesting a link between da and glutamate transmission in the cognitive effects of ethanol. focal application in the pfc of the competitive ampa/kainate receptor antagonist cnqx suppresses both da release and the improvement of working memory induced by low doses of ethanol. these results suggest that ethanol may increase da transmission in the pfc and enhance working memory functions by increasing the release of glutamate, thereby stimulating non-nmda glutamate receptors. the enhancing effect on working memory by low, excitatory doses of ethanol may be perceived as rewarding and could constitute an important neurobiological mechanism for excessive ethanol drinking. physiology department, faculty of medicine, al-quds university, jerusalem, palestine glutamate and asparate are considered as the main excitatory neurotransmitters in brain and spinal cord, in addition to their role in energy metabolism, synthesis of proteins and detoxification of ammonia. glutamate and aspartate are centrally involved in basic mechanisms generating epileptic seizures and in epileptogenesis. stimulated release of glutamate and aspartate was detected in vivo and in vitro following neuronal depolarization. photic stimuli has increased glutamate release from visual cortex, and afferent brachial stimulation has increased the endogeneous release of glutamate from contra-lateral sensorimotor cortex compared to ipsi-lateral side. similar results were achieved after local application of tityustoxin or veratridine to the sensorimotor cortex. implantation of cobalt powder over the right sensorimotor cortex of rats produced an epileptogenic lesions characterized by contra-lateral fore and hind limb jerks and an increase in the frequency of eeg spikes. the jerks started after days with maximum myoclonic activity ( jerks/min). the concentration of glutamate in the epileptogenic focus was decreased significantly by % (p Ͻ . ) compared to the non-epileptogenic area on the left sensorimotor cortex, which was dissected but not treated with cobalt. part of the decrease in glutamate could be related to the enhancement of in-vivo release from the epileptogenic lesion to the extra-cellular fluid. kindling is the best model for studying the development of the epileptic focus (epileptogenesis), it could be achieved by repeated intra-cerebral micro-injection of glutamate ( . µ mol), aspartate ( . µ mol) or nmda ( - n mol), or repeated electrical stimulations of specific brain regions. in addition, glutamate antagonists particularly those specifically acting on the nmda receptor type e.g. -amino- -phosphonovaleric acid (ap ) and -amino- -phosphonopheptanoic acid (ap ) have been found to inhibit seizures in epileptic animals and inhibit the development of electrically kindled epilepsy. pre-synaptic glutamate receptor agonists like ( s, s)-acpd the agonist of group ii, and l-ap the agonist of group iii receptors has reduced ca ϩϩ uptake and glutamate release, thus it has inhibited epileptogenesis by preventing the increase in both seizure score and after-discharge duration. injection into fully kindled animals has produced an anti-epileptic effect by reducing the mean seizure score and by increasing the mean generalized seizure thresholds. this results suggest the mechanism by which pre-synaptically active glutamate receptor agonists block the development of the chronically epileptic state induced by electrical kinding, and indicate that their anticonvulsive activity is due to inhibition of pre-synaptic glutamate and/or asparate release following blockade of pre-synaptic ca ϩϩ entry. testing the changes in glutamate release from hyperactive brain tissues, and the effect of different glutamate agonists and antagonists, supports the role of glutamate in initiating the process of epileptogenesis, and contributes in developing new anti-epileptic agents. (this project was supported by a grant from alexo) the functional roles of cl(Ϫ) and divalent cations in the na(ϩ)/cl(Ϫ)/gaba cotransport were examined in xenopus oocytes expressing the human gat- (hgat- ) gaba transporter cdna. our results showed that cl(Ϫ) was not absolutely required for na(ϩ)/gaba transport via the hgat- (loo et al., j biol. chem. : - , ) . the cl(Ϫ) interacted with the transporter to modulate the binding of external na(ϩ). although hgat- transported cl(Ϫ) across the membrane with a stoichiometry of na(ϩ) : cl(Ϫ) : gaba, the transported cl(Ϫ) did not contribute to the net charge translocated across the membrane, suggesting a cl(Ϫ)/cl(Ϫ) exchange mechanism during the gaba transport cycle. the gaba transport via the hgat- is also modulated by divalent cations. the uptake of [ h]-gaba was inhibited significantly when both ca( ϩ) and mg( ϩ) were removed from the uptake buffer. several divalent cations tested were individually able to sustain the gaba uptake. in contrast to uptake, the gaba efflux was enhanced significantly upon removal of both ca( ϩ) and mg( ϩ) from the efflux buffer. the gaba transporter inhibitor skf a blocked the enhanced efflux, suggesting that the hgat- operated faster in the reverse mode in the absence of external divalent cations. these results suggest a regulatory role for the divalent cations in gaba transport. merck sharp & dohme, neuroscience research centre, terlings park, harlow, essex, u.k. the role of alpha containing gaba a receptors in hippocampal synaptic function has been investigated using pharmacological and electrophysiological techniques, as well as following disruption of the alpha subunit gene in knockout mice (ko). in the ca region of the hippocampus the induction of long-term potentiation (ltp) is powerfully regulated by gaba mediated synaptic currents (ipscs). agents that inhibit gaba-mediated transmission potentiate ltp induction, whereas allosteric agonists such as benzodiazepine-site agonists which slow the decay kinetics of ipscs suppress ltp induction. in alpha ko mice paired pulse facilitation of the amplitude of excitatory synaptic potentials is selectively enhanced in the ca region but not dentate gyrus. likewise, the frequency and rise time of spontaneous ipscs were similar in wt and ko slices. however their amplitude was significantly smaller in ko mice. furthermore, a significantly greater proportion of ipscs were best fitted to a mono exponential function in ko mice compared to wt animals. thus alpha containing gaba a receptors contribute to functional postsynaptic receptors on ca pyramidal cells in the hippocampus and modulate a postsynaptic component of synaptic facilitation. pharmacological research institute, volgograd medical academy, volgograd, russia the purpose of the study is to investigate effect of phenil (karphedon, mephebut, gammoxin) and circle (pyracetam) gaba derivatives on reproductive function of stressed male rats. the adult male rats were stressed by immobilization exposure ( hours) twice in week during weeks. four from five groups of stressed males were given substances (daily) at doses: karphedon - mg/kg, mephebut - mg/kg, gammoxin - mg/kg, pyracetam - mg/kg. the treated males were mated with intact females during days. after the mate the treated males and more in days all the mated females were sacrificed and investigated. analysis of our data indicates that the time of spermatozoa motion and epididymal sperm counts were decreased . % (p Յ . ) and . % (p Յ . ) respectively when compared with their intact controls. gaba derivatives have a softening effect on functional parameters of spermatozoa stressed males. karphedon and pyracetam increased the time of motion spermatozoa . % (p Յ . ), karphedon and mephebut drew near sperm counts to intact control level. the result of mate show that pregnancy rate was increased (p Յ . ) by stress exposure and pregnancy rate of females mated with gaba stressed males was some more (p Ն . ) than that of intact controls. the general embryonic morality was increased twice by stress and so the number of embryos was reduced . %. the gaba derivatives exposure to stressed male rats reduced the embryonic mortality of their posterity and increased the number of embryos to intact control level. our findings demonstrate that gaba derivatives administration has a protective effect on reproductive function of stressed male. transmission on the brain. the realization that glutamatergic pathways are involved in such diverse processes in epilepsy, ischemic brain damage and parkinsons' disease, is of a great practical interest. there are at least three functional classes of ionotropic glutamate receptors: n-methyl-d-aspartate (nmda), α-amino- -hydroxy- methyl- -isoxazolepropionic acid (ampa) and kainate (ka). other central neurotransmitter systems are under nmda influence. some data point on neuroprotective action of nmda antagonist on nigrostriatal pathway. in the present study female wistar rats were exposed during pregnancy with daily injected mk- (dizocilpine) . mg/kg sc. control rats received tap water only. behaviour of month old male offsprings was investigated by several psychopharmacological methods. oral activity, yawning, locomotor activity, stereotypy and catalepsy were recorded following respective central dopamine receptors agonists and antagonists administration (skf , quinpirole, apomorphine, haloperidol). our results indicate that mk- applied during pregnancy modulate reactivity of the central dopamine receptors in adult offspring rats. [ the development of mammalian ingestive behavior is characterized by a transition from suckling to chewing, two distinct motor behaviors. we hypothesize that this transition is accompanied by changes in brainstem circuitry underlying these movements. since glutamatergic neurotransmission is critical for the proper functioning of brainstem circuitry responsible for mastication, we investigated the development of glutamate receptors in trigeminal motoneurons (mo ) and mesencephalic trigeminal neurons (me ); neurons comprising the circuitry responsible for jaw movements. we conducted a series of receptor immunohistochemistry experiments that characterized the expression of iontotropic and metabotropic glutamate receptors (mglurs) during early postnatal development. the functional roles of nmda, ampa and mglurs in neonatal mo were investigated using in vitro electrophysiological experiments. results demonstrated that the spatial and temporal expression of ampa, nmda and group i and ii mglurs are developmentally regulated within and between mo and me during early development. electrophysiological data demonstrate that mglurs function pre-and postsynaptically to modulate synaptic transmission between trigeminal premotoneurons and mo . furthermore, nmda induced bursting is developmentally regulated and coincident with the transition from suckling to chewing behaviors. our studies suggest that the transition from suckling to chewing is accompanied by changes in the composition and function of glutamate receptors. fetal life in down syndrome starts with normal neuronal density but impaired dendritic spines and synaptosomal structure r. weitzdoerfer , m. dierssen , m. fountoulakis , and g. lubec department of pediatrics, university of vienna, austria information on fetal brain in down syndrome (ds) is limited and there are only few histological, mainly anecdotal reports and no systematic study on the wiring of the brain in early prenatal life exist. histological methods are also hampered by inherent problems of morphometry of neuronal structures. it was therefore the aim of the study to evaluate neuronal loss, synaptic structures and dendritic spines in the fetus with down syndrome as compared to controls by biochemical measurements. dimensional electrophoresis with subsequent mass spectroscopical identification of spots and their quantification with specific software was selected. this technique identifies proteins unambiguously and concomitantly on the same gel. fetal cortex samples were taken at autopsy with low postmortem time, homogenized and neuron specific enolase (nse) determined as a marker for neuronal density, the synaptosomal associated proteins alpha snap [soluble n-ethylmaleimidesensitive fusion (nsf) attachment protein], beta snap, snap and the channel associated protein of synapse (chapsyn ) as markers for synaptosomal structures and drebrin (drb) as marker for dendritic spines. nse, chapsyn and beta snap were comparable in the control fetus panel and in down syndrome fetuses. drebrin was significantly and remarkably reduced and not even detectable in several down syndrome brain samples. quantification of snap revealed significantly reduced values in ds cortex and alpha snap was only present in half of the ds individuals. we conclude that at the time point of about weeks of gestation (early second trimester) no neuronal loss can be detected but drebrin, a marker for dendritic spines and synaptosomal associated proteins alpha snap and snap were significantly reduced indicating impaired synaptogenesis. early dendritic deterioration maybe leading to the degeneration of the dendritic tree and arborization, which is a hallmark of down syndrome from infancy. pathfinding of growing axons to reach their target during brain development is a subtle process needed to build up contacts between neurons. abnormalities in brain development in down syndrome (ds) are described in a couple of morphological reports but the molecular mechanisms underlying abnormal wiring in fetal ds brain are not yet elucidated. we therefore performed a study using the proteomic approach to show differences in protein levels involved in the guidance of axons between control and ds brain in early prenatal life. proteins obtained from autopsy of human fetal abortus were applied on -dimensional gel, identified and quantified. we quantified members of the semaphorin/collapsin family, the dihydropyrimidinase related proteins - and the collapsin response mediator protein- (crmp- ) in ds and control cortex samples. drp- and crmp- levels were comparable in the control and ds samples. evaluation of drp- , drp- and drp- revealed significantly decreased levels of of the spots assigned to drp- and increased levels of one spot assigned to drp- and increased drp- in ds brain. we conclude that as early as from the th week of gestation pathfinding cues of the outgrowing axons are impaired in ds. these findings may help to elucidate mechanisms leading to abnormalities in neural migration of ds brain. inflammatory processes play an important role in the degeneration of basal forebrain cholinergic cells alzheimer's disease. the proinflammagen lipopolysaccharide (lps) was infused chronically into the basal forebrain of young rats. we then determined whether the administration of two novel nonsteroidal anti-inflammatory drugs or a pancaspase synthesis inhibitor, zvad, could provide neuroprotection from the cytotoxic effects of the neuroinflammation. we also determined whether the administration of the non-competitive n-methyl-d-aspartate (nmda) receptor antagonist, memantine, could provide neuroprotection from the cytotoxic effects of the neuroinflammation. chronic lps infusions decreased choline acetyltransferase activity and increased the number of activated microglia within the basal forebrain. caspases , and activity was increased in ventral caudate/putamen. non-steroidal antiinflammatory drug therapy attenuated the toxicity of the inflammation upon cholinergic cells and reduced caspases , , and activity in the caudate/putamen. zvad significantly decreased the levels of caspases , and but did not provide neuroprotection for cholinergic neurons. memantine significantly attenuated the cytotoxic effects of chronic inflammation upon cholinergic cells. these results suggest that prostaglandins contribute to the degeneration of forebrain cholinergic neurons in alzheimer's disease and that the cytotoxic effects of prostaglandins occur upstream to nmda receptor activation. intracranial administration of n-methyl-d-aspartate (nmda) receptor antagonists block learning of classical and avoidance conditioning in goldfish. studies with goldfish have shown that nmda receptors are mostly dense in the telencephalon and telencephalon ablation impairs avoidance learning. the present study investigated amnestic effects of microinjection of nmda receptor antagonist ap to the goldfish telencephalon in avoidance conditioning. in experiment , fish received no injection or microinjections of saline or various doses of ap to their telencephalon minutes before three semiweekly training sessions. fish were tested without injec-tions in session . a one-way anova with multiple comparisons on the test scores showed that ap produced anterograde amnesia in a dose-dependent manner. in experiment , fish received several training sessions and a microinjection of various doses of ap minutes before testing. the test scores showed that ap did not decrease avoidance responses, suggesting that microinjection of ap did not impair performance processes. in experiment , fish received microinjections of ap or saline to their telencephalon immediately following three semiweekly training sessions and were tested without injections in session . a one-way anova on the test scores showed that ap did not produce retrograde amnesia. (supported by gvsu grant-in-aid.) tryptophan modulates striatal serotonergic activity relative to fatigue t. yamamoto and e. a. newsholme health science laboratory, tezukayama university, nara, japan department of biochemistry, university of oxford, u.k. we have been reported that mechanism of fatigue in the brain relates to enhanced extracellular tryptophan and serotonergic function. brain concentration of tryptophan is not only dependent on the change of tryptophan which originates from the centarl nervous system, but also enhance tryptophan entering the brain from the blood-brain barrier and peripheral circulating tryptophan which is a trigger. supplementation of ltryptophan ( um) into the incubation medium with the synaptosomal striatum causes tryptophan to the extrasynaptosomal release by high kϩ stimulation. injecting l-tryptophan ( mm/ min) into the left striatum by microdialysis method can induce early fatigue for running time of rats. on the other hand, tryptophan deficiency rats (body weight average g) were made by tryptophan free feeding for weeks, and the rat's running time increased (Ͼ min difference). these results suggests that tryptophan is a potent active substance for fatigue in the brain. the active zone may be presynaptic terminal and the tryptophan itself may be releasing neuromodulators. (we appreciate that tryptophan free diet was provided by ajinomoto co., inc., japan.) our recent studies on the distribution of free d-serine, together with the d-serine action on the glycine site of the nmda type glutamate receptor, suggest that the d-serine can be an endogenous modulator of the nmda receptor. to explore the possible removal systems for brain d-serine signaling, we have evaluated the uptake of [ h]d-serine into the synaptosomal p fraction from the rat cerebral cortex. the cortical p fraction was able to accumulate [ h]d-serine in a temperatureand ph-dependent and saturable manner. the kinetic analysis indicates that cortical d-serine transport occurs by an apparent single-component system with km value of µm and a vmax value of pmol/mg protein/min. depletion of na ϩ and cl Ϫ ions remarkably decreased d-serine uptake into the cortical p fraction. the pharmacological profile of the inhibition of dserine uptake by various amino acids was different from those of glycine uptake system and other amino acid transporters reported. d-serine uptake activity was preferentially observed in the brain tissues such as cerebral cortex and cerebellum to the peripheral tissues. the present data support the view that the endogenous d-serine is taken up mainly through a carriermediated transport system to regulate the extracellular concentration in the mammalian brain. a. bocheva et al. the mammalian brain contains all the urea cycle intermediates, whereas enzymes participating in the conversion of lornithine (l-orn) into l-citrulline (l-cit) are absent, resulting in an incomplete urea cycle. the discovery of nitric oxide (no) synthase that catalyses the formation of no and l-citrulline as a co-product from l-arginine (l-arg) in the brain has indicated an additional pathway for l-arg metabolism. l-canavanine (l-cav), is a potent antimetabolite and structural analog of larginine, produced by legumes such as the jack bean, canavalia ensiformis. l-canaline (l-can) is a potent inhibitor of ornithine aminotransferase. our previous results indicated that l-cav, l-cit, l-arg, and l-orn exerted an antinociceptive effect, whereas l-canaline induced hyperalgesia in rat. l-canavanine exert stronger antinociceptive effect than l-arginine, l-ornithine and l-citrulline. the aim of the present study was to investigate are d-arg, l-cav and naloxone reversed the analdesic effects of l-ornithine, l-citrulline and l-arginine. the experiments were carried out on male wistar rats. the changes in the mechanical nociceptive threshold of the rats were measured by the radall-selitto paw pressure test using and analgesimeter (ugo basile). the amino acids were applied intracerebroventricularly (i.c.v.) at a dose µg/rat. the present results shown that d-arg, l-cav and naloxone reversed antinociception. the regulation of lysine metabolism in cereal crops r. a. azevedo , p. j. lea , s. a. gaziola , a. p. pellegrino , and s. m. g. molina departamento de genética, escola superior de agricultura luiz de queiroz, universidade de são paulo, brazil department of biological sciences, university of lancaster, u.k. a major nutritional drawback of cereal seeds is a deficiency in some amino acids, in particular lysine. biochemical, molecular and genetic studies have considerably increased our knowledge concerning the regulation of the aspartate pathway, by which lysine is synthesized. among the enzymes involved in lysine metabolism, aspartate kinase (ak) and dihydrodipicolinate synthase (dhdps) control the regulation of lysine biosynthesis, whereas lysine: -oxoglutarate reductase (lor) and saccharopine dehydrogenase (sdh), have been shown to play a key role in the breakdown of lysine. in general, lysine overproduction can be obtained by altering the sensitivity of dhdps to lysine, but accumulation of this amino acid in cereal seeds requires further manipulation of lor and/or sdh. this suggestion is strongly supported by five main points: ( ) cereal mutant or transgenic plants do not exhibit any significant accumulation of lysine in seeds, but only in other tissues. ( ) the enzymes of lysine degradation, lor and sdh, are endosperm specific in cereals only. ( ) the opaque- mutant, which exhibits higher concentration of soluble lysine and protein lysine in the seed, contains several-fold lower lor and -fold lower sdh activity when compared to the wild-type maize. this reduction in activity in the opaque- mutant is due to a reduced protein lor-sdh concentration by reduction of the zlkrsdh gene transcript. furthermore, the opaque- maize gene has been shown to regulate ak and lor activity. ( ) intermediates of lysine catabolism accumulated in the seeds of soybean and canola lysine overproducing plants, suggesting the presence of reduced lor and/or sdh activities. ( ) among cereals and although still below the recommend values by fao, rice exhibits the higher concentration of lysine, but lor and sdh are present in much lower activities. also, in phaseolus vulgaris, lor and sdh activities were shown to be around fold lower then in maize endosperm. the regulation of the lor activity is complex and involves a calcium dependent phosphorylation/dephosphorylation mechanism. it remains to be seen whether this latter mechanism can be controlled, so as to allow the production of more crop plants that contain elevated concentrations of lysine in the seed. the genetic progress for nue can be accelerated with the use of secondary traits that possess high inheritance and correlation with productivity. several traits have been studied such as chlorophyll concentration, plant height, leaf senescence, anthesis-silking interval, kernel number, activities of enzymes of n assimilation and loci of quantitative traits for assisted selection. (we are grateful for financial support from fapesp, brazil and the british council.) (termed hyperaccumulators) that grow on metalliferous soils, are able to translocate cd from the roots and accumulate it in high concentrations in the shoots. cd may be detoxified in plants by combination with a family of sulphur rich peptides termed phytochelatins. cd has the capacity to inhibit a range of enzyme activities in plants, in particular those of the calvin cycle and chlorophyll biosynthesis. evidence that cd causes the production of reactive oxygen species (ros) has also been obtained. we have investigated the antioxidant responses of radish, soybean and sugarcane to cd treatment. seedlings were grown in increasing concentrations of cdcl , ranging from . - mm, for up to h in a hydroponic system. analysis of cd uptake indicated that most of the cd accumulated in the roots, but some was also translocated and accumulated in the leaves. roots and leaves were analysed for catalase (cat), glutathione reductase (gr) and superoxide dismutase (sod) activities. gr activity increased considerably in the roots of all plant species tested after exposure to the metal, indicating a direct correlation with cd accumulation. cat activity also increased in roots but to a much lesser extent when compared to gr and also varied depending upon the plant species. the analysis of native page enzyme activity staining, revealed several sod isoenzymes in leaves of all plant species, however, only in radish was a clear increase in activity observed. the results suggest that in these plants, the activity of antioxidant enzymes responds to cd treatment. the main response may be via the activation of the ascorbate-glutathione cycle for the removal of hydrogen peroxide, or to ensure the availability of glutathione for the synthesis of cd-binding proteins. (we are grateful for financial support from fapesp, brazil and the british council.) all plant cells, tissues and organs provide the biosynthetic machinery and capacity to synthesise aliphatic polyamines. however, in physiological conditions only some organs and tissues synthesise polyamines, such as apical buds and sprouts, root apex, lateral buds of branches and secondary roots, as well as superficial layers of young stems and leaves, like epidermis, subepidermis and parenchyma cells. apical roots can also synthesise polyamines, but these activities in physiological conditions are lower than that of the shoots. this patterns recalls the one of auxins. polyamines are accumulated in high concentrations in storage organs, such as seeds, but not in tubers like helianthus tuberosus, potato or tuberised roots such as the carrot. also some fruit, e.g. oranges, contain high level of free polyamines, putrescine in particular. all other organs obtain polyamines through translocation via phloem tubes and xylem vessels. in plants, in addition to free polyamines, many polyamines are conjugated to hydroxycinnamic acids, the hydroxycinnamic amines, that only rarely represented outside the plant kingdom. this compounds are paticularly abundant in solanaceae family, where they can represent as much as % of the total polyamine pool, but they can be detected in different concentrations in many other families. the role of free and conjugated polyamines and their importance in food is discussed. drought, salinity or other environmental stressors promote the accumulation of free amino acids, amines and other organic n-metabolites with low molecular weight. in this contribution the influence of drought on the accumulation of amino acids, polyamines and trigonelline in growing barley plants and barley grains was examined. in comparison to non-stressed plants we obtained in stressed plants, exposed to drought before flowering, a higher concentration of proline (increase: -fold), n-trimethylglycine ( -fold), histidine ( -fold), tryptophane ( , -fold), putrescine ( , -fold), spermine ( , -fold) and trigonelline ( -fold) in the dry matter of barley sprouts. in addition to this, drought caused an increase of the n-content in the plant biomass ( %) as a result of growth inhibition ( %). six weeks later the content of soluble n-metabolites and protein was analyzed in non-stressed and pre-stressed barley plants again. during this reproductive period of plant development all the test groups were cultivated under the same moisture conditions. the analysis of n-metabolites in the ripening grains showed, surprisingly an after-effect of the drought stress. for example, in grains of pre-stressed barley the concentrations of free proline, histidine, tryptophane and asxϩglx were threefold to fivefold higher than in grains of non-stressed barley. depending on the resistance of barley cultivars to drought the biochemical response was different: in plants with low resistance the increase of amino acids and amines was higher than in resistant cultivars. however, resistant cultivars have already high genuine concentrations of n-metabolities in non-stressed plants. by treatments with choline or -aminoethanol the stresspromoted accumulation of amino acids and trigonelline was diminished. consequently, different biochemical responses of cereals to drought result in changes of product quality and nitrogen use. our goal is to increase the lysine content in corn. we have used genetic engineering to increase lysine synthesis and to prevent metabolic breakdown of lysine. to increase synthesis we circumvented the normal feedback control of a key enzyme in the lysine biosynthetic pathway, dihydrodipicolinic acid synthase (dhdps). lysine-feedback-insensitive dhdps, encoded by the corynebacterium dapa gene, was expressed from seed-specific promoters in transformed corn seeds. expression of dhdps in the corn embryo, but not in the corn endosperm, resulted in a to -fold increase in the accumulation of free lysine in the seeds and the total seed lysine content nearly doubled. lysine breakdown products have been observed in transgenic seeds that accumulate high levels of free lysine. we isolated a corn gene for the bifunctional enzyme lysine ketoglutarate reductase (lkr)/saccharopine dehydrogenase (sdh), which catalyzes the first two steps in lysine breakdown. knockout of lkr/sdh in corn by either mutation or genetic engineering results in a -fold increase in seed free lysine. combination of feedback-insensitive dhdps with knockout of lkr/sdh results in to -fold higher levels of free lysine than dhdps alone. no adverse effects on seed or plant agronomic performance are associated with the high lysine trait. biotechnology center for agricultural and the environment and the plant science department, rutgers university, new brunswick, new jersey, u.s.a. Ј-adenylylsulfate (aps) reductase catalyzes a key reaction in the plant sulfate assimilation pathway leasing to the synthesis of cysteine and the antioxidant glutathione. in arabidopsis thaliana aps reductase is encoded by a family of genes. in vitro studies revealed that the enzyme product derived from one of the aps reductase genes (apr ) is activated by oxidation, probably through the formation of a disulfide bond. redox titrations show that the regulation site has a midpoint potential of Ϫ mv at ph . and involves a -electron redox reaction. exposure of a variety of plants to ozone induces a rapid increase in aps reductase activity that correlates with the oxidation of the glutathione pool and is followed by an increase in free cysteine and total glutathione. during the response to ozone the level of immuno-detectable aps reductase enzyme does not increase. treatment of a. thaliana seedlings with oxidized glutathione or paraquat induces aps reductase activity even when transcription or translation is blocked with inhibitors. the results suggest that a post-translational mechanism controls aps reductase. a model is proposed whereby redox regulation of aps reductase provides a rapidly responding, self-regulating mechanism to control the glutathione synthesis necessary to combat oxidative stress. in aspergillus nidulans the structural genes coding for nitrate reductase (niad) and nitrite reductase (niia), share a common promotor region of , bp. we have previously characterized in vitro and in vivo the physiologically relevant cisacting elements for the two synergistically acting transcriptional activators, nira and area. we have further shown that area is constitutively bound to a central cluster of four gata sites and is directly involved in opening the chromatin structure over the promoter region and thus making additional cis-acting binding sites accesible. here we show that the asymmetric mode of nira-dna interaction determined in vitro is also found in vivo. binding of the nira transactivator is not constitutive as in other binuclear c -zn ϩϩ -cluster proteins but depends on nitrate induction and additionally, on the presence of a wild type area allele. dissecting the role of area further, we found that it is required for intracellular nitrate accumulation and therefore could indirectly excert its effect on nira via inducer exclusion. but in a strain accumulating nitrate independently of area nira binding and chromatin rearrangement is not triggered by nitrate in the absence of area. v. nikiforova , m. zeh , o. kreft , s. maimann , h. hesse , and r. höfgen max-planck-institut für molekulare pflanzenphysiologie, potsdam, and institut für biologie, angewandte genetik, freie universität berlin, germany higher plants, being a source of reduced sulfur for animal nutrition, assimilate inorganic sulfate into cysteine which is subsequently converted to methionine, another sulfur-containing amino acid. in order to investigate the possible regulatory points of the cysteine and methionine biosynthesis pathway a series of transgenic potato plants was engineered using clones encoding enzymes of the branched pathway from serine to cysteine as a pathway intermediate and from aspartate further on to methionine. increased cysteine levels were obtained in the leaves of serine acetyltransferase (sat) sense and cystathionine -lyase (cbl) antisense transformants. furthermore, glutathione levels were elevated in sat plants while downregulation of cbl was desastrous for plant growth, eventually. increased methionine levels were successfully obtained in potato by antisense inhibition of threonine synthase (ts). accumulation of free methionine was not only observed in source leaf tissues but as well in tubers. this enzyme competes with cystathionine gamma-synthase for the common substrate o-phosphohomoserine at the branchpoint between threonine and methionine synthesis, respectively. important control points of the biosynthesis of cysteine and methionine in potato, thus, turned out to be sat and ts, while further studies on overexpression of cystathionine gamma-synthase, cbl and ms did not reveal any substantial effect on potato methionine biosynthesis. dniepropetrovsk national university, board of biophysics and biochemistry, ukraine amino acids in root exudates of plants may be chelate agents as an alpha-amino acid can act like a bidentate ligand, forming a five-membered heterocyclic ring with suitable metal cations thus increasing mobility of metals. recently we have showed that application of growth regulators led to sharp increase of root exudative activity of some cultural (zea maize l.) and wild cereals (festuca rubra l., lollium perenne l.) during first days of germination. in this work we present results obtained in experiments with lollium perenne l., grown on sterile sand and on soils contaminated with great quantities of zn. detailed analysis of amino acid content of root exudates of several types of maize (hybrid, several lines, an opaque- mutant line) showed that the specie had more certain amino acids (cysteine, aspartic and glutamic acids and their amides, serine) in root exudates than cultural ones. these amino acids has more possibility for chelation due to existance of one more polar or ionogenic functional groop. seeds of lollium perenne l. were treated with growth regulater and planted on soils contaminated with salts of zink. it was shown that during days of germination quantity of zn in primary leaves increased from , to , % and decreased in soil: in upper layer from , to , , midde layer from , to , , lower layer from , to , mkg/kg correspondingly. thus, it was shown that stimulation of root exudative activity by pretreatment with a growyh regulator may be succesful in cleaning of soils and basicly this is a good method for phytoremediation. erenol exerted the strongest effect. exercise completely abolished the levels of cysteine in the atrial heart muscle. propranolol, isoproterenol, caffeine and pentylenetetrazol increased the ratio of cysteine to the total free amino acids in the atrial muscle, while physical stress and all cardioactive drugs tested increased this ratio in the ventricle muscle. disappearance of cysteine from the heart's atrial muscle after intensive exercise may be attributed to its utilization for atrial natriuretic factor and/or for endothelin synthesis, during stress. on the other hand it seems that hypoxia and isoproterenol are strong stimulants of no production, and consequently decrease the tissue levels of l-arginine, which is the major endogenous donor of no acting as the endothelin antagonist. measurement of serum levels of vitamin b is a screening test for detection of deficiency of this vitamin but low levels do not always indicate a deficiency of the vitamin. measurements of serum homocysteine and methylmalonic acid (mma) are used to confirm this deficiency because two enzymes involved in their metabolism have been shown to require vitamin b , but these results can also be inaccurate. vitamin b deficient white cells exhibit ultrascopic nuclear appenages which have been shown to contain dna; this finding could possibly be used as another confirmatory test of vitamin b deficiency. twenty-seven patients (mean age - . years) with low serum b were studied by electron microscopic determination of the percent of neutrophils exhibiting these appendages and routine clinical parameters. only one patient did not have nuclear appendages; the others had a range of . %- . % of neutrophils examined. there was a significant correlation of homocysteine (r ϭ . , p Ͻ . ) and mma (r ϭ . , p Ͻ . ) with serum b levels but no correlation of appendage number (r ϭ . ) with serum b . there was no correlation of appendage number with homocysteine (r ϭ . ) or mma (r ϭ . ). these results suggest that b -deficient white cell nuclear appendages do not measure the same metabolic pathways as homocysteine and methylmalonic acid and may be useful in confirmation of vitamin b deficiency. further extensive clinical evalution would be necessary to explore this possibility. the hypothesis: "l-theanine has relaxing effects of central nervous system of human beings", was verified by electroencephalographical methods. methods: male, healthy sport-students, free of drugs or stimulants, participated weekly in a cross-over study. after exhaustive bicycle-ergometer test as an individual, reliable, stress model, the subjects recovered by lying in a segregated shaded room. three testdrinks with different l-theanine content (d ϭ placebo, d ϭ mg, d ϭ mg) were given in a randomised, double-blind order. all test-conditions were standardized strictly. eeg-recordings (closed eyes) were carried out (m ϭ min. after stress/before testdrink, m ϭ min.-, m ϭ min.-, m ϭ min.-, m ϭ min. after testdrink) with the cateem ® system. absolute and relative eeg-spectralpower were examined. results: significant reductions in all frequencies (exception theta-power) were found in early recovery, being not significant influenced by testdrinks. qualitative different behavior trends were found in frontal-, central-, occipital-regions with increased alpha , theta (frontal) and decreasing beta relative-power earlier in recovery with d . these findings were related to relaxing effects. after ingestion of l-theanine alpha -, beta -power at occipital regions decreased faster (m ) to placebo recovery levels (m /m ). thus it may be concluded that l-theanine has no pharmaceutical effect on the down regulation system but supports the physiological mechanisms during recovery after physical stress in human brain. arginine and cysteine in muscle cytosol of rats' heart after exercise, hypoxia or challenge with six selected cardioactive drugs r. brus , j. gabryś , j. konecki , and j. shani department of pharmacology and department of histology and embriology, medical university of silesia, zabrze, poland department of pharmacology, the hebrew university school of pharmacy, jerusalem, israel levels of the amino acid l-arginine (a major endogenous donor of nitric oxide-no), cysteine (sulfur-containing amino acid, important for atriopeptins and endothelins synthesis), and of total free amino acids, were assayed by gas-liquid chromatography in cytosols of rats' atrial and ventricular muscle cardiomyocytes. the tissues were assayed after the rats had been exposed to either exercise (swimming), hypoxia or one of six cardioactive drugs such as propranolol, digoxin, pentylenetetrazol, reserpine, isoproterenol and caffeine. physical stress and the examined drugs significantly reduced the total amount of cytosolic free amino acids in both cardiac muscles. in the cytosol of the heart atrial muscle, reserpine, propranolol and pentylenetetrazol increased the relative content of l-arginine, while hypoxia and digoxin decreased it. in the cytosol of the ventricular heart muscle, hypoxia and all six drugs used, decreased the relative levels of l-arginine. hypoxia and isoprot-addition of somatostatin- or of some of its analogs was found to cause a selective inhibition, up to %, of the uptake of large neutral amino acids by isolated brain microvessels. although the luminal and abluminal sides of brain endothelial cells are both capable of taking up large neutral amino acids, only the uptake from the abluminal side was apparently inhibited by somatostatin. the involvement of a type- somatostatin receptor was suggested by assays with a series of receptorspecific somatostatin agonists, and was confirmed by the inhibition release caused by a specific type- receptor antagonist. a type- specific mrna was indeed shown to be present both in bovine brain microvessels ex vivo and in primary cultures of endothelial cells from rat brain microvessels. hemorphins represent a bioactive peptide class which contents between and amino acids and generated from the proteolysis of an hemoglobin "strategic zone". many activities have been related to hemorphins such as in vitro anti tumour effect, analgesia effects in vivo, and a potential role in the renin angiotensin system (ras). as far as their activity towards the ras is concerned, it was demonstrated that they could inhibit angiotensin converting enzyme (ace) and aminopeptidase n activity. so they could reduce angiotensin ii formation and angiotensin iv degradation. moreover some hemorphins, lvv-hemorphin- and vvhemorphin- , could behave like angiotensin iv receptor binding competitor. further it could be interesting to study the angiotensin iv potentiality to interact with ace. inhibition studies showed that it was possible that angiotensin iv could behave like a competitive inhibitor of ace. so some hemorphins could interact at different ras steps to inhibit ace. additionally to their inhibition of angiotensin i conversion, they could inhibit angiotensin iv degradation and consequently cause ace feedback inhibition. inhibition studies have been checked with ras natural substrate (angiotensin i) and confirmed that angiotensin iv, vv-hemorphin- and mainly lvv-hemorphin- could be natural ace inhibitors. so the hemorphins regulatory role in the ras appears to be more and more probable. the role of administration of each of methionine and finastride on the testicular function of both normal and prostate precancerous old male rats was investigated. for normal animals, neither methionine nor finastride has exerted any significant change in the hormonal profile after teatment for days. however methionine alone could exert a significant change in both testosterone and prostatic specific antigen {psa} levels after treatment for days. on the other hand, both methionine and finasteride significantly increased the levels of testosterone and androstenedione, whileas markedly reduced the levels of dihydrotestosterone and prostatic specific antigen {psa} after treatment of prostate precancerous old male rats for a period of days. noteworthy, continuation of treatment for aperiod of days realized marked improvement of hormonal profile of the prostate precancerous old male rats. several observations in our department point to some role of glycine in fatigue and exercise. ) in the framework of a study on the involvement of one-carbon matabolism in patients suffering from a polymorphic episodic psychosis, amino acid loading tests with serine, glycine and alanine were performed. a few hours after the administration of glycine, approximately % of the patients reported overwhelming feelings of fatigue and/or showed vegetative symptoms. ) in patients suffering from chronic fatigue syndrome, we found increased plasma levels of glycine in % of the female patients. moreover, - % of these patients omplained about a distorted sensory perception of objects. ) young soccer players were observed during a period of months, while in the course of this period eight blood samples were taken for amino acid analysis. based on the number and severity of injuries this population was divided into injury-prone and not injury-prone soccer players. it was found that in injury-prone soccer players plasma glycine levels during the whole observation period were significantly lower than in subjects who were not injury-prone. the consequences of the above mentioned observations will be discussed. institute of sportsmedicine, university of paderborn, germany percent of amino acids in green tea leaves are represented by l-theanine ( -n-ethylglutamine). previous rat experiments demonstrated effects of l-theanine to act on metabolism of neurotransmitters. it was therefore suggested that is causes the relaxing effects of green tea. to examine its influence as a component of a drink on the sympathetic nervous system after maximal physical exercise skin resistance measurements through electrosympathicography (esg) were used. after individual maximal exercise on a bicycle-ergometer testdrinks with different amounts of l-theanine ( , and mg) were administered to healthy volunteers in a randomised cross-over double-blind distribution on a weekly base. esg was monitored before and immediately after exercise as well as , , , , and minutes after end of exercise. all testconditions were standardized strictly. a characteristic esgcourse with subsequent qualities could be shown: . decreasing skin resistances after exercise could be established in each volunteer. . esg-activation levels before exercise could not even be reached again after a period of regeneration of / hours. . maximal electrodermal activity did not appear immediately after exercise, but after minutes. however, l-theanine could not significantly influence peripheral sympathetic electrodermal activity during the regeneration after maximal physical exercise. a. mero and h. pitkänen , neuromuscular research center, department of biology of physical activity, university of jyväskylä, and rehabilitation center of kankaanpää, finland essential amino acid leucine has many important roles in the body. therefore the purpose of the present study was to investigate if leucine supplementation has effects on serum amino acid profile and performance following training period or following single training sessions. all experiments were carried out in a randomized double blind cross-over procedure during a training season. thirty six adult male track and field power athletes served as subjects. in experiment ten of them were given leucine ( mg/kg body weight per day) as tablets. the concentration of leucine decreased significantly ( %) in the placebo group (p; n ϭ ) during weeks but not when leucine was taken. also total amino acids (taas) decreased strongly ( %) during weeks when dally protein intake was . g/kg body weight. in experiment the subjects (n ϭ ) carried out a single strength training seasion (sts) and consumed a drink containing leucine mg/kg body weight. following sts leucine in serum increased by % (ns) when leucine was taken but decreased strongly ( %) in p, in experiment the subjects (n ϭ ) underwent at days interval two maximal anaerobic running exercise (mare) tests on treadmill (n ϫ s with a recovery of s between the runs) until exhaustion. the subjects consumed drinks containing leucine ( mg/kg body weight) or placebo min before the test runs. following mare the concentration of leucine strongly increased by % whereas isoleucine ( %) and valine ( %) strongly decreased with the supplementation but no changes occurred in p. there were no improvements in physical performance either in mare or in explosive strength (experiment ) with leucine supplementation. the date suggest that leucine supplementation during a training period and before single training sessions prevents decreases in serum concentration of leucine and may have also effects on some other single amino acids. this may be beneficial during intensive training although improvements in performance were not observed in this study. since there are only limited data regarding effects of training period or training sessions on serum amino acid profile, the purpose of this study was to investigate serum amino acid changes following training period and following three different training sessions. the subjects consisted of track and field adult male power athletes. in experiment eleven of them performed a -week training period including a training sessions per week, which included sprint work, speed endurance work, endurance work, weight training, and jumps. significant decreases in the fasting concentrations of total amino acids (taas) ( %), branched chain amino acids (bcaas) ( %), essential amino acids (eaas) ( %) and leucine ( %) were observed following training with the daily protein intake of . g/kg body weight. in experiment eleven subjects performed a short run session (srs) of ϫ ϫ m with recoveries of s and s, and a long run session (lrs) of s runs with recoveries of s until exhaustion. there were no significant changes in taas following the sessions but bcaas decreased by % in srs and by % in lrs. leucine decreased by % following srs but only by % (ns) following lrs. the peak blood lactate concentrations after srs and lrs were . Ϯ . mmol/l and . Ϯ . mmol/l, respectively. in experiment sixteen subjects carried out a strength training session (sts), which consisted of jumps and heavy resistance exercises (speed and maximum strength) during minutes. the taas decreased significantly by %, bcaas by % and leucine by % following sts, while the peak blood lactate concentration was . Ϯ . mmol/l. these data indicate that remarkable decreases occur in the concentration of amino acids during a training period with the daily protein intake of . g/kg body weight. the decreases in serum amino acids are more pronounced following a strength training session than following lactic anaerobic running sessions. glutamine acts as a multipurpose regulator of amino acid and peptide transport across the blood-brain barrier departments of cellular biotechnology and haematology and of biochemical sciences, university "la sapienza", rome, italy isolated brain microvessels, the in vitro equivalent of the blood-brain barrier, have distinct na ϩ -independent uptake systems for the uptake of large hydrophobic amino acids, of enkephalins and of deltorphins, as shown by the absence of reciprocal inhibition. both d-and l-glutamine were capable, if added to the extracellular buffer, of exerting a competitive inhibition on the uptake of all these substrates. a trans-stimulatory effect was instead induced, in all cases, by l-glutamine preloading of the microvessels -the d-stereoisomer being instead ineffective, probably because of only l-glutamine could be taken up, in a concentrative manner, by some na ϩ -dependent concentrative system(s). all the na ϩ -independent systems present in brain microvessels seem therefore to share some structural feature responsible for their common susceptibility to interference by l-glutamine. this amino acid, whose synthesis can take place in the astrocytes, in the pericytes and also in the endothelial cells of the microvessels, plays a critical role in regulating the movements of several different substrates across the blood-brain barrier. department of applied bioorganic chemistry, division of life science, graduate school of agricultural science, tohoku university, aoba-ku, sendai, japan isolation and structure analysis of two amino acids from bovine ligamentum nuchae elastin hydrolysates revealed the presence of pyridine cross-links in elastin. the structures of these amino acids were determined to have , , -and , , -trisubstituted pyridine skeletons both with three carboxylic acids and a mass of (c h n o ), identified as -( -amino- -carboxybutyl)- , -di-( -amino- -carboxypropyl)pyridine and -( -amino- -carboxybutyl)- , -di-( -amino- carboxypropyl)-pyridine. we have named these pyridine cross-links, desmopyridine (desp) and isodesmopyridine (idp), respectively. structure analysis of these pyridine crosslinks implied that the formation of these cross-links involved the condensation reaction between ammonia and allysine. the elastin incubated with ammonium chloride showed desp and idp levels increased as the allysine content decreased. desp and idp were measured by hplc with uv detection and were found in a variety of bovine tissues. the desp/desmosine and idp/isodesmosine ratios in aorta elastin were higher than in other tissues. desp and idp contents in human aorta elastin were found to be gradually increased with age. the concentration of idp was significantly elevated in aorta elastin of rat with chronic liver cirrhosis induced by carbon tetrachloride when compared with normal rats. the provision of glutamine to marathon runners has resulted in a decreased, self-reported incidence of illness. increasing evidence -in vitro; and in vivo suggests that neutrophils in humans may benefit from exogenous glutamine. the provision of glutamine in vivo should replete the marked decrease in the blood concentration observed after stress such as clinical trauma or prolonged, strenuous exercise. beneficial effects of glutamine supplementation include increased phagocytic activity and reactive oxygen intermediate production in vitro; decreased neutrophilia and il- production (a chemoattractant for neutrophils) in vivo and ex vivo. the aim of the present study was to establish whether glutamine supplementation in vitro and in vivo affects neutrophil function at rest and after exhaustive exercise. in addition, it was planned to establish the presence of glutaminase in human neutrophils, which has not yet been achieved, although glutaminase is present in rat neutrophils. methods: blood samples were taken from marathon runners receiving either glutamine or placebo, immediately after and one hour after a race. measurements included the plasma concentration of glutamine (enzymatic assay), il- production (elisa), and neutrophil activity. the latter was measured with two different techniques for measuring oxidative burst in whole blood, one of which was a novel chemiluminescence assay (knight scientific ltd, u.k.) with the fluorescent label, pholasin, and two different stimuli, f-met-leu-phe (fmlp) and phorbol-myristate-acetate (pma). in addition, isolated whole cells and subcellular neutrophil fractions were assayed for the presence of glutaminase. results: the plasma glutamine concentration was reduced overall by % hr after the race (p Ͻ . ). there was an apparent decrease (only close to significance, p Ͻ . ) in il- production in the glutamine group compared with the placebo group. neutrophil function did not change between groups at any stage. the incidence of illness was % higher in the placebo group than the glutamine group in the week after the race. neutrophils from four out of six subjects gave an increased response ( . %) to fmlp when incubated with glutamine compared with no glutamine, and four out of four gave an increased response to pma ( . %). in the fmlp experiments there were two individuals who did not respond to the addition of glutamine. however, the response was not diminished whether or not glutamine was present. in separate studies, the effect of glutamine on lipopolysaccharide-induced il- production was also monitored. conclusions: the provision of glutamine after prolonged, exhaustive exercise appears to modify exercise-induced neutrophilia via a reduction in il- production and to reduce the incidence of illness in the following week. in vitro data suggest a role for glutamine in neutrophil metabolism. disappointingly, little or no evidence of the presence of glutaminase was found in human neutrophils. the three different methods used, freeze-thaw, homogenisation, nebulisation were apparently not sufficient to break open the granules. current studies are addressing this problem. r. j. ward and l. m. castell departments of biochemistry, university catholique de louvain, belgium oxford university, oxford, u.k. it is essential that the developing muscle has adequate amino acids for the synthesis of actin and myosin as well as those required for a multitude of enzymes involved in muscle metabolism. with carbohydrates and lipids, the body is able to store a reserve as glycogen and triglycerides respectively; however this is not the case with amino acids creatine supplementation in increasingly being used as a dietary supplement by athletes during high intensity, short term exercise to improve physical performance since it is converted in the muscle to phosphocreatine. transporters which permit creatine to cross the muscle membrane namely crt and crt (a na ϩ and clЈ dependent mechanism) have now been identified. creatine uptake is enhanced by the ingestion of carbohydrate at the same time as supplementary creatine. this may be due to increased circulating levels of insulin or insulin-like growth factor . more recently attention has been focussed upon the various transporters for amino acids across the muscle membrane. certain criteria are needed for the amino acids to enter the blood which include the presence of specific carriers for its transport across cells of the gastrointestinal tract, such as enterocytes, as well as minimal metabolism within these cells. a wide number of different transporters has been identified, which include neutral amino acids and cationic amino acids. despite the evidence which suggests that supplementation with some amino acids can influence metabolism, and therefore athletic performance, much more experimental work is still required in this area. m. weiss , t. barthel , r. schnittker , k. e. geiss , w. falke , and l. r. juneja university of paderborn, germany taiyo kagaku co., yokkaichi, japan, isme gmbh mörfelden, germany in animal studies l-theanine was shown to influence neurotransmitter systems. thus it may be helpful in managing stress regulation. so we observed the down regulation after physical stress in the brain (measured by eeg-mapping) and in peripheral hormonal systems (plasma levels of catecholamines, cortisole, prolactine, serotonine, measured by hplc). n ϭ healthy students consumed drinks containing either , or mg l-theanine in randomized double-blind trials in the min - after a near maximal bicycle step test. measurements were done directly after exercise (m ) and (m ), (m ), (m ), (m ) min after the drink. l-theanine seemed to accelerate the normalization of eeg spectral power in high frequency waves (barthel in this congress). the physiological return of increased hormon levels to basal levels / the circadianic rhythm up to m (catecholamines) or m (cortisole, serotonine, prolactine) was not influenced by the drinks. but in the l-theanine trials correlations between eeg spectral power and some hormones were altered (slow wave power/some catecholamines except norepinephrine/delta disappeared and new correlations with prolactine appeared). thus we conclude that l-theanine acts at the switch from the brain to the peripheral stress regulation and thereby supports physiological relaxing after severe exercise. polyamines the development from callus to plantlets, both activities increased, reaching the maximum at this latter stage. also sadenosylmethionine decarboxylase activity displayed a similar trend. all the activities were present in supernatant and in particulate fraction. higher activity of enzymes assayed in the small embryos rather than in the embryo with higher shape, was consistent with following polyamine accumulation. department of biology, laboratory of cell biochemistry, university of rome "tor vergata", rome, italy intracellular transglutaminase (tg, ec . . . ), which catalyzes the formation of ε-(γ-glutamyl)lysine isopeptide cross-links between polypeptides, has been related to a variety of important biological processes and in the development of senile cataract. the majority of the dry weight of the eye lens is composed of protein called crystallins. in the mammalian lens, these proteins are divided into three major classes: α-, -and γcrystallins. native -crystallins are oligomers, which elute in two or more size classes during gel filtration, ranging from - kda. they contain different types of subunits, named b , b , b , a , a , a , ba , ranging from - kda. in the rabbit eye lens two -crystallin subunits ( b and b ), among the water soluble proteins, have been shown to act selectively as acyl donors substrates for lens tg. calpains are cytoplamic ca ϩϩ -dependent cystine proteinases. the cleavage of αand -crystallins, the main substrates of lens calpain ii, has been associated to the increase of lens turbidity, due to insolubilization of peptides. we observed that tg-induced post-translational modification of b -and b -crystallins with polyamines, enhances their cleavage by calpain ii. this finding suggests that the enhancement of calpain ii activity, after conjugation of polyamines into -crystallins, could represent an important regulatory mechanism which may contribute to the opacification process of the eye lens, conducting to cataract formation. transglutaminases represent a family of enzymes, widely diffused in nature, from bacteria to plants and higher animals. the present discussion will focus on isoenzymes in mammals, which have been well characterized from the structural and functional point of view. they act on tissular proteins catalyzing crosslinkage through isopeptide bonds at peptidyl glutamine and lysine residues or incorporation of small molecular weight primary amines, usually polyamines, in an irreversible, calcium dependent reaction. in several instances the expression of transglutaminases is regulated at the transcriptional level. these enzymes help in maintaining structural integrity of tissues intervening in wound repair and in cellular homeostasis at the levels of cell activation, receptor signaling, cell proliferation, differentiation and death. these general roles involve bis(guanylhydrazones) are a class of compounds known to interfere with the metabolism of polyamines by virtue of their ability to inhibit s-adenosylmethionine decarboxylase (samdc), a key enzyme of polyamine biosynthesis. this property has made them useful tools to study the biological functions of these compounds. a curious feature of bis(guanylhydrazones) is their structural relationship with two molecules involved in polyamine biosynthesis, namely spermidine and s-adenosylmethionine. the methylglyoxal derivative of bis(guanylhydrazones), mgbg, has been actively studied both in animal and plant systems. in the present work the male pollen from actinidia deliciosa has been utilized to investigate the role of polyamines on the pollen tube growth. the effect of several bis(guanylhydrazones) was tested on pollen germination, length of pollen tube, levels of free and conjugated polyamines and samdc activity. all bis(guanylhydrazones) tested (glyoxal-bis-guanylhydrazone, gbg, methylglyoxal-mgbg, methylpropylglyoxal-mpgbg, ethylmethylglyoxal-emgbg) inhibit pollen germination and their effect is dose-dependent. a clear reduction of spermidine, both in free and conjugated form, was observed, as well as a pronounced decrease in samdc activity. these results suggest that the mechanism by which bis(guanylhydrazones) reduce the germination of kiwi pollen is related to their effect on spermidine biosynthesis. molecules structurally related to polyamines ( , diaminooctane, , -diaminononane, , -diaminodecane) and other inhibitors of their metabolism (cyclohexylamine, cha) are also tested on kiwi pollen germination. n. bagni , d. bertoldi , , e. candioli , l. martinelli , and a. tassoni istituto agrario, san michele a/adige, and dipartimento di biologia, università di bologna, italy in the frame of the study aiming to enlighten developmental programs during regeneration in grapes, polyamine content (free and conjugated to hydroxycinnamic acids) and biosynthetic enzyme activities were assayed during somatic embryogenesis. aliphatic polyamines are growth regulators affecting plant growth and development both in vivo and during in vitro cultures, being involved in several morphogenic processes related ti their action in cell division. the study was conducted on samples of callus, embryogenic callus, embryo at different stages and plantlets of vitis vinifera brachetto and chardonnay cultivars induced from anthers and ovaries. polyamine content (putrescine, spermidine and spermine) free plus conjugated to percloric acid soluble fraction, referred to unit, was higher in the cv. brachetto than in the cv. chardonnay, and reached the higher levels in the fullydeveloped embryo stage. besides, ornithine decarboxylase activity resulted higher than arginine decarboxylase and during multiple catalytic processes: the receptor signaling activity (demonstrated only for isoenzyme ) is related to an intrinsic gtp-ase activity of type transglutaminase; the processes leading to control of cell proliferation, differentiation and death are mainly related to the protein crosslinking activity, while the cell activation is tentatively considered dependent on the polyamidation of endogenous proteins at glutamine residues. the knowledge on this last aspect lies far back in comparison to the other roles of transglutaminases and requires further accurate investigation, which must further extend to the role of the enzyme in human pathology. the examination of polyamine metabolism at the present time suggests that vitamin b is implicated in polyamines metabolism. literature data speak that spermine and spermidine stimulate activity of cobalamin-dependant methionine synthase, the enzyme that catalyses the recycling of homocysteine to methionine; polyamines inhibit methionine adenosyltransferase. beside the wellknow significance of vitamin b , in transmethylation reaction, the significance of ,-deoxyadenozyl cobalamin, except the conversion of methylmalonyl-coa to succinyl coa, is not well elucidates. methionine as s-adenosylmethionine (sam) is essential amino acid for polyamine biosynthesis. sam has frequently usage in treatment of liver diseases. according the mentioned facts the aim of our experiments is to exanimate the significance of application of vitamin b alone and altogether with methionine to rats without and with experimentally induced cholestasis. our preliminary results speak about the disturbance of polyamine metabolism in hepatic tissue of rats with cholestasis. application of methionine alone increases the amount of polyamine in rat liver tissue, in-group without cholestasis and with bile duct obstruction. the animal treatment with cobalamin has higher amount of polyamines and lower activity of polyamine oxidase in liver tissues in both groups. the effects of vitamin b may be in direct relation with the formation of ,-methylthio deoxyadenosine (mta), the by-product of spermidine and spermine biosynthesis. the explanation the exact roles of vitamin b in polyamine metabolism of liver tissue need the futher investigations. department of molecular genetics, the weizmann institute of science, rehovot, israel exposure of mouse myeloma cells that massively overproduces ornithine decarboxylase (odc) but not of parental cells to ornithine results in a massive increase in the intracellular concentration of putrescine, followed by rapid cell death. the treated odc overproducing cells display fragmented nuclei, chromatin condensation and an oligonucleosome-size dna "ladder"; consequently, their death can be described as apoptosis. the apoptotic process induced by the accumulated putrescine involves the release of cytochrome c from the mito-chondria, activation of caspases cascades demonstrated by the cleavage of caspase- and parp, a substrate of caspase- . the general inhibitor of caspases, bd-fmk, effectively inhibited parp cleavage but failed to inhibit cell death. the intracellular ca ϩ chelator bapta/am and the antioxidant bha inhibit parp cleavage. however, only bapta/am inhibit the induction of cell death. it seems that bha subverted the death into caspase independent pathway. treatment with bapta/am did not interfere with the accumulation of putresine following ornithine treatment, suggesting that the accumulated putrescine induces the elevation in the concentration of intracellular ca ϩ which then activates the apoptotic process. the dominant anti-apoptotic effect of bapta/am over egta suggests that internal stores are the main source of the elevated ca ϩ , but that putrescine is also capable of inducing influx of extracellular ca ϩ . extensive small intestine resection results in the loss of absorptive surfaces, acceleration of intestinal transit and, as a consequence, in malnutrition, weight loss, diarrhoea and other complications of short bowel syndrome. the availability of human recombinant growth hormone rgh and its stimulatory effects on gut growth suggested its use in the treatment of short bowel syndrome. the trophic response of gi tract epithelium to hormones such as growth hormone is mediated by polyamines, which are vital in cell proliferation. this study was undertaken in rats to: / evaluate the effects of rgh by monitoring polyamine and amine metabolism parameters in the adapting short bowel and / determine whether erythrocyte (rbc) polyamine concentrations reliably reflect the proliferative activity of the remaining bowel. seventy per cent resection of the small intestine of wistar rats was performed under ether anesthesia leaving equidistant lengths of bowel from pylorus and ileocecal valve. recombinant human gh ( . iu, s.c., saizen, serono, switzerland) was administered once daily for or days, to randomly selected rats on the second postoperative day. animals were sacrificed , and days after the operation. enzyme activities were measured with radioassays or fluorimetry. polymines were determined as dansyl derivatives by hplc/fluorimetry. gh treated animals had significantly higher intestinal odc and sat and lowel dao activities; higher (non-significant) mucosal growth index and polyamine concentrations than in untreated counterparts on th postoperative day. thereafter the two groups did not differ in the investigated parameters. rbc polyamine concentrations were higher in operated verses control rats; rgh treatment had no significant effect. however, rgh treatment significantly reduced hepatic mao a and b activities. our results suggest gh accelerated the adaptive growth of the bowel remnant. they justify use of erythrocyte polyamine concentration measurement as the marker of small bowel proliferative activity. however, side-effects of this treatment must be considered. tissue transglutaminase (ttg) activity has been evaluated in different neural tissues, such as brain, spinal cord and peripheral ganglia, and appears to be expressed in cerebellar granule cells (cgn) as well as in astrocytes. the role of ttg in neuronal functioning is likely to be quite complex. other than the role during development, significant changes of enzyme activity have been evaluated in different neurodegenerative conditions. it is well known that nmda receptor activation may be able to trigger excitotoxicity. the nmda-induced injury is mainly associated to ion influx and subsequent calcium overload. the effects of nmda application to both, cerebellar granule cells and glial cell cultures, have been assessed. in cgn, ttg activity increased rapidly after a brief stimulation with µm nmda, whereas in glial cell cultures, high levels of enzyme activity was obtained after incubation of h in presence of the same concentration of nmda. such results rule out the possibility that excitotoxicity can modify numerous proteins making them better substrates of ttg, and this could contribute to enhanced ttg-modifications of proteins in response to excitotoxicity. the pote protein can catalyze both uptake and excretion of putrescine. the km values of putrescine for uptake and excretion (putrescine-ornithine antiport) are . µm and µm, respectively. amino acid residues, cys , trp , glu , trp and tyr are strongly involved in both activities, and that glu , tyr , cys , cys , cys and glu are moderately involved in the activities. mutations of tyr , trp and trp mainly affected uptake activity, indicating that these amino acids are involved in the high affinity uptake of putrescine by pote. mutations of lys and tyr mainly affected excretion activity, indicating that these amino acids are involved in the recognition of ornithine. the putrescine and ornithine recognition site on pote was found to be located at the cytoplasmic surface and the vestibule of the pore consisting of twelve transmembrane segments. the cadb protein has % sequence homology with pote protein. cadb can catalyze both uptake and excretion of cadaverine. the km values of cadaverine for uptake and excretion (cadaverine-lysine antiport) are µm and µm, respectively. it was found that two glutamate residues (glu and glu ) and four tyrosine residues (tyr , tyr , tyr and tyr ) are involved in the both activities. the difference of the substrate recognition site on pote and cadb is discussed. a. lentini, b. provenzano, and s. beninati department of biology, laboratory of cell biochemistry, university of rome "tor vergata", rome, italy tissue transglutaminase (ttg, e.c. . . . ) is a protein cross-linking enzyme which catalyzes an acyl transfer reaction where the carboxamide group of a peptide-bound glutamine is the acyl donor, and a lysine residue the acyl acceptor. polyamines may act as acyl acceptors, leading to the formation of mono-and bis-(γ-glutamyl)derivatives. we provided evidence that ttg activity is directly associated to differentiation markers, and inversely related to cell proliferation and invasion. we have shown the in vivo reduction of experimental melanoma metastasis by i.v. injection of a plasmid (psg ) carrying the ttg gene sequence to c bl /n mice. tumor cell metastatization requires specific interactions with subendothelial basement membrane (bm) and migration through the endothelial wall, allowing the colonization of the target tissue. therefore, the investigation on the possible mechanisms responsible for ttg effects is focused on the posttranslational modification of bm proteins. we detected that "matrigel", a tumor-derived complex of bm proteins, modified with polyamines after ttg catalysis, reduces both melanoma cell adhesion and invasion in an in vitro metastatic assay. similar results were obtained using polyamines conjugated to laminin, one of the major bm components, as unique substrate. our findings suggest that the increase of bm proteins conjugated to polyamines may be responsible for impairments of the invasive properties of melanoma cells. we demonstrated that interferon-α (ifnα) induces apoptosis in human epidermoid cancer cells. tissue transglutaminase (ttgase) is an enzyme involved in the regulation of apoptosis through the inactivation of some cell components. among these eukaryotic initiation factor- a (eif a) is peculiar because its activity is modulated by the formation of the amino acid hypusine. recently, we found that growth inhibition induced by ttgase is paralleled by reduced hypusine levels. here we report the effects of ifnα on the apoptosis, ttgase modulation and eif a activity in human epidermoid lung h cancer cells. we have found that h exposure to , iu/ml ifnα induces % growth inhibition and % apoptosis in h cells. moreover, ifnα induced a -fold increase of ttgase activity and expression that already occurred after h of exposure to the cytokine. this effect was paralleled by a . -fold enhance of ttgase mrnas. ifnα induced also a % increased eif a expression while an about % decrease of hypusine levels was observed. increased ttgase activity was paralleled by a decrease of hypusine content and of eif a activity. therefore, ifnαinduced apoptosis could occur through an increase of ttgase activity and the mechanism by which ttgase regulates biological functions can be the reduction of eif a activity. adometdc deficient mice k. nishimura , f. nakatsu , , k. kashiwagi , h. ohno , t. saito , and k. igarashi graduate school of pharmaceutical sciences, chiba university, chiba, graduate school of medicine, chiba university, chiba, and cancer research institute, kanazawa university, kanazawa, japan the amd gene encodes s-adenosylmethionine decarboxylase (adometdc) that is one of the key enzymes of polyamine biosynthesis. to examine the physiological role of polyamines, we performed the targeted disruption of the gene in mice to generate spermidine-and spermine-free mice. although the level of adometdc mrna decreased by % in amd ϩ/Ϫ mice, adometdc activity reduced only by % and spermidine and spermine contents did not change significantly. they showed normal phenotype and life span. to obtain amd Ϫ/Ϫ mice, we intercrossed amd ϩ/Ϫ mice and determined the genotype of the resulting offspring. however, we could not obtain any amd Ϫ/Ϫ mice from heterozygous intercrosses over. amd Ϫ/Ϫ embryos died early in development, between e . and e . days post coitum. in culture of blastocysts at e . , the shapes of all cell lines were normal, but amd Ϫ/Ϫ cells appeared to arrest the cell proliferation at day after the onset of cell culture. the arrest of amd Ϫ/Ϫ cell proliferation was rescued by addition of spermidine. these data indicated that the lethal phenotype of amd Ϫ/Ϫ mice was caused by growth retardation by polyamine depletion at early developmental stage. the formation of active species such as h o and aldehydes during the oxidative deamination of biogenic amines by amine oxidases (ao) suggests for these enzymes a key role in cellular processes. the ability of bovine serum amine oxidase (bsao) to oxidase free amino groups of lysozyme and ribonuclease a has been observed indicating a possible ao involvement in the post-translational protein modification. furthermore, bsao inhibition by h o formed during substrate oxidation under limited turnover conditions was demonstrated, which may be relevant to cellular physiopathology. we have also observed that some inhibitors of mitochondrial amine oxidases (mao) protected human melanoma cell line (m ) against apoptosis. the protection by catalase of mao-substrates induced membrane permeability transition was also obtained in isolated rat liver mitochondria, thus confirming a role of mao-derived h o in apoptosis. enrichment in ao activity by treatment with vegetal ao has been obtained in a erythroleukemia cell line (k ), substaining the possibility to modulate the intracellular ao activity. an antiarrhythmic and cardioprotective effect of bsao has been also observed on isolated rat heart in reperfusion; a protective effect during anaphylaptic crisis has been shown "in vivo", thus suggesting aos as a possible therapeutic agents. tetrakis( -aminopropyl)ammonium, a unique polyamine produced by an extreme thermophile, stabilizes nucleic acids at high temperature t. oshima and y. terui department of molecular biology, tokyo university of pharmacy and life science, hachioji, tokyo, japan an extreme thermophile, thermus thermophilus, produces tetrakis( -aminopropyl)ammonium; a novel polyamine containing a quaternary ammonium nitrogen. to clarify the roles of the unique polyamine in thermophily, the effects of tetrakis( aminopropyl)ammonium on biochemical reactions related to nucleic acids have been investigated. the unique polyamine stabilized both double and single stranded dnas and rnas. tm of a double stranded dna was raised by °c by the addition of . mm of tetrakis( -aminopropyl)ammonium. at around the boiling temperature of water, depurination of dna takes place. other long polyamines produced by the thermophile such as caldopentaamine also stabilized dnas and rnas. we found that tetrakis( -aminopropyl)ammonium prevents depurination most effectively. tetrakis( aminopropyl)ammonium activated the protein biosynthesis catalyzed by a cell-free extract of the thermophile at high temperature. the effects of this unique polyamine on dna and rna polymerases are also being investigated and the results will be presented. tissue transglutaminase (ttg) catalyses the cross-link formation between glutamine (q) residues and nh -donor molecules present in the cells (polyamines, lysine-donor proteins). recently, it has been correlated to neurodegenerative disorders characterised by polyglutamine (q n ) expansion, like huntington's disease. studies carried out on cell extracts revealed that glyceraldehyde -phosphate dehydrogenase (gapdh) was found covalently linked to q n domains. however, to date no structural data are available to solve the issue of which residues of gapdh are substrates for ttg. by coupling classical protein chemistry procedures and mass spectrometric techniques we achieved this goal by using as ttg substrates the substance p, an -aa peptide bearing the simplest q n domain (q ), and polyamines of different size and shape as q-and nh -donor, respectively. in the present study we report that out of the lysines present in gapdh only three are sites of ttgasedependent cross-link formation in vitro. moreover, to characterize the ttg catalysed cross-link between gapdh and polyq protein we used a synthetic q -peptide as ttg substrate in the catalysed reaction with polyamines. we found that any q residue is a potential ttg substrate, no matter the specific position in the sequence or the steric hindrance of the specific amine under investigation. cjf inserm - , institut contre les cancers de l'apareil digestif (ircad), strasbourg, france as soon as the key role of odc in polyamine metabolism was recognised, it became the major target for selective inhibi-tion. s. harik presented in the first potent odc inhibitor, α-hydrazino ornithine. although efforts continued until today, with the aim to improve odc inactivation, -(difluoromethyl)ornithine (dfmo) remained the most important compound among all polyamine-directed drugs. a known anti-leukaemic drug, methylglyoxal-bis(guanylhdrazone), was recognised early on by g. williams-ashman and his collaborators as an inhibitor of adometdc, the other highly regulated biosynthetic decarboxylase, and served as matrix for more recent developments. in the course of the years selective inhibitors for all enzymes involved in polyamine biosynthesis and degradation were synthesised. moreover, a series of polyamineuptake inhibitors were reported. however, only some of these numerous compounds reached a stage above evaluation as growth inhibitors of cancer cells. owing to the sophisticated homeostatic regulation of the polyamines in cells and organs by de novo synthesis, degradation, uptake and release, and due to the fact that exogenous polyamines (i.e. gut polyamines) can be utilised by the vertebrate organism, the efficacy of selective enzyme and uptake inhibitors remained modest in cancer therapy. the fact the dfmo became the most important drug for the therapy of west and central african sleeping sickness relies on differences of vertebrate and parasite biochemistry. a novel approach, initiated by carl porter, involved the design and synthesis of structural analogues of spermidine and spermine, which do not share the growth-promoting effects of the natural polyamines. a very large variety of homologues, mostly of spermine, with different alkyl-substituents on the primary amino groups, have been studied systematically with regard to their ability to alter enzyme and polyamine patterns, and to inhibit cell growth. in addition polymine-like chains with interposed heteroatoms ( , s, si etc.), and analogues with rigid aliphatic chains (due to inbuilt double and triple bonds, or of small rings) have been explored. the structural analogues either mimic regulatory functions of the natural polyamines, and thus lead to the depletion of endogenous pools of putrescine, spermidine and of spermine, or they prevent growth effects of the natural polyamines by displacing them from functionally important binding sites. the later type may be considered as polyamine antagonists. the actual drugs usually exhibit to some extent polyamine mimetic and antagonist properties. at present several polyamine analogues are in clinical trial. however, after more than years of active research, a polyaminerelated anticancer drug is still not available. one may conclude from this fact that the polyamines are an inappropriate target for cancer treatment. however, it is more likely that polyamine metabolism is a difficult target, because the differences between normal and cancer cells are mainly of quantitative nature. moreover, numerous mechanisms have developed in the course of evolution, which enable the vertebrate organism to prevent lethal polyamine losses. nine novel chemically modified polyamine (pa) analogs were evaluated for their ability to inhibit the pa biosynthesis in rat hepatoma g- cell-free system as well as the growth of caov tumor cells. the final concentration of oxy-and aminoadenosine pa analogs or two uracils modified pa analogs were . mm in the reaction mixture. bis(uracilyl)-analogs and -( -oxyethyl)ami-no- --d-xylofuranosyladenine supressed pa and putrescine synthesis and in the same conditions were more effective than dl-α-difluoromethylornithine (dfmo) -strong specific inhibitor of ornithine decarboxylase (odc). the other adenosine modified compounds could act both as activators of odc and inhibitors both diamine and polyamine oxidase activities in regenerating liver test system. in contrast to those mentioned above two uracils modified agents as well as dfmo were able to inhibit odc and to increase the rate of oxidative deamination of pa in the same system. thus bis(uracilyl) pa analogs were the most active and may be useful for further investigation as substances having potential antitumor and antiproliferative properties. several studies concerning the periodontal status in adult and adolescent patients treated with fixed ni-ti archwires have been performed, but until now it is not yet available any information about the influence of patient age on gingival tissue responses to ni-ti alloy. recently, researches by us demonstrated that the prolonged use for over months of ni-ti appliances may contribute to local pathological proliferative processes early detectable only through salivary polyamine concentration increase. although other data from our laboratory showed that salivary polyamine amounts are age and sex-independent, nothing is known about the influence of the age on salivary polyamine content m subjects wearing ni-ti appliances. eighty patients, under orthodontic treatment for months, were divided into four groups: the pre-, the mid-, the late-and the post-pubertal. salivary polyamine concentrations were determined by hplc. only the late pubertal group revealed a significant increase in both the spermine and spermidine content, while the other groups showed no modification. the results suggest that gingival pathological responses to a long-term appliance's use may be related to the endocrine modifications that occur in the late-pubertal age. sexual hormones appear to be in synergy with ni-ti alloy in promoting proliferative activity of gingival cells. the effects of polyamines on the synthesis of various σ subunits of rna polymerase were studied to determine how polyamines influence the functional specificity of transcription using western blot analysis. synthesis of σ was stimulated . -fold and that of σ was stimulated . -fold by polyamines, whereas synthesis of other σ subunits was not influenced by polyamines. stimulation of σ synthesis by polyamines occurred at the level of transcription. since our hypothesis is that polyamines regulate macromolecular synthesis mainly at the translational level, we searched for a target protein, related to the polyamine stimulation of σ synthesis, whose translation is altered by polyamines. stimulation of σ synthesis was due to an increase in the level of camp, which occurred through polyamine stimulation of the synthesis of adenylate cyclase at the level of translation. polyamines were found to increase the translation of adenylate cyclase mrna by facilitating the uug codon-dependent initiation. analysis of rna secondary structure suggests that exposure of the shine-dalgarno (sd) sequence of mrna is a prerequisite for polyamine stimulation of the uug codon-dependent initiation. antitumor quinones are approved for clinical use and others antitumor quinones are in different stages of clinical and preclinical development. the efficiency of the quinonic compounds in inhibiting cancer cells growth is believed to stem from their participation in key cellular redox mechanisms with consequent generation of highly reactive oxygen species (ros). the ros is turn modify and degrade nucleic acids and proteins within the cells. recently, quinonic drugs were attached to the neurodecapeptide lh-rh and evaluated as potential drugs in the treatment of different tumours. we have synthesized several series of n-quinonyl amino acids in which five ω-amino acids are attached to p-quinones with different values of redox potentials. the attachment was made via michael-like reductive addition of the amino acids to the quinonic ring or via substitution of a chlorinated atom. the n-ω-quinonyl amino acids were characterized as to their ability to form semiquinone anion radicals by epr and cyclic voltammetry technique. the preparative methods, the redox potentials as well as the physical and spectral data ( h-nmr, ir, uv-vis and hrms) of these n-ω-quinonyl amino acids will be presented. the de novo design of biologically active peptides and proteins, mostly has involved consideration and design of backbone conformations (secondary structures) such as α-helix, -sheets, -turns, etc. (η/ψ space). however, for many bioactive peptides and proteins, especially those critical for information transduction such as neurotransmitters, hormones, antigens, neurocrines, etc. molecular recognition via side chain moieties is of paramount importance. thus far, the specific three dimensional orientations of side chain groups ( angles; chi space) in terms of biological activity has received only modest attention. in part this may be due to the energetics of chi space compared to ramachandran space. in order to overcome the current limitations of evaluating the importance of chi space in critical biological functions related to disease and behavior, we have designed amino acids with novel structures and unique constraints in chi space ( , , etc.), with special attention to their ability to mimic the chi space of native proteins and peptides. we have developed novel and simple asymmetric synthetic methods for such amino acids, often with ees greater than %. incorporation of these novel amino acids into bioactive polypeptide neurotransmitters has provided ligands with unique biological activities that effect unique behaviors including feeding, sexual, pain, and addictive behaviors. (supported by grants from the usphs and nida.) protein technology, wallenberg laboratory ii, lund university, lund, sweden we describe a method for comparative quantitation and de novo peptide sequencing of proteins separated either by standard chromatographic methods or by one and two-dimensional polyacrylamide gel electrophoresis. the approach is based on the use of an isotopically labelled reagent to quantitate (by mass spectrometry) the ratio of peptides from digests of a protein being expressed under different conditions. the method allows quantitation of the changes occurring in spots or bands that contain more than one protein, and has a greater dynamic range than most staining methods. since the reagent carries a fixed positive charge under acidic conditions and labels only the n-terminal of peptides, the interpretation of tandem mass spectra to obtain sequence information is greatly simplified. the sequences can easily be extracted for homology searches instead of using indirect mass spectral based searches and are independent of post-translational modifications. dehydroamino acids and their derivatives play important roles as constituents of various natural products and as synthetic intermediates for the preparation of optically pure amino acids. a large number of amino acid derivatives containing a pyrazol- -yl, isoxazol- -yl and other heterocyclic moieties has been prepared as potential agonists or antagonists for central glutamate receptors in connection with (r,s)- -amino- -( hydroxy- -methylisoxazol- -yl)propanoic acid (ampa), a bioisostere of (s)-glutamic acid. -hetaryl-α, -didehydroalanines might be considered as conformationally constrained ampa analogs and might be potential candidates for the synthesis of novel types of ampa analogs, for example, via their hydrogenation. compounds containing h-pyran- -one ring are also very useful synthons in selective synthesis. recently we have shown their use for the preparation of (e)-α, -didehydroα-amino acid derivatives containing a pyrazolyl moiety (vraničar l, polanc s, kočevar m ( ) tetrahedron : ). as a continuation of our investigation in this field we report here a detailed study of the transformation of h-pyran- -one derivatives with hydroxylamine ( , x ϭ o) and various hydrazines ( , x ϭ nr ) towards novel types of (e)-and (z)α, -didehydroamino acid derivatives . in most cases, the reactions were performed under basic conditions in a mixture of ethanol and pyridine. depending on the substrate and the reagent used the reaction could be controlled to give either (e)-or (z)-isomers; in some cases decarboxylation to the corresponding enamines also occured during the reaction course. some attempts to hydrogenate compounds towards α-amino acid derivaties by homogeneous or heterogeneous catalysis were also performed. analogs of endomorphin and were prepared to investigate the effect of the positional and c-terminal amide replacements and modifications on the biological activity. modifications in position and were studied. in position several hydroxy-and serine related amino acids were incorporated, whereas in position the amide bond was replaced by hydroxymethyl and allyl group. protected peptide derivatives were synthesized on chlorotrityl resin and further transformed to the corresponding derivatives in solution phase. among the analogs tested, in in vitro tests the most effective compound found was d-ser -endomorphin Ϫ . quite surprisingly, the partial agonist/antagonist properties of the derivatives in receptor binding and g-protein stimulation tests have been shown behave differently. the differences in efficacy and receptor binding properties of the compounds may explain the discrepancies between the in vitro and receptor binding tests. we have been assessing the possible applications of substituted h-pyran- -ones containing α, -didehydroamino acid unit in their structure as dienes in [ ϩ ]-cycloaddition reactions. as dienophiles we have been using different acetylene derivatives as well as n-phenylmaleimide and maleic anhydride. as it is evident from the structure of h-pyran- -ones upon the cycloaddition of acetylene derivatives the first intermediate formed ( ) still contains the carbon dioxide bridge. in many cases easily expels co and substituted benzene derivative is produced. when the alkenes are used, the first part of cycloaddition is the same as when acteylene derivatives are used, but after the extrusion of co from the adduct there are two possible paths: so formed cyclohexa- , -diene ( ) is either aromatized into benzene derivative ( ) or it acts as another diene with favourably positioned double bonds and unusual double cycloadducts ( ) are formed. since co -containing adducts are thermally unstable it is advantageous to use high pressure techniques. with the acetylene derivatives we have not been able to isolate co -containing adducts ( ), while with alkenes we have isolated, depending on the structure pattern of the compound , all three types of products: aromatized , co containing and double adducts . especially the type is suitable for further transformations into other heterocycles containing amino acid moiety. research group of peptide chemistry, hungarian academy of sciences, budapest, hungary among the opioid receptors family, the cloning of µ, k and δ receptors was followed by another member, named lc or orl . searching for an endogenous ligand for this receptor resulted in successful identification of a peptide (fggft garksarklanq) called noc or ofq. in vitro and in vivo studies have demonstrated that noc mediates a variety of biological actions. results from structure-activity experiments suggest that the whole sequence of noc is not required for binding to the lc receptor and for full biological activities. noc( - )-oh seem to be the minimum and essential sequence for good interaction with the receptor. this neuropeptide, similarly other peptides, are unresisting for enzymatic degradation and the releasing metabolites are very weakly active or inactive. some previous experiments refer to that the c-terminal amidation may protect the peptide from degradation. we purposed to synthesize carbamoyl analogues of noc( - )-nh , hoping that these derivatives retain the ability to bind lc receptor and are resistant against biological degradation: phe-nh-co--ala-noc( - )-nh phe--ala-nh-co-phe-noc( - )-nh phe-gly-nh-co--hphe-noc( - )-nh the first step in the synthesis of the carbamoyl analogues was the preparation of the building block [r-co-nh-co-nh-hc(rЈ)-cooh] by the classical method and then it was incorporated into the peptide by solid phase peptide synthesis. [ nonproteinogenic amino acids and their derivatives are valuable compounds from their pharmacological and biochemical effects. they can be used also in synthesis of peptides, as biomarkers, as the ligands in catalitically active transition metal complexes and so on. it is possible to prepare such amino acids by asymmetric hydrogenation of their prochiral precursors. however high enantioselektivities was reached only in the case of chiral phosphine-rhodium catalysts. recently we showed that high diastereoselectivity in the hydrogenation of linear dehydrodipeptides may be achieved over achiral catalyst in the catalytic system substrate -salts of ca ore mg -pd/c due to formation of dehydrodipeptides complexes with ions Ñà ϩ or mg ϩ and hence increasing of the conformational rigidity of substrates. this phenomenon may as well happen in other dehydrodipeptides, containing nonproteinogenic amino acids. among unnatural amino acids those bearing heterocyclic rings have attracted considerable attention due to the possibility of the heteroatoms participation in coordination with ions of metals. we have received some n-acyldehydrodipeptides, containing in the prochiral unit of dipeptides nonproteinogenic dehydroamino acids. all this n-acyldehydrodipeptides form in alcohol solution complexes with cax and mgx where one metal ion binds together several (up to ) substrate molecules. this kind of complexation leads to the increase of conformational rigidity and to the diastereoface shielding of cϭc bond. moreover the combination of cations (ca ϩ or mg ϩ ) and anions (x) and the sequence of their mixing with a substrate determine the assembly inside complex particles and hence the sign and degree of asymmetric induction. indeed hydrogenation of these complexes formed in situ over achiral heterogeneous catalyst (pd/c) gives two diastereomers of corresponding n-acyldipeptides with the substantial increase of the reaction diastereoselectivity (up to %). in living cells, glutamine represents one of the main storage forms of nitrogen and is a major physiological source of ammonia for the biosynthesis of aminoacids, aminosugars, purine and pyrimidine nucleotides and coenzymes. glutamine-dependent amidotransferases perform nitrogen transfer from the amide group of glutamine to various electrophiles. when the latter is fructose- p, the product of the reaction catalysed by glucosamine- p synthase is d-glucosamine -phosphate, a structural building block of peptidoglycane (bacteria) and of chitin and mannoproteins (fungi). fluorinated analogues of glutamine are expected to interfere with this biological process due to the strong electron withdrawing effect of fluorine atom (without significant steric consequence), inducing modulation of binding and/or electronic properties. these compounds might therefore behave as reversible or irreversible active site-directed enzyme inhibitors. synthesis of optically active from d-serine will be described and first results in the biological evaluation on glucosamine -phosphate synthase will be included. o. melnyk , d. bonnet , e. loing , l. bourel , and h. gras-masse -umr , -sedac-therapeutics, biological institute of lille, france lipopeptides, owing to their ability to cross passively the cell membrane or biological barriers, are unique tools for the intracellular delivery of bioactive peptides. the structure of the lipophilic moiety is known to have a profound effect upon the interaction with the membrane and its alteration. the stepwise solid phase synthesis of lipopeptides is limited by the necessity to perform a complex rp-hplc purification following the cleavage and deprotection step. in addition, the harsh conditions used during the final acidolysis procedure does not allow the introduction of unsaturated or sensitive fatty acids. to speed up the access to large lipopeptides modified by various fatty acid moieties or cholesterol derivatives, we have designed novel synthetic methods which involve the chemoselective reaction of fully deprotected and purified hydrazinopeptides with fatty acid succinimidyl esters or glyoxylyl derivatives. application of these methodologies to the c-terminal - portion of interferon (ifn)-γ allowed the selection of the optimal lipopeptide ifn-γ agonist, as determined by its ability to induce the expression of surface mhc-ii molecules through interaction with the intracellular components of ifn-γ receptor. graduate school of science, osaka city university, osaka, japan glutamate receptors in mammalian cns are implicated in the construction of memory and early learning as well as in the pathogenesis of neuron damage to cause various neuronal diseases. in recent years, we have studied the conformational role of l-glutamate when it binds to the receptors through the synthesis of l- -(carboxycyclopropyl)glycines (ccgs) and their related analogs. the works have demonstrated that not only the receptors require a specific conformation of l-glutamate, but also these analogs can be used as important tools for the neuropharmacological research. among them, dcg-iv, a Јsubstituted analog of ccg-i, is used as a potent and selective agonist of mglur . as an extension of these works, next program was focused on the synthesis of α-substituted glutamate analogs which would enable to develop potent and subtype-selective ligands for mglurs and transporters. α-alkoxymethylglutamate and ly and its c epimer were chosen for the synthetic targets, since the former slightly restricts the glutamate conformation to an extended form and the latter rigidly fix to an extended or a folded form on its bicyclo[ , , ]hexane skeleton. the key to the synthesis was a stereoselective construction of the quarternary carbon center, which was efficiently performed based on an asymmetric version of the strecker synthesis. details of the synthesis and their neuropharmacological activities will be described. using a genetically modified organism a broad variety of linear unsaturated amino acids are now accessible in enantiomerically pure form via this methodology, which can be used as starting materials for the synthesis of highly functionalized pipecolic acid derivatives. these compounds can be used to restrict conformations in polypeptides or can serve as scaffolds in synthesizing libraries for drug discovery. the synthetic approach involved both a pd-catalyzed amidopalladation reaction of alkoxy-allenes, in which the nh is added across one allene double bond and a ruthenium catalyzed ring closing metathesis step, to form a benzyloxypipecolic acid. further reaction of this n-sulfonyl-iminium-ion precursor with a nucleophile results in the formation of cis-substituted pipecolic acids. due to the unique electronic properties of fluorine, incorporation of α-fluoroalkylated amino acids is a new approach to design biologically active peptides with increased metabolic stability and defined secondary structure and provides a powerful nmr label for spectroscopic investigations. the application of proteases especially for cn-ligations is an attractive alternative to chemical methods, because the enzymatic formation of peptide bonds is highly regio-and stereospecific and, therefore, does not require large efforts to protect side chains of trifunctional amino acids. recently, the enzyme-catalyzed incorporation of α-fluoromethyl amino acids into the p , p and p Ј-position (nomenclature according to schechter and berger) of peptide fragments has been successfully performed. carboxypeptidase y was now shown to be suitable to catalyze the incorporation of α-trifluoromethyl alanine into the p position of peptides. furthermore, the general applicability of the substrate mimetic concept in enzymatic peptide synthesis was expanded to the transfer of c-terminal α-fluoroalkyl substituted amino acids. generally, each trifluoromethyl-and difluoromethyl amino acid guanidinophenyl esters can be applied as acyl donor in trypsin and chymotrypsin catalyzed peptide bond formation independently of the acyl moiety and the natural enzyme specificity, respectively. via these two approaches, incorporation of αfluoroalkylated amino acids into the p position of peptides using enzymatic methods was successfully applied for the first time. this investigation was performed in search of new Јdeoxynucleoside analogues modified at Ј-and Ј-positions with amino acids and possessing antiviral activity. substrate mimetics strategy: an efficient approach to protease-catalyzed peptide ligation n. wehofsky and f. bordusa , max-planck-society, research unit "enzymology of protein folding", halle, and institute of biochemistry, university of leipzig, germany two main drawbacks seriously restrict the synthetic value of proteases as reagents in peptide fragment coupling: ( ) native proteolytic activity and, thus, risk of undesired peptide cleavage; (ii) limited enzyme specificities restricting the amino acid residues between which a peptide bond can be formed. the latter can be overcome by the use of substrate mimetics. contrary to common acyl donors, substrate mimetics bear a binding site specific ester leaving group instead of having a specific amino acid moiety at the c-terminus of the acyl residue. this replacement mediates the acylation of the protease by nonspecific acyl residues. deacylation of the artificial acyl enzyme intermediate by the amino component added results in peptide bond formation regardless of the primary specificity of proteases enabling nonspecific coded and noncoded amino acid derivatives and even non-amino acid-derived acyl moieties to be coupled. the successful application of these artificial substrates for model peptide ligations catalyzed by the argspecific trypsin, the glu-specific staphylococcus aureus strain v protease (v protease), and α-chymotrypsin, which is specific for aromatic amino acid moieties, will be demonstrated. new development in the tritium labelling of peptides and proteins using solid state catalytic isotopic exchange with spillover-tritium yu. a. zolotarev , a. k. dadayan , b. v. vaskovsky , and n. f. myasoedov institute of molecular genetics, russian academy of sciences, and shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, moscow, russia the reaction of high temperature solid state catalytic isotope exchange (hscie) of hydrogen in peptides and proteins with spillover-tritium was studied. the reaction ability of amino fragments in hscie was shown to depend both of their structure and on the availability and the mobility of the polypeptide chain. [ h] peptide analysis using h nmr spectroscopy was carried out, and the modified fragment [ h]actc - (met-glu-his-phe-gly-pro), with molar activity of ci/mmol and [ h] zervamicin iib (ac-trp-ile-gln-iva-ile-trh-aib-leu-aib-leu-hyp-gln-aib-hip-aib-pro-phl, where aib ϭ amino-isobutyric acid) with molar activity of ci/mmol was produced. the obtained preparations completely retained their biological activity. with the -galactosidase protein from termoanaerobacter ethanolicus as example, the interrelation between the protein's tertiary structure and the isotopic label distribution incorporated due to the hscie reaction was used. the labeled protein with the molecular mass of kda was brought to fragmentation by glu-proteinase. peptide fragments were separated by hplc and were identified by maldi mass spectrometry. a correlation between the position of the amino acid fragment in the protein tertiary structure and its reaction ability in the hscie reaction was obtained. data on the retention of thegalactosidase enzymatic activity in condition of tritium label introduction are supplied. taurine chloramine modulates cytokine production by peripheral blood mononuclear cells m. chorą z . y , e. kontny , j. marcinkiewicz , and w. maśliń ski institute of rheumatology, warsaw, and jagiellonian university, cracow, poland objective. proinflammatory cytokines are produced in a cascade fashion, where monocyte-derived tnfα and il- trigger production of il- and il- also in the other cell types. we reported recently that taurine chloramine (tau-cl) inhibits production of the latter cytokines in fibroblast-like synoviocytes. in present study the effect of taurine (tau) and tau-cl on tnfα, il- and il- production was examined. methods. peripheral blood mononuclear cells from healthy volunteers were stimulated with lps ( h) in the presence of tau or tau-cl ( - µm). cytokine production was measured in culture supernatants (secreted) and cell lysates (intracellular) using elisa. results. in lps-stimulated cells both secreted and intracellular il- and il- were inhibited by tau-cl with ic Ϸ µm and µm, respectively. however, tau-cl exerted dual effect on tnfα production, raising it slightly ( . times) at low ( - µm) while reducing it (ic Ϸ µm) at higher concentration. tau did not significantly affect cytokine production. tau-cl modulates proinflammatory cytokine cascade and eventually might down-regulate it when present at high (Ͼ µm) concentration. department of biology, division of general physiology, university of oslo, blindern, norway every living cell must deal with osmotic and hydrostatic pressure changes between its environment and its interior and counteract volume changes. swelling activated channels is one group of effectors in the cell membrane that is important in preventing excessive volume increases by releasing inorganic ions and organic solutes that include taurine. such channels are associated with several physiological processes, but little is known about their activation mechanisms. we have used a rat thyroid cell line (frtl- ) to investigate the activation of a swelling sensitive [ h]taurine efflux pathway. hypo-osmolality and thyrotropin (tsh, µm) increased transiently the rate coefficient for [ h]taurine efflux with a similar pattern of activation. the phosphodiesterase inhibitor -isobutyl- -methyl xanthine ( µm) increased the swelling activated efflux rate coefficient . times above the control level and the camp analogue dibutyryl-camp ( µm) activated the pathway. these results indicate that both swelling and tsh activation of the taurine efflux pathway are mediated by camp. other aspects of the signal transduction pathway will be discussed. based on the inclination of n-chloroamines to disproportionate, the endogenous bactericidal agent n-chlorotaurine (nct), mainly at ph Ͻ , is accompanied by n, ndichlorotaurine (ndct). since pure ndct could be synthesized as crystalline sodium salt, a first evaluation of its chemical and bactericidal properties was possible. ndct-na (melting point: - °c, decomp.) is very well soluble in water and poorly in ethanol where it can be recrystallized from. on storage the initial ph of the aqueous solution decreases which correlates with a decrease of oxidation capacity of . % per day, probably originated by the elimination reaction r-ch -ncl ae r-chϭncl ϩ h ϩ ϩ cl Ϫ as a first step. contrary to nct-na an immediate decomposition occurs when ndct-na comes into contact with undiluted dmso. in aqueous solution, however, ndct does not react with dmso. the bactericidal activity of mm ndct at ph and against the gram-positive bacteria s. epidermidiand two strains of s. aureus was the same as with equimolar nct though ndct bears twice the oxidation capacity. against the gramnegative bacteria e. coli, p. aeruginosa, and p. mirabilis, however, a significantly higher activity of ndct was observed at both ph. the mechanism of taurine chloramine inhibition of fibroblastlike synoviocytes growth e. kontny , m. kurowska , j. kowalczewski , i. janicka , j. marcinkiewicz , and w. maśliń ski institute of rheumatology, warsaw, and jagiellonian university, cracow, poland objective. in rheumatoid arthritis (ra) enhanced proliferation of fibroblast-like synoviocytes (fls) leads to hyperplasia of synovial membrane (sm). therapeutic approaches to inhibit an excessive growth of these cells are not satisfactory. thus, we investigated the effect of taurine (tau) or taurine chloramine (tau-cl) on ra fls growth. methods. fls isolated from sm of ra patients were stimulated for hours with rhpdgf or rhtnf-α. tau or tau-cl were added at - µm concentrations. cell proliferation was determined by incorporation of h-thymidine into dna. expression of proteins regulating cell-cycle progression or apoptosis, was estimated by western blotting. results. at µm concentration tau-cl inhibited (by Ϸ %) both pdgf-and tnf-α-triggered cell proliferation and similarly reduced expression of pcna (a cofactor for dna polimerase δ). however, tau-cl affected neither the expression of cell-cycle inhibitors (p , p ) nor anti-apoptotic bcl- protein. tau has no effect on tested responses. conclusion. we report that tau-cl inhibits proliferation of ra fls by affecting expression of pcna, that is critical for cell cycle progression. e. kontny , k. szczepań ska , j. kowalczewski , m. kurowska , i. janicka , j. marcinkiewicz , and w. maśliń ski institute of rheumatology, warsaw, and jagiellonian university, cracow, poland objective. proinflammatory cytokines play critical role in the pathogenesis of rheumatoid arthritis (ra). we reported recently that taurine chloramine (tau-cl), but not taurine (tau), inhibits production of il- and il- by fibroblast-like synoviocytes (fls). in present study the mechanism of tau-cl inhibitory action was investigated. methods. fls isolated from synovial membrane of ra patients were stimulated with rhil- . tau or tau-cl were added at - µm concentration. after . - h or h the dna binding activity of nfkb and ap- (emsa) and the expression of il- and il- mrnas (rt-pcr) was examined, respectively. results. il- raised nfkb and ap- activity, followed by the elevation of cytokine mrnas expression. tau-cl, but not tau, reduced both the expression of cytokine mrnas (il- Ͼ il- ) and the activity of transcription factors (nfkb Ͼ ap- ). conclusion. tau-cl inhibits transcription of il- and il- genes due to its ability to diminish the activity of key transcriptional factors, that regulate these proinflammatory cytokine expression. institut für organische chemie, universität bremen, germany the synthesis of taurine and hypotaurine from cysteine can be followed up in astroglia-rich primary cultures obtained from brain of neonatal wistar rats. using h and c nuclear magnetic resonance spectroscopy cell extracts of glia cells incubated with c labelled cystein show the label subsequently in hypotaurine, taurine, lactate and gluthathione. within h, % of the total intracellular hypotaurine and . % of taurine were newly synthesized from cysteine. both metabolites were also released to the medium. neurones are capable to take up both metabolites from glia media to recruit their organic osmolite. part of newly synthesized glutathione and lactate are also exported to the medium. by this means lactate may serve as an energy substrate for neurons. in-vivo mrs of lactate is obscured by line splitting and signal overlay. using various two dimensional pulse sequences as spin preparation sequences prior to localized single voxel, in-vivo mrs or spectroscopic imaging sequences will provide homonuclear non-coupled resonance signal of taurine. these singlet signals are detectable and quantified. diffusion weighted spectroscopy is used to characterize the mobility of taurine in living tissue. department of pharmacology and ophthalmology and visual sciences, texas tech university health sciences center, lubbock, texas, u.s.a. taurine depletion, whether by removing taurine from the diet or by using a taurine transport inhibitor, has demonstrated various pathologies in various animal models including man. the first reported pathology associated with dietary taurine depletion was in the retina of the cat. in this animal model, taurine deficiency resulted in disorganization of the tapetum (the light reflecting membrane), disruption of the outer segments, photoreceptor dysfunction, and cell loss. when allowed to proceed for a number of months the result was blindness. subsequent studies demonstrated that taurine deficiency also had a profound effect on cardiac physiology. echocardiograms of the left ventricle of the cat heart depleted in taurine showed a dilated cardiomyopathy reflected in an extended end-diastolic diameter and an extended end-systolic diameter. dietary taurine supplementation resulted in the above parameters returning to normal. the cat is a difficult animal model to use for a variety of reasons and thus the rat was chosen to further probe the consequences of taurine depletion. unfortunately, the tissue taurine levels in the rat do not respond to dietary taurine depletion, and thus other experimental means had to be designed. guanidinoethanesulfonic acid (ges), an analogue of taurine and a taurine transport inhibitor, has been utilized for the last years to deplete rat tissues of their taurine content (j. e. shaffer and j. j. kocsis, methods of reducing tissue taurine levels, and r. j. huxtable, h. e. laird, and s. lippincott, rapid depletion of tissue taurine content by guanidinoethyl sulfonate. in: the effects of taurine on excitable tissues, spectrum publications, new york, ) . ges, when administered to rats in their diet in the drinking water as a - . % solution, usually produces a significant decrease in the taurine content of all tissues within one week of treatment. within - weeks the levels of taurine reach their nadir ( - % of control) and continued feeding of ges does not further reduce the levels of taurine. unfortunately, ges replaces taurine and thus one must always consider the effects of ges on physiological events that occur within the tissues in question. again, as in the cat, taurine depletion manifested itself in retinal pathology: disruption of the photoreceptor structure, dissociation of the disc membranes, and abnormal electroretinograms (erg). other animals models such as the monkey have also demonstrated structural disorganization of the photoreceptors and abnormal ergs. finally, the ultimate test is whether taurine deficiency has an effect in man. in , koppel and associates (geggel et al., n. eng. j. med. : - , ) demonstrated that children on long term parenteral nutrition devoid of taurine had abnormal erg. supplementation of the parenteral nutrition with taurine restored the ergs to normal in the majority of the children. because of these definitive studies, all infant formulae in the united states, europe and japan now contain taurine. (supported in part by a grant from the taisho pharmaceutical co., ltd., tokyo, japan.) department of pharmacology and ophthalmology and visual sciences, texas tech university health sciences center, lubbock, texas, u.s.a. it has been demonstrated previously in our laboratory that taurine inhibits the phosphorylation of an ϳ kdalton protein present in the mitochondrial fraction of the rat heart (j. mol. cell. cardiol. : - , . upon administering . % guanidinoethanesulfonic acid (ges) in the drinking water of rats for weeks, the taurine levels in cardiac tissue decline by %. however, the phosphorylation of a ϳ kdalton protein in the mitochondrial fraction of the heart tissue increased by % (j. mol. cell. cardiol. : - , ) . reversal of these effects could be accomplished by feeding the rat . % taurine in the drinking water for weeks. the ϳ kdalton protein was isolated by -dimensional polyacrylamide gel electrophoresis (page) using traditional glycine buffers followed by re-electrophoresing the cut out portion of the gel, which corresponds to the ϳ kdalton protein, on a tricine-buffered gel resulting in sufficient pure protein for digestion and sequence analysis. it was determined that the ϳ kdalton was pyruvate dehydrogenase (amino acids : - , ) which indicates a significant regulatory role for taurine in energy metabolism in cardiac tissue. these data are of significant interest in that taurine may be an additional effector of this enzyme or of the enzyme complex. studies are in progress to determine if taurine has a direct effect on either the kinase (inhibition) or the phosphatase (stimulation) associated with the pyruvate dehydrogenase complex. it has also been demonstrated and now reported that taurine depletion utilizing ges in vivo in rats affects the phosphorylation of myelin basic protein (mbp). in these experiments the animals were given ges ( %) for weeks in their water and then killed; the hearts were removed and homogenized. the homogenate was then incubated with buffer containing mbp ( ì) and radioactive atp for minutes. animals were also treated with taurine ( %) in their drinking water for - weeks or treated with taurine following the ges treatment. page of the incubation mixture, autoradiography on the dried gel, and densitometry of the mbp band gave the following results: relative % activity Ϯ sem (normalized to mg protein) control Ϯ (n ϭ ) ges-treated Ϯ (n ϭ ) p Ͻ . (paired -test) control Ϯ (n ϭ ) taurine-treated Ϯ (n ϭ ) p Ͼ . control Ϯ (n ϭ ) ges followed by taurine Ϯ (n ϭ ) p Ͼ . these data confirm previous reports that it is easier to deplete animals of their cardiac taurine content than it is to raise the levels of taurine. these data on the effects of taurine depletion (increase in mapkinase activity) and taurine supplementation (no change in mapkinase activity) on mapkinase activity reflect these past observations. (supported in part by a grant from the taisho pharmaceutical co., ltd., tokyo, japan.) act additively, or in the case of mpo negated each other's effects. regarding our results there is significance to pharmacological regimens which enhance the supply of propofol or taurine in whole blood. these regimens influence considerably pmn intracellular amino acid concentrations and it is this pmn "labile free amino acid pool" which may be one of the determinants in cell nutrition positively or adversely affecting pmn immune functions. taurine supplementation to pmn seems to interfere independently from the effects of propofol on pmn free amino acids and on immune functions tested. institut für hygiene, universität innsbruck, austria n-chlorotaurine (nct) is a long-lived oxidant produced by activated human leukocytes during the oxidative burst. it has activity against a broad spectrum of pathogens including bacteria, fungi, viruses and helminths. as a special feature, the killing of microbes by nct can be increased significantly in the presence of ammonium and also of some amino acids (alanine, glycine). this is explained by transfer of the active chlorine ("transhalogenation") from nct to ammonium and amino acids to form the corresponding, stronger microbicidal n-chloro derivatives monochloramine and n-chloro amino acids, respectively. especially addition of ammonium to nct provokes rapid inactivation of fungi and even mycobacteria. because of its good tolerability, nct solution can be applied to human tissue to treat infections. in ammoniumcontaining body fluids like nasal mucus and urine, fungi and bacteria are killed within minutes. therefore, amino compounds of human secretions can be transformed to the above quoted endogenous and highly microbicidal chloramines by nct via transhalogenation -a unique property of an antimicrobial agent. successful treatment in cases of urinary tract and otorhinolaryngological infections and conjunctivitis in phase iia clinical trials provides strong support for this concept. the endogenous sulfonated amino acid taurine has numerous functions in the central nervous system, including positive modulation of gaba a receptor function. recently we found that mice lacking protein kinase c -epsilon (pkcε) are behaviorally and biochemically supersensitive to ethanol and other positive allosteric modulators of the gaba a receptor. in addition, these mice consume - % less ethanol and wildtype controls in two separate self-administration paradigms. microdialysis studies in pkcε-deficient mice revealed elevated extracellular levels of taurine, which may account for the supersensitivity of gaba a receptors in these mice and resulting decreases in ethanol intake. in light of the fact that the taurine derivative acamprosate (calcium acetylhornotaurinate) is moderately effective in reducing craving and relapse in detoxified alcoholics, we examined the effect of taurine-related compounds on acute ethanol consumption in a two-bottle choice paradigm in rats. taurine ( - mg/kg ip) was only slightly effective in reducing ethanol intake but not preference, while the highest dose of taurine ( mg/kg) also suppressed water intake. the taurine precursor hypotaurine ( - mg/kg ip) was also weakly effective in reducing ethanol intake but not preference or water intake. the most effective compound tested was homotaurine ( - mg/kg ip), which suppressed ethanol intake and preference by approximately % without altering water intake. these data indicate that endogenous taurine may regulate sensitivity to ethanol and subsequent ethanol self-administration, and that taurine-related compounds may be effective in reducing alcohol intake in humans. we are currently exploring whether taurine and related compounds are able to suppress ethanol-stimulated mesolimbic dopamine release, a primary neural substrate of ethanol reinforcement. (this work was supported by funds provided by the state of california for medical research on alcohol and substance abuse through the university of california at san francisco.) organic osmolytes, such as taurine, regulate a cell's osmotic balance without directly altering either the cell's ionic composition or the membrane potential. this property of the organic osmolyte often renders the cell resistant to damage during a pathological insult. indeed, ischemia is associated with a massive efflux of taurine from the cell, an event that minimizes the severity of the osmotic imbalance that develops from the accumulation of lactate, inorganic phosphate and sodium. however, taurine depletion also activates specific signaling pathways that provide further protection to the cell. among the signaling pathways activated by taurine depletion is a pi -kinase (phosphatidylinositol -kinase) linked pathway that catalyzes the phosphorylation and inactivation of the pro-apoptotic factor, bad. taurine depletion also activates protein kinase c, which in turn elevates the intracellular content of the antiapoptotic factor, bcl- . increases in the extracellular osmolality by either addition of mm taurine or mm mannitol to the incubation medium activates similar pathways. however, pi kinase assumes a more important role in the mannitol treated cell than the taurine depleted cell. moreover, p map kinase is activated by mannitol treatment but not by taurine depletion. despite these differences, both taurine depletion and mannitol treatment protect the cell against hypoxia-induced apoptosis. the data suggest that osmotic stress protects the cell against apoptosis by increasing cellular levels of bcl- and promoting the inactivation of bad. this work was supported by a grant from the american heart association. a dummy or a protagonist on the stage of inflammation? r&d department, zambon group, bresso, milan, italy amino acids are usually present in large excess in healthy and the excess is used as source of calories. however, metabolic alterations are observed in ill patients and preferential retention of sulphur amino acids (saa) occurs during the inflammatory response. the metabolism of cysteine is modified during the acute phase of sepsis in rats. sulphate production is lower, whereas the higher liver production of taurine seems to play a protective role; glutathione concentration is greater in liver, kidney and other organs and cysteine incorporation into proteins was higher in spleen, lung and plasma (acute phase proteins) while albumin level decreases. another important phenomenon is the impairment of methionine conversion to cysteine during stressed condition. premature infants or hiv patients synthesise cysteine from methionine at a much lower rate. thus, the metabolic flow through the trans-sulphuration pathway may be insufficient to meet the glutathione and cysteine requirement in critical conditions. the pro-inflammatory cytokines, interleukin- , interleukin- and tnf-α are the main initiates that alter protein and amino acid metabolism. in this complex picture, saa supply may contribute to the immune system regulation. department of applied biological chemistry, the university of tokyo, japan the intracellular level of taurine is maintained not only by the taurine transporter that transports extracellular taurine inside cells but also by endogenous synthesis from methionine and cysteine. we therefore investigated the regulation of both the taurine transporter and the cysteine dioxygenase, one of the main taurine biosynthetic enzymes, in hepg human liver cells. the intracellular taurine content of hepg cells was extremely increased by culturing in a hypertonic medium. the activity of taurine transport was increased by hypertonic conditions, which was due to the increased expression of the taurine transporter gene. the expression level of the cysteine dioxygenase gene was also increased, suggesting that the expression levels of both the taurine transporter gene and the cysteine dioxygenase gene were regulated in harmony by hypertonic conditions to accumulate taurine inside cells. on the other hand, the activity of taurine transport in hepg cells was down-regulated on culturing the cells in taurine-rich medium, the expression level of the taurine transporter gene being also markedly decreased. however, the expression level of the cysteine dioxygenase gene was not significantly altered under taurine-rich conditions, indicating that the gene expression of the taurine transporter and that of the cysteine dioxygenase was independently regulated by extracellular concentration of taurine. the amino acid, taurine, is found in very high concentration in the heart. although its most important putative function is osmoregulation, it also serves as a regulator of cell growth. isolated cardiomyocytes exposed to medium containing nm angiotensin ii undergo hypertrophy, a process blocked by mm taurine. the amino acid also inhibits angiotensin iiinduced activation of c-fos, upregulation of atrial natriuretic factor and induction of tgf-betal. central to virtually all of these actions of angiotensin ii is the translocation and activation of key protein kinase c (pkc) isoforms. therefore, we proposed that taurine inhibited the hypertrophic actions of angiotensin ii by interfering with the translocation of one or more of the pkc isoforms. indeed, taurine and angiotensin ii exhibited different effects on the translocation of several pkc isoforms. while taurine promoted the translocation of pkcalpha, pkcdelta and pkcepsilon from the particulate fraction to the cytosol, the levels of the three isoforms in the particulate fraction were elevated following treatment with angiotensin ii. by contrast, both taurine and angiotensin ii increased the pkczeta content of the particulate fraction and the pkcbeta content of the cytosol. when the isolated cardiomyocytes were incubated with medium containing both angiotensin ii and taurine, the effects on pkc distribution were largely additive. these data support the notion that taurine prevents the hypertrophic effects of angiotensin ii by interfering with the translocation of either pkcalpha, pkcdelta, pkcepsilon or a combination of more than one of the isoforms. (the study was supported by a grant from taisho pharmaceutical co.) main final metabolites of l-cysteine in mammals are sulfate and taurine, and they are excreted in the urine. our previous studies in rats have shown that the ratio of urinary sulfate and taurine in rats fed diet containing sufficient methionine and cysteine is : - . in the present study, we determined urinary sulfate and taurine in urine samples of healthy japanese women after h starvation following usual meal. free (inorganic) and total (free ϩ ester) sulfate were determined with ion chromatography, and taurine by reversed-phase hplc after dabsylation. average excretions (micromols per mg of creatinine) were: total sulfate, . Ϯ . ; free sulfate, . Ϯ . ; ester sulfate, . Ϯ . ; taurine, . Ϯ . ; urea, . Ϯ . . the ratio of total sulfate and taurine was : . . this suggests that sulfate formation in humans is more dominant than taurine formation as in rats and this tendency is more evident in humans than in rats, which is in accordance with low cysteinesulfinate decarboxylase activity in humans. sum of sulfate and taurine excretions was significantly correlated with that of urea: correlation coefficient, . . this indicates that sulfur metabolism in humans is in the state of sulfur equilibrium similar to that of nitrogen and reflects protein metabolism. h. yokogoshi and h. oda laboratory of nutritional biochemistry, school of food and nutritional sciences, the university of shizuoka, and department of applied biological sciences, nagoya university, nagoya, japan the effect of taurine on hypercholesterolemia induced by feeding a high-cholesterol (hc) diet to rats was examined. when various amounts of taurine ( . - g/kg) were supplemented to hc for wk, serum total cholesterol gradually and significantly decreased in a dose-dependent manner, compared with the control (cholesterol free) diet group. by contrast, serum hdl-cholesterol was elevated by taurine supplementation. in the hypercholesterolemic rats fed the hc diet, the excretion of fecal bile acids and hepatic cholesterol α-hydroxylase (cyp a ) activity and its mrna level increased significantly, and the supplementation of taurine further enhances these indexes, indicating an increase in cholesterol degradation. agarose gel electrophoresis revealed that, in hypercholesterolemic rats fed the hc diet, the serum level of the heavier vldl increased significantly, but taurine repressed this increase and normalized this pattern. significant correlations were observed between the time-and dose-dependent increases of cyp a gene expression and the decrease of blood cholesterol concentration in rats fed the hc diet supplemented with taurine. these results suggest that the hypocholesterolemic effects of taurine observed in the hypercholesterolemic rats fed the hc diet were mainly due to the enhancement of cholesterol degradation and the excretion of bile acid. in vitro studies have shown that ammonia, which is responsible for neurological symptoms associated with hyperamonemia, causes a massive release of taurine from cultured cns cells and brain slices. in this study, taurine (tau) release was measured in vivo in rat striatum following direct application to the microdialysis tube of mm ammonium chloride which renders the final ammonia concentration in the extracellular space of ϳ mm. various in vivo stimuli evoke taurine efflux either by opening osmosensitive anion channels and/or by a mechanism secondary to glu accumulation and its interaction with nmda or ampa receptors. the following compounds were coadministered with ammonia to distinguish between these mechanisms: anion/cation transport inhibitors -dids and furosemide, a glu transport inhibitor-pdc, and nmda and ampa/ka receptor antagonists dizocilpine and dnqx. ammonia stimulated tau accumulation in the microdialysates to ϳ % of basal value. dids and furosemide moderately inhibited the effect of ammonia (furosemide by ϳ %), albeit dids added alone induced massive accumulation of tau with a delayed onset as compared to ammonia. ammonia-dependent tau accumulation was increased by ϳ % in the presence of pdc and reduced in an equal degree (ϳ %) by dizocilpine and dnqx. none of the agents affected tau accumulation in the absence of ammonia. the results show that ammonia in vivo evokes tau accumulation both via anion channels, possibly secondary to cell volume changes, and in consequence of stimulation of both nmda and ampa/ka receptors. (supp. by a scsr grant no p a and cimo, the acad. of finland) biosciences department, university of hertfordshire, hatfield herts, u.k. the discovery in that endothelium-derived relaxing factor is nitric oxide (no) was followed a year later with reports that the cationic amino acid l-arginine is the physiological precursor for nitric oxide. it has since been established that the terminal guanidinium nitrogen of l-arginine is metabolised via a series of oxidation reactions resulting in no production, with citrulline being formed as a co-product. of interest was the parallel observation that uptake of l-arginine was enhanced in inos expressing cells and that this was due to de novo synthesis of carrier proteins. the precise signaling pathway that regulates the enhanced expression of these carriers has been the subject of intense studies in recent years. current literature suggests that activation of upstream signaling molecules such as protein kinase c may be critical. in addition, downstream kinases thought to be points of convergence for various signals originating from cell surface receptors have also been implicated. two of these downstream targets include the and kda forms of mitogen-activated protein kinase (mapk) and the stressactivated kda mapk. it is worth noting however that the involvement of these different transduction pathways in the regulation of the induction of l-arginine transporters is not universal, and likely to be different from system to system. as a result there has been conflicting data on the relevance of these signaling proteins in inducing l-arginine transport in different cell. these issues will be discussed and the individual signaling pathways assessed on a cell type and species basis. moreover, the role of downstream signaling molecules will be examined in more detail, looking in particular at the critical dependency on the p mapk. this kinase currently exists in four different isoforms which are p α, , γ and δ. the involvement of individual isoforms of p in enhancing the expression of carrier proteins for l-arginine will be discussed. gw is an acetamidine derivative of heterosubstituted lysine which has been shown to have a marked selectivity for the human inducible nitric oxide synthase isoform (young et al. . bioorg. med. chem. lett., : , - ) . the systems associated with transport of this compound have been investigated using the macrophage cell line j . prior to each study, j cells were seeded in -well culture plates and allowed to adhere for h in dulbecco's modified eagle's medium (dmem). transport studies were carried out using hepes buffered krebs solution ( µl; °c) containing l-[ c]gw ( µciml Ϫ ) in the presence of either . mm or . - mm unlabelled substrate. in parallel studies transport ( µciml Ϫ , . mm) was monitored in the presence of mm excess of various other amino acids known to be substrates for distinct transport systems. time course experiments revealed that transport of . mm of l-[ c]gw occurred in a time-dependent manner and was linear for up to min. in addition, uptake was only marginally dependent on extracellular na ϩ . kinetic studies revealed that transport was saturable, and michaelis-menten analysis revealed single affinity entry with an apparent k t of . mm and v max of . pmol·µg protein Ϫ min Ϫ . at mm, -methylaminoisobutyric acid (meaib), lalanine, l-valine and - -amino-bicyclo-( , , )-heptane- carboxylic acid (bch) caused little or no inhibition of l-[ c]gw ( . mm) uptake. in contrast, transport of l-[ c]gw was inhibited markedly by l-arginine, llysine, l-leucine, l-methionine, -diazo- -oxo-l-norleucine (don) and l-glutamine. with the exception of l-arginine and l-lysine, the inhibition caused by the other substrates was critically dependent on extracellular na ϩ and was completely reversed when extracellular na ϩ was replaced with choline. in parallel kinetic inhibition experiments, transport of . mm l-[ c]gw was inhibited in a concentration dependent manner by l-arginine (ki ϭ . mm), l-leucine (ki ϭ . ), don (ki ϭ . mm) and l-glutamine (ki ϭ . mm). taken together, these data suggest that gw may be transported, at least in part, by system y ϩ . however, the marked inhibition caused by l-leucine, l-glutamine and l-methionine, substrates for the relatively high affinity cationic amino acid transporter system y ϩ l, would suggest that this system may also contribute to the uptake of gw ; if so, the monophasic substrate kinetics imply that the two systems handle gw with similar affinity. other systems such as b ,ϩ could be ruled out on the grounds that this transporter is critically na ϩ -dependent while uptake of gw is largely (ϳ %) na ϩ -independent. similarly, b ,ϩ , another broadspectrum aminio acid transporter that may be capable of transporting gw does not interact with l-glutamine and thus unlikely to be involved in transport of gw , at least in j cells. although a large number of different amino acid transporters have been identified on a molecular basis, some of themfunctionally described in mammalian cells -are still missing. in search of mammalian est sequences, which contain the signature of the aaap (amino acid/auxin permease) family, we identified a murine full length cdna, which encodes a membrane protein with - putative transmembrane domains. the transporter mrna is expressed in various murine tissues, including lung, heart and kidney. for functional characterization we used the xenopus laevis oocyte expression system and employed flux studies and electrophysiological analysis. oocytes injected with the crna showed an increased uptake of h-l-alanine and h-l-proline. detailed electrophysiological analysis revealed an electrogenic transport mode, independent of sodium and chloride ions. lowering the extracellular ph increased significantly substrate induced currents in crna injected oocytes. out of the proteinogenic amino acids the transporter recognizes only small amino acids, such as gly, ala, pro and ser. distinct structural analogues of these amino acids also interact with the transporters substrate binding site. in conclusion, we describe the molecular and functional characteristics of the first electrogenic proton driven amino acid transporter of mammals. pharmacology department, dr. willmar schwabe gmbh, karlsruhe, germany it is now well established that transport of amino acid neurotransmitters (like glutamate, aspartate, gaba and glycine etc.) from and to the neurones is essential for their proper functioning. like in the case of other neurotransmitters, specific pre-and post-synaptic as well as vesicular transporters are involved in such processes. extensive efforts to clarify the mechanisms and processes involved in the control and/or proper functioning of the amino acid transporters are now, therefore, being made in numerous laboratories. such efforts have not only led to the identification of a few specific ligands and/or modulators of neuronal amino acid transporters, but also have started unravelling the complex and diverse processes regulating their functions. aim of this communication is to point out potential usefulness of some neuroactive constituents isolated from therapeutically used medicinal herbs for clarifying the mechanisms involved in neuronal amino acid transport. our interest in such studies was initially triggered by the observations made with hyperforin, i.e. quantitatively the major neuroactive component of hypericum perforatum extracts widely used for the treatment of mild to moderate depressive disorders. this acyl phloglucinol derivative not only modulate synaptic transports of biogenic amines but also of glutamate, aspartate and gaba. since it does not interact with any of the till now described transporters for these neurotransmitters, efforts were made to clarify the mechanisms involved in their observed effects (both in vitro and as well as in vivo). the results of the in vitro studies available to date strongly suggest that its effects on neuronal amino acid transport processes is mediated via some novel extracellular mechanism controlling the h ϩ (and/or other ionic) concentrations of neurones. these observations not only demonstrate that hyperforin represent a structurally and mechanistically novel class of therapeutically useful agent but also suggest that it could be useful tool for clarifying the complex mechanisms involved in the control of neuronal amino acid transport. these observations stimulated us to screen other putative psychoactive herbal extracts and their active constituents on neuronal amino acid transport and on the consequences of disturbances caused by malfunction of specific transporters. observations made with several such agents indicate that either modulation of mechanisms and/or processes involved in neuronal amino acid transport or reversal of pathologies caused by anomaly of transporter functions could be involved in their modes of actions. these observations reinforce our conviction that studies directed towards clarifying the effects of herbal constituents on neuronal amino acid transport might not only be a feasible way for identifying novel types of therapeutically interesting molecules but also could expedite our knowledge on these complex processes. glutamate-regulated sodium dynamics in cortical astrocytes: implications for cellular bioenergetics j.-y. chatton, p. marquet, and p. j. magistretti the mode of na ϩ entry and the dynamics of intracellular na ϩ concentration (na ϩ i ) changes consecutive to the application of the neurotransmitter glutamate were investigated in mouse cortical astrocytes in primary culture by video fluorescence microscopy. an elevation of na ϩ i was evoked by glutamate, whose amplitude and initial rate were concentration-dependent. the glutamate-evoked na ϩ increase was primarily due to na ϩ -glutamate cotransport. the rate of na ϩ influx decreased during glutamate application, with kinetics that correlate well with the increase in na ϩ i and which depend on the extracellular concentration of glutamate. a tight coupling between na ϩ entry and na ϩ /k ϩ atpase activity was revealed by the massive na ϩ i increase evoked by glutamate when pump activity was inhibited by ouabain. during prolonged glutamate application, na ϩ i remains elevated at a new steady-state where na ϩ influx through the transporter matches na ϩ extrusion through the na ϩ /k ϩ atpase. a mathematical model of the dynamics of na ϩ i homeostasis will be presented which precisely defines the critical role of na ϩ influx kinetics on the establishment of the elevated steady-state and its consequences on the cellular bioenergetics. indeed, extracellular glutamate concentrations as low as µm approximately doubled the energetic demands of the astrocytes. department of biochemistry and molecular biology, faculty of biology, university of barcelona, spain in the last years a new family of amino acid transporters composed by two different subunits has been defined. two heavy subunits (rbat and f hc) and seven light subunits are known. rbat and the light subunits b ,ϩ at and y ϩ lat are responsible for the inherited aminoacidurias type i cystinuria, non-type i cystinuria and lysinuric protein intolerance, respectively. the heavy subunits are highly glycosylated type ii proteins, while light subunits are very hydrophobic unglycosylated membrane proteins, displaying a polytopic (generally transmembrane domains) predicted structure. the specificity of the amino acid transport activity depends on the light chain expressed. this, together with its topology, indicates that the transport function mainly relies on the light subunits. i will summarize some of our current studies directed to the understanding of structure-function relationships of these heteromeric carriers, specially concerning their oligomeric structure and initial attempts to reconstitute them. ongoing work on the isolation of new rbat-associated light subunits and new b ,ϩ at-associated heavy subunits, which could also play a role in cystinuria, will also be discussed. department of pharmacology, joh. gutenberg university, mainz, germany mammalian cationic amino acid transporters (cats) catalyze the transport of basic amino acids through the plasma membrane. the cat family comprises at least five related carrier proteins (cat- , - a, - b, - and - ) with cat- a and - b being splice variants. in humans, only the "old" members of the family have been characterized (hcat- , - a and - b). hcat- and - b exhibit high affinity for cationic amino acids and are sensitive to trans-stimulation, consistent with the classical system y ϩ . in contrast, hcat- a is a low affinity carrier relatively insensitive to trans-stimulation. interestingly, hcat- a and hcat- b differ only in a region of amino acids. cat- , so far only identified in rat and mouse, exhibits also system y ϩ activity. however, the substrate recognition and maximal transport activity seems to differ from other y ϩ transporters. cat- expression has been reported to be restricted to the brain in adult animals. a cdna encoding for human hcat- has recently been isolated, however, the transport activity of hcat- has not been characterized. when optimally aligned, the amino acid sequence of hcat- shows only about % identity with the other hcat isoforms. in contrast, the amino acid sequences of hcat- , - (a or b) and - are about % identical. to elucidate which amino acids are responsible for the difference in the transport properties of the hcat proteins, we constructed chimeric proteins between hcat- and hcat- a and performed site directed mutagenesis. using this approach, we identified two amino acid residues that are responsible for the different transport properties of hcat- a compared to the high affinity cat-isoforms. to characterize the human cat- , we cloned a cdna encoding hcat- . when expressed in xenopus laevis oocytes, hcat- had a similar transport activity and affinity for l-arginine as hcat- or - b. hcat- mediated l-arginine transport was trans-stimulated and independent of extracellular na ϩ ions. expression studies demonstrated that hcat- is not only expressed in different regions of the human brain, but also in peripheral tissues. to investigate if hcat- also functions as an amino acid transporter, we measured the transport of cationic, neutral and acidic amino acids in xenopus laevis oocytes expressing hcat- , but could not detect an transport activity for any substrate tested. a bright fluorescence could be detected in the plasma membrane of oocytes expressing hcat- with the green fluorescent protein attached to the c-terminus. therefore, hcat- might either need a complementary protein to function as an amino acid transporter or serve as a transporter for a yet unidentified substrate. renal amino acid reabsorption in immature and adult rats as a sensitive marker of heavy metal-induced nephrotoxicity (pt, cr, tl) institut für pharmakologie und toxikologie, klinikum der friedrich-schiller-universität jena, germany the effects of cis-platinum (cp; . mg/ g b. wt. i. p.), sodium dichromate (cr; mg/ g b. wt. s. c.) and tl so (tl, mg/ g b. wt. i. p.) on renal amino acid excretion and plasma amino acid composition were investigated in -(both sexes) and -day-old (female) anaesthetised wistar rats (han : wist). on the basis of diuresis experiments on conscious rats (determination of urinary volume and protein excretion) the mentioned doses and times ( st day after cr in both age groups and in -day-old rats after cp and rd day after cp in adult rats; nd [ -day-old rats] and th - th day [ -day-old rats] after tl) were found out to be optimal for the characterisation of amino acid transport after heavy metal poisoning. interestingly, in conscious -day-old rats cr nephrotoxicity is not detectable after mg/ g b. wt. whereas all of the other experimental groups showed nephrotoxic effects of cr, tl and cp in conscious rats. urine volumes were lower, but not significantly, in anaesthetised immature rats, independently of the administered nephrotoxin. glomerular filtration rate (gfr) is significantly lower in -day-old rats compared to adults. after cp, cr and tl gfr is significantly reduced only in adult rats and age differences disappeared nearly completely. in principle the renal fractional excretion (fe aa ) of amino acids was distinctly higher in immature rats as a sign of lower amino acid reabsorption capacity. nevertheless, the amino acid plasma concentrations were relatively high in immature control rats. however, both cr and cp did not distinctly influence molecular cloning and functional characterization of ata , a novel subtype of the amino acid transport system a medical college of georgia, augusta, georgia, u.s.a. recent molecular cloning studies have revealed that the amino acid transport system a consists of more than one subtype. two different system a subtypes, called ata and ata , have been cloned and functionally characterized. ata is expressed primarily in the brain and placenta whereas ata is expressed ubiquitously. heterologous expression studies have shown that these two subtypes cannot be distinguished functionally based on substrate affinity nor substrate specificity. we have now cloned a third subtype of system a, designated ata . it is expressed primarily in the liver. apart from the liver, detectable level of expression is noted only in the skeletal muscle. interestingly, ata can be easily differentiated from the other two subtypes of system a based on functional characteristics. we first isolated rat ata cdna from a skeletal muscle cdna library using rat ata cdna as the probe. rat ata consists of amino acids and exhibits a high degree of homology in amino acid sequence to rat ata ( % identity) and rat ata ( % identity). interestingly, this new transporter also has a comparable degree of homology to sn and sn , the two known subtypes of the amino acid transport system n. however, when expressed heterologously in xenopus laevis oocytes, rat ata transports α-(methylamino)isobutyric acid (meaib), a specific model substrate for system a, confirming that this transporter is definitely a subtype of system a. system n does not transport this system a model substrate. with two-microelectrode voltage-clamp technique, we have shown that exposure of rat ata -expressing oocytes to neutral, short-chain aliphatic amino acids induces inward currents. the amino acid-induced current is na ϩ -dependent and phdependent. analysis of the currents with alanine as the substrate has shown that k . for alanine (i.e., concentration of the amino acid yielding half-maximal current) is . Ϯ . mm and that the na ϩ : alanine stoichiometry is : . subsequently, we have cloned the human homolog of rat ata from a liver cell line (hepg ) cdna library. human ata also contains amino acids and shows % identity in amino acid sequence with rat ata . the sequence identity of human ata with human ata and human ata is % and %, respectively. the homology of human ata with human sn and sn is also similar ( % and % identity, respectively). the gene coding for human ata contains exons and is located on chromosome p . in the human, ata is expressed almost exclusively in the liver. when expressed in mammalian cells heterologously, human ata mediates the transport of neutral amino acids, including meaib, in a na ϩ -dependent manner. interestingly, while characterizing the function of this clone, we have uncovered a unique feature of this system a subtype. human ata is capable of mediating the transport of cationic amino acids. in fact, the affinity of human ata for cationic amino acids is higher than for neutral amino acids. however, the human ata -mediated cationic amino acid transport is na ϩ -independent. in this respect, ata is similar to transport system y ϩ l that also transports neutral amino acids in a na ϩ -coupled manner and cationic amino acids in a na ϩindependent manner. in contrast, ata and ata have not been shown to interact with cationic amino acids. in addition to this difference in substrate specificity, ata also differs from ata and ata in substrate affinity. ata and ata interact with meaib with a k t of ϳ . mm whereas the affinity of ata for this model substrate is comparatively at least -fold lower (k t , ϳ mm). but, ata interacts with arginine with a k t value of . mm. since liver does not express any of the previously known high affinity cationic amino acid transporters, amino acid plasma concentrations. but in both age groups the administration of cr and cp significantly decreased amino acid reabsorption capacity (increase in fe aa ) as a sign of nephrotoxicity, most pronounced in adult rats after cp. on the other hand, after tl, the fe of amino acids was distinctly higher only in adult rats as a sign of lower amino acid reabsorption capacity and, thus, as a sign of higher nephrotoxicity. in immature animals fe aa was increased only for few amino acids. however, in both age groups tl administration significantly decreased plasma amino acid concentrations, more pronounced in immature rats. the investigation of renal amino acid handling confirmed: ( ) cr, cp and tl were more nephrotoxic in -day-old animals compared to immature rats as could be demonstrated previously using other parameters for nephrotoxicity testing. ( ) the extent of toxic effects of heavy metals on the kidney is related to the maturity of renal functions involved in the enrichment of the respective metal in renal tissue and in its toxicity mechanism. ( ) changes in the fractional excretion of amino acids (reduction in renal amino acid reabsorption capacity, e.g. increase in fe aa ) and in amino acid plasma concentrations (especially decreases as a consequence of enhanced renal loss of amino acids) are early indicators of nephrotoxicity. ( ) therefore, the determination of renal amino acid handling is a highly sensitive marker for nephrotoxicity testing, both in immature and in adult rats. the mammalian h ϩ /peptide cotransporter pept was initially identified in the brush border membrane of renal proximal tubular cells as a high affinity type ptr -family member. here we describe the synthesis and functional analysis of novel high affinity inhibitors for pept that will be useful in further studies on structure and functions. starting from lys[z(no )]-pro a series of different lysine-containing dipeptide derivates were synthesized and studied for interaction with pept based on transport competition assays in pichia pastoris yeast cells and in epithelial skpt cells, both expressing pept . the twoelectrode-voltage-clamp technique in x. iaevis oocytes expressing pept was used to determine whether the compounds are transported electrogenically or block the uptake of dipeptides. synthesis and functional analysis of lys-lys derivates containing z(no ) side chain protections provided a set of inhibitors that reversibly inhibited the uptake of dipeptides by pept with k i values as low as nm. this is the highest affinity of a ligand of pept ever reported. moreover, based on the structure-function relationship we can conclude that the spatial location of the ε-amino protecting group in a lys containing dipeptide and its intramolecular distance from the alpha catom are key factors for the transformation of a substrate into an inhibitor of pept . ata is likely to provide the major route for the uptake of arginine in this tissue. institute of pharmacology and therapeutics, faculty of medicine, porto, portugal the present study examined the nature and regulation of the l-dopa transporter in two functionally different clonal subpopulations of opossum kidney (ok lc and ok hc ) cells. the inward transfer of l-dopa was largely promoted through an energy-dependent and sodium-insensitive transporter, though a minor component (ϳ %) was found to require extraceilular sodium. l-dopa uptake was insensitive to meaib, but competitively inhibited by bhc (ok lc , ic ϭ µm; ok hc , ic ϭ µm). l-and d-neutral amino acids and basic amino acids markedly inhibited l-dopa accumulation. l-dopa, lleucine, l-arginine, bhc or l-arginine plus bhc stimulated [ c]-l-dopa efflux. the accumulation of l-dopa was significantly higher at an acidic ph, and incubation of cells with l-dopa ( µm) resulted in marked intracellular acidification. modulators of pka, pkg, pkc and ptk failed to affect the accumulation of l-dopa. only the ca ϩ / calmodulin inhibitors inhibited l-dopa uptake. it is likely that system b ,ϩ might be responsible for the sodium-dependent uptake of l-dopa in ok cells, whereas sodium-independent uptake of l-dopa may include systems b ,ϩ and lat , the activation of which results in trans-stimulation of l-dopa outward transfer. the trans-stimulation of l-dopa inward transfer by an imposed h ϩ gradient suggest that ok cells are provided with an l-dopa-h ϩ cotransport system. amino acids are essential nutrients for cell growth and maintenance. the essential amino acids arginine and lysine, are mainly transported via the cationic amino acid transporter protein (cat ). the regulation of translation of the cat mrna during amino acid starvation was studied. an adaptive response to amino acid starvation and stress is a global decrease of protein synthesis, by phosphorylation of the translation initiation factor eif a. translation of the transporter mrna increases when eif a is phosphorylated, allowing synthesis of the essential for survival arginine/lysine transporter protein. the mechanism of increased translation of this mrna involves the induction of activity of a uorf-containing internal ribosomal entry sequence (ires). translation of the uorf and phosphorylation of eif a are required for increased activity. we propose that eif a phosphorylation triggers translational attenuation within the uorf, converting a relatively inactive, to a high activity ires. this study demonstrates that like yeast, mammalian cells have developed a sophisticated response to stress conditions: when expression of most genes decreases, synthesis of stress response proteins increases to support cell survival. amino acid transport, cell volume and the regulation of cell death f. lang, s. fillon, i. setiawan, p. lang, v. tanneur, d. häussinger, and s. bröer department for physiology, university of tübingen, germany cell volume regulatory mechanisms participate in a wide variety of cellular functions including regulation of epithelial transport, excitability, hormone and transmitter release, metabolism, migration, cell proliferation and apoptotic cell death. besides ion transport, polyols, betaine and glycerophosphorylcholine, cells utilize amino acids including taurine to balance extracellular osmolarity and regulate their volume. cells counteract shrinkage by uptake and swelling by release of amino acids including taurine. moreover, cell swelling stimulates synthesis and cell shrinkage favours breakdown of proteins which are osmotically less active than the sum of the amino acids thus generated. conversely, amino acid transport does influence cell volume. concentrative uptake of amino acids leads to cell swelling, amino acid release to cell shrinkage. through alterations of cell volume the amino acids participate in the regulation of protein metabolism. thus, concentrative amino acid transport inhibits and release of amino acids favours proteolysis. these mechanisms participate in the regulation of cell death. cd induced apoptotic death of jurkat t lymphocytes is paralleled by the release of taurine. the taurine release occurs with a delay of some min following cd receptor triggering but immediately preceedes apoptic cell shrinkage and dna fragmentation. the signaling leading to taurine release is in large part elusive but requires at some stage activation of caspases. moreover, taurine release and apoptotic dna fragmentation are strongly inhibited by lowering of temperature. preloading of the cells with taurine retards cd induced dna fragmentation pointing to an active role of taurine in the regulation of apoptosis. peptide transporters of the ptr-family are integral plasma membrane proteins, that mediate the electrogenic protoncoupled transport of di-and tripeptides and peptide-like drugs across cell membranes. the physiological role of pept , one member of this family in mammals, is mainly the uptake of small peptides into intestinal and renal tubular epithelial cells. in caenorhabditis elegans a homologue to mammalian pept is encoded by the pep- gene, which is expressed in the intestinal cells and a subset of sensory neurons in the head of the animal. to study the physiological role of the pep- transporter in vivo, a c. elegans pep- mutant was constructed. the animals deficient in pep- show a remarkable phenotype with pronounced signs of malnutrition, characterised by a delayed development, less eggs in the uterus, a smaller brood size and a prolonged mean life-span compared to wild-type animals. we rescued the phenotype by the expression of the wt pep- gene in the mutant. the observed starved phenotype in pep- mutants might be best explained by the reduced intestinal absorption of peptide bound amino acids that are required for protein synthesis and energy metabolism and provides the first direct evidence for the predominant role of the intestinal peptide transporter in amino acid absorption. adenosine is a potent vasodilator in many vascular beds and modulated tone via elevation of intracellular camp and/or release of nitric oxide (no). we have previously reported that adenosine (ado) stimulates l-arginine transport and no production in human cultured umbilical vein endothelial cells (sobrevia et al., j. physiol. , - , ) , and here further characterise the signalling cascades. rt-pcr demonstrated that fetal endothelial cell possess mrna levels for a a , a b and a -adenosine receptor subtype, whereas negligible levels were detected for the a -receptor. adenosine ( µm, min) induced increases in l-arginine transport and no production were ca ϩ and camp independent and stimulated transport was abolished in cells depolarised with mm k ϩ . whole-cell patch clamp experiments revealed that adenosine activated inward k ϩ currents, resulting in a membrane hyperpolarization and enhanced influx of the cation substrate l-arginine. adenosine induced l-arginine transport and no production were also abolished by inhibitors of tyrosine kinases (genistein), mek / (pd , u ) but unaffected by inhibitors of pkc (calphosin c) and pi- kinase (ly ). these data suggest that adenosine induces membrane hyperpolarization by activating inward k ϩ currents, increasing the driving force for cationic amino acid influx via system y ϩ . the discovery of nocardicine a by aoki et al. and aztreonam showed that monocyclic -lactams, collectively known as monobactams, can have antibiotic activity. this activity is poor but compensated by the unique effect they can induce on certain microbial cell membranes. our quest for new non-conventional surfactants for various biomedical applications led us to synthesize bioactive compounds with structural similarities to nocardicins. we present here the preparation and the study of original trimodular biosurfactants of type i: spermine and amine oxidase induce a cytotoxic effect on multidrug resistant chinese hamster ovary cells e. agostinelli , s. lord-fontaine , e. przybytkowski , and d. a. averill-bates department of biochemical sciences "a. rossi fanelli", university of rome "la sapienza" and cnr, centre of molecular biology, rome, italy department de chimie/biochimie and toxen (centre de recherche en toxicologie de l'environnement), université du québec à montréal, canada the occurrence of resistance to cytotoxic agents in tumor cells is a major obstacle to successful anticancer chemotherapy. multidrug resistance (mdr) is associated with several phenotypic alterations. cells with the mdr phenotype display decreased drug accumulation due to overexpression of pglycoprotein (p-gp), encoded by the mdr- gene, which acts as an energy-dependent pump involved in extrusion of drugs. we studied a new strategy to eliminate mdr cells using an enzyme, bovine serum amine oxidase, capable of forming cytotoxic products, h o and aldehyde(s), from polyamines (spermine). the involvement of both toxic products, formed by the bsao/spermine enzymatic system, in causing cytotoxicity was investigated in multidrug resistant chinese hamster ovary cells, ch r c , at and °c. we observed that hyperthermia, depletion of intracellular glutathione (by l-buthionine sulfoximine) and inhibition of glutathione s-transferase (by ethacrynic acid), sensitized ch r c cells to the cytotoxic effect of spermine enzymatic oxidation products. mdr cells showed no resistance to h o and aldehyde(s) relative to their drug-sensitive counterparts, auxb cells, in experimental conditions of: higher temperature, higher spermine concentration and longer incubation time. the inhibition of cellular detoxification systems led to increased cytotoxic effects of spermine enzymatic oxidation products on both mdr and sensitive cell lines. these results might be of great interest and suggest that toxic oxidation products formed from spermine and amine oxidase could be used in anticancer therapy, mainly against multidrug resistant tumor cells. [acknowledgements: this work was supported by cnr "target project on biotechnology", ministero della sanità tar these compounds present a hydrophobic part introduced by an ester or amide linkage with an aminoacid, a junction modulus which corresponds to -lactam, and a hydrophilic part which contains a triazole, well-known in pharmaceutical industry for its inhibitor effect against -lactamase. the compounds are synthesised from -hydroxymethyl- methyl propionic acid in five steps. selective activation of one of the primary hydroxyl groups was accomplished by the formation of alkoxy tris(dimethylamino)phosphonium (atdp) salts from the corresponding diol. treatment of with excess potassium carbonate in refluxing anhydrous acetone yields the monobactams . activation by atdp salts followed by treat-ment with sodium azide and reflux in toluene gives the azido compound. the reaction with acetylenic derivatives allows to obtain the surfactants. the compounds show classical surfactant behavior and the evaluation of their biological properties give evidence for their antibacterial and antiviral activity, which corresponds apparently to antiprotease activity. a prodrug approach to glutathione derivatives with in vitro antiparasitic activity department of chemistry and materials manchester, faculty of science and engineering, metropolitan university, manchester, u.k. the potential chemotherapeutic activity of peptides are lost in many cases in vitro, due to their inability to cross cell plasma membranes. the recent identification of a series of glutathione diesters with high antiparastic activities in vitro against t.b.brucei (african sleeping sickness) lead us to investigate the determinants associated with their activity. a qsar study on some twenty-five diester derivatives against t.b.brucei and t.b. rhodesiense lead us to conclude that the mechanism of action of these compounds is related to membrane penetration and hydrolysis, controlled by hydrophobicity and steric factors. a hplc and sensor study have confirmed the de-esterified diacid as the active agent of these prodrugs. dietary taurine prevents oxidative stress and morphological alterations in the retina of diabetic rat f. franconi , m. a. s. di leo , s. caputo , n. gentiloni silveri , and g. ghirlanda department of pharmacology, university of sassari, and department of internal and geriatric medicine, catholic university, rome, italy diabetes mellitus can cause various complications including retinopathy, which is the earliest and most common complications of diabetes mellitus, affecting % of diabetics and progressing to blindness in about %. considerable evidence implicates oxidative stress in the pathogenesis of diabetic retinopathy. in fact, hyperglycemia generates reactive oxygen species and free radical defense is reduced in diabetic patients. thus, the prevention of oxidative stress may have important implications for pharmacological attempts to prevent diabetic retinopathy. at this regard, it has been found that taurine, a semi essential amino acid with antioxidant activity, is decreased both in type and type diabetes mellitus. moreover, taurine seems to have a peculiar role of taurine in terms of cellular physiology and pathophysiology of the retina. among others, taurine is thought to produce important physiological effects through osmoregulation, calcium modulation and antioxidant effects. therefore, we examined the effect of dietary chronic ( months) taurine ( % and %) supplementation in diabetic rats in comparison with vitamin e ( and ui). dietary taurine supplementation, for months, does not influence conjugated dienes (cd), lipid peroxides (lp) and na/k atpase activity in the retina of non diabetic rats. using rats streptotozocin (stz) induced diabetes of -month duration, we found that cd, lp are significantly increased and they remained elevated for months. while, the na/k atpase is significantly decreased during the whole experimental time ( months). moreover, an inverse correlation has been found among the cd and lp and atpase activity. in the retina of stz rats, these biochemical alterations are accomplished with marked profound morphological changes. in stz rats, taurine enriched diets decrease the lipid peroxidation and preserve the atpase activity, being % taurine more effective than % diets. the morphological examination reveals that in rats feed with % taurine no proliferative changes are present. moreover, the beneficial effects of taurine are more marked than of those of vitamin e. these results and previous findings encourage new investigations to evaluate the efficacy of taurine as an adjunctive agent ch ch (ch ) n xco iran applicated be ( mg/kg - days) and the third -control. enzyme activities were determined spectrophotometrically in brain homogenate. results: polyamine oxidase activity decreased significantly lower dose of be didn't induce any significant change in diamine oxidase activity gaba-transaminase activity increased significantly (p Ͻ . ; p Ͻ . ) and dose dependently upon be treatment we have been examined the effects of propofol, taurine and propofol combined with taurine on free intracellular amino acid (aa) profiles, superoxide anion formation (o Ϫ ), hydrogen peroxide production (h o ) and released myeloperoxidase activity (mpo) in polymorphonuclear leucocytes (pmn). propofol led to significant changes in pmn free taurine, glutamine, glutamate, aspartate, methionine, basic, neutral (naa) and branched chain amino acid concentrations. exogenous taurine reduced pmn naa while increasing intracellular taurine. taurine supplemented to propofol significantly reversed the changes in taurine, naa and alanine only. regarding pmn immune functions propofol significantly decreased o Ϫ , h o formation and mpo. taurine decreased o Ϫ and h o production, while increasing released mpo. when propofol and taurine were combined they appeared to by reacting tyrosine with -nitroso- -naphthol in the presence of nitric acid - -benzyo- -(alanyl)- -phenoxazone (blp) an analog of actinomycin d is produced. the structural similarity of blp to actinomycin d prompted the national cancer institute (nci) to investigate its antitumor activities. the nci investigations revealed that blp exhibits growth inhibitory effects on various cancer cells and as a result blp has received the u.s. patent from the u.s. patent office. the purposed of this investigation was to synthesize similar benzo phenoxazone derivative by reacting -nitroso- -naphthol with -(α-hydroxy -methylaminopropyl)phenol in the presence of nitric acid. during the study, it was found out that , -benzo- phenoxazone derivative is not produced but a hydrogenated form of , -benzo- -phenoxazone which is probably , -benzo- -(α-hydroxy -methylaminopropyl)- -hydroxyphenoxazine (bhmhp) which has been suhhested from mass spectra obtained by electron ionization, ei, chemical ionization, ci and electro-spray ionization, esi, methods. bhmhp was screened against various cancer cell lines by nci and has shown promising effect against three ( ) breast cancer cell lines: mda-mb- , mda-n and hs- t. the % growth inhibitory (gi ) concentrations for these three cell lines were . ϫ Ϫ , . ϫ Ϫ and . ϫ Ϫ molar respectively. a. bocheva and t. pajpanova institute of molecular biology, bulgarian academy of sciences, and institute of physiology, bulgarian academy of sciences, sofia, bulgariathe histamine is an endogenous substance with neurotransmitter and neuromodulator functions in the organism. its antagonists are used in the therapy of allergic diseases and inflammatory reactions and as antiulcer drugs.the limited potentialities of the antihistamine therapy together with the increasing number of the people suffering from allergic diseases give rise to the design and synthesis of new histamine analogues as a perspective area in the chemistry of therapeutic drugs.additionally, compounds containing the guanidine, oxyamino and sufonamide moieties are known to elicit a variety of pharmacological responses and are present in several marketing drugs or drug candidates.on the other hand, similar compounds, being a part of bigger structures (for instance peptides), can imitate the molecules of already known at ii-receptor antagonists.having in mind these data we aimed to synthesize new analogs of histamine containing sulfo-and oxy-guanidino groups with common formula: a. bocheva , s. pancheva , and t. pajpanova institute of physiology, bulgarian academy of sciences, and institute of molecular biology, bulgarian academy of sciences, sofia, bulgariathe problem of the efficient therapy of pain is important not only from clinical but from social and economic point of view. the great achievements in medicine are connected with the research on the development of antinociceptive drugs.melanocyte-inhibiting factor (mif) is a tripeptide (pro-leu-gly-nh ) that was discovered in hypotalamus.the mif- exerted a weak analgesic effect. the synthesis of non-protein amino acids and their incorporation into biologically active peptides might become a powerful method for the design and development of modified analogues of natural peptides. having in mind these data we synthezied a number of new mif-analogues, containing unnatural amino acids such as cav, slys, sleu, slle and snie and in vivo experiments were performed to study their action on the nociception. the changes in nociceptive effects were examined in male wistar rats by the tail-flick (tf) and hot-plate (hp), as well as, the randall-seitto paw-pressure tests. the peptides were applied intaperitoneal (i.p) injection at a does mg/kg. the results show that the newly sinthesized analogues exert an antinociceptive effects in all tests used. naloxone at a dose mg/kg (i.p) antagonized the antinociceptive effects of mif-analogues. the interaction between platelets and fibrinogen is known to be mediated by the intergrin gp iib/iiia. the arg-gly-asp (rgd) sequence located on fibrinogen and other proteins of blood and extracellular matrix is the minimum requirement for cell attachment and adhesion. it has been found that peptides containing the rgd sequence can effectively inhibit the binding of fibrinogen to gp iib/iiia. in addition aspirin has been shown to be beneficial in the treatment of stable and unstable angina, acute myocardial infraction. aspirin acetylates and inhibits the enzyme cyclooxygenase, the first enzyme involved in thromboxane a (txa ) synthesis, an activator of platelet aggregation and adhesion.we have already reported that the combination in the same molecule of dipeptide amides, containing amino acid(s) of rgd sequence, with salicylic-residue -ro-c h -coϳ, {where rϭh or ch co} at their n-terminal amino group have shown inhibitory activity on human platelet aggregation. continuing this research project on salicyl-peptides we have synthesized a series of rgd analogs, incorporating salicylic acid derivatives, by conventional solution techniques and/or by solid phase. the synthesized rgd analogs were identified by ir, nmr and es-ms spectra and tested for inhibitory activity on human platelet aggregation in vitro, by adding common aggregation reagents (collagen, adp, thrombin) to citrated platelet rich plasma (prp). platelets were obtained from venous blood of healthy donors and the prp was isolated by centrifugation at g for min at °c. the aggregation was determined using a dual channel electronic aggregometer. malonyl dialdehyde (mda) production was measured using thiobarbituric acid reagent. in order to confirm these results, flow cytometry with monoclonal antibodies against gpib, gpiib/iiia, gpiiia and gmp was used. the ic values of the synthesized and tested compounds, as well as their mda production and flow cytometry results will be discussed. amino acids have a long tradition as building blocks, chiral auxiliaries and/or ligands in advanced organic synthesis and catalysis. at dsm an enzymatic kinetic resolution process has been developed, based on an aminopeptidase catalyzed stereoselective hydrolysis of racemic amino acid amides to form a mixture of l-amino acid and unchanged d-amino acid amide.several small peptides currently are under investigation as possible anti-tumor agents. neuropeptides such as substance p (sp) and neuropeptide y (npy), have been studied for their ability to prevent tumor growth or the proliferation of several cancer cell lines. these neuropeptides have been investigated for their effect to prostate cancer, small cell lung cancer (sclc) and breast cancer. the synthetic sp analog [d-arg , d-phe , d-trp , , leu ]sp (antagonist d) and the c-terminal analog [arg , d-trp , , mephe ]sp - (antagonist g) inhibit sclc cell proliferation in vitro and in vivo, while the analogs [glp , glu(bu t ) ]sp - and [glp , glu(bu t ) ]sp - showed significant inhibition in the proliferation of the cancer cell lines hela and t d.in the present study the c-terminal analogs of sp [glp , d-trp , glu(bu t ) ]sp [ ] [ ] [ ] [ ] [ ] [ ] ( ), [glp , d-trp , , glu(bu t ) ]sp [ ] [ ] [ ] [ ] [ ] [ ] ( ), [glp , d-trp , , mephe , glu(bu t ) ]sp - ( ), [glp , d-trp , mephe , glu(bu t ) ]sp - ( ), [glp , trp , mephe , glu(bu t ) ]sp - ( ), [glp , mephe , d-trp , glu(bu t ) ]sp - ( ), [glp , d-trp , mephe , glu(bu t ) -oh]sp - ( ), [glp , d-trp , cys(acm) -oh]sp - ( ), [glp , d-trp , mephe , cys(acm) -oh]sp [ ] [ ] [ ] [ ] [ ] [ ] ( ), [glp , d-trp , , mephe , cys(acm) -oh]sp - ( ) have been synthesized and tested for their antineoplastic properties in several cancer cell lines. they were also examined for their cytotoxicity to normal cells.the analogs - are peptide amides whereas the analogs - are peptide acids. they were performed using the stepwise synthesis either in solution, using the method of mixed anhydrides with carbonic acids or in spps using the fmoc/bu t methodology. the fragment condensation method in solution, using phosphonium reagents, such as pybop, was also applied. the analogs were purified (hplc) and identified (ft-ir, es-ms, h-nmr).the antineoplastic properties of the analogs were studied using sister chromatide exchange (sce) and proliferation rate index (pri). as it is known the sce method is an indicator of dna damages or its repair mechanism, while the method of pri is a sensitive marker of cytotoxicity. the experiments were carried out using cultured human lymphocytes from healthy donors and these results will be discussed.semiempirical quantum chemical investigation of some thymidine derivatives modified with amino acids and peptides at Ј, Ј-positions j. velkov , i. stankova , a. ivanova , and a. tadjer department of chemistry, south-west university "neophit rilski", blagoevgrad, and department of chemistry, sofia university "st. kl. ohridsky", sofia, bulgaria optimized geometry and electron charge distribution for some thymidine derivatives ( Ј, Ј-bis-o-n-α-benzyloxycarbonyl-alanyl-, Ј, Ј-bis-o-n-α-benzyloxycarbonyl-valyl , Ј, Ј-bis-o-n-α-benzyloxy-carbonyl-glycyl-glycyl-glycyl, Ј, Јbis-o-n-α-benzyloxycarbonyl-phenylalanyl, Ј, Ј-bis-o-n-αbenzyloxycarbonyl-glycyl) were calculated at the semiempirical (am ) level. the choice of method is limited by the molecular size. in addition, the differences between the ground state energy of the compounds and that of the hydrolysis reaction intermediates were compared to the experimentally found stability towards hydrolysis.with a few notable exceptions, attempts to crystallise integral membrane proteins have failed due to the difficulties in finding appropriate conditions for proteins that have both hydrophobic and hydrophilic domains. thus structural information is largely limited to predictions of secondary structure from the amino acid sequence and computer modelling, neither of which can as yet give high resolution detail. thus alternative approaches are required, and one that we have employed is to look at the substrate binding/transport characteristics of compounds and predict what features the binding site might have. the membrane transport protein that we are interested in is the proton-coupled di/tri-peptide transporter, which has a wide range of natural substrates and is known to transport therapeutically important non-peptides such as ᮀ-lactam antibiotics and angiotensin converting enzyme inhibitors.the initial question that interested us was what makes a di/ tri-peptide a substrate, but not an amino acid? while the obvious answer is the peptide bond, studies with 'space mimic' compounds (which have the space filling properties of a dipeptide but no peptide bond) gave the surprising result that the peptide bond was not essential for binding and translocation. although these space mimics had n and c termini, studies from our laboratory and others have shown that the presence of free amino or carboxyl groups are not a prerequisite for binding or translocation either. this leaves the question of what does distinguish a pept substrate from a non-substrate?computer modelling of a large number of pept substrates has allowed the development of a substrate template, whereby potential substrates can be scored according to their predicted binding affinity. from this it is clear that it is a sum of energies derived from a number of substrate-transporter interactions that determine binding affinity, including the n-and c-termini, the peptide bond components and the substrate side-chain groups. further studies aim to refine this model through the complimentary approaches of novel substrate design and sitedirected mutagenesis of the transporter protein.why are we interested in this? a large number of promising therapeutic compounds are found to have little or no bioavailability. compared with most membrane transporters pept has a wide range of potential substrates, and amongst its non-peptide substrates are a range of peptidomimetic therapeutic compounds. the recent finding that a peptide bond is not a prerequisite for transport opens up the possibility of designing prodrugs to be substrates for pept , and this has found to be an effective strategy for example with the antiviral drug valacyclovir.(we thank the wellcome trust for their generous support.) nuklearmedizinische klinik und poliklinik der technischen universität münchen, germanyaim: the high amino acid metabolism of tumor cells allows tumor imaging with radiolabeled amino acids as c-methionine (met) by positron-emission-tomography (pet). however in recent experimental and clinical studies met uptake was also found in inflammatory tissue thus leading to false positive results. the aim of the study was to compare [ f]fluoroethyltyrosine (fet), a new amino acid analogue, with met to assess their suitability for differentiating between tumor cells and inflammatory cells in vivo and in vitro.methods: popliteal lymph nodes of balb/c and dba/ mice were stimulated either by streptocotocin (stz), causing chronic lymphadenitis, or by concanavalin a (con a), causing in acute lymphadenitis. tumor infiltrated lymph nodes were induced by inoculating cells from a lacz transfected t-cell mouse lymphoma line into the footpads of syngenic dba/ mice. the uptake of met and fet was determined quantitatively in tumor infiltrated and inflammatory lymph nodes as well as in the lymph nodes of untreated mice. in vivo imaging of tracer uptake in mouse lymph nodes was performed using a high resolution ( . mm) small animal pet (madpet). in vitro the uptake of the amino acids met and fet was investigated in different cells, such as sw human colon carcinoma cells and c rat glioma cells, stimulated human lymphocytes and macrophages. about ϫ cells of each cell line were incubated in a buffered medium containing either different concentrations of unlabeled amino acids or con a (stimulation of lymphocytes) or the transport inhibitors amino-norbornane-carboxylic acid (bch, l-system), α-(methylamino)-isobutyric acid (meaib, a-system) or l-serin (asc-system). . mbq of each amino acid tracer were added and incubated. uptake was stopped by using ice-cold pbs, cells were washed three times and uptake was analyzed.results: in tumor infiltrated lymph nodes uptake of both tracers was higher than in control lymph nodes. met showed an increased uptake in both lymphadenitis models, whereas fet did not accumulate significantly. met and fet uptake in tumor infiltrated lymph nodes was also seen in madpet images, however inflammatory lymph nodes could only be detected in met images.the amount of tumor uptake was different in the various cell types investigated. c cells showed the highest uptake of all cells investigated and a slightly lower uptake was found in sw cells. in con a stimulated lymphocytes, the uptake of fet was negligible, while met uptake was significantly higher than in both tumor cell lines. since bch reduced the uptake of fet and met to approximately %, fet seems to be also predominantly transported into tumor cells by the l-system. the results indicate, that fet appears to differentiate between tumor and inflammatory tissue, as a result of the low uptake of fet in inflammatory cells. nuklearmedizinische klinik und poliklinik der technischen universität münchen, germanyover the past few years numerous studies have documented the high diagnostic accuracy of positron emission tomography (pet) using the glucose analogue f- -fluordeoxyglucose (fdg) for detection and staging of malignant tumors. a significant limitation of fdg-pet, however, is that increased uptake is not only observed in malignant tumors but also in activated inflammatory cells. due to the high glucose utilization of the normal brain and the lower protein synthesis in the normal gray matter the radiolabelled amino acid c- -methionine (met) gives higher contrast between brain tumors and normal tissue than fdg-pet. rapid uptake of met has been documented for several malignant tumors like gliomas, lung cancer, bladder cancer and malignant lymphomas since amino acid transport and protein synthesis are generally increased in malignancies. the application of met-pet however has been limited by the short half life of the radioactive label c- ( min) in contrast to f- ( min). amino acid analogous labeled with f- like f- -fluoro-α-methyltyrosine (fmt), f- -fluoro-ethyltyrosine (fet), f- -fluoro-phenylanaline, f- -fluore-proline will allow a more widespread application of amino acid pet in oncology. an other amino acid analogue i- -iodo-α-methyltyrosine (imt) is of clinical interest because the radionuclid i- allows it applicability for single-photoemission-computer-tomography (spect). the uptake of the amino acid analogues can only be regarded as a measure for the increased amino acid transport in the tumor cells because they are not incorporated into proteins. clinical data show that radiolabelled amino acids that are only transported into the cells are not inferior to those that enter protein synthesis. this tracers may also help to differentiate tumor lesions from inflammatory lesions when the expression of the transport systems for amino acids in tumor cells and inflammatory cells is different.lysinuric protein intolerance: understanding the pathophysiology of a multi-system disorder of dibasic amino acid transport m. p. sperandeo , , v. fiorito , a. pietrosanto , a. pepe , g. andria , and g. sebastio telethon foundation, rome, and department of pediatrics, federico ii university, naples, italy lysinuric protein intolerance (lpi; mim ) is an autosomal recessive disease, mainly found in finland and italy. clinical findings of lpi include: vomiting, diarrhea, failure to thrive, hepatosplenomegaly, osteoporosis, episodes of coma, and mental retardation. a life-threatening lung involvement (alveolar proteinosis) and renal insufficiency were also reported. metabolic derangement of lpi includes: reduced intestinal absorption of cationic amino acids (lysine, ornithine, arginine, caa), increased renal excretion of caa and dysfunction of the urea cycle leading to hyperammonemia and orotic aciduria. most of the clinical findings cannot be explained by a selective deficiency of amino acid transport, as indeed observed for cystinuria (mim ), a cognate disease of lpi. the molecular basis of lpi resides in an abnormal caa carrier functioning at the level of basolateral membrane of epithelial cells in the intestine and the kidney. caa transport is mediated by y ϩ l system, that is exerted by heterodimers consisting of the f heavy chain ( f hc) and a light chain represented by either the solute carrier family a, member (slc a ) or (slc a ). after excluding the f hc as the causative gene of lpi, we identified slc a as the lpi gene and characterized mutations in twenty-five patients from families ( italian, japanese, moroccan, greek, and pakistani; independent alleles) affected by lpi. thirty-two of the independent alleles ( . %) were characterized and fourteen mutations were identified. only five mutations (namely insatca, w x, delctct, ivs ϩ gaea, s r) were identified in more than one independent family. most mutations are located in the slc a coding region, except for two splicing mutations. the pathogenesis of some clinical findings of lpi, namely alveolar proteinosis and renal involvement, remains mostly unknown. we are currently investigating the role of slc a gene in lpi, which, in addition to slc a , is responsible of the y ϩ l activity. in fact, the regulation of the y ϩ l system, exerted by either f hc/ slc a or f hc/slc a , is still unknown. hypothetically, the activation of f hc/slc a in all tissues might be the "simple" way to a lpi gene-therapy.[acknowledgements: m. p. s. is supported by telethon-italy (grant n. cp) and is an assistant telethon scientist.] pre-eclampsia (pe) is a potentially life threatening complication of pregnancy and is one of the leading causes of maternal and fetal morbidity and mortality. pe is associated with endothelial cell dysfunction and inadequate placental perfusion. fetal plasma l-arginine levels are decreased in pe and there is controversy as to whether nitric oxide (no) production is altered. we have investigated whether the kinetics of l-arginine transport via system y ϩ and no production are altered in fetal umbilical vein endothelial cells (huvec) from pe pregnancies. kinetics of l-arginine transport were similar in huvec isolated from normal, preterm and pe pregnancies, however nethylmaleimide inhibited transport in normal but not pe huvec. basal and histamine-stimulated no production was similar in normal and preterm huvec, whereas pe increased basal ( Ϯ vs . ϫ pmol/ cell/ min) and histaminestimulated ( Ϯ vs Ϯ pmol/ / min) no production. whole-cell patch clamp measurements revealed similar inward rectifying k ϩ currents in normal and pe huvec, with resting membrane potentials of Ϫ Ϯ and Ϫ Ϯ mv in normal and pe huvec, respectively. increased enos activity in pe endothelial cells may serve as a compensatory mechanism to counteract the hypertension observed in pe, however, elevated no production is apparently not associated with enhanced larginine transport. department of pharmacology, university of cambridge, u.k.over the past years, concerns have heightened over the escalating numbers of pathogenic microorganisms that are resistant to multiple antibiotics. this phenomenon poses major problems in the treatment of patients with hospital or community-acquired infections caused by bacteria, yeast, fungi and parasitic organisms. particularly intriguing are the so-called multidrug transporters, which have specificity of compounds with very different chemical structures and cellular targets. this lecture will focus on the molecular properties of the atpbinding cassette multidrug transporter lmra in the lactic acid bacterium lactococcus lactis. lmra is a close homolog of the human multidrug resistance p-glycoprotein, overexpression of which is one of the major causes of resistance of human cancers to chemotherapy. surprisingly, lmra can even substitute for pglycoprotein in human lung fibroblast cells. recent biochemical and pharmacological studies on lmra suggest that the protein may operate by a two-cylinder engine mechanism to transport amphiphilic drugs from the inner leaflet of the plasma membrane. this mechanism will be discussed in more detail. bone and bone marrow are important sites of metastasis formation in breast cancer; so, we studied the level of bone sialoprotein (bsn) and fibronectin (fn), two key connective tissue antigens, in patients with metastatic breast carcinoma. our data reveled that bsn have a statistically significant association with bone metastases in that disease. fn level was also significantly changed in metastatic breast carcinoma when compared to the non metastatic cases. kharkov national university, radiophysical department, chair of molecular and applied biophysics, kharkov, ukraine * present address: institute of cell and molecular biology, university of edinburgh, edinburgh, scotland, u.k.in the work the temperature dependencies of dielectric parameters of human serum albumin (hsa) and fibrinogen solutions ( . m nacl, ph . ) were obtained in the temperature interval - c degrees. the measurements of the dielectric parameters were carried out at the frequency of . hhz, i.e. in the range of free water molecules dispersion. in contrast to dependencies for poor solvent, temperature dependencies of dielectric parameters for protein solutions are of nonmonotonous character; they have a number of peculiarities in the temperature ranges of - , - and - c degrees. this fact means that at these temperatures redistribution of free and bound water in protein-water system occurs due to structural changes in protein molecules. the dependencies of hydration of hsa and fibrinogen on temperature were obtained as well.in the work the mechanism of temperature changes of spatial organisation of protein molecules was proposed. perhaps, this mechanism is responsible for maintenance of thermal stability of the functionally active conformation of native proteins. as peculiarities on temperature dependencies of dielectric parameters of solutions of globular (hsa) and fibrillar (fibrinogen) proteins were in the same temperature regions, one may to assume that the mechanism of proteins thermal stabilisation in physiological temperatures interval has a general character. laboratory of cell pharmacology, university of leuven, medical school, campus gasthuisberg (o&n), leuven, belgium n-pomc was purified from conditioned medium of att cells using a sequence of concentration, fractionation by ion exchange, rp-hplc and gel-filtration. twenty isoforms of n-pomc, for both and kda, were identified by means of mass spectrometry and n-terminal sequencing. these isoforms are assumed to be pomc - or pomc - with heterogeneous glycosylation.the n-pomc isoforms were tested on prolactin (prl) gene expression and lactotroph mitosis in pituitary cell aggregate cultures. prl mrna content was quantified by means of real time rt-pcr. three kda n-pomc fractions enhanced prl mrna levels by - %, while all other isoforms were inactive. this effect was abolished by immunoneutralization with n-pomc monoclonal antibody. only one fraction stimulated lactotroph proliferation ( . Ϯ . %) as assessed by brdu incorporation in prl-immunoreactive cells. several (but not all) kda n-pomc fractions stimulated prl mrna level and lactotroph mitosis. on the other hand, all and kda isoforms activated the mc- and mc- receptor in cell lines in which these receptors were transfected. thus, att cells produce various n-pomc isoforms, only a part of which display an effect on prl mrna expression. even fewer isoforms affect lactotroph proliferation. since all isoforms activate the mc- and mc- receptor, it is suggested that the effect of the few isoforms on lactotrophs is mediated by (a) different receptor(s). are widely prescribed for the treatment of mild to moderate depression and the putative antidepressant constituent is probably hyperforin. in this study the effect of hyperforin was investigated on the release of neurotransmitter amino acids.coronal cortical slices ( µm) were cut and perfused with gassed ( % o , % co ) acsf at °c. two-minute samples of perfusate were collected and aspartate and glutamate were assayed by hplc. potassium-and veratridine-stimulated release was elicited by administering pulses of k ϩ ( mm) or veratridine ( µm) minutes apart.in control experiments the second k ϩ pulse elicited glutamate release which was % of the first pulse. hyperforin ( µm) perfused for minutes prior to, and during, the second k ϩ pulse significantly increased glutamate release to % (p Ͻ . , n ϭ - ). release elicited by the second veratridine pulse was % of the first pulse for both glutamate and aspartate. hyperforin ( µm) increased this release to the second pulse to % and % respectively (p Ͻ . , n ϭ - ). when perfused on its own for minutes, hyperforin ( µm) increased the basal release of glutamate (p Ͻ . , n ϭ - ).in conclusion, the increase in the release of neurotransmitter amino acids observed following hyperforin is possibly mediated through a facilitatory action on voltage-operated ca ϩ or na ϩ channels.glucagon-like peptide- ( - ) amide (glp ) is the main product of the glucagons gene expression in intestinal l cells into the cirulation in response to the ingestion of food and is the most potent stimulator of glucose-induced insulin secretion. glp receptors have also been detected in discrete areas of rat brain and intracerebroventricular glp has been shown to inhibit feeding in fasted rats. in this study hplc techniques were employed to evaluate the effects of glp on serotonin ( -ht) and γ aminobutyric acid (gaba) metabolism in rat brain. glp ( . µm) produced a significant decrease in levels of -ht by % after minutes of incubation with combined hypothalamus and brain sterr. synaptosomes. levels of hydroxyindolacetic acid ( -hiaa), the principal metabolite of -ht, and tryptophan the amino acid precursor of -ht, were also decreased significantly by % and % respectively. gaba and its amino acid precursor glutamic acid were both measured at the same conditions as above, but a precolumn derivatization hplc technique was used. the increase in levels of gaba ( %) and glu ( %) by glp was not significant.the results suggest that decreased synaptosomal levels of -ht and -hiaa caused by glp are due to diminished availability of typtophan by the peptide. in experimental model of iron overload we obtained the following results: the concentration of carbonyl groups tended to increase, while mda level significantly increased after feso treatment ( . Ϯ . vs control . Ϯ . µmol/mg prot.) and ( . Ϯ . vs control . Ϯ . nmol/mg protein p Ͻ . ) respectively. it was associated with significantly increased iron content ( . Ϯ . µg/mg prot. vs control . Ϯ . p Ͻ . ). it is clear that oxidative stress occurs in experimental iron overload, if sufficiently high levels of iron within hepatocytes are achieved. in group treated with feso and spermine, iron content was significantly decreased ( . Ϯ . p Ͻ . compared with fe treated only) and carbonyl group content tended to be lower in comparison to feso treated only ( . Ϯ . ), but mda level didn't change ( . Ϯ . ). in addition, treatment with spermine alone resulted in increase of mda level ( . Ϯ . vs control p Ͻ . ), iron content didn't change ( . Ϯ . ), but carbonyl groups were decreased ( . Ϯ . vs control p Ͻ . ). feso treatment increased gsh level ( . Ϯ . nmol/mg prot. vs control Ϯ . ; p Ͻ . ) while in combination with spermine this increase was more profound ( . Ϯ . ; p Ͻ . vs control, p Ͻ . vs feso ). spermine alone produced similar increase of gsh level ( . Ϯ . , p Ͻ . vs control; p Ͼ . vs feso ). the results emanating from the human genome programme have required a reappraisal of protein science and have led to the rapid upsurge in interest in the area of proteomics. this sudden re-emergence of protein science, in fact, was predictable and should not have been surprising.recent experience of protecting group design with respect to lysine and aspartic acid will be discussed together with aspects of chemical synthesis of small proteins of biological significance and in the context of chemical synthesis methodology making contributions to the general field of proteomics. using a cell line permanently expressing the mouse taurine transporter (mtaut) as a fusion protein, we investigated the underlying mechanism by which the immunosuppressive drug cyclosporin a (csa) inhibits taurine transport. csa inhibited the recombinantly expressed mtaut function both in dose and time dependent manner. the inhibitory effect of csa was reversible. thus, washing out the csa resulted in almost complete recovery of taurine uptake. to obtain further insight, we examined the surface abundance of the mtaut as a function of csa treatment using a surface-labeling assay. our results demonstrated that csa treatment altered the surface expression of the mtaut without significantly altering its total expression level, and the reduction in the cell surface expression paralleled the decrease in taurine uptake. upon removal of csa, the virtual recovery in taurine uptake was due to the concomitant increase in the number of taurine transporters on the cell surface. taken together, our results suggest that csa induced inhibition of taurine uptake was either due to the impaired targeting of the taurine transporters to the cell surface or due to the removal of the transporters from the cell surface. polyamines are neuromodulators in a number of physiological and pathological conditions in cns. since application of ethylene glycols causes hypoactivity and lethargy of experimental animals, depression of cns and various neurological symptoms, the aim of this study was to examine the effects of butoxyetanol on polyamine and gaba catabolism, taking in account an alternative pathway of gaba synthesis from putrescine. methods: male wister rats were allocated into three groups: first treated by be ( mg/kg - days), second key: cord- -xgikbdns authors: Ühlein, e. title: Übersicht Über neue ernährungswissenschaftliche publikationen date: - - journal: z ernahrungswiss doi: . /bf sha: doc_id: cord_uid: xgikbdns nan shoe~taxer, w. c., yanof, h. m., tui~k, l. n., u. wilson, t.h.: glucose and fructose absorption in the unanesthetized dog. gastroenterology [ ] nr. , s. ff . ( s.) . * sieaei~, p. s., u. correia, iv] . j. : speed of resumption of eating following distraction in relation to number of hours food-deprivation. physiol. ree. [ ] nr. i, s. ff. ( s.) . si~eth, r. n., u. christie, it. j. : studies on the absorption of insulin from the gastrointestinal tract of the rabbit. diabetes [ ] nr. , s. ff. ( s.) . *st~oh~.x~r, g.: ~iessung der ksrperradioaktivitiit zur bestimmung der eisenresorption beim mensehen. nuclear med., suppl. [ ] s. ] . tallis, g. ] ~., moore, ~. w., u. gream, b. d. : specific gravity of live sheep. nature [ ] nr. , s. . tanaka, s., nu-kasawa, a., yoshikawa, h., u. yoneyama, y. : protein nutrition and radiation damage in mice. nature [ ] nr. , s. / . tho~tson, a. m., u. billv, wlcz, w. z. : nutritional status, maternal physique, and repro- ductive efficiency. prec. i~utrition see. [ ] nr. , s. / . toothill, j. : the effect of certain dietary factors on the apparent absorption of magnesium by the rat. brit. j. nutrition [ ] nr. , s. . *trail, j. c. m. : upgrading the indigenous poultry of uganda. i.: the growth rates and feed conversion from hatching to maturity of indigenous poultry crossed with four imported breeds. j. agrie. sei. {} [ ] nr. , s. ft. ( s.) . van i~iekerk, b. d. h., i~eid, j. t., be~s~uoun, a., u. paladines, . l.: urinary ereatinine as an index of body composition. j. nutrition [ ] nr. , s. / . voigtl~ndeg, h. : der rhythmus des brotessens. lebensmittel u. ern~hrung [ ] nr. , s. ] . wadsworth, g. r. : nutrition surveys: clinical signs and biochemical measurements. prec. l~utrition see. [ ] nr. , s. ] . watson, w. c., gordon, r. s., kx~en, a., u. jove~, a. : the absorption and excretion of castor oil in man. ft. pharmacy pharmacol. [ ] nr. , s. / . wkeel~r, r. r. : evaluation of various indicator techniques in estimating forage intake and digestibility by range cattle. diss. abstr. [ ] nr. , . williams, w. p., dawxs, r. e., u. couch, j. r. : the utilization of earotenoids by the hen and chick. poultry sei. [ ] nr. , s. ft. ( s.) . wolford, j. i-i., ringer, ~. k., col~_ai% t. h., u. zrsdel, h. c. : individual feed consumption of turkey breeder hens and the correlation of feed intake, body weight, and egg production. poultry sci. [ ] nr. , s. ff. ( s.) . youl~o, g. m., tensuan, r. s., saimt, f., u. hol~es, f. : estimating body fat of normal young woman. visualizing fat pads by soft-tissue x-rays. j. amer. dietetic assoc. [ ] nr. , s. ff . ( s.) . zuck~r, t. f., u. ziicker, l. hi. : fat accretion and growth in the rat. j. nutrition [ ] nr. , s. / . intermediary metabolism c - kohlenhydrate -garbohydrates +n. n. : enzymatic defects in the hereditary fructosurias. nutrition i~ev. [ ] i~r. , s. / . *agosi~, hi., scar~elli, n., di~amarca, iv] . l., u. arav~i~a, l. : intermediary carbohydrate metabolism in triatoma infestans (insecta: hemiptera). ii. : the metabolism of c-glucose in triatoma infestans nymphs and the effect of ddt. comp. bioehem. physiol. [ ] nr. , s. ff . ( s.) . baens, g. s., lv-~deen, e., u. cornblxth, m. : studies of carbohydrate metabolism in the newborn infant. vi. : levels of glucose in blood in premature infants. pediatrics [ ] nr. , s. ff . ( s.) . * b~akeb, j., u. mapson, l. w. : studies in the respiratory and carbohydrate metabolism of plant tissues. xil : l~tlrther studies of the formation of co~ and the changes in lactate, alcohol, sucrose, pyruvate, and cr in potato tubers in nitrogen and in air following anaerobic conditions. prec. royal soc. b. [ ] nr. , s. ff. ( s.) . *bean, r. c., porter, g. g., u. bur, b. k.: carbohydrate metabolism of avocado. il: formation of sugars during short periods of photosynthesis. plant physiol. 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( s.) . *randle, p. ft., garland, p. b., hales, c. n., u. newsholme, e. a.: the glucose fattyacid cycle. lancet [ ] nr. , s. ff. ( s.) . rossi, f., z~ri, m., u. g] av.enbau~, a. l.: evidence for the existence of ~he hexose monophosphate pathway for glucose metabolism in the normal and denervated skeletal muscle of rats. biochcm. j. [ ] nr. , ] . *ruiteb, j., wei-i~bei~o, f., u. morrisoi~, a. : the stability of glucose in serum. clinical chem. [ ] bit. , s. ff. ( s.) . $sac]ter, j. a., ]: l~tch, ~r d., u. glasziou, k. t [ ] nr. , s. ff. ( s.) . *scm~ass~k, h.: metabolite des kohlenhydratstoffwechsels der isoliert pcrfundierten rattcnleber. biochem. z. [ ] iqr. , s. ff. ( s.) . *sch~reiu, c. : relation between fructose content of semen and fertility in man~ j. reproduction fertility [ ] i~ir. , s. ff. ( s.) . * schlubac~, it. h., u. g~em~, m neptune, e. ~., u. sudduth, h. c. -" toxic effects of oxygen at hight pressure on the metabolism of d-glucose by dispersion of rat brain. biochem. j. [ ] nr. , s. / . *waaei~, h. g.: pathways of utilization of ( - c) glucose and ( -x c) glucose in slices of peas. j. exper. botany [ ] nr. , s. ff. ( s.) . weic-~r, h., schs~r~cl, h., u. re~schle~, h. e.: ~ber physiologische glucose-ansscheidung im urin yon stoffwechsel-gesunden. klin. wschr. [ ] nr. , s. . w~ovse, s., fr~ed~ia~r~, b., u. reicm~d, g. : effects of insulin on hepatic glucose production and utilization. diabetes [ ] nr. i, s. lff i~ou~, l~i., u. kum~e~ow, f. a. : fatty acid composition of lymph lipids from rats fed fresh and thermally oxidized fats. j. dairy sci. [ ] i~r. , s. ff. ( s.) . * blou~rr, a. w., u. co~, m.: tissue lipid pattern in a case of xanthoma disseminatum. arch. intern. ivied. lu [ ] nr. , s. ] . bortz, w., anr~m~, s., u. c~o~, i. l.: localization of the block in lipogenesis resulting from feeding fat. j. biol. chem. [ ] nr. , s. / . bronsert, u., hartmani~', f., u. mitzkat, ii.-j.: die wirkung yon milchs~ure im fettstoffwechsel der leber bei experimenteller fettlcber der ratte. naturwisscnschaften [ ] i~r. , s. . br w~, m. l. : effect of a low dietary level of three types of fat on reproductive performance and tissue lipid content of the vitamin b -deficicnt female rat. j. nutrition [ ] nr. , s. . *busfm~k, e. r., tho~rrson, r. h., u. weedo~, g. d. : metabolic response to cold air in men and women in relation to total body fat content. j. appl. physiol. [ ] nr. of mast cells to fat transport. ann. new york acad. sci. [ ] ar~. , s. ff. ( s.) . katorsx~, b. a.: the influence of growth hormone on fat and protein metabolism. diss. abstr. [ ] nr. , -- ( s. amer. j. clinical nutrition [ ] nr. , s. ff. ( s.) . mxmo~, j. e.: the influence of diet and age on lipid metabolism of chickens. diss. abstr. [ ] nr. , - ( s.) . *me, i, f. : a study on the lipid metabolism in the patients with atherosclerosis especially on the cholesterol in serum lipoprotein fl-fraction. japan. heart j. [ ] nr. brown, l. d., grev~es, %. m., u. duncan, c. w. : effect of protein level in milk replacers on growth and protein metabolism of dairy calves. j. dairy sci. [ ] nr. , s. ff. ( s.) . *ma~i)elst~, j. : protein turnover and its function in the economy of the cell. arm. new york acad. sci. [ ] nr. , s. ff. ( s.) . mast~as, c. j. : nucleic acids and protein stores in the merino sheep. australian j. biol. sci. [ ] nr. , s. ff. ( s.) . noack, r.: zur biochemischen funktion des proteins als grundlage der bedarfsaormen. ernehrungsforschung [ ] nr. , s. / . *par.r. anin, a. u : le m tabolisme pro~ique dans le systems nel-ceux. studii si cercet~ri de biochimie [ ] nr. , s. ft. ( s.) . roouski, j., ~i[akowska, k., i~sn~, j., stankowska, a., hryniewiecki, l., u [ ] nr . [ ] . suppl. to nr. , s. ff. ( s.) . co~solazio, c. f., ~ixtous~, l. ., nv. lson, r. a., ~[a~drng, r. s., u. can" a~, j. e.: excretion of sodium, potassium, magnesium, and iron in human sweat and the relation of each to balance and requirements. j. nutrition [ ] nr . goodwin, a. f., u sci. [ nr. , s. ff. ( s.) . *gerlinger, p., much, j.-v., u. clukvert, j.: note pr liminaire sur raction du eyclophosphamide (endoxan) sur le d veloppement de l'embryon. c. r. acad. sci. [ nr. , s. ff. ( s. [ nr. , - ( s.) . nelson, f. e., jensen, l. s., u *caruzzo, c., tartara, d., pagano, p. g., pellegrini, a., costanzo, f., u [ ] nr. , -- ( s.) . [ ] nr. , s. ll ff. ( s.) . gal/~bos, j. t., asada, m., u. s~ks, j. z. : the effect of intravenous ethanol on serum enzymes in patients with normal or diseased liver. gastroenterology [ ] nr. , s~ ff. ( s.) . gall, c., u. weissw~.~le~, p. : un~ersuchungen fiber die labf~higkeit der milch und ihre beziehung zur mineralsteff-fiitterung der kfihe. milchwissenschaft [ ] nr. , s. ff. (? s.) . zitat: dt. lebensmittel-rdsch. [ ] nr. , s. . gasic, g., u. morrison, a.b.: mucopolysaccharides of renal collecting tubule ceils in potassium deficient rats. prec. soc. exper. biol. med. [ ] l~r. , s. ff. ( s.) . gear~-, t. f. : oak wilt development and its reduction by growth regulators. i. production and activity of oak wilt fungus pectinase, cellulase, and auxin. ii. effect of halogenated benzoic acids on oak trees, the oak wilt diseases, and the oak wilt fungus. diss. abstr. [ ] nr. , -- ( s.) . *g~l~,~cs~,r, f., g~ti, t., gy~g~, k., u. s s, j.: effec~ of cardiopathogenic diet on the thiopental anaesthesia. acts physiologica aead. sci. hung. suppl. nr. , s. . gar~'ls, j., piazu~lo, e., u [ ] nr. , -- ( s.) . ikir~a, h., u. t~, k. v. : activity of gibberellin 'd' on the germination of photosensitive lettuce seeds. nature ] nr. , s. / die wirkung des glucagons auf den blutzuckerspiegel in abh~ngigkeit yore alter. z. aiternsforsch. [ ] nr. , s. / . *loosli, j. k. : primary signs of nutritional deficiencies of laboratory animals. j. amer. veter, reed. assoc. [ ] nr. , s. ft. ( s.) . lowrey, r. s., pond, w. g., lo sli, j. k. u. barnes, r. h.: effect of dietary protein and fat on growth, protein utilization, and carcass composition of pigs fed purified diets. j. animal sci. [ ] nr. , s. ft. ( s.) . lvnd, c. c., u. ) [ ] nr. , s. ff. ( .) . nr~cleod, l. b. : effect of liming and potassium fertilization on soil solution and on yield and composition of alfalfa and orchard grass mixtures. dlss. abs~r. [ ] nr. , -- ( s.) . ~ds~n, k. ., u. edmonds, e. j. : prolonged effect on caries of short-term feeding of rice hulls to cotton rats. j. dental res. [ ] nr. , s. ff. ( s.) . m~a~.~, a. c. : biological responses of young rats fed diets containing genistin and genistein. j. nutrition [ ] nr. , s, ] . ~_ajaj, a.s., dnc~g, j.s., azzam, s.a., u. da_~by, w. j.: vitamin e responsive megaloblastic anemia in infants with protein-calorie malnutrition. amcr. j. clinical nutrition [ ] nr. , s. ft. ( s.) . v~jcm~owicz, e., u. quxstel, j. h. : effects of aliphatic alcohols on the metabolism of glucose and fructose in rat liver slices. canad. j. bioehem. physiol. [ ] nr. , s. ff. ( s.) . m_al~otra, o. p., n~a-wl)ov, a.v., r~ber, e. f., u. norton, h . w., effects of rat strain, stilbestrol, and testosterone on the occurrence of hemorrhagic diathesis in rats fed a ration containing irradiated beef. j. nutrition [ ] nr. , s. ff. ( ,) . --, u. r~ber, e. f. : effect of methionine and age of rat on the occurrence of hemorrhagic diathcsis in rats fed a ration containing irradiated beef. j. nutrition [ ] nr. , *misga, u. k. : effect of corn oil feeding on the lipids of dog bile. indian j. exper. biol. [ ] nr. , s. / . * miyao, m., tsvn•isnz, m., nagano, k., u. ttosogi, t.: experimental studies on the digestibility and absorbability of milk proteins. . effects of carbohydrate addition on the digestibility and absorbability of cow's milk proteins. tokushima j. exper. med. [ ] nr , nr. , s. ff. ( s.) . nagra, c. l., brerrenbach, r. p., u. meyer, ~. k. "-influence of hormones on food intake and lipid deposition in castrated pheasants. poultry sci. [ ] nr. , s. ff. ( s.) . *+na~:ler, w. g.: the significance of calcium ions in cardiac excitation and concentration. amer. heart j. [ ] nr l~, b. l., u. re(~a~, w. s., u. dobozy, a.: effect of vitamin a on the nucleic acid metabolism of rats. acts biologics acad. sci. hungariae , suppl. , s. . ~itz, k., u. loeser, a. : uber den einflufl appetithemmender substanzen auf das fettgewebe. klin. wschr. [ ] nr. , s. . * rstadius, k., nordstrom, g., u. lannek, n. : combined therapy with vitamin e and selenite in experimental nutritional muscular dystrophy of pigs. cornell veterinarian [ ] nr. , s. ft. 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[ ] nr. , -- ( s.) . petuet,y, f.: diskussionsbemcrkungen zu rcferaten fiber sauermilchprodukte auf einer vortragsveranstaltung der gesellsehaft fiir erniihrungsbiologie e.v., miinchen, . juni . milchwissensehaft [ nr. , s. ] . pfei~ter, ( . j., gass, g. h., u. schw~rz, ( . s.: reduction by chlorpromazine of ulcem due to acute starvation in mice. nature [ ] nr. , s. . prrrllros, a. w., newc m~, tl r., u. sha.wklrs, d. r.: long-term rat feeding studies on irradiated chicken stew and irradiated cabbage. toxicol. appl. pharmacol. [ ] nr. , s. ] . prrmt~s, p. h., sutti~, j. w., u. ze~rowski, e. j. : effects of dietary sodium fluoride on dairy cows. vii.: recovery from fluoride ingestion. j. dairy sei. [ ] l~r. , s. ff. ( s. browi~, t. h., u. levee, r. v.: the effect of milk intake on nematode infestation of the lamb. prec. nutrition oc. [ ] nr. , s. / . * srnwrvasan, m., n~o~bhusha_~a~, a., u. sm~rrvas~, k. s.: changes in serum inorganic phosphate following ingestion of protein. curr. ci. [ ] nr. , s. . stallwoth, h. : some effects of . -dichlorophen-oxyacetie acid on sweet corn (zea mays rugosa l.) with emphasis on yield, tillering, root development, and exudation of electrolytes from roots and stems. diss. abstr. [ ] nr. , --- ( s.) . starcher, b., u. i~.ratzer, f~ h.: effect of zinc on bone alkaline phosphatase in turkey poults. j. nutrition [ ] nr. , s. / . stei~-~off, d., u. ~rqu~lrdt, p. : kombination yon kaliumpyrosulfit und ~xthy]alkohol ira tr~nkungsversuch an ratten. arzneimittelforschung [ ] nr. , s. / . stevermer, e. j. : influences of level of nutrition of the boar and of ionic environment of the spermatozoa on the properties of boar semen. diss. abstr. [ ] nr. , -- ( s.) . stn~p~., f., u. c~warz, k.: incorporation of valine-l-x*c into serum and tissue proteins of rats fed torula yeast diets. j. nutrition [ ] nr. , s. / . ton~., d. b., u. co~or, w. e. : the prolonged effects of a low cholesterol, high carbohydrate diet upon the serum lipids in diabetic patients. diabetes [ ] nr. , . ft. ( s.) . *stormont, j. •., u. waterhouse, c. : effect of variations in previous diet on fasting plasma lipids. j. labor. clinical med. [ ] nr. , s. ff. ( s.) . * stuart, a. e., u vity of the heart extract of rats. medlcina exporimentalis [ ] nr. , s. ff. ( s.) . szepsenwol, j.: carcinogenic effect of egg white egg yolk and lipids in mice. prec. soc. exper. biol. med. [ ] nr. , s. ff. ( s.) . *t~cs, l, u. ny~i, i.: effect of saline infusion, acth infusion, and blood transfusion on the hormone excretion of patients with hypereme~is. act~ medics aead. sei. hun. garicae [ ] nr. , s. ff. ( s.) . tanner, j. w.: an external effect of inorganic nitrogen on nodulation. diss. abstr. [ ] nr. , -- ( s.) . --, u [ ] nr. , s. ff. ( s.) . *t~i~er, l., m~az, m., u. cs:menafiov , m. : the effect of glucose and glucose together with insulin on the resistance of fasted rats to trauma in the noble-collip drum. physiologia bohemoslovenica [ ] nr. , s. ff. ( .) . ude~iend, s. : factors in amino acid metabolism which can influence the central nervous systems. amer. j. clinical nutrition [ ] nr. , s. ff. ( . .) vahouny, g. v., moede, a., silver, b., n . treadw~ll, c. r. : nutrition studies in the cold. iv. effect of cold environment on experimental atherosclerosis in the rabbit. j. nutrition [ ] nr. , s. / . van p lsum, j. f., olsen, b., taylor, d., rozyc'ki, t., u voss, r. d.: yield and foliar composition of corn as affected by fertilizer rates and environmental factors. i)iss. abstr. [ ] nr. , -- ( s.) . *vyval,ro, i. g. : the effect of gibberellin on the transformation of substances in germinating corn seeds. i)oldady akad. nauk sssr [russ.] [ ] nr. , s. ff. ( s.) . wao~ei~, g. r., cl~mk, a. j., hays, v. w., u amer. j. clinical nutrition [ ] lgr. , s. ff. ( s.) . the assessment of marginal protein malnutrition. prec. nutrition soc. [ ] mr. , s. ] . --, u. stnp~, j. m. l.: the free ly~ine and amino nitrogen content of liver, muscle, and serum in normal and protein-depleted rats. prec. nutrition soc. [ ] mr. i, s . viii/ix. *watson, w. c.: the morphology and lipid composition of the erythrocytes in normal and essential-fatty-acid-deflcient rats. bri . j. haematology [ ] lgr. , s. ff. ( s.) . waite, r., u. : blackburn, p. s. : the relationship between milk yield, composition and tissue damage in a case of subclinical masticis. j. dairy res. [ ] , nr. , s. ff. ( s.) . *n. n.: paraty ~hoid fever from frozen chinese eggs. brit. reed. j. . ( s.). *n. n. : radioactivity and human diet. chem. ind. , nr. , . . ~q. ~.: eh~digungen dutch konservlerungsmittel bei zitrusfrfichten? dr. reed. wschr. [ ] nr. , s. . *n. n. : salt-poisoning in infancy. lancet [ ] l~r. , s. . n.n.: kouoquium des arbeitskrcises hamburg der gdch-fachgruppe lebensmittelchemic und gerichtliche chemic am . januar . lebensmittelchem. u. gerichtl. chem. [ nr. , s. . *n. n. : smoking and heath disease. new england j. med. [ ] nr. , s. . +hi. n. : toxic components of lathyrus peas. nutrition rev. [ ] nr. , s. / . +n. n. : cariogenic ability of different diets. nutrition roy. [ ] nr. , s. ] . +n. ~.: aminoaeiduria in lead intoxication. nutrition rev. [ ] nr. , s. / . +n. n. : pyridoxine and dental caries. human studies. nutrition rev. [ ] nr. , s. . +n. n.: pyridoxine and dental caries. animal studies. i~utrition rev. [ ] nr. , s. . +hi. n. : rcporb: the prophylactic requirement and the toxicity of vitamin d. pediatrics [ ] nr. , s. ff. (? s.). axrkroa, a.: caesium- from fall-out in human milk. nature [ ] nr. , s. ff. ( s.) . *allcroft, r., u. car~agha~, r. b. a. : groundnut toxicity: an examination for toxin in human food products from animals fed toxic groundnut meal. veteri. rec. [ ] nr. , s. ff. ( s.) . *--, l~wis, g., u. hill, k. ~.: groundnut toxicity in cattle: experimental poisoning of calves and a report on clinical effects in older ca~tle. ve~er. rcc, [ ] nr, , s. ff. ( s. nr. , , nr. , , , , nr. , nr. , influence of calcium and ouabai bain upon potassium influx in human erythrocytes the enzymatic assimilation of nitrate in the tomato plant translocation of '~p, i~n, and ~c in plants einige neue gesiehtspunktr zum caleiumstoffwechsel dis~ibution of water, sodium, and potassium in resting and stimulated mammalian muscle. canad the influence of vitamin bi, on calcium ('sca) metabolism of maxillodental tissues kidney, water, and electrolyte metabolism intermediiirer elektroly~toffwechsel und zellgrenzfl~chenphysiologie im theoretischen zusammenhang mit der krebsentstehtmg. tell i dcr einflul~ yon vollkornbrot auf den calcium-stoffwechsel bei schulkindern recherches sur le m tabolisme du soufre. x. : la non- quivalence de la eystine et de la eyst ine dans la couvcrture des besoins sufr~s du rat adulte potassium-magnesium antagonism in soils and crops low serum iron levels in obese adolescents metal content of human organs studies on the requirements and interaction of copper and iron in broad breasted bronze turkeys to weeks of age iron absorption and excretion in experimental iron deficiency the measurement of exchangeable magnesium in dogs the copper metabolism of warmblooded animals with special reference to the rabbit and the sheep comparative studies of the metabolism of strontium and barium in the rat the utilization of iron in erythropoiesis binding of strontium in blood ~iolybdenum, copper, and zinc contents of mouse liver and sarcoma treated with molybdenum compounds biochemical effects of zinc deficiency in tomato plants excretion of sodium, potassium, magnesium, and iron in human sweat and the relation of each to balance and requirements turnover rate of zinc in the body as determined by the study of szn in rats a study of the iron absorption in mice as modified by various agents funktionsteste des radiojodstoffwechselstudiums und ihre bedeutung in der diagnostik der sehilddrfisenerkrankungen. ~rztl. laboratorium the fate of radioiodine after parcnteral administration a possible humeral regulator of iron absorption beitrag zur kl~rung der ursachen der anreicherung yon caesinm- im rganismus blood-and serum-level of watersoluble vitamins in man and animals significados metabslicos do ~cido ~-lip ico. . o ~cido r e o metabolismo do ferro observations on a magnesium-fluoride interrelationsip in chicks prevention of ,meat anemia" in mice by copper and calcium iron metabolism in experimental pyridoxine deficiency aluminium in soils and plants on the coastlands of british guinea physiology of adolescence. ii. : nutrition -basal oxygen consumption -energy expenditure and balance -nitrogen metabolism -calcium metabolism -iron metabolism -red cell mass and hemoglobin dietary strontium and calcium, and deposition of sr and asca in the bones of rats -~iengenelementansatz wachsender sehweine bei unterschicdiichen cuso -zulagen differences in copper retention in two strains of chickens untersuchungen fiber anreicherung und verteilung yon rubidium in gerstenkcimpflanzen [ ] nr. , s. / . *+lv~ointyr~, i.: an outline of magnesium metabolism in health and disease. a review uptake by the root and subsequent distribution within the potato plant of strontium- leached from the foliage nor zinkstoffwechsel in der schwangerschaft foetal metabolism of caesium- in the rat magnesium metabolism of chickens zinc metabolism in patients with the syndrome of iron deficiency anemia hepatospenomegaly dwarfism, and hypogonadism. labor. clinical mcd c~isium- trod kalium in menschlichen organen und in der milch / l~ole of the genotype in controlling accumulation of strontium- by plants copper and zinc interrelationships in the pig effect of chromium, cadmium, and other trace metals on the growth and survival of mice studies in the metabolism of zinc. iv. some observations on the urinary zine-porphyrin relationship in non-porphyries and in a patient with aeutm intermittent porphyria aastrontium balances in man studies on zinc metabolism. ii.: effect of the diabetic state on zinc metabolism: an experimental aspect effect of diabetic state on zinc metabolism: a clini. cal aspect copper metabolism and the liver iron metabolism and the liver with particular reference to the pathogcnesis of haemachromatosis studies on iron metabolism uber den eisenstoffwechsel. (bemerkung zu t. su~di~, miinchener reed yifinchener reed. wschr. [ ] nr. , s. . c - wirkstoffe -biocatalysts +n. n. : vitamin a in human livers. nutrition biological half.llfe of vitamin b~ in plasma hypervitaminosis a and mast cells. a study of the interrelationship of mast cells and vitamin a in vivo and in vitro evidence concerning the human requirement for vitamin bi~. use of the whole body counter for determination of absorption of vitamin blz vitamin c in plasma and leucocy~s of smokers and non-smokers some factors affecting the absorption of vitamins the determination of vitamin a in animal tissues and its presence in the liver of the vitamin a deficient rat metabolic activities of vitamin a and related compounds in animals. i.: role of vitamin a in intestinal muscular contraction zum vitamin-b--haushalt dcr ratte bei sorbitfiitterung contribution a l'~tude do la relation entre la vitamine big et la glande thyrolde effects of deficiencies of certain b vitamins and ascorbic acid on absorption of vitamin blv amer ascorbic acid metabolism in plants. ii. : biosynthesis dietary and thyroid interrelationships affecting vitamin a status of feedlot beef cattle die wirkung gesteigerter kupferzufuhr auf den vitamin-c-haushalt vom meerschweinchen bei parental zugeffihrter ascorbins~ure kritische auswe~ung der naeh erschienenen arbeiten fiber gebundene ascorbins~ure im tierischen gewebe metabolism and biological activity of vitamin a acid in the chick biochemical studies of vitamin metabolism in poultry urine. ii. : on the excretion of thiamine in poultry urine after subcutaneous and oral administrations of some thiamine derivatives untersuchungen fiber die speiehcrung und fiber die ansscheidung yon vitamin a nach ungeniigender vitamin-a-versorgung bei legenden hfihnern studies on metabolism of vitamin a. .: the biological acticity of vitamin a acid in rats vitamin a and cholesterol absorption in the chicken studies on the interaction of vitamin bi~, intrinsic factor, and receptors. il the possible absorption of intrinsic factor human growth hormone in infant malnutrition macro-and micromethods for the determination of serum vitamin a using trifluoroacetic acid the activation of sulphate by extracts of cornea and colonic mucosa from normal and vitamin a-deficient animals the importance of blood as a pool of vitamin d studies on metabolism of vitamin a. .: enzymic synthesis ~nd hydrolysis of phenolic sulphates in vitamin-a-deficient rats human metabolism of l-ascorbie acid and erythorbic acid tissue distribution and storage forms of vitamin b~, injected and orally administered to the dog relation between vitamin a, tocopherol, and cholesterol serum levels in the elderly interrelationships of vitamin bi~, folio held, and ascorbie acid in the megaloblastie anemias zum wirkungsmechanismns des vitamins e. helvetica physiologica et pharmacologica effect of some physiologic factors on the absorption of vitamin bi~ in rats transport of dietary nitrogen mitochondrial fatty acids of fish and fish-eating birds the biosynthesis of fatty acids influence of age and dietary protein on cerbain free amino acids in chick blood plasma nitrogen metabolism in coldexposed rats ~ber des vermehrte auftreten yon fettsiiuren mit bis c-atomen in den depotfetten siiugender ratteu und den ubergang der linolsi~ure yon den mfittern auf die jungen die bildung yon antiksrpcrn gegen verschicdene kuhmilehproteine bei neugeborenen, kindern, erwachsenen und graviden the myocardial arterio-venous differences of free amino acids and of free fatty acids in healthy individuals, patients with diabetes and with essential hypercholesterolemia urinary amino acids on phenylalanine-tyrosine-supplemented diets difference in the metabolic fate of acetate and ethanol fed to higher plant tissues iiemodynamic relationships of anaerobic metabolism and plasma free fatty acids during prolonged, strenuous exercise in trained and untrained subjects effects of palmitate on the metabolism of leukooytes from-guinea pig exudate the dynamics of plasma free fatty acid metabolism during exercise ~ber die retention, den abbau und die ausseheidung yon -thion-tetrahydro-l. . -thiadiazinen transport systems for amino acids effects of protein intake and cold exposure on selected liver enzymes associated with amino acid metabolism quantitative studies on tryptophan metabolism in the pyridoxine-deficient rat effect of desoxypyridoxine-induced vitamin b~ deficiency on polyunsaturated fatty acid metabolism in human beings studies on the wheat plants using carbon- compounds. xix.: observations on the metabolism of lysine- c. canad genetic defects of amino acid metabolism. pediatric clinics north america metabolism of tryptophan in diabetes mellitus the two-carbon chain in metabolism der einfluss yon vitamin a auf den citronens~urestoffwechsel ~)ber phenolspeicherung und phenolabbau in wasserpflanzen. naturwissensehaften effect of physical exercise on nitrogen balance in obese subjects metabolism of nitrogen compounds in the rumen of ruminants. izvest. acad der intermedli~r-stoffwechsel s~ffweehsel der carotine im hiihnerembryo nucleic acid metabolism of germinating corn seedlings carbon metabolism of ~ c-labelled amino acids in wheat leaves. ii.: serine and its role in glycine metabolism bovine metabolism of insecticides. the metabolism of ~evin in dairy cows stoffweehsel der carotine. wiss. veroff. dr. oes. ern~hrnng metabolism of labelled linolcic-l-tac acid in the sheep rnmen nonessential nitrogen supplements and essential amino acid requirements. nutrition rev vitamin c requirements of man re-examined. new values based on previously unrecognized exhalatory excretory pathway of ascorbie acid studies on the requirements and interaction of copper and iron in broad breasted bronze turkeys to weeks of age water requirements of men as related to salt intake evidence for a high zinc requirement at the onset of egg production effect of lysine and glyeine upon arginine requirement of guinea-pigs the cobalt requirement of subterranean clover in the field fluid and electrolyte, requirements of newborn infants with intestinal obstruction the requirement and availability of dietary iron for young pigs studies on the protein and methionine requirements of young bobwhite quail and young ringnecked pheasants phenylalanine requirement of women consuming a minimal tyrosine diet and the sparing effect of tyrosine on the phenylalanine requirement effects of starvation on the cardiovascular system of the chicken calcium and phosphorus requiremeats of finishing broilers using phosphorus sources of low and high availability water intake of normal children sex differences in the ~ oeopherol requirement of rats as shown by the haemolysis test further studies on protein and energy requirements of chicks selected for high and iow body weight smoking and blood dotting dental caries and trace elements a statement approved by the board of directors of the canadian heart foundation the question of fats. il : fats and disease moldy peanuts and liver cancers vitamin c and healing of wounds berieht fiber die vortragstagung des fachverbandes lehensmittelchemie der chemisehen gesellschaft in der d])r veto . his diet and human depot fat ethanol and plasma free fatty acid in man dietary water and protein efficiency in rats. nutrition rev. [ ] nr. , s. / . +n. n. : nature of the coagulation defect in rats fed diets producing thrombosis or experimental atheroselerosis factors affecting growth depression by raw soybeans bones in undernourished animals. nutrition rev. [ ] nr. , s. . +n. n. : vitamin e and the etiology of muscular dystrophy in the rabbit riboflavin eoenzymes and congenital malformations the thyroid gland in infant malnutrition. nutrition rev. [ ] nr. , s. . +n. n. : a proposed mechanism for the effect of fats on serum cholesterol effect of varying levels of dietary protein on synthesis and excretion of urea dietary phosphates and dental caries folic acid restriction and cancer inhibition changes induced by lipoie acid in normal rat liver vitamin b deficiency and tryptophan metabolism effect of ubichromenol on development of encephmomalacia in vitamin e deficient chicks leucine-induced hypoglycemia nutritional muscular dystrophy in lambs. nutrition rev. [ ] nr. , s. / . +n. n.: amino acid imbalance in cold-exposed rats. nutrition rev. [ ] nr. , s. . +n. n.: milk and athletic performance effect of vitamin a deficiency on the ubiquinone content of rat liver idiopatjaie stea~orrhea gastrointestinal protein loss in iron-deficiency anemia nutritional cirrhosis of the liver. nutrition rev. [ ] nr. , s. / . +n. n. : exercise and heart disease calcium deficiency in the etiology of osteoporosis the relation of dietary fat to the fatty acids in the intestinal wall clot-strength and elot-lysis in rats fed hyperlipemic diets effect of potassium iodide and duodenal powder on the growth and organ weights of goitrogen-fed rats ver~nderungen im stoffwechsel und wachsturn junger tomatenpflanzen nach giberillins~,urebehandiung effect of restricted feeding during the growing period on reproductive performance of large type white turkeys keys, a. l glucose, sucrose, and lactose in the diet and blood lipids in man ambient temperature and survival on a protein-deficient diet some considerations of changes in total body composition in relation to nutritional status the effect of variations in the energy and protein levels of the ration upon performance in the pig studies in choline deficiency. fate of injected - ~c-pal. mitio acid and fatty acid spectra in fasting and refed rats bartou i~ vitamin a requirements of chicks at moderately elevated temperature influence of age and dietary protein on eer~ain free amino acids in chick blood plasma the effects of nicotine on weight increment, activity, food intake, and water intake in weanllng albino rats effect of pyridoxine deficiency upon delayed hypersensitivity in guinea-pigs vitamin a deficiency in chickens ccrebellar encephalomalacia produced by diets deficient in toeopherol vitamin b deficiency in indian infantm plasma liplds in scurvy: effect of ascorblc acid supplement and insulin treatment effect of gibberellin on the variations of the growth-point in winter wheat uptake of dinitrophenol and its effect on transpiration. calcium accumulation in barley seedlings l~[ethylmalonate excretion in vitamin b~ deficiency reduction of plama cholesterol levels in atherosclerosis by diet and drug treatment. australasian ann effects of reduced dietary intake on the activities of various enzymes in the livers and kidneys of growing male rats the effect of feeding d-methionine on the d-amino acid oxidase activity of chick tissues l~etabolic effects of dietary protein level in cold-exposed rats relative effects of rapeseed oil and corn oil on rats subjected to aclrenalectomy, cold, or pyridoxine deprivation metabolic effects of dietary protein level with caloric restriction in coldexposed rats. canad metabolic effects of three dietary protein levels fed isocalorically to coldexposed rats. caned die nolle versehiedener fette im eiweis des organismus. nahrung the bursa of fabricius and xanthine oxidase activity of liver and kidney following dietary supplementation of iodina~d casein to chickens effects of linolsate and dietary fat level on plasma and liver cholesterol and vascular lesions of the cholesterol-fed rat a comparative study of the effect of bile acids and cholesterol on cholesterol metabolism in the mouse, rat, hamster, and guineapig the effects of ruminal and plasma sodium concentrations on the sodium appetite of sheep effect of level and sequence of feeding and breed on ovulation rate, embryo survival, and fetal growth in the mature ewe inhibitory effects of carbohydrates on histamine release and mast cell disruption by dextran fett-s~urestoffwechsel bci the effect of calcium infusions, parathyroid hormone, and vitamin i) on renal clearance of calcium nutritional supplementation during pregnancy effect of level of dietary protein with and without added cholesterol on plasma cholesterol levels in man die schilddrfisenfunktion bei enteralem eiweiflverlust effects of pelleting and varying grain intakes on milk yield and composition fatty acid composition of lipids of serum and aorta in the chicken on different diets rat intestinal suerase. ii.: the effects of rat age and sex and of diet on suerase activity the effect of selenium administration on the growth and health of sheep on scottish farns die wirkung yon vitamin b bei leukopenien effect of energy source and level of alfalfa pellets on growth and tissue hpids of beef calves effect of magnesium deficiency on mast cells and urinary histamine in rats histamine-liberating effect of magnesium deficiency in the rat zur wirkung des wassers bei der seitenwurzelbildung an luftwurzeln influence of the aqueous potato extract and its fractions on growth and spore formation of the b. pumilus and the production of the antibiotic tetaine influence of mineral nutrition on the resistance of peach tree to fusicoceum amygdali de la croix the effect of dietary protein on the course of various infections in the chick bifidus intestinal flora in infants fed on mamysan b. acta paediatriea effect of food fats on concentration of ketone bodies and citric acid level in blood and tissues is there a hemostatie effect of peanuts in hemophilioid disorders? milk allergy in infancy dental effects of fluoridation of water with particular reference to a study in the united kingdom influence of previous feeding with a high-fat diet on liver steatosis produced by acute starvation of growth hormone in mice effect of amino acid imbalance on nitrogen retention. ii.-interrelationships between methionine, valine, isoleucine, and threonine as supplements to corn protein for dogs supplementation of cereal proteins with amino acids. v. : effect of supplementation lime-treated corn with diffe. rent levels of lysine, tryptophane, and isoleueine of the nitrogen retention of young children the interrelation of nutrient supply, leaf nutrient content, and vegetative growth of ilex crenata gastric content of fasted primates. a survey serum cholesterol in vitamin c deficiency in man the effect of carbohydrates on the production of staphylococcal pigment effect of a low dietary level of three types of fat on reproductive performance and tissue lipid content of the vitamin b -defieient female rat effect of magnesium deficiency on synthesis of hear~ and liver mit~chondria phospholipids ~ber den einflub l~nger dauernder ksrperlicher inaktivit~t auf die blutzuckerkurve nach oraler glucosebelasttmg. helvetica medica acts action of trace elements on the metabolism of fluoride zur frage des einflusses yon kondensmilch und einer protein-valerina-polyphosphat-komplexverbindung auf die kreislaufwirktmg des kaffees beim menschen modifications de la croissance de la plantule de lapin blanc (lupinns albns l.) provoqu es par une diminution exp rimentale des r serves influence of the dietary protein level on the magnesium requirement effects of high levels of copper and chlorotetracycline on performance of pigs effect of dietary calcium lactate and lactitic acid on faecal escherichia coli counts in pigs die entwickhing von calcium-mangelsymptomen. z. pflanzenern~hrung, diingung, bodenkde. [ ] nr. , s. / . --calcium-mangelsymptome an hsheren pfianzen copper deficiency in relation to swayback in sheep. i. : effect of molybdate and sulphate supplements during pregnancy long-term, low fat, low protein diets and their effect on normal trappist subjects further studies of the influence of diet on radiosensitivity of guinea-pigs, with special reference to broccoli and alfalfa effects of the infusions of ammonia, amides, and amino acids on excretion of ammonia answirlmngen langfristig fettreicher ern~ihrung auf das plasma-cholesterin the relationship of dietary energy level and density to the growth response of chicks to fats influences of dietary carbohydrate-fat combinations on various functions associated with glycosis and lipogenesis in rats. i. effects of substituting sucrose for rice starch with unsaturated and with saturated fat compensatory carcass growth in steers following protein and energy restriction fatty acid composition and glyceride structure in rats fed rapeseed oil or corn oil. canad influence of selective and nonselective hydrogenation of rapeseed oil on carcass fat of rats. canad studies in serum lipids. with special reference to spontaneous variations and the effect of short-term dietary changes experimentelle untersuchungen zur wirkung yon kaffeefett evaluation of the effect of breed on vitamin be requirements of chicks -iigh salt content of western infant's diet: possible relationship to hypertension in the adult the effect on the serum cholesterol levels of the consumption of a special dietary fat with a high content of unsaturated fatty acids in elderly people effect of dilution of the diet with an indigestible filler on feed intake in the mouse effect of tea and its tannins upon capillary resistance of guinea-pigs food input and energy extraction efficiency in carassins auratns effect of calcium and magnesium upon digestibility of a ration containing corn oil by lambs effects of calorie restriction during the growing period on the performance of egg-type replacement stock effects of insulin on hepatic glucose metabolism and glucose utilization by tissues cellulase and polygalacturonase in tomato fruits and the effect of calcium on fruit cracking effect of nutritional muscular dystrophy and of starvation on amino acid penetration in rabbit tissues plasma protein synthesis in nutritional muscular dystrophy inulin and sucrose distribution in tissues of vitamin e-deficient and control rabbits protein-bound dyes in the serum and liver of rats fed aminoazo dyes vanadium. excretion, toxicity, lipid effect in man the influence of vitamin a status on the proteoly~ic activity of lysosomes from the livers and kidneys of rats disauxie metainfettive e da malnutrizione induced drinking in dogs: comparative effects of hypertonic sodium chloride and sorbitol the influence of early nutrition on brain cholesterol accumulation during growth changes in composition of the saliva of cows on grazing heavily fertilized grass. res. veter changes in composition of the saliva of sheep on feeding heavily fertilized grass efect of varying alfalfa: barley ratios on energy intake and volatile fatty acid production by sheep the influence of calcium on the secretory response of the submaxillary gland to acetyi-choline or to noradrenaline clinical dentistry and fluoride food allergy as a cause of abdominal pain the effect of various dietary levels of ddt on liver function, cell morphology, and ddt storage in the rhesus monkey effect of natural and purified diets on survival of x-irradiated mice effect of autoclaving and ])'sine supplementation of skimmilk-powder diets on growth and caries in rats effects of alcohol intake on subjective and objective variables over a five-hour period nitrogen metabolism of african cattle fed diets with an adequate energy feeding value of fl-caroteno following treatment with n~o~ the relationship of the quantity and quality of dietary fats to serum cholesterol levels in men of different ages and weights effects on girls of greater intake of milk, fruits, and vegetables effect of antioxidant, protein, and energy on vitamin a and feed utilization in steers the growth-maintaining activity of ascorbic acid the effects of an induced pyridoxine and pantothenie acid deficiency on excretions of oxalic and xanthurenic acids in the urine influence of lactose and dried skim milk upon the magnesium deficiency syndrome in the dog. i.: growth and biochem chronic malnutrition in turkey. v. study on serum fatty acids in malnourished children the effect of nutrition conditions on the growth of and nitrogen accumalation by fodder beans when sown together with indian corn effects of a diet high in polyunsaturated fat on the plasmalipids of normal young females citrate and action of vitamin d on calcium and phosphorus metabolism beeinflussung der sportliehen lelstungsf/~higkeit durch eine geeignete er-nehrung effect of dietary erotic acid on liver proteins effect of barbiturie acid and ehlortetraeyeline upon growth, ammonia concentration, and urease activity in the gastrointestinal tract of chicks effects of feeding low levels of dimethoate on milk and whole blood eholinesterase activity of dairy cattle changes in serum lipoproteins after a large fat meal in normal individuals and in patients with isehemic hear~ disease the relationship of specific nutrient deficiencies ~) antibody response in swine. i. : vitamin a. ii. : pantothenie acid, pyridoxine or riboflavin. i)iss. abstr relationship of specific nutrient deficiencies to antibody production on swine. i. : vitamin a relationship of specific nutrient deficiencies to antibody production in swine. il : pantothenic acid, pyridoxine or riboflavin histechemistry of dietary cardiac lesions effect of various levels of fluorine, stilbestrol, and oxytetraeycline, in the fattening ration of lambs uptake of copper and its physiological effects on chlorella vulgarls the effects of a small dose of ethyl alcohol on certain basic components of human physical performance. i. the effect on cardiac rate during muscular work. arch. internat some effects of feeding stilbestrol, chlortetracycline and penicillin with alfalfa soilage on steer performance and carcass quality experimental induction of ciguatera toxicity in fish $hrough diet effects of potassium fertilizer, age of ewe, and small magnesium supplementation on blood magnesium and calcium levels of lactating ewes the influence of higher volatile fatty acids on the intake of urea-supplemented low quality cereal hay by sheep un~emuchungen fiber den umsatz wachsender schweine ab geburt. . mitt eczema and cow's milk. brit. med. j. , nr. , s. . isaacso~, a. : the effects of zinc on responses of frog skeletal muscle effects of zinc on responses of skeletal muscle effect of diet on work metabolism carbohydrate-phosphorus metabolism in the skeletal muscles of epinephrectomized animals durlng treatment with cortisone and vitamins c and p. ukrainskii biokhimichnii zhurnal composition of dietary fat and the accumulation of liver lipid in the choline-deficlent rat nutrition and palatability the incidence of protein-calorie malnutrition of early childhood theories on the mode of action of fluoride in reducing dental decay saccharase deficiency, familial entailing intolerance ~ cane sugar. acts paediatrica raw and heat-treated soybeans for growing-finishing swine and their effect on fat firmness effect of the administration of isoniazid and a diet low in vitamin be on urinary excretion of oxalic acid dietary and thyroid interrelationships affecting vitamin a status of feedlot beef cattle ration effects on rumen acids, ketogenesis, and milk composition. i.: unrestricted roughage feeding the effect of supplements of groundnut flour or groundnut protein isolate fortifed with calcium salts and vitamins or of sklmmilk powder on the digestibility coefficient, biological value and net utilization of the proteins of poor indian diets given to undernourished children a comparison of skin-testing with natural foods and commercial extracts die wirkung yon hungern auf den ammoniakgehalt und das ph der pansenfi(issigkeit sowic auf die harnstoff-, cholesterin-und zuckerkonzentration im blu~. acts veterinaria acad increase of plant virus infection by magnesium in the presence of phosphate effect of intramuscular injection of magnesium sulphate solution on the growth rate and serum composition in rats diskussionsbemerkungen zu referaten fiber sauermilchprodukte auf einer vortragsveranstaltung der gesellschaft fiir eru~ effect of massive sodium bicarbonate infusion on renal function excessive insulin response to glucose in obese subjects as measured by immunoehemical assay die wirkung gesteigerter kupferzufuhr auf den vitamin-c-hanshalt yon meerschweinehen bci paren~ral zugef'dhrter ascorbinsrure the influence of growth hormone on fat and protein metabolism. dies. abs~r. [ ] nr phenolearbons~iuren in mensehlichen nahrungsprodukten. zum vorkommen yon phenolcarbons~uren in mensehlichen nahrungsprodukten und ihr einflul~ auf den intermedit~ren stoffwechsel influence of pregnancy and an oxidized lipid diet on the fatty acid composition of blood and tissue an experimental approach to the mechanisms of weight loss. ii. a comparison of effects of thyroxine, fat-mobilizing substance (fms) and food deprivation in achieving weight loss in mice fat accumulation in the regenerating media of arteries in rats given an atherogenic diet the effect of nutrition on the growth of faseiola hepatica in its snail host the effects of specific viruses, virus complexes, and nitrogen nutrition on the growth, flowering, and mineral composition os strawberry plants body weight and food intake as initiating factors for puberty in the rat the role of catecholamines in the free fatty acid response to cigarette smoking die renale steroidausseheidung bei experimentellem vitamin-e-mangel peculiar features of respiration and redox processes in the rice plants grown under different nutritional conditions der bohnenkaffee und die migrrne repeatability of litter size and weight in the laboratory rat as affected by selection and plane of nutrition wirkungen yon muskelextrakt auf den stoffwechsel. arzneimittelforschung is [ ] nr. , s. / einflul] der silageftitterung auf die qualit~t der butter einflul] der silageftitterung auf die qualit~t yon milch und milchproduk. ten. . mitt.: einflul] von silagefiitterung auf die organoleptischen eigenschaften dcr milch effect of protein intake and cold exposure on selected liver enzymes associated with amino acid metabolism body iron levels and hematologic fin. dings during excess methionine feeding der einttul~ des kulturmediums auf die bildung von streptolysis s durch ruhcnde zellen influence of polyphosphates in chilling water on quality of poultry meat influence of breed-type, feed level, and sex on characteristics of the lamb carcass, and some relationships among live animal and carcass measurements the toxic action of phenothiazine and some disturbances of intermediary metabolism in undernourished sheep efficiency of feed use in beef cattle uber die wirkung der nalmmgsfette auf die blutlipoide, teil i. ernahrungs-umsehau [ ] nr. , s. / . --~ber die wirkung der nahrungsfette auf die blutlipide fette und blutgerinnung. bibliothcca hacmatologiea effect of heavy cigarette smoking on postprandial triglycerides, free fatty acids, and cholesterol role of calcium in fibrin formation glucose- -phosphate dehydrogcnase and aldolase in lenses of lactose-fed rats -effect of riboflavin, choline, pantothenic acid and vitamin a on the excretion of sodium in urine of rats the effect of waterwashed rice in the diet on the growth, excretion of sodium in urine and blood pressure of rats effect of aseorbic acid, vitamin b, and milk on the dark adaption effect of single deficiency of vitamin a, thiamine der einflub yon vitaminen auf die psyehomotorisehe leistungsi'~higkcit beim menschen. naunyn-schmiederberg's arch. exper feeding response of adult tribolium to carbohydrates in relation to their utilization nutritional secondary hyper hieronymi: influence of nutritional conditions on the cellular rna metabolism in rive and in vitro diffusible auxin increase in a rosette plant treated with gibberellin transamination in muscular dystrophy and the effect of exogenous glutamate: a study on vitamin e deficient rabbits, and mice with hereditary dystrophy. canad effect of auxin on the emergence of lateral roots in p. mungo seedlings the effect of nutritive status on oestrus, ovulation, and graafian follicles in merino ewes biologische wirkungcn autoxydierter, epoxydierter und bestrahlter fette s~ure-basen-gleichgewicht mad chronische acidogene und alkalogene eruehrung effect of protein level in milk replacers on growth and protein metabolism of dairy calves effect of sodium bicarbonate in the drinking water of ruminants on the digestibility of a pelleted complete ration sucrose diet and bfliary chelate excretion in rats: with note on procedure for chelate determination eirdlub sauerer milcherzeugnisse auf die darmflora untersuchungen fiber die speicherung und die ausscheidung yon vitamin a nach ungenfigender vitamin-a-versorgung bei legenden hiihnern the effects of low magnesium intake on lactating ewes effect of vitamin a on the content of pyridinnucleotides, pyrovic, and lactic acid and on anaerobic phosphorylation. ukrainskii biokkimichnii zhurnal the pyridine nucteotides. a study of a method of measurement. a study of the alterations in rat fiver under the conditions of diabetes and starvation. a preliminary study of various marine fish tissues with the emphasis on the islet of langerhans iron uptake-transport of soybeans as influenced by other cations hshe und zeitpm~kt der dfingung yon sommerweizen mit chlorcholinchlorid zur vcrkiirzung der halmlange nutritional significance of soluble nitrogen in dietary proteins for ruminants effects of long-term feeding of vegetable fats on atherosclerosis effects of feeding various mile, corn, and protein levels on laying performance of egg production stock some observations on the influence of a magnesiumdeficient diet of rats, with special reference to renal concentrating ability effect of gibberellic acid on flowering of apple trees the effects of dietary fat and energy levels on the performance of caged laying birds motivational producing properties of the feeding system of the rat hypothalamus the influence of diet and age on lipid metabolism of chickens effect of age on the response of chickens to dietary protein and fat absorption and excretion of biotin after feeding minced liver in achlorhydria and after partial gastrectomy variations de la calcsmie du chien normal apr~s ingestion de cholesterol eristallis~ dans l' thanol ou dans rhexane changes in activity of rat epididymal adipose tissue in vitro due to time elapsed since last feeding differences in rat strain response to three diets of different compositions l'alcoolisme ~ l'hspital psychiatrique de colsou (martinique). ann. m~dico-psychologiques nitrogen, lipid, glycogen, and deoxyribonu. eleie acid content of human liver. the effect of brief starvation and intravenous administration of glucose some metabolic and nutritional factors affecting the survival of erythrocytes erythrocyte survival of rats deficient in vitamin e or vitamin b . j. nutrition [ ] nr. , s. / nutrition and lactation the effect of administering sodium chloride, sodium bicarbonate, and potassium bicarbonate to newly born piglets strontinm- and calcium in milk of miniature swine further studies on cariostatic effect of organic and inorganic phosphates urinary excretion of magnesium in man following the ingestion of ethanol the effects of magnesium compounds and of fertilizers on the mineral composition of herbage and on the incidence of hypomagnesaemia in dairy cows behavioral, dietary, and autonomic effects of ehlordiazepoxide in the rat the effect of a high and low sodium diet in a patient with familial periodic paralysis the effect of quaternary ammonium anion exchange resin on plasma and egg yolk cholesterol in the laying hen vitamin e deficiency and ion transpor~ in rat liver slices effect of level of nitrogen on growth and reproductive physiology of young buus and rams influence of low protein rations on growth and semen characteristics of young beef bulls, if treatment of nutritional cirrhosis in rats with choline and methionine; with special reference to fibrogenesis and fibroelasia probleme der beurteilung yon sauermilcherzeugnissen. milchwissen. schaft [ ] nr. , s. / . --antwort auf die diskussionsbemerkungen auf einer vortragstagung der gesellschaft ffir ern~hrungsbiologie e. v., miinehen, am response of early-weaned pigs to variations in dietary calcium level with and without lactose effect of low calcium diet on bone crysta]linity and skeletal uptake of sca in rats response of rural guatemalan indian children with hypocholesterolemia to increased crystalline cholesterol intake source of plasma free fatty acids in dogs receiving fat emulsion and heparin alcoholic intoxication in the newborn infant. bril mcd dental caries of rats fed a rice diet and modifications a study of zinc deficiency in the dairy calf effects of different levels of zinc and phosphorus on the growth of subterranean clover (trifolium subterraneum l) lrber den einflu yon fluor auf den wassergehalt des knochens gegevens ovvr vitamine b,-deficientie, -behoefto en -voorziening the liquefying action of pancreatic, cereal, fungal, and bacterial co-amylases ern~ihrung und endokrines system . mitt.: der einfiul der erniihrung auf die schilddriise the effect of saline water on kidney tubular function and electrolyte excretion in sheep zinc and iron deficiencies in male subjects with dwarfism and hypogonadism but without ancylostomiasis, schistosomiasis or severe anemia die yextriiglichkeit yon xyli~ beim diabetiker einflni der laevulose auf die fu~ionsbrelte. sport~tl. praxis der einflul~ yon vitamin a auf den zitronensi~urestoffwechsel studies on the growth-promoting value and digestibility of passion fruit seed oil ration effects on drylot steer feeding patterns dextrothyroxine on metabolism of cholesterol some effects of feeding lactates to dairy cows copper deficiency problems in south-east scotland bone changes in iron deficiency anaemia a preliminary report on nucleic acid levels in mineral deficient plants metabolism of histidine in protein malnutrition ttypoglycemie effect of l-leucine during periods of endogenous hyperinsulinism nutritional studies with the guinea-pig. viii. : effect of different proteins, with and without amino acid supplements, on growth some effects of sulphur-containing amino acids on the growth and composition of wool effects of hunger and vi value on vi pacing potency of conditioned reinforcem based on food and on food and punishment sfibwaren und karies in theorie und praxis effect of magnesium on the changes of myocardial potassium confent untersuchungen fiber den einflub oral verabreiehten oxytetraeyclins auf leberlipide und serumeholesterin der weil~en ratte further studies on manganese nutrition of tobacco in relation to virus infection and synthesis aminobutyric acid (),-aba)-vitamin be relationships in the brain serum lipids and diet: a comparison between three population groups with low, medium, and high fat intake effects of light and gibberellin on elongation of intact wheat coleoptiles experimental magnesium deficiency in the cow thyroid function in chickens and rats: effect of iodine content of the diet and hypophysectomy on iodine metabolism in white leghorns cockerels and long-evans rats kuhmilehallergie beim sgugling und a rapid method for assessing drug inhibition of feeding behavior variation in, and the effects of vitamins on vertieillinm albo-atrnm influence of high level vitamin a supplement on semen characteristics and blood composition of breeding bulls influence of diet on viral hepatitis der einflul] der stiekstoffdfingung auf die zusammcnsetzung yon karf~ffeleiweib insulin response to fructose and galactose the effect of excess vitamin a on the oxygen consumption of young female rats effect of dietary amino acid pattern on plasma ~mino acid pattern and food intake gibbere/lln: effect on diffusible auxin in fruit development effect of intravenous versus oral fat administration in fat-deiicient dogs plasma vitamin bi~ levels in some nutritional deficiency states nutritional factors influencing the conversion of tryptophan to niacin pancreatic hypertrophy and chick growth inhibition by soybean fractions devoid of trypsin inhibitor production, interior egg quality, and some physiological effects of feeding raw soybean meal to laying hens effect of palm jaggeries on the growth and blood and liver composition of albino rats kept on rice and jowa effects of polyphosphates on water uptake, moisture retention, and cooking loss in broilers untersuchungen fiber den ern~hrungsphysiologischen wer~ yon kasein entgegnung zu diskussionsbemerkungen auf einer vortragsveranstaltung der gesellschaft for ern~ihrungsbiologie e. v., mfinchen, am . juni die erg~nzungswirkung yon dl-methionin allein oder in kombination mit l-lysin beim wachsenden schwein der einflul~ der nahnmg auf den kauapparat der einflu der nahrung auf den kauapparat. teil il changes in bone mass and density in living rats during the manipulation of calcium intake effect of chromium, cadmium, and other trace metals on the growth and survival of mice a study of fermentation in the production of mahewu, an indigenous sour maize beverage of southern africa the vitamin b~ deficiency syndrome in human infancy, biochemical and clinical observations lipids in chick urine: the influence of dietary rapeseed oil effects of dietary nitri~ on the chick" growth, liver vitamin a stores, and thyroid weight influence of radioactive sodium- on higher nervous activity of dogs, when chronically administered into the organism in comparatively small doses the change of erythroeyte blood composition in persons with prolonged complete alimentary starvation (without limiting the water intake) and subsequent feeding dental abnormalities in rats attributable to protein deficiency during reproduction the effect of environment, and nutrition of pathogen and host, in the damping off of seedlings by rhizoctonia solani effect of dietary protein on fructose, citric acid, and -nucleotldase activity in the semen of bulls the effect of fluorine on dairy cattle. v. : fluorine in the urine as an estimator of fluorine intake some effects of diet and therapy on the survival and metabolism of adrenalectomized rats effect of methonine and choline deficiency on liver choline oxidase activity in young rats untersuchungen fiber die wasserlssliehen hemmstoffe aus dem chnittholz der weinrebe (vi~is vinifera l.). i. zur wirkung der hemmstoffe auf die keimlmg mad entwicklung yon rebs~mlingen nutrition and palatability. lancek the effect of feeding fluoride on some enzymes of bovine tissues diet and histamine in the rmninant effect of food reflexes on cholinestera~e activity of cortical tissue. federation essential fatty acid deficiency and rat liver homogenate oxidations the effect of vitamin and antibiotic injections on early turkey poult growth and mortality alimentary production of gallstones in hamsters. . studies with rice starch diets with and without antibiotics nitrogen studies with apple and cranberry the influence of diet on the quality of faecal fat in patients with and without steatorrhoea uber die unterschiediiche beeinflussung des tryptophansteffwechsels dutch vitamin b -mangel in der ratte. hoppe-seyler's influence of vitamin b and its coenzyme upon incorporation in rive of amino acids into tissue proteins in rats relativer vitamin b -mangel hei erkrankungen der schilddriise strukturanomalien der z/ihne bei vitamin d-mangel-raehitis und der vitamin d-resistenten renalen rachitis the effect of fluoride on bone effects of insulin on hepatic glucose production and utilization prevention by hydrocortisone of changes in connective tissue induced by an excess of vitamin a acid in amphibia acute hypervitaminosis a in guinea-pigs. i. : effect on acid hydrolases der einfiu~ yon vollkornbrot auf den calciumstoffwechsel bei schulkindern effect of feeding milk replacers with varying amounts of f~ for hothonsc lamb production egg yolk and serum cholesterol levels: importance of dietary cholesterol intake effect of protein intake on glutamic dehydrogenase and amino acid desruination in rive observations in experimental magnesium depletion effect of gibbercluc acid on growth, gibberellin content, and chlorophyll content of leaves of potato ~ physiological factors influencing growth, reproduction, and production of well-fed dairy heifers. i. age at first breeding. ii. feeding of diethytstilbestrol tm~ influence of diet on the development of parotid salivation and the rumen of the lamb bericht fiber den wissenschaftlichen kongreb der deutschen geseuschaft fiir erniihrung influence of variations in dietary calcium: phosphorus ratio on performance and blood constituents of calves the lack of a consistent chick growth response to norwegian kelp meal some effects of kinctin on the growth and flowering of intact green plants incorporation of [, p] orthophosphate into phospholipids of frog tissues during feeding and stmrvation growth-modifying and antimetabolite effects of amino acids in chrysanthemum a study of techniques used by advertisers in dealing with weight control. a national health problem lipid excretion. .: examination of faecal lipids of rats injected intravenously with serum lipoprotcin containing ~ac-labelled cholesterol effect of diet and diabetes on plasma glucose, fatty acid, and insulin effect of cigaret smoking during pregnancy: study of cases. obstetrics gynecology respiration and phosphorylation in live homogenates from rats exposed to hypoxia and food restriction the influence of mi]l~ fat depressing rations on the yield and composition of bovine milk phosphatides and cholesterol in the rat bed: effects of growth, diet, and age the effect of plant nutrients and antagonistic microorganisms on the damp. ing-off of cotton seedlings caused by rhizoctonia solani kurus l~utrition of gram-negative anaerobic bacilli. nutrition rev. effects of glucose on the production by escherichia coli of hydrogen sulphide from cysteine. j. general iylierobiol. enumeration of psyehrophilie microorganisms vitamin requirements of three pathogenic fungi calorie requirements of rat intestinal microorganisms specificity of the salt requirement of halobacterium cutirubrum influence of the aqueous potato extract and its fractions on growth and spore formation of the b. pumilus and the production of the antibiotic tetaine the relationship between hormonal activity and sugar metabolism in protoparce scxta (joka~sen) and blabcrus craniifer bur~ieister apparent incorporation of ammonia and amino acid carbon during growth of selected species of ruminal bacteria l~ber die wirkung anorganischer st~ube auf das wachstum yon mikroorganismen effect of dietary calcium lactate and lactic acid of faecal eseherichia coli counts in pigs uber den einflul des n~ihrsubstrats auf die hemmung des bakterienwachstums durch cyanid autoradiographic studies of the differential incorporation of glycine, and purine and pyrimidine ribosides by paramecium aurelia correlation between the essential amino-acid requirements of staphylococcus aureus, their phage types, and antibiotic patterns nutrition and metabolism of marine bacteria. xii.: ion activation of adenosine triphosphat~se in membranes of marine bacterial cells carbon dioxide fixation in bacillus anthracis bacterial growth under conditions of limited nutrition interrelationship between temperature and sodium chloride on growth of lactic acid bacteria isolated from meat-curing brines morphological variation and nutrition of a new monoeentric marine fungns feeding stimulants required by a polyphagons insect, schlstocera gregaria vitamin requirements of root nodule bacteria phcnotypic, genotypic, and chemical changes in starving populations of aerobacter aerogenes studies on the d-amino acids. ii.: utilization of d-amino acids by lactic acid bacteria role and formation of the acid phosphatase in yeast der einflub yon co~-partia]druck und glucose-konzentration auf wachsturn und stiekstoffbindung yon azotobacter chroococcum bei~ inorganic polyphosphate metabolism in chlorobium thiosulfatophilum effects of molybdenum, vanadium, tungsten, and cobalt on growth of rhizobia and their hosts nutrition of leptospira pomona. ii.: fatty acid requirements sterilization by beta-propiolactone of solid nutrient media for eultivation of moulds the digestion of natural food protein by the elearnose skate raja eglanteria (bose.) utilization of amino acids during metabolism in escheriehia coil the effect of nutrition on the growth of fasciola hepatica in its snail host cobamide coenzyme contents of soybean nodules and nitrogen fixing bacteria in relation to physio]ogical conditions determination of carbohydrate metabolism of marine bacteria the amino acid requirements of various types of shigella mushroom culture. factors affecting the growth of morel mushroom myecelium in submerged culture lebensmittelzusatzstoffe und mutagene wirkung. vii. : priifung einiger xanthen-farbs~offe auf mutagene wirkung an escherichia coll the biological control of glycogen metabolism in agrobaeterium tumefaeiens the maintenance requirement of escheriehia coll methionine requirement for growth of a species of mieroeoecus ~iorphogenesis and nutrition in the memnionella-stachybotrys group of fungi viable organisms from feces and food-s~uffs from early antarctic expeditions the metabolism of yeas~ sporulation. v. : stimulation and inhibition of sporulation and growth by nitrogen compounds the effect of lipids on citric acid production by an aspergillns niger mutant relationship between deuterium inhibition of growth and glucose catabolism in saecharomyees cerevisiae function of trehalose in baker's yeast (saccharomyces cerevisiae). arch. biochem preparation and lyophilization of colicine suspensions. i. production of eolicines in liquid nutrient media lvber den einflub der kulturbedingungen auf die stramenempfmdliehkeit der glueoseoxydation in baeterium cadaveris nutritional requirements and metabolism of myeoplasma laid-lawil j. gen. microbiol. die wirkung subletaler konzentrationen yon sorbinsi~ure auf escherichia coli und aspergillus niger the genetic analysis of carbohydrate utilization in aspergillus nidulans the amino acid nutrition of respiration deficient and respiration competent saecharomyces. a. van leewenhoek nutritional studies of some membem of mucorales. iv.: . sugars, amino-, and organic acids of the myceaium selektivn~hrboden fiir staphylokokken effects of certain amino acids in anthranilate production in neurospora crassa studies on the polysaccharide-fermenting lactic acid bacteria. in. : nutritional requirements and the existence of fermentation promoting factors for sucrose and inulin the catabolism of proteins and nucleic acids in starved aerobacter aerogenes aerobic fermentation and the depletion of the amino acid pool in yeast cells influence of hydrogen ion concentrations on the utilization of sodium nitrite by fungi oxidative metabolism of glucose in leaf tissues infected with tobacco mosaic virus differential effects of amino acid deficiencies on bacterial cytochemistry nutritional requirements of an aspergiuus niger mutant for citric acid production culture de tissus d'insectes ~, l'aide d'extrait d'embryon de poulet en l'absenee d' h molymphe. c. r. acad utilization of some carbon and nitrogen sources by pseudomorms fluorescens on the selection of microorganisms for use in bacterial fertilizers in vitro and -rive uptake of carbon- labelled alanine and glucose by ascaridia galli, parasitic nematode of chickens growth of psychrophiles. i. : lipid changes in relation to growgh-temperature reductions vitamin requirements of listeria monoeytogenes parasitism and nutrition of gonatobo~rys simplex the effect of alkali metals on the growth of staphylococcus pyogenes the uptake of potassium and rubidium by staphylococcus pyogenes metabolism of nucleic acids and of nucleotides in the course of synchronous development of azotobacter vihelandii studies on the biotin-oleie acid requirements of a lactobaeillus plantarum variant isolated from chick feces unusual response to iron-dextran. brit. *ned. j. , nr. , s. . +in'. n.: tissue trypsin and trypsinogen levels in pancreatitis skeletal development of suckling kittens rate of liver regeneration atherogenesis in the monkey the significance of serum triglyeerides anaemia and parasitism in man physiological mechanisms in nutritionally-induced differences in ovarian activity of mature ewes bone development in suckled pigs production performance of artificiauy and non~r~ifically sired herd-mates in wisconsin search for an unidentified nutrient in mammalian liver. part i.: growth studies with various ox liver preparations proline control of the feeding reaction of cordylo-phora relationship between longevity and production in holstein-friesian cattle circadian adrenal cycle in c mice kept without food and water for a day and a half metabohc pmduc~s form labelled ethanol. iv. : disappearance of ethanol-carbon from morphological fractions and lipids of rat tissues acetate utilization by maize roots vajda, ] . : i~c~sll-rcs of body fat and hydration in adolescent boys some characteristics of a proteolytic enzyme system of pseudomonas fluorescens some metabolic relationships between host and parasite with particular reference to the eimcriae of domestic poultry nucleotide degradation in the muscle of iced haddock (gadns aeglefinns), lemon sole (pleuronectes microcephalus), and plaice (pleuronectes platessa) effect of starvation and -mcthylprednisolone (m_edrol) on the acid phosphatase of rat liver and muscle metabolic patterns in preadolescent children. vii. : intake of niacin and tryptophan and excretion of niacin or tryptophan metabolites biochemical changes in fish muscle during rigor morris studies on ornithine synthesis in relation to benzoic acid excretion in the domestic fowl effect of manual total collection of feces upon nutrient digestibilities polarographie studies on storage of meats. xxii. : influence of proteolytic enzymes on the polarographie wave of beef protein solutions post-mortem changes in chilled and frozen muscle genetic-nutritional interactiions as affecting the early growth rate of chickens effect of unequal milking intervals on lactation milk, milk fat, and total solids production of cows changes with age in glutamic oxalacetic transaminase activity of sonically oscillated tureen juice compared to total steam volatile fatty acids in calves fed different roughages catecholamine metabolism and some functions of the nervous system a study of some of the conditions affecting the rate of excretion and stability of creatinine in sheep urine changes in feeding behavior after intracerebral injections in the rat kanzcrogene substanzen in wasser und boden food additives and contaminants and cancer milk allergy in infancy food poisoning due to salmonella cnteritidis vat the mineral element content of spring pasture in relation to the occurrence of grass tetany and hypomagnesaemia in dairy cows insecticide residues in meat. residues in body tissues of livestock sprayed with sevin or given sevin in the diet over de giftigheid van solanum-alkaloidcn toxic products in groundnuts zur beziehung zwischen lipidcn hepatotoxicity of foods: a consideration of the hepatotoxicity of a few phanerogams and eryptogams. their possible influence in the pathogenesis of cirrhosis and hepatoma food-poisoning potential of the enterococei vanadium. excretion, toxicity, lipid effect in man an institutional food-poisoning outbreak examination of market milk of novokuznctsk for brucellosis an outbreak of food poisoning in a mental hospital food allergy as a cause of abdominal pain radioactivity in the diet the effect of microbial contamination on the requirement of chicks for certain nutrients the acute oral toxicity of cottonseed pigment glands and intraglandular pigments sur l'absorption du edsium radioactif par rorge. c. r. hebd. s ances aead die experimentelle alimentiire lebernekrose a]s empfindlicher indikator bei thermiseher belastung der milch. uber die magermilehtroekntmg the comparative toxicity of ethylene dibromide when fed as fumigated grain and when administered in single daily doses repository polyvalent insect antigen treatment for patients sensitive to hymenoptera. a clinical evaluation precursors of carcinogenic hydrocarbons in tobacco smoke toxin production in naturally separated liquid and solid components in preparations of heated surface-ripened cheese inoculated with clostridium botulinum allergieversuehe am tier zur ~tiologie der sogenannten margarinekrankheit. dr. reed. wschr. [ ] nr. , s. / . --, allergenwirkung oder immunologisohe adjuvanswirkung in der ~tiologie der sogenarmten margarinekrankheit radium- in human diet and bone miodine in the thyroids of north american deer experimental groundnut poisoning in pigs cholesterol as carcinogen safety factors in water fluoridation based on toxicology of fluorides entelo epidemiologische gegevens over her ,planta-exantheen" te rotterdam, verkregen door enquetc-onderzoek planta-~x~ntheem" epidemie te rotterdam in de m~nden ~ugnstu~ en september salmonella-verontreiniging van plantaardige grondstoffen veer voedingsmiddelen van mens en dier increase of strontium- and caesium- sodium fluoride intoxication salmonellosis in the netherlands zwei seltene salmonellenfunde untersuehungen fiber die chronisehe toxizit~t der ascorbins~ure bei der ratte captan in green vegetables rfickst~nde yon pflanzenschutzmitteln, insektiziden mid dergleichen in der nahrung und ihre bedeutung ffir die gesundheit der gehalt der milch an j, ,~co ' u ba _{_ la in der deutschen )iileh in der zeit yon langfristige nutritive anwendung yon antibiotika in der tierern~hrung im hinblick auf die menschliche gesundheit mi~ besonderer beriicksichtigung yon chlortetrazyklin a milk-borne outbreak of food poisoning due to salmonella heidelberg ergebnisse yon schwebversuehen an farbstoffen zur farbmattierung yon tabakwaren nutritional secondary hyperparathyroidism of the cat insecticide residues in fat. a screening method for chlorinated pesticide residues in fat without cleanup untersuehungen fiber die quantitative verteilung radioaktiver falloutprodukte in milch too many vitamins radios~ron$ium removal from milk. determination of apparent equilibrium constants of the exchange reactions of sodium, potassium, calcium, and magnesium with amberlite ir- ern~hrungsphysiologische eigenschaften der margarine. fette, seffen, anstrichmittel smoking and cancer: retrospective studies and epidemlologieal evalution beobachtungen fiber den verlauf der alkoholkrankheit am krankengut einer heilanstalt die verschmutzung yon trinkwasser dutch i)etergentien grain fumigant residues. occurrence of bromides in the milk of cows fed sodium bromide and grain fumigated with methylbromide insecticide persistence. the disappearance of endrin residues on cabbage lebensm ittelchem u. gerichtl reproductive performance of female miniature swine ingesting strontium- daily toxicity of nitrate nitrogen to cattle methods of residues of phosphated insecticides and miticides in foods on bacillary excretion in food toxinfections of salmonella etiology relationships between the deposition of strontium- and the contamination of milk in the united kingdom staphylococci in cottage cheese is~cs and potassium in people and diet. -a study of finnish lapps effect of treatment of seeds with -chloroethanol on the resistance to boron toxicity in wheat seedlings desferrioxamin, eine neue das eisen bindende und eliminierende substanz zur behandlung der rim~rcn und sckund~ren i-i~mochromatose akuter eisenvergiftungen toxic products in groundnuts smoking, arteriosclerosis, and age the incidence of milk sensitivity and the development of allergy in infants einflul] yon fluor und jod auf den stoffwechsel, insbesondere auf die schilddrtise quelques exemples illnstrant la valour et l'utllit des m~thodes de lysotypie clans certaines salmonelloses humaines d'origine alimentaire food poisoning caused by panthogenic halophilic bacteria (pseudomonas enteritis txkikawa): %ep rt of four autopsy cases procaine penicillin g in milk following intramuscular injections comparative subacute toxicity for rabbits of citric, fumaric, and tartaric acids distribution of pesticides in fermentation products obtained from artificially fortified grape musts mercurial fungicidal seed protectant toxic for sheep and chickens the problem of salmonella food poisoning dietary factors in the pathogenesis and treatment of cirrhosis of the liver. med. clinics of north america an outbreak of salmonella food poisoning in l~ehmadabad town, kaira i)istxiet, gnjaxat ~a~e la tossinfezionl alimentarl da salmonella nell' spedale maggiore di milano dal al water intoxication due to oxytocin: reporb of a case c~sium- und kalium in menschlichen organen und in der milch / caesium- in dried milk produets in relation to phytoellmatic zones smoking and oral cancer occurrence of hepatomas in rats fed diets containing peanut meal as a major source of protein nachweis yon mangan- und kobalt- in pflanzen als fo]ge russischer kernwaffenvcrsuche e-ruhr-epidemie durch speiseeis bcricht fiber eine arbeitstagung bei der internationalen atomenergic-behsrde in wien vom . bis . i)ezember . i)t. lebensmittel-rdsch the development of microbiological standards for foods. j. milk food technol a case of breslau salmoneliosis caused by eating chicken internationales rundgespr~ch fiber lebensmittelchemische probleme in wiesbaden und eltvllle a. rh. ( vortragsreferate) staphylococcal infection of raw milk as a cause of food poisoning. monthly bull. ministry health pub carcinogenic effect of egg white, egg yolk, and lipids in mice eczema and cow's milk exitus letalis nach lebensmittelvergiftung dutch bacillus cereus repression of staphylococcus aureus by food bacteria. ii. : causes of inhibition a further report on the radioactive contamination of milk and milk products in japan. determination of strontlum- and cesium- concentrations in milk powder in japan concerning sporadic salmonelloses insecticide residues. extraction and cleanup studies for parathion residues on leafy vegetables salmonellosis epidemiology zur frage der deponierung yon nutrltiven allergenen im organismus. allergic, _~sthma allergic children with various symptoms caused by cows' milk messungen der umweltsradioaktivit~t und der radioak-tivit~t yon lebensmitteln im jahre ein cxperimenteller bcitrag zur tabakrauehkanzerogenese. dr. reed. wschr. [ ] nr. . u n. : contamination of leaves by radio active fall-out insecticide residues in milk and meat. residues in butterfat and body fat of dairy cows fed at two levels of kelthane ( . and insecticide residues in milk. residues in milk from dairy cows fed low levels of toxaphene in their daily rations tier-und pfhnzenerniihrnug _anlrnal and plant nutrition summary of ,tropical crops soil, analysis, and its relation to plant composition and growth fertilisers and plant nutrients ulcers in swine tnfluence of chelating agents on the concentration of some nutrients for plants growing in soil under acid and under alkaline conditions nutritional evaluation of permanent pastures with dairy cattle in louisiana the herbage intake of eattle grazing lucerne and cocksfoot pastures terminology and methods for feeding and weighing animals the effect of feeding on evaporative heat loss and body temperature in zebu and jersey heifers studies on the requirements and interaction of copper and iron in broad breasted bronze turkeys to weeks of age some factors affecting iron uptake by strawberry plants feed consumption during lactation and involution in sprague-dawley-rolfsmeycr rats the effect of variations in the energy and protein levels of the ration upon performance in the pig use of barley in high-efficiency broiler rations. . poultry sci tierarzncimittcl und aufzuchtmittel in der landwirtschaftlichen praxis. gesund. heitliche erwggungen zum schutze des konsumenten bei der anwendung yon tierarzneimitteln und aufzuchtmitteln in der landwirtschaftlichcn praxis, tell ii mechanism for movement of plant nutrients from soil and fertilizer to plant root growth rate of the tea leaf as determined by shade and nutrients. empire j. exper note on induction of flowering in ~railing shoots of clones of saccharum spontaneum effect of level and sequence of feeding and breed on ovulation rate, embryo survival, and fetal growth in the mature ewe evidence for a high zinc requirement at the onset of egg production aufnahme und wirkung des mikronghrstoffs knpfer in ionogencr und ehelatisierter form bei gerste effects of pelleting and varying grain intakes on milk yield and composition the relationship of gibberellic acid to flower initiation in column stock, math~ela incana the effect of selenium administration on the growth and health of sheep on scottish farms the horsebean (vicia faba l.) as a vegetable protein concentrate in chick diets size and feeding of different types of fishes the influence of age of chicks on their sensitivity to raw soybean oil meal influence of the mineral nutrition on the resistance of peach trees to fusicoceum amygdali de la croix granular fertilizer. influence of associated salts on plant response to dicalcium phosphate a comparison of feeding growing pigs once or twice daily the interrelation of nutrient supply, leaf nutrient content, and vegetative growth of ilcx crenata 'green island' effect of rationing grass on the growth rate of dairy heifers and on output per acre, with a note on its significance in experimental design experiments on the nutrition of the dairy heifer. iv.: protein requirements of -year-old heifers grass silage vs. hay for lactating dairy cows hay vs. silage for two to six months old dairy calves weaned at or days effects of high levels of copper and ehlortetracycline on performance of pigs seedlings resistance of corn to leaf feeding of the european corn borer ostrina nubflalis ease of hydrolysis of the hemieeiluloses of forage plants in relation to digestibility bodenkde. [ ] mr. , s. / . --caleium-mangelsymptome an h heren pflanzen effects of frequency of feeding on urea utilization and growth charae%oristics in dairy heifers factors affecting the voluntary intake of foods by cows. . : a preliminary experiment with ground, pelleted hay the relationship of dietary energy level and density to the growth response of chicks to fats salt tolerances of pinus thunbergii compensatory carcass growth in steers following protein and energy restriction a guide to production, care, and use of laboratory animals. federation prec. estimation of essential fatty acid intake in swine automatic dispensing at frequent regular intervals of liquid diet for piglets chelation in nutrition. chelates and the trace element nutrition of corn der einflul yon humuss~ure auf wachstum und ver~inderungen des freien zuekergehaltes bci winterweizenpflanzen, die im dtmkeln kultiviert wurden a comparison of the growth of chicks in the gustafsson germ-free apparatus and in a conventional environment, with and without dietary supplements of penicillin an evaluation of weed competition and the effects of weed extracts and leachates on the development of field corn (zea mays l.) and oats (arena sativa l digestibility of rations containing different sources of supplementary protein by young pigs the effects of urea supplements on the utillzation of straw plus molasses diets by sheep production performance of artificially and nonartifieiallysired herd-mates inwiseonsin dietary phosphorus for laying hens tolerance to acid soil conditions in barley effect of calcium and magnesium upon digestibility of a ration containing corn oil by lambs effects of caloric restriction during the growing period on the performance of egg-type replacement stock untersuchungen fiber die verwertung yon calcium-und phosphorsalzen aus fisehgr~itenmehl einigcr frischfische und fischkonserven bei der verffitt~rung a nut~rient requirement for optimum water absorption by intact root systems preparation of feed for animal nutrition experiments responses of winter wheat to nitrogen and soil nitrogen status studies on calcium requirements of broilers znm problem dcr nahrungspflanzenwabl der aphiden some factors affecting leaf development and longevity and the subsequent yield of corn grain efficiency of energy utilization by young cattle ingesting diets of hay, silage, and hay supplemented with lactic acid the effects of a plant steroid on body weight and feed efficiency of broilers feeding troughs for guinea-pigs beitrag zur ]~ackfruchtmast mit schweinen unter besondercr beriicksichtigung des n~hrstoffgehaltes der beifuttermischungen und der the feeding of thyroprotein to lactating sows zur planung, durchfiihrnng und auswertung yon schweinemastvcrsuchen bei gruppenfiitterung the influence of barbituric acid derivatives on the development of plant roots and root hairs factors affecting the voluntary intake of food by cows. .: the relationship between the voluntary intake of food, the amount of digesta in the retieulo-rumen, and the rate of disappearance of digesta from the alimentary tract with diets of hay, dried grass or concentrates artificial food for oak-silkworm raising the comparative toxicity of ethylene dibromide when fed as fumigated grain and when administered in ingle daily do~ ~ gibberellin at the vineyard oak wilt development and its reduction by growth regulators. i. production and activity of oak wilt fungus pectinase, ecmulase, and auxin. ii. effect of halogenated benzoic acids on oak trees, the oak wilt diseases, and the oak wilt fungus regulating nutrient intake in laying hens with diets fed ad libitum some effects of different soils on composition and growth of sugar beet production of fodder yeast from barley. i. preliminary studies on the use of the waldhof fermenter development and nutrition of new species of thranstochytrium studies on the effect of frequency of feeding upon the biology of a rabbit-adapted strain of pediculns humanus the influence of previous vitamin k nutrition on thromboplastie activity of brain extract the effect of nutrition conditions on the growth of and nitrogen accumulation by fodder beans when sown together with indian corn. i)oklady akad. nauk sssr dutch phenylbors~ure induzierte fragen der resistenzsteigerung in der modernen gefliigelhaltung chelation in nutrition. metal chelates in plant nutrition beziehungen zwischen dcm kupfergehalt und dem zeitlichen auftreten yon kupfermangelsymptomen an hafer in wasserkultur mit kleincn bodenmengen increased tolerance of bean plants to soil drought by means of growth-retarding substances effect of monocaleium and diammonium phosphates on crop yield, and their influence on soil solution movement and characteristics when associated with different salts effect of barbituric acid and ehlortetraeyeline upon growth, ammonia concentration, and urease activity in the gastrointestinal tract of chicks effects of feeding low levels of dimethoate on milk and on whole blood cholinesterase activity of dairy cattle die ziichtung von fleischschweinen und die folgeerscheinungen, die sich besenders im hinblick auf die qualit~t yon fleiseh und fett ergeben the relationship of specific nutrient deficiencies to antibody response in swine. i.: vitamin a. ii.: pantothenie acid, pyridoxine or riboflavin further studies of diet composition on egg weight effect of various levels of fluorine, stilbestrol, and oxytetraeycline, in the fattening ration of lambs studies on the properties of l~ew zealand butterfat. viii. the fatty acid composition of the milk fat of cows grazing on ryegrass at two stages of maturity and the composition of the ryegrass lipids soil potassium and the growth of vegetable seedlings artificial feed for silkworm, bombyx mori some effects of feeding stilbestrol, ehlortetracyeline, and penicillin with alfalfa soflage on steer performance and carcass quality food agric. [ ] nr. , s. / . --, mineral supplements for sheep the influence of higher volatile fatty acids on the intake of urea-supplemented low quality cereal hay by sheep untersuehungen fiber den umsatz waehsender sehweine ab geburt. . mitt growth of edible emorophyllous plant tissues in vitro chelates in agriculture. metal chelation by glucose-ammonia derivatives economic analysis of high-level grain feeding for dairy cows evaluation of the dacron bag technique as a method for measuring cellulose digestibility and rate of forage digestion n~ihrlssungen fiir zuckerriiben in wasser-und sandkultur activity of gibbereuin:'d' on the germination of photosensitive lettuce seeds raw and heat-treated soybeans for growing-finishing swine, and their effect on fat firmness ration effects on tureen acids, ketogenesis, and milk composition. i.: unrestricted roughage feeding a new growth stimulant, ~ growth hormone the effects of specific viruses, virus complexes, and nitrogen nutrition on the growth, flowering, and mineral composition of strawberry plants peculiar feature of respiration and redox processes in the rice plants grown under different nutritional conditions einflul] der silagefiitterung auf die qualit~t yon milch und milchprodukten. . mitt. : einflul] der silagcfiitterung auf die organoleptischen eigenschaften dcr milch effect of grinding and pelleting on the utilization of coastel bermuda grass hay by dairy heifers langfristige nutritive anwendung yon antibiotika in der tierernlihrung im hinblick auf die menschliche gemmdheit mit besonderer beriicksichtigung yon chlor-te~azyklin mode of action of growth retarding chemicals yield of sugarcane in louis-ana as influenced by soil moisture status and climate diss effect of auxin on the emergence of lateral roots in p. mungo seedlings compound mouse diets a semipurified caries-test diet for rats present status of feeding antibiotics to htctating dairy cows effect of sodium bicarbonate in the drinking water of ruminants on the digestibility of a pelleted complete ration semi-purified diets for sheep effect of vacuum-drying, freeze-drying, and storage environment on the viability of pea pollen. ii. : effect of boron, sucrose, and agar on the germination of pea pollen hshe und zeitpunkt der diingung yon sommerweizen mit chlorcholinchlorid zur verkiirzung der halml~nge nutritional signifieance of soluble nitrogen in dietary proteins for ruminants primary signs of nutritional deficiencies of laboratory animals ]~ffe[~b of dlctary pro~in and fat on growth, protein utilization, and carcass composition of pigs fed purified diets trans-fetts~iuregehalt yon schweineschmalz nach fiitterung yon sehweinen mit rindertalghaltigem kraftfutter. (ein beitrag zur quantitativen infrarotspektroskopischen bestimmung yon trans-fetts~uren in fetten effect of liming and potassium fertilization on soil solution and on yield and composition of alfaffa and orchard grass mixtures effects of feeding various milo, corn, and protein levels on laying performance of egg production stock effect of gibberellie acid on flowering of apple trees the effects of dietary fat and energy ]evels on the performance of caged laying birds effect of age on the response of chickens to dietary protein and fat chemical control of flowering. concentration of a floral-inducing entity from plant extracts strontium- and calcium in milk of miniature swine studies on the properties of newzealand butterfat. vii. effect of the stage of maturity of ryegrass fed to cows on the characteristics of butterfat and its carotene and vitamin a contents new radioactive tests show how termites feed mechanisms regulating the feeding rate of daphni~ magna straus influence of low protein rations on growth and semen characteristics of young beef bulls a study of zinc deficiency in the dairy call effects of different levels of zinc and phosphorus on the growth of subterranean clover (trifolium subterraneum l.). australian j. agrie absorption, translocation, exudation, and metabolism of plant growth-regulating substances in relation to residues the effect of the performance of growing pigs of the level of meal fed in conjunction with an unrestricted supply of whey increase in yield of legumes by fer~iliser mixture with lime chemically defined medium for growth of animal cells in suspension dis sieherung der eiweiljwrsotgung in dor l~ndwirt influences of previous calcium and phosphorns intake and plant phosphorus on the requirement of developing turkeys for calcium and phosphorus relationship between isotopicauy exchangeable calcium and absorption by plants effect of adding buffers to all-concentrate rations on fcedlot performance of steers, ration digestibility, and intrarumen environment lysine supplementation of corn -and barley-base diets for growing-finishing swine the effect of gibbereliin on the germination of seeds of arboreal plants effect of physical state of coastal bermuda grass hay on passage through digestive tract of dairy heifers nitrate reduction and carotene stability. effect of nitrate and some of its reduction products on carotene stability, d. agric. food chem chemical preparations for plant protection untersuchungen fiber die ksrperzusammensetzung und den stoffansatz waehsender mastschweine und ihre beeinflussung dutch die erniihrung. . mitt the cobalt requirement of sub-~erranean clover in the field comparing mile and corn in broiler diets on an equivalent nutrient intake basis effect of mineral nutrition on the invasion and response of turnip tissue to plasmodiophora brassicae wor the relation of chlorogenic acid and total free phenols in potato plants to resistance to infection by verfieillium alboafxum nitrogen and potassium as variables influencing soluble nitrogen and organic acid accumulation in soybean (glyoine max). di~s. abstr. bett~r british beef and barley feed. veter effect of nitrogen fertilization upon yield and digestibility of aftermath timothy forages fed to dairy heifers ration effects on dltlot steer feeding patterns effects on zea mays seedlings of a strontium replacement for calcium in nutrient media evaluation of albumen quality in a poultry breeding program nutritional studies with the guinea-pig. viii. : effect of different proteins, with and without amino acid supplements, on growth some effects of heredity and environment on appetite in dairy animals further studics on manganese nutrition of tobacco in relation to virus infection and synthesis amillo acid supplementation of pig diets chelation as a basic biological mechanism der einflu~ der stiekstoffdiingung auf die znsammensetzung yon kartoffeleiweiil z studies on photosynthesis. i. : biosynthesis of sucrose from glycolate. par~ ii. : bicarbonate utilization by washed algae production, interior egg quality, and some physiological effects of feeding raw soybean meal to laying hens alteration of post-mortem changes in porcine muscle by preslaughter heat treatment and diet modification chelation in nutrition. soft microorganisms and soil chelation. the pedogenie action of lichens and lichen acids die ergiinzungswirkung yon dl-•ethionin allein oder in kombination mit l-lysin beim wachsenden schwein untersuchungen iibcr den einflub unterschiedlicher wasservemorgung auf ertr~ge, gehalte an ~therischem , trenspirationsquotienten, biattgrsl~en und relative ~)ldriisendichtsn bei einigen arten aus der familie der labiaten. . teil gehalte an iitherischem , transpirationsquotienten, blattgrsflen und relative -driisendichten bei einigen arten aus dcr familie der labiaten. iii.: blattgrsflen, relative driisendichten, anzahl am haupttricb inseriertcr blattpaare und internodien. liingen cadmium: uptake by vegetables from supcrphosphate in soil studies on the protein and methionine requirements of young bobwhite quail and young ringnecked pheasants chelation in nutrition. evidence for natural chelates which aid in the utilization of zinc by chicks selective fertilization of apple-trees some soils and fertilizer relationships of the cavendish banana (muss cavendlshl lambert) on three different soils in costa rice soil organic phosphorus and the phosphorus nutrition of plants the effect of heat treatment on the nutritive value of milk for the young calf. . : a comparison of spray-dried skim milks prepared with different preheating treatments and roller.dried skim milk, and the effect of chlortetracyclinc supplementation of the spray-dried skim milks the effect of heat, ~reatmen~ on the nutritive value of milk for the young calf. . :the effect of the addition of calcium a biological assay for metabolizable energy in poultry feed ingredients together with findings which demonstrate some of the problems associated with the evaluation of fats feed additives in livestock rations: part i. : urea in dairy rations. part.i/: use of thyroprotein in cattle nutrition diet and histamine in the ruminant synthetic ion-exchange resins as a medium for plant growth nutrition of vibrio fetus theoretical basis of unicellula algae cultivation amino acid supplementation of barley diets for growing swine some effects of . -dichiorophen-oxyacetic acid on swect corn (zea ]~ays rugosa l.) with emphasis on yield, tillering, root development, and exudation of electrolytes from roots and stems. i)iss. abstr feeding and management of broiler strain breeder hens relationships among seven elements in the nutrition of corn in sand culture an external effect of inorganic nitrogen on nodulation influence of enzyme supplements in lamb fattening rations gravenstein and jonathan apples produced with giberellle acid the role of carotene in the dairy cow. wiss. ver ff. dr. ges. ern~hrung vitamin a-wirksamkeit der carotine bei versehiedenen tierarten. wiss. ver- ff. dt. ges. eru~hrung upgrading the indigenous poultry of uganda. i. : the growth rates and feed conversion from hatching to maturity of indigenous poultry crossed with four imported breeds effect of different kinds of litter on growth and feed efficiency in chick rearing investigation of the mineral nutrition of datura innoxia the effect of flooding in the availability of phosphorus and on the growth of rice nutrition of the boll weevil larva ascorbic acid in the nutrition of plant-feeding insects effects on the s~maeh worm, i-iaemonehus contortus, of feeding lambs natural versus semipuriiicd diets yield and foliar composition of corn as affected by fertilizer rates and environmental factors. i)iss. abstr. ~ [ ] nr praktische erfahrm]gen in der carotinoidversorgung yon vsgeln effect of protein-energy relationship on the performance and carcass quality of growing swine the use of quarter samples in the assessment of the effects of feeding treatments on milk composition * calcium and phosphorus requirements of finishing broilers using phosphorus sources of low and high availability amino acid supplementation of peanut meal diets for broiler chicks the effects of feeding various levels of vitamin a on chicks with cecal coccidiosis chelation in nutrition. review of chelation in plant nutrition water use by irrigated arabia coffee in the failure of certain dietary ingredients to affect the incidence of blood spots in chicken eggs dcr einflul~ der anbauverh~ltnisse auf die eigensehaften der kartoffelknolle und der st~rke effect of feeding milk replacers with varying amounts of fat for hothouse lamb production l~hysiologieal factors influeneelng growth, reproduction, and production of wcll-fed dairy heifers. i. ago at first breeding. ii. feeding of diethylstflbestrol results of an experiment ot rothamsted testing farmyard manure and n, p, and k fertilizers on five arable crops. i. : yields results of an experiment at rothamsted testing farmyard manure and n, p, and k fertilizers on five arable crops. ii.: nutrients removed by crops the utilization of carotenoids by the hen and chick some effects of potassium and lime on the relation between phosphorus in soil and plant, with particular reference to glasshouse tomatoes, carnations, and winter lettuce the lack of a consistent chick growth response to norwegian kelp meal further studies on protein and energy requirements of chicks selected for high and low body weight some effects of kinetin on the growth and flowering of intact green plants individual feed consumption of turkey breeder hens and the correlation of feed intake, bocly weight, and egg production crop analysis technique for studying the food habits and preferences of chickens on range supplemental value of turkey protein for wheat herbicides and plant growth regulators preparation of purified ration for chick. parg iv. : preparation of crystalline amino acid diet evaluation of algae as a food for human diets the influence of milk fat depressing rations on the yield and composition of bovine milk the effect of plant nutrients and antagonistic microorganisms on the damping-off of cotton seedlings caused by rhizoetonia solani kukn ki;-;sehe ern~ihrung und di~tetlk clinical nutrition and dietetics a statement approved by the board of directors of the canadian heath foundation radioactivity and human diet probleme der ern~hrung durch gefrierkost. sympomum der d utschen gcgell~ehaft ffir ernghrung veto . bis . mgrz klinische ernghrungslehre" und wissenschaftlicher kongreb der deutschen gesellschaft flit ern~hrung an der johannes-gutenberg-universit~t mainz veto . bis wissenschaftlicher kongreb der deutsehen gesellschaft ftir ern~hrung an der johannes-gutenberg-universit~t mainz am . und ernghrung nnd digit" . deutseher kongreb ffir ~irztliehe fortbildung in berlin veto . bis arbeitstagung fiber klinisebe ernghrungslehre. ern~ foods of the future (forts.). problems in space foods and nutrition. foods for extended space travel and habitation the question of fats. ii.: fats and disease behandlung fettbedingter gerinnungsstgrungen mit lipostabil sugar and dental caries obesity and sugar addiction hunger and malnutrition lancet nutrition and general practice bericht fiber die vortragstagung des fachverbandes lebensmittelchemie der chemischen gesellschaft in der ddr vom arbeitstagung fiber kommission ffir volksern~hrung, lebensmittelgesetzgebung und -kontrolle (eek) zu yi~inden des eidg the national diet-heart study low fat diet in familial mediterranean fever the thyroid gland in infant malnutrition evaluation of fao amino acid reference pattern studies on the physiology of nutrition in surinam rickets in southern israel diet and heart disease maiskeims in ernt~hrung und ditltetik apha conference report safe and nutritious food supply malnutrition and disease expert committee on medical assessment of nutritional status protein malnutrition the fat tolerance curves of patients with hyperllpidcmia and athcrosclcrosis die lactose im rahmen der ernt~hrung effect of environment on nutritional status zur theorie und praxis der zuckerkrankheit. wiener z dietetically induced experimental flous of rats physikalisch-diiitetische therapie yon hautkrankheiten. arch. phys. therapie err~hrungsforschung [ / ] :nr. , s. / . +bo rtiw~l, p. w. : milk-borne disease consumers' reactions to instand foods de voeding van woonwagenbcwoners experimental investigations on nutrition and human behavior. a post-script. amer di~tetische therapie der chronischen herzinsuffizienz construction and validation of the food attitude scale why we have a safe and wholesome food supply use of food in a psychiatric setting stature and nutrition in cystinuria and hartnup disease ])as endokrinologisehe syndrom des proteinmangels urinary excretion of . -dlhydroxyphenylalanine (dopa) in two children of short stature with malnutrition current problems affecting consumption of milk and indnstry's response to them preparedness for emergency feeding fluoridation and public relations dieetprodukten in vlaanderen incorporation of labelled glycine into erythrocyto glutathione of rabbits; effect of nutritional muscular dystrophy hot wcreldvoedselvraagstuk sniker -glycogcen -tandbederf dietary in take in patients with arthritis and other chronic diseases a clinical trial of iron-fortified bread effects of freater intake of milk, fruits, and vegetables joint fao/w/to expert committee on nutrition" fiber eine sitzung in genf vom . his serum cholesterol in a military population. its relation to obesity and the military diet ). ~z~, a. c. some nutritional problems of older age groups neuere biochemisehe untersuchungen zur diagnostik und therapie yon b-vitamin-mangelzust~nden call-harvard nutrition project. iii.: the erythroid atrophy of severe protein deficiency in monkeys cali-harvard nutrition projeet. ii. : the erythroid a~rophy of kwashiorkor and marasmus zur hshe des erwiinsehten fottverbrauehs ern~hrung des sportlers voeding moderne ern~hrungsbedarfsnormen. i. mitt. z. ges. hyg. [ ] nr. , s. . *--~ioderne ern~hrungsbedarfsnormen. . mit~.: z. ges. hyg. a comprehensive home-care program for the chronically ill fat-modified foods for serum cholesterol reduction besonderheiten der ern~hrung alter menschen chronic malnutrition in turkey. v. studies on serum fatty acids in malnourished children prevention of ,meat anemia" in mice by copper and calcium beeinflussung der sportlichen leistungsfdhigkeit dutch eine geeignete er-n~ihrung physiology of adolescence. ii, l~u-trition -basal oxygen consumption -energy expenditure and balance -nitrogen metabolism -calcium metabolism -iron metabolism -red cell mass and hemoglobin ern~hrungsproblemc bei chirurgisehcn kranken. wiss. versff. dr. ges. ern~hrung moderne vitamin b~-therapie: oral, rektal oder parenteral ? a palatable diet for producing experimental folate deficiency in man smoking in hospital was ist hungern und was heibt the cultivation of tflapia. this prolific fish as a fine source of proteinrich food in underdeveloped areas pyridoxine supplementation during pregnancy. clinical and laboratory observations on japanese foods nutritional sequelae of ga~trio surgery koehsalzarme kost und nierenerkrankungen theoretische und praktische grundiagen der ernilhrung in der fett in der diabeteskost foods or supplements? g zur hygiene und ,di~tetik des rauehens zur dis behandlung der uremic anaphylactie shock of the lungs triggered by mieroaspiration of cows' milk: a form of sudden unexpected death in early infancy the food service industry and its relation to the control of foodborne illness die konservative therapie des peptischen gesehwiirs gezondheid op reis addictive aspeeta in heavy cigarette smoking die ern~hrung im rahmen de~ heilvers im kurort the diet in renal faiiure is the rationale for gaetrointestinal diet therapy sound? familie-beruf-ern~hrung die dfiitbehandiung der leberkrankheiten. i)t. reed vom hunger his zum ~beritul -weltweite ern~hrungsprobleme die bedeutung der vitamine in der t~tglichen erniihrung s~ure-und baseniibersehiissige naln'ung. therapiewoehe [ ] nr. , s. / . --ss und chronische acidogene und alkalogene erns z. em~hrungswiss industrial lunches and public health the assessment of nutritional status in man: chairman's opening remarks therapie der essentiellen hypertonie speeifieke voedings-en voorlichtingsproblemen in tropische landen wie kann man unsere kos~ und unsere kostgewohnheiten beeinflussen? neue konzeptionen in der wasser-und salzsubstitution some aspects of the relation of nutrition and pregnancy is coronary heart disease preventable? world hunger demineralization of whey. use of its protein in infant feeding elaidinized olive oil and cholesterol atherosclerosis soziatrischo aspekte dee genul)-und arz~aeimittelkonsums verwendung yon htilsenfriichten in der diabetiker-di~t sanitation and dishes sanitation and dishes. aspects old and new. part ii smoking, arteriosclerosis, and age neue weg~ zur erni~hrungsphysiologi~chen aufwertung yon getreide-erzeugnissen diphyliobothrium latum and human nutrition, with particular reference to vitamin bli deficiency milk and diverticulosis dietary factors in the pathogenesis and treatment of cirrhosis of the liver. ivied. clinies north america zur frage der quanti~tiven charakteristik der ern~hrung der berufst probleme der gemeinschaftsverpflegung aus der sieht des ern~hrungsphysio-logen gegevens over vitamine b,-deficientie, -behoefte en -voorziening use of government-donated foods in a rural community fluoride, teeth, and the analyst eiweil bedarfsnormen im rahmen unserer ern~hrungsrichts~tze physiologic discomforts in navy protective shelter tests di~tvorschl~ige : akute nierenentziindtmg siii]waren und karies in theorie und praxis ernehrtmgsberatung im krankenhaus use of a low-sodium formula as an improved karell diet, with emphasis upon the outpatient management of heart failure and lymphedema pflanzliches eiweil~ fiir die erniihrung des menschen influence of diet on viral hepatitis influence of siblings on student smoking patterns voeding yon leerlingen van een lagere technische school. ii. calorie~n-en nutri~ntenwaarde early use of circulating blood volume, weights, and normal diet in acute renal failure fette in tier nahrung. dr. recd. wschr. [ ] nr. , s. / . *scm~-idt-b~bach, a.: ~j~ber die di~tetik der sauermilch begriff und anfgabe di~tetischer lebensmittel zur ursache yon geschmackskalamit~ten in trinkwassertalsperren psychologische motive im wandel des brotverzehrs improving levels of nutrition through better food practices the vitamin b~ deficiency syndrome in hun~n infancy. biochemical and clinical observations treatmen~ of ,refractory obesry" with ,formula die~ nr. , s. ff. ( s.). elwzea, c. c.: morphologie constitution and smoking prediction the outcome for obese dieters symposium fiber probleme der ern~hrung durch gefrierkost in karlsruhe yore coordination of long-term care of :pku children die di~t bei diabetes meuitus a nutritional supplement (nutrament) for elderly patients dietary intake of five groups of subjects. -hr. recall diets vs. dietary patterns the prolonged effects of a low cholesterol, high carbohydrate diet upon the serum lipids in diabetic patients stand und perspektiven der eiwei~versorgung. zur zielsetzung des verhandiungsthemas de voeding van rijswerkers signification des standards calorieo-azotes utilists en france erg~nzungen veto standpunkt des lebensmittelehemikers zu (dem beitrag) official acceptance of homogenized milk in the united states advances in nutrition and dietetics nutrition of naval recruits during a shelter habitability study hot ombuigen van voedingsgewoonten de voeding van schippemkinderen san boord en in de internaten voeding [ ] iqr. , s. / . --le traitement de rinsuffisance rtnale her zoutarme-eiwitarme dicer ein beitrag zur allgemeinen el problematik, ausgehend yon einer anorexia nervosa rroblem~ in nutrltlon~l supplementation an ~mri~hnl~ut detection of nutritional imbalances theorie und praxis der schwangerenern~hrung die pharmakologisehe beeinflussung yon hunger mad s~ttigung problems in the evalutaion of nutritional status in chronic illness europ~ische di~ttagung in amstercl~m ( . bls entwieklung des brot-und gctreidevcrzehrs in der neuercn zeit nutrition and palatability coehac dis~tse. -biochemical and technological aspects die di~itetik der :fettsucht kinderemll}~mg nutrition of infants and children report on infant feeding childhood nutrition in lapland. :nutrition rev the thyroid gland in infant malnutrition. nutrition rev. [ ] nr. , s. . +n. n.: physical activity of obese girls appraisal of nutritional adequacy of infant formulas used as cow milk substitutes isomaltose intolerance causing decreased ability to utilize dietary starch passagere hypereh]or~mische azidose bei zwei ausgetragenen s~uglingen wghrend s~uremflch-em~hrung ober erfahrungen in der frfihgeborenonaufzucht mit einer neuen bedarfsangepa ten friilmahrung nutritional defects in adolescence g p intravenous glucose tolerance in the normal newborn infant: the effects of a double dose of glucose and insulin ! attitudes towards physical ac. tivity, food, and family in obese and nonobese adolescent girls urinary excretion of . -dihydroxyphenylalanine (dopa) in two children of short stature with malnutrition high salt content of western infant's diet: possible relationship ~o hypertension in the adult vitamin e to premature infants des enzym yon ~'leming (lysozym) und seine bedeutung flit die si~ugllngsern~ihrung. ann investigation on the relation of between-meal eating and dental caries of sixth-year molars in school children studies in infantile malnutrition. i.: nature of the problem in peru chronic malnutrition in turkey. v. studies on serum fatty acids in malnourished children emi~hrnng mad faekalc lysozymaktivitiit beim s~iugiing role of linoleio acid in infant nutrition factors related to the eating behavior and dietary adequacy of girls to years of age. dies. abstr. des vitamin c im jugendalter. ii. mitt.: uber die wirkung yon natiirlichem und synthctisehemvitamin c bei l~ngeren zugaben the incidence of protein-calorie malnutrition of early childhood ein besonderer znsammenhang zwischen dem bedarf an nahrungsfett und dem stoffwechsel in den ersten lebensjahren the effect of supplements of groundnut flour or groundnut prorein isolate fortified with calcium salts and vitamins or of skim-milk powder on the digestibility coefficient, biological value, and net utilization of the proteins of poor indian diets given to undernourished children lysine fortifications of wheat bread fed to haitian school children lrber den -stunden-rhythmus der kalorienerzeugung bei friihgeborenen beitriige zur frage der spezifisch-dyrmmi~ehen wlrkung auf grund yon glykokoll-belastungen bei friihgeborenen. acta paediatriea acad carotine in tier stiuglingserni~hrung. wiss. versff. d$. ges. ernkhrung liver and depot lipids in children on normal and high carbohydrate diets response of rural guatemalan indian children with hypocholesterolemia to increased crystalline cholesterol intake feeding value of soy milks for premature infants early feeding and birth difficulties in childhood schizophrenia. a brief study zusammenh~nge zwischen stoffwechsel und fl~issigkeitsbedarf beim s~ug-ling kuhmilchauergie beim s~ugling und ,cot death". die unspezifische kumulative sensibilisicrung malnutrition and the health of children practical aspects of infant feeding breast-feeding, weaning, ~nd acculturation appetithemmer in der ]~ehandlung der fettsucht bei kindern. miinchener med the effect of different amounts of vitamin d on growth and serum levels of calcium, inorganic phosphorus, and alkaline phosphatase in premature infants partition of urinary nitrogen in children with kwashiorkor treated with animal and vegetable proteins der einflud yon voukornbrot auf den caleiumstoffweehsel bei schulkindern ist eine rektale vitamin blz-behandlung vertretbar? dr ethyl alcohol in the pathogenesis of gout c|e~rance of infused fat emulsion in diabetic dogs praktische durehffihrung der parentcralon ern~hrung die parcntcrale ern~hrung chirurgischor paticntcn. wiss. ver ff. dr. ges. em~hrung verwertung intravenss verabfolgter aminos~,urengemische. wiss. versff. dr. ges die erkcnnung yon fe~-transportstsrungen und ihre bedeutung ftir die intra-ven se fettzufuhr intravenous glucose tolerance in the normal newborn infant: the effect of a double dose of glucose and insulin new intravenous fat emulsion indikationen und kontraindikationen der intraven sen fettzufuhr in der chirurgie anwendung intraven s gegebener aminos~urengemische in dcr p~diatrie ymtravensse fettinfusionen. wiss. versff. dt. ges. ern~rung ~qotwendigkcit und erfolge der parenteralen mad sonder-ern~hrung moderne vitamin blz-therapie: oral, rektal oder p~renteral? mcd anwendung intravenss gegebener aminos~urengemische in der gyn~kologie und geburt~hilfe aminos~ureninfusionen. schweiz. reed. wsehr. klinische anwendung mad erfahrungen bei der verabreiehung intravensser fettemulsionen an ehirurgischen patienten diskussionsbemerkung zum thema: die parenteralo ern~hrung ~tude exp~rimentale de la tolerance d'une solution de graisse vsgstale d'administration intraveineuse ern~hrungsphysiologische grundlagen der parenteralen ern~hrung erfahrungen mit der parenteralen ern~hrung mittels fettinfusionen. helvetica chirurgica aeta nitrogen, lipid, glycogen, and deoxyribonucleic acid content of human liver. the effec~ of brief starvation and intravenous administration of glucose techn~ und indikationen der parenteralen ern~hrung des neugeborenen die praktische organisation der klinisehen infusionstherapie mit zuckerund elektrolytlt)sungen. l ~ed untersuchungen und bcobachtungen fiber intravensse fettinfusionen in der inneren klinik. wiss. versff. d$. ges. ern~hrung l'alimentation parenttrale, mulsions lipidiques. (a suivre) ann intravcnsse ern~hrungstherapie mit fettemulsionen parenteral-und sondeneru~hrte patienten zur rekt~len kaliumsubstitufion parenteraie ern~hrtmg mit fettemulsionen konservierung mad zubereitung yon lebens-und futtermitteln nutritional hygiene, preservation and preparation of foodstuffs and feeds n. n.: vortragsveranstaltung ,fleischhygieno" der th seminar on the use of radioisotopes in nutrition science and of ionising radiation in food technology. strasbourg, st - th october progress of food irradia~on work and programmes in o.e.c.d. member countries ( berichte) safe heat processing of canned cured meats with regard to bacterial spores the role of food science i and technology on the freeze dehydration of foods public health aspects of handling animal products in the txopics fack)rs affecting bacfcrial spoilage of animal products at elevated temperatures. food technol sterilized concentrated millr. food teehnol. [ ] nr. , s. / , . n.n.: foods of the future. now opportunities for flavor modification unrestricted approval for irradiated bacon -a sanitary standards for multiple-use rubber and rubber-like materials used as product contact surfaces in dairy equipment -a sanitary standards for batch and continuous freezers for ice cream, ices, and similarly-frozen dairy foods bericht fiber die vortrag~tagung des fachverbandes lebensmittelchemio der chemischen gesellsch~ft in der ddr vom . bis lebensmittelchem. u. geriehtl. chem. fluoridation in great britain die enzymatische phycinspaltung in geschrotetem getreide in abh~ingigkeit yon der relativen lufffeuchtigkeit the effect of certain antioxidants during freezer storage of pork chops and sausage the mechanics of treating hatching eggs for disease prevention beitrag zur sfil~gerinnung yon kakaotrunk jodophore als desinfektionsmittel in der milchwirtschaft. milchwissenschaft [ ] nr. , s. ff. (? s.). zitat: dr. lebensmittel tierarzneimittel und anfzuchtmittcl in der landwirtschaftllehen praxis. gesundheitliche erwrgungen znm sehutze des konsumenten bei der anwendung yon tierarzneimitteln und aufzuchtmitteln in der landwirtschaftlichen praxis n~ihrwertminderung dureh zubereitung dcr nahrung suue modifieazioni della flora mierobiea dei mollusohi eduli par effetto di eonservazione impropria verlinderungen des inhaltes yon dosenkonserven w~hrend lgngerer lagerung digtbrote aus der sieht ihrer praktischen gestmtung. wiss. versff. dr. ges. ern~hrung l erniihrungshygienische untersuchungen in kindergarten an budapester kindern im alter yon bis jahren. z. ges. hyg. die eignung der bakteriologischen untersuehung yon kannenmilchproben als grundlage eines eutergesundheitsdienstes adequacy of cooking procedures for the destruction of salmonellae zur revitaminierung des mehles bzw. brotes. wiss. versff. dr. ges. er-n~hrung conventionele verwarningsmethoden beitrag zur kenntnis der wechselwirkungen zwischen proteinen und poly. phenolcn der kakaobohnen wtihrend der fermentation nihydrazone feed medication ag~ins~ ar~iiieiaily induced escheriehia eoli air-sac infection foam-mat dried orange juice. i. time-temperature drying studies lebensmittelhygicnische probleme bei der herstellung yon gemeinschaftsverflpegung. . mitt. : z. ges. hyg. lebensmittelhygienische probleme bci der herstellung yon gemeinschaftsverpflegung. . mitt.: z. ges. ttyg. untersuchungen fiber die temperaturvorg~nge im innern yon lebensmittein w~hrend ihrer thermischen zubereitung, erl~uter~ am kochen yon kartoffelklsl~en. z. ges. hyg. zur gewinnung yon niederverestertem pektin aus toehnisehen apfelpektinextrakten mit ammoniak: einflul] der entesterungsbedingungen auf das geliervermsgen hygienisohe beurteilung einer dutch clostridlum verursaehten massen. lebensmittelvergiftung [ungar neuartige teehnik der lebensmlttelverpaekung filr ge. schmaeks-aromastoffe und andere artikel bei ~berdruek studies with a natural source of xanthophylls for the pigmentation of egg yolks and skin of poultry die problematik der tuberkulosebeurteilung in der sehlachttier-und fleischuntersuchung freezing rate of beef as affected by moisture, fat, and wrapping materials ~ber mit komblnier~en konservierungsmitteln hergestellte konfitiiren und consumers' reactions to instant foods. food teclmol effect of supplementing lime-~reated corn with different levels of lysine, tryptophane, and isoleueine on the nitrogen retention of young children effect of freezing on autoxidation of oxymyoglobin solutions the control of gloecsporium album rot of stored apples by orchard sprays which reduce sporulation of wood infections bakteriologische befunde bei der spelseeisuntersuchung im sommer the microbiology of vacuum packed sliced bacon the stability of canned foods in long erm storage the effect of proofing and baling on concentrations of organic acids, carbonyl compounds, and alcohols in bread doughs prepared from pre-ferments nutrients in raw vs. cooked globe artichokes effect of gamma-radiation, chemical, and packaging treatments on refrigerated life of strawberries and sweet cherries. food teehnol beeinflussung der wirkung yon kaffeeinhaltsstoffen dutch be-s~immte behandiungsverfahren der l~hbohne. (eine tierexperimentell-toxikologische studie influence of surface pasteurization and ehlortetraeycline on bacterial incidence on fryers the hydrolysis of grass hemicelluloses during ensilage post-harvest storage studies with selected fruits the science of food technology in venezuela beitrag zur aufbewahrung von sti~rkesirup in verzinkten ge-f~ben inactivation-rate studies on a radiation.resistant spoilage microorganism. ii. : thermal inactivation rates in beef the oceurrence and growth of staphylococci on packed bacon, with special reference to staphylococcus aureus zur verhiitung yon lebensmittelinfektionen in grobklichenanlagen dutch desinfektionsmabnahmen. ~rztl the influence of selected bacteria upon the flavor of a precooked frozen poultry product flour maturing and bleaching with aeyclie acetone peroxides effect of processing conditions on dry-heat expansion of bulgar wheat zur vcrwendung von pentachlornitrobenzol bei der lagerung yon kohl. dr. lebensmittel-rdsch. [ ] nr. , s. los mati~res f cales des pores et les selles des ouvriers d'aba~toir constituent une source permanente de diss mination des salmonella studies on cooking fats and oils aspect sanitaire et l~gal aetuel des aliments conserv&s. rev. d'hyg over de betekenis van postduiven als besmettingsbron van levensmiddelen met salmonella-kiemen. ti]dschr. v. diergeneesk over bet voorkomen van salmonella-kiemen bij slagerijen. tijdschr. v. diergeneesk ursache und entstehung yon brotfehlern les salmonella des oeufs et ovoproduite frangais eg trangers the microstructure of baked products and doughs. food technol tomsto powder by foam-mat drying zitat: dt. lebensmittel-rdsch. [ ] nr. , s. . --die verwendung yon gefrosteter saline zur butterherstellung. teil iv. die ergebnisse der in d~nemark, frankreich und in den l~iederlanden durchgefiihrten praktischen versuche und ihre bedeutung ffir die in der deutschen molkereiwirtschaft erfolgendo verwendung yon gefrosteter sahne bei der butterherstellung tenderne~ of the turkey meat as influenced by pre-cooling before proce~ing and hand masv~ging vortragsmaterialien flit die ern~hrungsproplldeutik. (erlruterungen zu insgesamt groben sehautafeln.) . mitt. : behandlung der tafeln iv bis vi. ernahrungsforschung die unterschiedliehe problematik und ihre konsequcnzen bei der bekrmplung der rinder-und schwefnefinnen salmonellae from flies in a mexican slaughterhouse factors affecting quality of pies prepared from frozen bulkpack red sour pitted cherries zur bedeutung antimikrobiellcr stoffc in der nahrung modified equipment for pasteurizing and deodorizing market milk and for pasteurizing, deodorizing, and slightly concentrating cheese milk adhesion of coatings on frozen fried chicken oob~age eheeae problems in production and sanitation. publle health aspects ~ber den einflub yon licht, auerstoff und tempera~ur auf die hal~barkoib yon verpaektem emmentaler ks in scheiben aktuelle notwendigkeiten -gesetzliche m glichkeiten zur fischkfihlung in eis und seewasser techniques de recherche des salmonella dans les oeufs frais et de conserve food hygiene on board ship safety factors in water fluoridation based on toxicology of fluorides the effect of oiling before and after cleaning in maintaining the albumen condition of shell eggs lebensmi~t~l-aerosole. fette preservation of the natural color in processed sweetpotato products. i.: flakes. food technol temporary inhibition of fermentation in apple juice preservatives and artificial sweeteners the mechanism of the development of rancidity in frozen fresh pork sausage and practicable methods for its inhibition die entwicldung der trinkwasseriinoridierung in den usa microbiological principles in prcpaeking meats st~ndard-kapazit~tstest ffir die bestimmung der desinfektionswirkung yon desinfektionsmitteln in der milchwirtsehaft. internationaler standard fil/ii)f - die anwendung einiger arteu, bzw. st~mme, yon propions~urebakterien zur herstellung bestimmter k~scsorten mit hohem vitamin b -gehalt the extraction of pectins from apple marc preparations zur hygiene und ,di~tetik des rauchens studies on control of respiration of mcintosh apples by packaging methods. food teehnol effects of ingredients used in condermed and frozen dairy products on thermal resistance of potentially pathogenic staphylococci der frisehkllse und seine verpackung studies on the viscosity of mayonnaise. ii.: the influence of addition of vinegar on the vi~co~isy of mayonnaise e~'ec~ of chemical v~lditives on the spreading quality of butter. ii. laboratory and plant churnings studies on browning mechanisms of fruit juice products. i.: changes in chemical composition which accompany browning of commercial concentrated lemon juice during storage ergebnisse der dlg-qualit~tspriifung fiir speiseeis. dr. molkerei-ztg beeinttnssung versehiedenartig verpaekter lebensmittel dureh desinfektion mit formaldehyd grunds~itzliches zur stabilisierung und solubilisierung yon carotin und carotinoidpr~paraten riickst~nde yon pflanzensehutzmitteln, insektiziden und dergleichen in der nahrung und ihre bedeutung fiir die gesundheit absehliebende stellungnahme lebensmittel-rdsch langfristige nutritive anwendung yon antibiotilm in der tierern~hrung im hinbliek auf die menschliehe gesundheit mit besonderer beriieksiehtigung yon chiortetrazyklin nutritional studies on the utilization of distiller's stillage. part l: insolubles of me]lasses-butanol distiller's stillage auswertung der dlg-priifung fiir frischk~ise in verbraueherpaekungen zu den fermentativen eigensehaften der milchs~urebakterien (,laetobacillus meijerinek"), zugleieh ein beitrag zur vermeidung yon fehlfabrikaten bei roh-nnd briihwurst. arch. lebensmittel-hyg microbiological aspects of one-trip glass bottles as used by the carbonated beverage industry de bereiding van bouillon aktnelle milchhygienische aufgaben und zicle des organisation der ~berwachung der umweltradioaktivit~t unter besonderer beriieksichtigung der l~berwaehung des gehaltes yon lebensmitteln an radioaktiven stoffen. dr. lebensmittel bakteriologie der auermilcherzeugnisse l~ber die italtbarkeit yon lebensmittelkonserven preparation of aeid-modifid flour for tub sizing radiostrontium removal from milk. determination of apparent equilibrium constants of the exchange reactions of sodium, potassium, calcium, and magnesium wish amberlite ir- probleme der vitaminierung yon brot the effect of several operational variables on the rate of freeze-drying of beef studies on beef quality. x. effect of temperature, freezing, frozen s~orage, thawing, and p~ on the rate of hypoxanthine production. div. food preservation techn retardation of gelation in high temperature-short-time sterile milk concentrates with polyphosphates nonenzymatic bread browning and flavor. changes in amino acids and formation of earbonyl compounds during baking ttinweise fiir konservierende wirkungen synthetiseher senfslbildner naeh versuehen an fisehen neuo wege zur herstellung haltbarer fisch-pr~iserven behavior of ethylene dibromide, methyl bromide, and their mixtures. i. : in columns of grains and milled materials der einflui~ des wksserns auf die kartoffel irradiation of fruits and vegetables in india effect of storage in nitrogen on the soluble sugar and dry matter contents of ryegrass drying of seaweeds and other plants. v. throughcirculation drying of asophyllum nodosnm in a semi-continuous dryer niacin, thiamin, and riboflavin in fresh and cooked pale, soft, watery versus dark, firm, dry pork muscle nouvelles observations concernant la survie des salmonellae clans les fromages pyroearhonie acid diethyl ester as a potential food preservative the effect of phosphates on moisture absorption, retention, and cooking losses of broiler carcasses gur hygienisehen beur~ilung der trinkwasserverh~ltnisse des oberon vogtlandes -eine hydrobiologische s~udie. z. ges. hyg. studies on preserving quality in market eggs rapid detection of faecal coliform bacteria in the food processing plant. j. milk food technol the relationship between the loss of water and carbon dioxide from eggs and the effect upon albumen quality plastic pacckaging of eggs. study on improvement of digestibility of milk protein. i.: the effect of heating, adjustment of activity of calcium ion, addition of whey protein, homogenization, and elimination of coarse casein micelle from milk by ultracentrifuge on the digestibility of milk especially on the coagulability of it part iii.; the digebtibility of slightly hydrolized milk with proteinase and the preparation of rnill~ which has same eoagulability as human milk. g. agrie, chem. see study on improvement of digestibility of milk protein. part iv. : the nature of coagulation of casein of milk preparation which has same coagulability as human milk the influence of added microorganism on the quality of margarine. i. : the influence of mold inoculation die ilberung yon tafelw/~ssern. dr. lebermmittel-rdsch technological aspects of the radiation pasteurization of foods rapid hydration of dried fruits. food technol untersuchungen fiber polygalakturonase-enzyme aus sehimmelpilzen. . mitt.: eigenschaften der polygalakturonasen aus schimmclpilzen role of individual phospholipids as antioxidants association of veterinary food hygienists symposium on the marketing, transport, and slaughter of calves. iil: scientific aspects. ve~cr ricerche sulla resistenza della brucella abor~us helle salsicce. riv pr senee des salmonelles dans les viandes. donn es frangaises et tran-gbres biochemisehe vorg~nge inl fleisch bei der lagerung einflul des r stgrades yon kaffee auf die extinktion w~briger extrakte und die menge der trockensubstanz die herstellung yon quark und weillk~e unter ansnfitzung ss eiweibstoffe der milch vcrluste yon vitamin b~ und c beim kochen und turmkoehen yon gemfise effect of chilled storage on the frozen storage life of whiting salmonellenfunde in einer importsendung amerikanischer tiefgefrierhiihner. arch. lebensmittel-hyg iron sulfide blackening in canned protein foods: oxidation and reduction mechanisms in relation to sulfur and iron raft research on food preservation by irradiation in poland no~: gas chromatography of chicken and turkey volatiles: the effect of temperature, oxygen, and type of tissue on composition of the volatile fraction l'inaetivation dens l'eau de meret l'eau d'alimentation de eertains entdrovirus de voedingswaarde van aardappelen van versehiuende re, sen en de invloed daarop van bemesting en bewaring effecb of l-arab]nose and d-xylose on dough fermentation and crust browning gelation of egg yolk corn carotenoids: effects of temperature and moisture on losses during storage salmonellenfunde in einer importsendung amerikanischer tiefgefriorhiihner. arch. lebensmittel-ttyg bacteriological examination of unbottled soft drink ~berbliek fiber kunststoff-folien und -kombinatlonen ale verpackungsmaterial in der mflchwirtschaft pigmentierung des eidottem bei gettiigel. wiss. versff. dr. ges. eruiilu'ung s~ beltrag zur bedeutung wasserlsslieher hochmolekularer kohlenhydrate f'tir die verkleisterung der st~irke einfiul ehemischer verbindungen auf die antimikrobielle konservierungs-~toffwirkung. . mitt.: einflub verschiedener stoffgruppen auf die konservierungswirkung gegen aspergillus niger a quantitative s~udy of changes in dried skim-milk and lactose cnscin in the 'dry' state during storage the role of the major sugars of potatoes in ~he browning roa tion during chipping probleme der zuverl~sigkeit yon kunststoffen zur lebensmittelverpackung in europi~ischer sicht bakteriologiseh-hygienisehe beur~ilung yon speiseeis weizenkeime ale wertvoller rohstoff-einige ~ragestellungen und probleme probleme der frischhaltung und haltbarmachung yon brot end backwaren the effect of selected polymers upon the albumen quality of eggs after storage for short periods preparation and quality evaluation of processed fruits and fruit products with sucrose and synthetic sweeteners the microflora within the tissue of fruits and vegetables changes in carbohydrate and phosphorus content of potato tubers during storage in nitrogen preparation of "natural" cow-milk fat globules; preliminary investigation of materials adsorbed at their surfaces lethal doses of gamma radiation of some fruit spoilage microorganisms alteration of post-mortem changes in porcine muscle by preslaughter heat treatment and diet modification ober die msglichkeiten end grenzen eines effects of polyphosphates on water uptake, moisture retention, and cooking loss in broilers flavors imparted to dairy products by phenol deriva aromatisehe crackproduktc yon sterinen. (i). z. ern~hrungs-wiss dose requirements for the radiation sterilization of food berichb fiber eine arbeitstagung bei der internationalen atomenergie-beh rde in wien vom zur bok~mpfung d r rinderfinne zum einfiu]~ handelsfiblicher, in lebensmittelbetrieben gebr~uch-]icher desinfektionsmittel auf lactobakterien; zugleich ein beitrag zur desinfektion in der marinadenindustrie einflu~ chemischer umsetzungen bei trockenen lebensmittelgemischen in hinsich~ auf die lagerfestigkeit. vi. mitt. : lebensmittelgemische mit troekenmagermileh als hauptkomponente. z. lebensmittel-untersuchung u die technologie yon sauren milcherzeugnissen, insbesondere der sauermilcharten und sauerrahmarten effects of several edible coatings on poultry meat quality how to control insects in stored foods. part die antibiotika und die ans ihrer anwendung fiir die ~iilehwirtsehaft sich ergebenden probleme zur haltbarkeitsverli~ngerung empfindlicher l~ahrungs-und genuflmittel dureh abpaeken unter vakuum. fette, seifcn the diffusion of hydrogen through tinplate containers packed with grapefruit juice effect of ice cream stabilizem on the freezing characteristics of various aqueous systems ist die infektion mit trichinen aus amtlich untersuchtem schweinefieisch im liehte der mathematisehen analyse der bestimmungen der fleischbcschau yon schweinefleisch msglieh? hygiene in milk production, processing, and distribution effect of pre-eooling eggs and cartons upon quality after storage a biological after-effect in radiation-processed chicken muscle accounting for farm tank milk factors related to the flavor stability during storage of foam-dried whole milk. iii. effect of antioxidants untersuchungen zur hygienischen beurteihing yon ~ietallverunreinlgungen in lvben~mitteln the effect of bleed time prior to scald and refrigerated storage upon bacterial counts in the axillary diverticula of the interclavicular air sac of chickens techniques de recherche des salmonella darts les viandes the serotypes of salmonella isolated from foods carotinverluste beider zubereitung der nahrung. wiss. vcrsff. dr. gcs. er-n~hrung stability of ascorbie acid in a liquid multivitamin emulsion containing sodium fluoride the effect of storage time and holding temperature on egg interior quality in uganda uber den einflul versehiedener fangverfahren auf die qualit~t und lagerreserve der fische zerst~ubungstrocknung yon tomatenkonzentraten. dr. lebensmittel radiation pasteurization of fresh fruits and vegetables a bacteriological survey of certain processed meat~. part l population studies at packet and retail levels association of veterinary food hygienists symposium on the marketing, transport, and slaughter of calves. l : marketing and slaughter die kombinierte verarbeitung yon kartoffeln auf st~rke und alkohol (fortschrittsbericht) usaec program in radiation research preservation of certain fish and fruits biochemical and quality changes in chicken meat during storage at above-freezing temperatures inleidend onderzoek naar strnctuurveranderingen die ontstaan bi] verhitten van plantaardige produkten veranderingen van hot vetgehalte bij de bereiding van vlces a study on the relationship between the factors influencing the time of cheese salting maple sirup. xxi. : the effec~ of temperature and formaldehyde on the growth of pseudomonas geniculata in maple sap vacuum-tempering corn for dry. milling organoleptische eigenschappen, thiamine-en ascorbinezuurgehalte van enige week-en diepvriesgroenten growth of psyehrophiles. il : growth of poultry meat spoilage bacteria and some effects of chlortctracycline tests of corn stored four years in a commercial bin association of veterinary food hygienists symposium on the marketing, transport and slaughter of calves. ii. : the slaughter and inspection of calves. veter the effect of processing conditions upon the nutritional quality of vegetable oils berieht fiber den wisscnsehaftlichen kongrel der dcutschen gesellschaft ftir ern~hrung ( vortragsrcferate) an objective measurement of the freshness of ready-to-cook broilers the fieldman's responsibilities in milk quality and procurement studies on the bacteria found in the wine during its making. i.: multiplication of bacteria in wine and male-lactic fermentation hydrophilic colloids as additives in white layer cakes the acceptability of cooked poultry protected by an edible acetylated monoglyeeride coating during fresh and frozen storage istes zu vertreten, dal das fleiseh sehwachfinniger rinder auch in gebriitetem zust~nd eingcfrorcn wird? arch. lebensmittel-hyg =[efenlnfit.ierte kondensmilch als ursache yon fehlfabrikation bei schokolade. arch. lebensmittel.hyg. [ ] nr. , s. . m, ober die bedeutung aerober sporenbildner als bombageerreger yon wfimtehenkonscreen. arch. lebensmittel-hyg aluminiumfolio zur verpaekung tiefgekiihlter und gefriergetrockneter lebcnsmittel. fette fiber die milcldtuorierung. bull. schwciz. akad. reed. wiss vitamin stability in diets sterilized for germfree animal~ die trinkwasserversorgung ernliln-ungsstatistlk nutritional statistics african nutrition problems der vcrbrauch yon alkoholisehen getr~,nken in sterreich childhood nutrition in lapland overweight children in stockholm iron deficiency in the finnish population vitamin b deficiency in indian infants the indices of nutritional change in great britain dietary values from a h recall compared to a -day survey on elderly people her vaststellen van de voedingstoestand van sen bevolldng in de tropen en subtropen dental effects of fluoridation of water with particular reference to a study in the united kingdom de voedlng van woonwagenbewoncrs nutritional attitudes of some london housewives de vocdingsgewoonten van bcjaarden in amsterdam community studies of drinking behavior fats and carbohydrates as factors on atherosclerosis and diabetes in yemenite jews nutritional beliefs among a low-income urban population algemene gezondimidsaspccten van de vocding in de ontwikkelingsgcbieden a comparative study of the nutritional adequacy of the morning intake of women clerical workers and women factory workers serum cholesterol in a military population. its relation to obesity and the military diet nutrient intakes of healthy older women analysis of the structures of food consumption by groups in japan thiamin (vitamin b,)-untercru~hrung in deutschland? lvied vortragsmaterialien ftir die ern~hrungsprops (erli~uterungen zu insgesamt groben sehautafeln.) . mitt.: behandlung der tafein iv bis vi. erns [ ] nr. , s. zur ern~ihrungssituation in arbeitexfamilien aus verschiedcnen bezirken der ddr. . mitt. : ern~hrungssoziologischc answertung der lebensmittelverzehrungen in itaushaltungen mit erwachscnen und kindern~ stand studies in infantile malnutrition. i. : nature of the problem in peru zur ~ethodik yon ern~ihrungserhebungen bei der gemeinschaftsverpflegung weight changes in relation to birthweight of papuan, indonesian, and chinese children during the first two weeks of life numbers of tasters required to determine consumer preferences for fruit drinks onderzoek naar de menupatronen in de noordoostpolder smoking habits of medical and non-medical university staff i**cs and potassium in people and diet. -a study of finnish lapps. ann. acad. scientiarum fennicae a ~berbliek fiber die radioaktivit~t der in sterreich im jahre konsumierten lebensmittel diet and plasma cholesterol in bank men der mengenmi~bige getr~inkeverbrauch je einwohner im bundesgebiet predictors of human food consumption use of goverument.donated foods in a rural community bericht fiber die durum-und teigwarentagung der arbcitsgemeinschaft der oetreldeforschung e. v. vorn . bis . miirz voeding van leerlingen van con lagere teehni~eho school. ii. calorie~in-en nutriiintenwaarde nutritional deficiencies in developing countries dietary survey in surinam vitamine a-tekor~en op de aarde schwierigkeiten beim erreichen einer vollwertigcn ern~ihrung in ausgew~hl-ten vcrbrauchergruppen die entwicklung des brot-und getreideverzehrs in der neueren zeit. wiss. ver ff. dr. ges. ern~hrung n~rwert und zusammensetzung yon lebens. und futtermltteln nutritive values, composition of food~tuffe and f physical chemistry of ice cream vitamin e in human nutrition appraisal of nutritional adequacy of infant formulas used as cow milk substitutes enkele gegevens betreffende de calorisehe waarde van klsine hepjes, gerechtcn en maaltijden the public health aspects of the use of antibiotics in food and foodstuffs. report of an expert committee niihrwertminderung dutch zubereitung der nahrung metals and other elements in foods die farbe der nahrungsmittel in anthropologischer sicht the enzymatic destruction of carotene and carotenoids foodstuff flavors. some factors affecting the flavor of sodium caseinate chemical and radiochemical composition of the rongelapese diet the organic constituents of food. i. : lettuce the influence of dehydration of foods on the digestibility and the biological value of the protein a stfidy of two methods of assessing vitamin b nutriture mincraisalze mad spurenelemente in der nahrung distribution of the bound form of nicotinic ac|d in natural material~ the distribution of c~rotenoids in nature and their biological significance zur bedeutung antimikrobieller stoffe in der mahrung die konsistenz yon margarine mad fetten an improved nutrient solution for diploid chinese hamster and human cell lines extraneous materials in foods and drugs the composition of food flavors studies on pantothenic acid intake. i. pantothenic acid content in japanese foods haben wir mangel an essentiellen fettsiiurcn? phonolcarbons~uren in menschlichen nahrungsprodukten. zum vorkommen yon phenolcarbons~uren in menschlichen nahrungsprodukten und ihr einttud auf den intermedi~ren sboffwechscl drugs in feeds relationship between the sulphur/nitrogen ratio and the protein value of diets gums in foods zur definition der begriffe ,aroma" und begriff und aufgabe dii~tetischer lebensmittel valettr vitaminique des carot nes pour rhomme. wiss. versff. dr. ges. ern~hrung internationales rundgespriich fiber lebensmittelchemische probleme in wiesbaden und eltville a. rh. ( vortragsreferate) consumer awareness of texture and other food attributes organisehe und organisierte substanz in der lebensmittelchemie frost resistivity of fruit plants californ'ia association of chemistry teachers : inorganic nutrients in the sea fluoride in food antibiotics in feeds and other products planktorm as foods relation between color of cranberries and color and stability of sauce berieht fiber die vortragstagung des fachverbandes lebensmittelchemie der chemischen gesellsehaf$ in der ddr vom . bis bulletin on tobacco evaluation of algae as a food for human diet~ digestibility of high-amylose corn starch. nutrition red. [ ] nr. , s. / . ~. n. : comparative evaluation of corn mesa and steam-processed whole corn flours the major anthocyanin pigments of vitis vinifera varieties flame tokay, emperor, and red ~alaga peonidin- -monoglucoside in vinifera grapes formation and distribution of amylosc and amylopectin in the starch granule nutrient in seeds. amino composition of some seeds sugar levels in fruits of the lowbush blueberry estimated at four physiological ages the relation of pectic substances to firmness of processed sweet potatoes (ipomoea berates) processed vegetable produc~s the protein composition of different flours and its relationship to nitrogen content and baking performance relationship between 'antitryptic factors' of some plant protein feeds and products of proteolysis precipitable by trichloroacetic acid. g. sci. food agric a thermostable haemolytic factor in soybeans foam-mat dried orange juice. i. time-temperature drying studies bound" growth inhibitor in raw soybean meal leaf analysis as a guide to the nutrition of fruit crops. ii. : distribution of total n, p, k, ca and mg in the laminae and petioles of raspberry (rubns idaeus l.) as influenced by soil treatments nutritive value of pumpkin seed. essential amino acid content and protein value of pumpkin seed (cueur bi~a farinoaa) effect of cooking and of amino acid supplementation on the nutritive value of black beans (phaseolns vulgaris l.) supplementation of cereal proteins with amino acids. iv.: lysine supplementation of wheat flour fed to young children at different levels of protein intake in the presence and absence of other amino acids uber den chemischen total ascorbic acid in potatoes. raw, fresh, mashed, and reeonstituted flakes moisture contents of hard red winter wheat as determined by meters and by oven drying, and influence of small differences in moisture content upon subsequent deterioration of the grain in storage rheological studies with canned tomato juice the chemical composition of maple sugar sand soluble carbohydrate content of varieties of te~raploid ryegrass natiirlicher gehalt und stabilit~t yon carotine und carotinoiden in citrnss~ften ~ber wein und weinuntersuchungen fatty acids and other lipids in mayonnaise cyclic fatty acid yields from linseed oil factors ~ffecting enzymatic solubilization of beef proteins weibulls original ring, en ny medeltidig varvetesort. (a new medium early variety of spring wheat, weibull's ring.) agri hortique genetiea ober die askorbins~uresynthese in zerschnittene kartoffeln die bestimmung der amylaseaktivit~t und einige studicn fiber amylaseaktivit~t in gekeimtem roggen effect of processing conditions on dry-heat expansion of ~ulgar wheat oils, fats, and waxes ~.~ber das der johannisbro~.kerne. fetto, seifen fruit and fruit products uber die variabili~it einiger eigcnschaften der kartoffelstilrke in abhiingigkcit yon witterung a short.term effect of weather on malie acid in pineapple fruit the specific surface of flour and starch granules in a hard winter wheat flour and in its five subsieve-size fractions oranges and lemons factors affecting quality of pies prepared from frozen bulk-pack red sour pitted cherries studies on the consish~ncy of thiamin and protein contents of pure.bred strains of rice a comparison of the nutritional value of protein from several soybean frac ions zum vitamin-und aminos~uregehal~ yon maisquellwasser dark discoloration of canned all-green asparagus. i. chemistry and related factors enzymatic enhancement of flavor peetinestcrase in normal and abnormal tomato fruit storage effects on winter squashes. varietal differences and storage changes in the ascorbie acid content of six varieties of winter squashes grape pigments. concord grape pigments corn meal as a source of ribonuclease banana odor components. volatile components of bananas. part i. isolation of an odor concentrate. part il separation and identification lipids of algae. ill. : the components of unsaponifable matter of the algae chlore]la volatile esters of bartlett pear. ii studies on the nutritive value of raw and cooked soybeans for growing rats and swine and their effect on fat firmness characterization of fruit juices by acid profiles nitrate content of beets, collards, turnip greens lysine fortifications of wheat bread fed to haitian school children studies on the flavor of green tea. par~ iv. : dimethyl sulfide and its preeursor funktionelle eigenschaften yon lehens-mittclst~rken ~)ber das vorkommen yon xylit im speisepi]z champignon ein beitrag zur znsammensetzung yon apfeisinens~ften aus spanischcn und l~iarokko-apfelsinen an examination of the free amino acids of the common onion (allium cepa) a trypsin inhibitor in wheat flour the protein quality, digestibility, and composition of algae, chlorella chemical and color changes in canned apple sauce digestibility of the a-cellulose and pentosan components of the cellulosic mieelle of rescue and alfalfa safeness and serva. bility of meringued pie alcoholic beverages diurnal-nocturnal changes in the starch of tobacco leaves sugar and sugar products modification of flour proteins by dough mixing: effects of suffhydryl-blocking and oxidizing agents ~ber das haferprotein. z. lebensmittel-untersuchung u sodium and potassium in wines and distilled spirits non-volatile organic acids of the dwarf cavendish (chinese) variety of banana~ biological evaluation of soybean meal and cottonseed meal by amino acid digestibility and protein efficiency ratio studies. oiss. abstr. [ ] l~r oxidation-reduction potentials of sak~f and synthetic sak . xi.: on the relationship of the various fermenting processes of sak~ to oxidereduction potentials and indicator time test (i.t.t.) values of sak mash neue wege zur ern~hrungsphysiologischen aufwcrtung von getreideerzeugnissen cereal products ascorbie acid in dehydrated po~es die eiweibqualit~t yon ,getoastetem" (dampferhitztem) und ungetoastetem sojaextraktionssehrot color studies on processed dried fruits studies on the basic amino acid of the soy sauces and the seasoning liquids. ii.: the quantitative changes of l-arginine in the process of soy sauces brewing studies on the flavorons substances in soy sauce. xxii. : the differences between the soy sauce made from soy bean and wheat and that made from defatted soy bean and wheat feeding value of soy milks for premature infants flavors and non-alcoholic beverages fat content and fatty acids in some commercial mixes for baked products sensory examination of four organic acids added f~ wine ber die inhaltsstoffe der robktmtanie und versuehe zu ihrcr gehaltsbestimmung. diss. univ. hamburg, . malt beverages, sirups, extracts, and brewing materials vitamin b and niacin in potatoes. retention after storage and cooking chemical investigation of some wild indian legumes zusatzstoffe der margarine. forte componen~ glyeerides of an indian fresh-water fish fat einflub des rsstgrades yon kaffce auf die extinktion w~$riger extrakte und die v[enge der trockensubstanz gaschromatographische untersuehungen yon fusclslen aus vcrsehiedchen g~irprodukten. . mitt.: problemsteilung und literaturfibemieht de voedingswaarde van aardappelen van versehillende rassen en de invloed daarop van bcmesting en bewaring eleetrophoretic separation of beet pigments studies on the growth-promoting value and digestibility of passion fruit seed oil polycyclisehe und aliphatisehe kohlenwasserstoffe dc~ tabakrauehes carotenoid, oil, and tocopherol content of corn inbreds location and possible role of esterified phosphorus in starch fractions untersuchungen fiber die chemischen u beim a]tern yon r stkaffee. . mitt.: gaschromatographische analyse der leichtflfichtigen aromabestandteile nature, origin, and prevention of hydrogen sulphide aroma in wines the magnesium contents of soil and crops versuehe zur ehemischen differenzierung der eiweibst~ffe des weizens und roggens lemon juice composition. iii.: characterization of california-arizona lemon juice by use of a multiple regression analysis weizenkeime als wertvoller rohstoff -einige fragestellungen und probleme isolation of gram quantities of a rhamnoglucoside of apigenin from grapefruit determination of distribution of water in wheat grains by interference microscopy preparation and quality evaluation of processed fruits and fruit products with sucrose and synthetic sweeteners changes in carbohydrate and phosphorus content of potato tubers during storage in nitrogen chemical composition of some natural and processed orange juices effects of various factors in the candy test zum stand der kenntnlsse fiber die v{eclmelwir-kung zwischen nativer sts und wasser some volatile compounds from cooked potatoes firmness of canned apple slices as affected by maturity and steam-blanch temperature nutritive values of ten samples of western canadian grains proteins of wheat and flour. the separation and purification of the pyrophosphate-soluble proteins of wheat flour by chromatography on dear-cellulose changes in quality and composition produced in wine by s~ gamma irradiation citrus essential oils. iii. evaluation of silician natural lemon oils mineral analysis of plant tissues a. : nature of colloids in clarified cane juices studies on amino acid content of rice. i. : amino acid composition of polished rice glutelln estimated by beckman amino acid analyzer yliichtige carbonylverbindungen in honig neue aminos~iuren in h heren pflanzen studium der wirkung der gammastrahlung auf die l-ascorbins~iure flour liplds and oxidation of sulfhydryl groups in dough zur kenntnls eincr weiteren in der sti~rke vorkommenden kohlenhydrat-komponente. ern~ihrungsforschung lemon juice composition. ii. : characterization of california.arizona lemon juice by its polyphenolic content lemon juice composition. i.: characterization of california-arizona lemon juice by its total amino acid and -malic acid content safflower amino acids amino acid composition of safflower kernels, kernel protein, and hulls, and solubility of kernel nitrogen citrus fruit enzymes. i. : ascorbie acid oxidase in oranges untersuehungen fiber die verf~rbung gekochter kartoffeln an den sorten des kulturlrartoffelsortiments ides instituts ffir pflanzenziiehtung groi~-liisewitz nutritive value of red kidney beans (phaseolns vulgaris) for chicks instability in potable spirits. ii.: rum and brandy effect of size classification and maturity on the protein content of alaska and perfection peas ma~tr~, d. c.: ascorbie acid retention and color of strawberries as related to low-level irradiation and storage time historical aoac data on four fema samples of vanilla extract the acid-extracted pentosan content of wheat as a measure of milling quality of pacific northwest wheats petroleum ether extraetables in tobacco isolation, origin, and synthesis of a bread flavor constituent the protein composition of airclassifled flour fractions * studies on the flavor of green tea. v. : examination of the essential oil of the tea-leaves by gas lipid chromatography nutritive value of starches. iv.; comparison of digestibility of natural starches estimated by a new procedure o c lebensmittel tierischen ursprungs _foodstu~$ o/animal origin n.n.: gdch-faehgruppe ,lebensmib~el. und gerichtliche chemie bericht fiber die vortragstagung des fachverbandes lebensmittelchemie der chemischen gesellsehaft in der ddr yore . bis a taxometric study of the propionic acid bacteria of dairy origin comparisons of the caseins of buffalo's and cow's milk matiirlicher gehalt und stabilit~t der carotinoide in fetten und milchprodukten. wiss. versff. dr. ges. ernhhrung antibiotics in milk vitamin a and d enrichment of nonfat dry milk some characteristics of yolk solids affecting their performance in cake doughnuts. i. effects of yolk type, level, and contamination with white proteine des eidotters post-mortem changes in the muscles oflandrace pigs t~ber den fettgehalt yon flcischkonserven. dr. lebensmittel der einflul~ der verfahrenstechnik auf die ks jahreszeitliche einfliisse alff den gehalt der fischmuskulatur an freien ami-nos~iuren u. deren bedeutung fiir die qualitiit fangtechnik und fisch-qualit~t. fette spcei~c distribution of fatty acids in marine lipids a comparison of pigs slaughtered at three diffcrcn~ weights. i. : carcass quality and performance a comparison of pigs slaughtered at three different~ weights. ii. : association between dissection results, various measuremen~ and visual assessments a note on the effect of heat on the colour of goat's milk chemical and nutritional changes in stored herring meal. .: nutritional significance of oxidation of the oil relation of pork muscle quality factors to zinc con~ent and other properties the chemical nature of the characteristic flavor of cultured buttermilk occurence of vaniuin in hea~ed milks the electrophoretie properties of the proteins in cottage cheese curd the influence of post-mortem glycolysis on poultry tenderness seasonal variations in cod liver oil isolation and characterization of the flavor components of rancid pork some problems in the evaluation of egg albumen quality quality evaluation studies of fish and shellfish from certain northern european waters indices for lamb carcass composition subjective and objective evaluations of prefabricated cuts of beef ils, fats, and waxes die bildung yon eiskristallen in diinnen milchschichten. milchwissensehaft the incidence of bacteria in cheese milk and cheddar cheese and their association with flavour body composition of market weight pigs some factors affecting tenderness of turkey meat ovine bioenergetics and nutritional efficiency, with special reference go forage utilization die ziichtung yon fleisehsehweinen und die folgeerseheinungen, die sich insbesondere im hinbliek auf die qualit~t yon fleisch und fett ergeben. arch. lebensmitgel-ityg n the structure of highly unsaturated fatty acids of fish oils by high resolution nuclear magnetic resonance spectral analysis the fatty acid composition of the milk fat of cows grazing on ryegrass at two stages of maturity and the composition of the ryegrass hpids effect of intrauterine infusion of penicillin-streptomycin and furacin and vaginal deposition of furacin on chemical residues tevei~ in millr beziehungen zwisehen l-aseorbinsaure und milch comparison of chemical and organoleptic data obtained on thawed and unthawed frozen cod, haddock, and perch fillets die ~ m~uosfiurenzusammensetzung der ziegenmflch und des ziegenmileh-caseins food flavors and odors. meat flavor: lamb dairy products de ehemische samenstelling van visen visprodukten chemical studies on the herring (clupen harengus). vii.: collagen and cohesiveness in heat-processed herring and observations on a seasonal variation in collagen content growth and pro~eolycie activity of pseudomonas fluoresccns in eggs and egg products the fatty acid composition of some perirenal and subcutaneous beef depot fats inactivation of peroxidase in milk by homogenization a study of the "cured meat" color producing reaction and the effects of some curing adjuncts t~yoer den fettgchalt yon br'tihwiirsten stress effects, eareass composition, and carcass quality in lambs effect of chemical additives on the spreading quality of butter. ii. laboratory and plant ehurnings preliminary studies on protein and moisture relationship in fresh and proeessed hams die zusammensetzung des kuhmilchfettes in abh~ngig-keit yon der ffitterung. fette chemical characterization of off-flavors in concentrated and nonfat dry milk zur ausseheidung yon xanthindehydrase mid molybdiin in der kuhmilch copper distribution in milk during early lactation die physikaliseh-ehemischen ursaehen der hitzestabilitet yon mileheiweil]stoffen. milehwissenscha sobn~a: ~oer einige verenderungen in den caseinfraktionen normaler und anomaler milch (colostralmilch und milch an lviastitis erkrankter kiihe). milchwissensehaft some characteristics of yolk solids affecting their performance in cake doughnuts. ii. variability in commercial yolk solids zum problem des znsammenhanges zwisehen der konsistenz und der physikalischen struktur der butter. fette relationships between milk fat acidity, short-chain fatty acids, and rancid flavors in milk induced and natural inhibitory behavior of milk and significance to antibiotics disc assay testing. j. dairy sei. [ ] nr. , s. ff. ( s.). --some distribution patterns of cottage cheese particles and conditions contributing to curd shattering natural inhibitory eharacteristies of some irish manufacturing milks thermophylie aetinomycetes in milk and dairy products. mikrobiologiya the free fatty acids of purdue swiss-type eheese die zusammensetzung yon siilzen lind ihre beurteilung im regierungsbezirk diisseldorf not~ on tyrosine production in frozen stored liver studies on the muscles of meat animals. hi. : comparative composition of various muscles in pigs of the three weight groups studies on beef quality. x. effect of temperature, freezing, frozen storage, thawing, and pe on the rate of hypoxanthine production. die. food preservation t~chn increased iodine in milk as a countermeasure for ~liodine time-temperature studies of baked, loaves. meat, fish, and poultry vergleichende untersuehungon an butter und einem butter~hnli-chen umgeesterten fort. forte, seifen, anstrichmittel zur beziehung zwisehen fettgehalt und wassergehalt bei ungewa~ohener s/il~rahmbutter (fritzbutter) variation of ovine fat composition within the carcass studies on the properties of new zealand butterfat. vii. effect of the stage of maturity of ryegrass fed to cows on the characteristics of butterfat and its carotene and vitamin a contents studies on turkey body composition. . poultry sci thermal conductivity of beef studies on turkey body composition. free fatty acid, tyrosine, and ~ changes during ripening of blue cheese made from variously treated milks l : factors influencing the nutritional value of fish flour. il: availability of lysine and sulphur amino acids. canad characterization of flavor compounds isolated from evaporated milk effect of egg yolk size on yolk cholesterol concentration ~tudo immuno~lectrophor~tiquo du lair dans los divers types de mammites. i. milchwissenschaft rdsultats hemmstoffe in der anlieferungsmflch und methoden zu ihrem nachweia eiweibverenderungen gefriergetroekneter muskulatur meat and meat products effect of high doses of vitamin a palmitate on vitamin a aldehyde, esters, and alcohol and carotenoid contents of hen's eggs. brig. j. nutrition [ ] nr. , s. / . --the amounts of vitamin a aldehyde, esters, and alcohol and of earotenoids in hen's eggs and in day-old chicks nutritive value of marine oils i. : ]~lenhaden oil at varying oxidation levels, with and without antioxidants in rat diets a quantitative study of changes in dried skim-milk and lactose-casein in the 'dry' state during storage physico chemical characteristics of canadian milk fat. unsaturated fatty acids collagen content and its relation t~ tenderness of connective tissue in two beef muscles ~ber eine braune verf~rbung yon mariniertem hering. dr. lebensmitbel versuche fiber den sonnenlichtgesehmaek in mit ass markierter milch effect of unequal milking intervals on lactation milk, milk fat, and total solids production of cows wei~re untersuehungen fiber die nitratreduktase verschiedener an der reifung yon rohwurst beteiligter mlkroorganismen nutritive value of leg of lamb roasts. l~oisture, energy, protein, fat, and iodine values ~ber einige gul~sigkai~s~renzen bei konsistenzfehiern yon butter influence of linoleic acid content of milk lipids on oxidation of milk and milk fat fish hydrolysates-iii.: influence of degree of hydrolysis on nutritive value einige beobaehtungen fiber die bildung des durch lieht verursaehten oxydationsgesehmackes s.). zitat: dt. lebensmittel comminuted meat emulsions: factors affecting meat proteins as emulsion stabilizers factors related to the flavor stability during storage of foam-dried whole milk. iil effect of antioxidants upgrading the indigenous poultry of uganda. hi. : shell and egg interior quality relation between carcass composition and live weight of sheep diethylstilbestrol occurence in eggs of subcutaneously injected hens biochemical and quality changes in chicken meat during storage a~ above-freezing temperatures investigations on the allerged goitrogenie properties of milk fish and other marine products bioehemieal properties of pork muscle in relation to curing potassium content of dried milk vergleiehende histometrische untersuehungen fiber den kollagengehalt yon brfihwfirsten a comparison of the volatile compounds of fresh and decomposed cream by gas chromatography studies on the volatile carbonyl compounds in ladino clover and their influence on the flavor of milk aut~xidation of fish oils. ii.: changes in the carbonyl distribution of autoxidizing salmon oils the public health aspects of the use of antibiotics in food and feedstuffs. repor~ of an expert committee composition and digestibility of corn silage as affected by fertilizer rate and plant population factors influencing the nitrate content of forage the component sugars and rate of hydrolysis of forage hemicelhflose as related to digestibility digestibility trials on forages in trinidad and their use in the prediction of nutritive value acetyl-(para-nitrophenyl)-sulfanilamide in feeds distribution of major and trace elements in some common pasture species -dimcthyl -( , , -trichlorophcnyl)phosphorothioate) in feeds feeding value of low-moisture alfalfa silage from conventional silos ovine bioenergeties and nutritional efficiency, with special reference to forage utilization a system for naming and describing feeds, energy terminology, and the use of such information in calculating diets supplemental methionine in a sixteen percent protein diet for laying chickens l : organic arsenicals in feeds cellulose degradation by enzymes added to ensiled forages influence of corn distillers dried grains with sohbles on the feeding value of wheat silage zinc content of certain feeds, associated materials, and water der natiirliche gehalt und die stabilit~it yon carotin und carotinoiden in lieu und silage forage digestibility. benzene-ethanol extracts of forage and faeces as indicators of digestibility the prediction of the metabolizable energy content of poultry fcedingstuffs from a knowledge of their chemical composition factors affecting the metabolizable energy content of poultry feeds. facters affeeting the metsbohzablo energy content of poultry feeds unidentified chick growth factor in fish solubles diet and histamine in the ruminant. occurrence of histamine in silage ethopabate in feeds die isolierung yon apocarotinalen aus luzernemehl isoflavone contents of red and subterranean clovers metabolizable energy of some oil seed meals and some unusual feedstuffs ii methoden der untersuchung yon lebens-und futtermitteln techniques analysis of foodstuffs and feeds n.n.: gdch-faehgruppe analysis of foods by neutronaetivation techniques continuous measurement of dissolved solids in food processes by critical-angle refractometry berieht fiber die vortragstagung des fachverbandes lebensmittelchemie der chemischen gesellschaft in der ddr vom . bis a rapid test for anionic detergents in drinking water fortschritte in der lebensmittelehemio dureh moderne analysenmethoden direct potcntiometrio determination of chloride in cheese modification of the polarimetrie starch determination on hig-hamyloso corn ~iethoden der ]~berwachung des wassers auf radioaktiviuit. btmdes prediction of quality in protein concentrates by laboratory procedures involving determination of soluble nitrogen direct chromatographic analysis of milk die eignung der bakteriologischen untersuehung yon kannenmilchproben als grundlage eines eu~ergesundheitsdienstes. arch. lebensmittel-i~yg nachweis fremder carotinoide in rangensiiften mittels diinnschichtchromatographie. i)t. lebensmit~el-l~dseh determination of phosphate composition of stock food calcium phosphate. . assoe. off. agric. chemists molybdenum in plants and animals. determination of molybdenum in biological materims with dithiol control of copper interference the determination of dissolved oxygen in canned drinks using a vibrating mercury-plated platinum electrode determination of chromium and lead in periodic acid solution and dialdehyde starch the utilization of infrared and ultraviolet spectrometric procedures for assay of pesticide residues ultramicro determination of potassium and sodium in biologic fluids gum quautativen naehweis des pektins und der alginsiiure ein schliffapparat zur abtrennung yon flfichtigcn stoffen mittel~ wasscrdampfdcstillation ~tude par chromatographic on phase gazeuse des acidea gras du beurre fabriqu en italic et clans d'autres pays. application ~, la recherche des falsifications clans le bcurre commercim. ann. falsiticatiorm expertise chim note on the determination of caffeine in coffee a comparison of the press method with taste-panel and shear measurements of tenderness in beef and lamb muscles zur diehtebestimmung der milch mit der neuen milehspindel. lebensmittelchem, u. geriehtl. chem. [ ] nr. , s. / . --aufschlu -.i~thercxtrakt und titrationswert bei der teigwarenunbersuehung als beispiel einer dynamisehen lebensmittelanalyse quantitative measures of carcass composition and qualitative evaluations fruit preservatives analysis. determination of calcium in cherry brines by versenat~ titration: elimination of anthocyanin interference by means of carbonyl reagents, g. agric. food chem allgemeine prinzipien der analytik yon carotinen und carotinoiden zur bestimmung yon ethoxyquin kolorimctrische bestimmung yon dipterex-riickst~nden yon lebensmittein the direct determination of shear stress-shear rate behavior of foods on the presence of a yield stress methods for evaluating bhe feeding quality of meat-andbone meals a paper chromatographic method for determination of vanillin and ethyl vanillin in vanilla flavorings ~drber die anwendung der papierchromatographisehen analyse auf dem fettgebiet. . mitt.: uber ls~iurereiehe samenole. ermittlung der konsti~uierenden fettsiiuren der samensie yon margosa (azadiraehta indies), cashewkern (anacardicum oecidentale) und putranjiva roxburghli. nahrung die jodzahl des riickenspeeks im verhiiltnis zu der qualit~t des futterfettes, dem alter der schweine und dem fettungsgrad bei sehweinen der ditnisehen landrasse determination of parathion, methyl parathion, epn, and their oxons in some fruit and vegetable crops an improved chromatographic method for determining trace elements in foodstuffs determination of guthion residues on fruits techniques used in meat flavor research the analysis of edible oils contaminated with synthetic ester lubricants colorimetrische bestimmtmg yon nitrat und nitrit in biologisehem material confrontation de quelques proe~d s de dosage iodometrique de l'anhydride sulfureux dans les vins zur anwendung der massenspektroskopie zur strukturermittlung yon naturstoffen, mit besonderer berfieksiehtigung der lebensmittelanalytik z lebensmittel cellulose solubility as an estimate of cellulose digestibillty and nutritive value of grasses the -thiobarbiturie acid reagent for determination of oxidative rancidity in fish oils bet die eignung yon daphnia magna zur ermittlung yon riieksti~nden auf frisehem bst und gemfise bemcrkung zum diinnschiehtchromatographischen kakaoschalennaehweis nach the determination of vitamin a in animal tissues and its presence in the liver of the vitamin a-deficient rat elution column preparation of leaf sample for flame photometry. ii. : determination of calcium in tobacco enzymatic-ultraviolet method for determination of uric acid in flour die bestimmung der amyiaseaktivit~t und einige studien fiber amylaseaktivit~it in gekeimtem roggen identification and determination of ascorbic acid (vitamin c) with janus green and its localisation in mitochondria cho]estehnbestimmung im kleinen laboratorlum chick edema factor. iii.: application of mieroeoulometric gas chromatography to detection of chick edema factor in fats or fatty acids bioassay of chick edema factor. collaborative study determination of nih te and nitrate in meat products the measurement of the surface areas of milk powders by a permeability procedure sedimentbeurteilung und sehaim-~iastitis-test als sortierverfahren zur ermittlung yon sekretionsstsrungen bei der ~iassenuntersuchung yon milchproben aoac methods for nutritional adjuncts die ermittlung der ribonucleins~ure im pflanzenmaterial beitrag zur analytischen beurteilung des frischezustandes der pharmazeutisch verwendeten e und fe~te sehnellbestimmung yon kupfer in fe~n applications of oscillographic polarography to the determination of organophosphorus pesticides. ii. : a rapid screening procedure for the determination of parathion in some t~its and vegetables zur standardisierung der vitaminb~-bestimmung in getreide und getreideprodukten eine mikromethode zur bestimmung des fettgehaltes der milch kleiner laboratoriumstiere the determination of organophosphato pesticides and their residues by paper chromatography die l~fikrochemie beim studium yon nahrung und erni~hrung aearicide residues. an improved method for kelthane residue analysis with applications for determination of residues in milk collaborative study of the determination of ethoxyquin in feeds ~rber den naehweis yon quellstoffcn in fleischwaren und m gliche stsrungen durch andere polysaccharide ersatz manueller labormethoden der l sungsspektralanalyse durch den automaten determination of total acids in wines: american society of enologists determination of aldehydes in wines and spirit~ by the direct bisulilte method ~fber einen vereinfachten nachweis des vitamin b mit poteriochromonas slipitata extraction of nys~tin in animal feeds for microbiological analysis zur quantitativen bestimmung der sorbinsi~ure mit dem thiobarbitur-s~urereagenz cottage cheese problems in production and sanitation. quality control in cottage cheese sitzung des arbeitskreises berlin der gdch-fachgruppe lebensmittelchemie und gerichtliche chemie am . . in ]~erlin-dahlem ( vortragsreferate) ein neues elektronisches sctmeuverfahren zur ermittlung der ~risehe yon seefischen fatty acids of lard. a. identification by gas.liquid chromatography oxydative abbauprodukte der l-aseorbins~ure. . mitt. : papierchromatographischer l~achweis analysis of orange juice for total earotenoids, carotenes, and added betacarotene. food technol. [ ] nr. , s. / . u. r~ck~, a. a. : refractometrio measurement of soluble solids in orange juice analysis of iron chelates in plant extracts. il: ferric ethylenediamine'bis'(~ acid) determination of n-aeetylglucosamine- -phosphate and n-aeetylglucosamine in milk arsenic in foods: collaborative comparison of the aminemolybdenum blue and the silver diethyldithiocarbamate methods improved method for testing macaroni products neuere beitri~ge zur chemic der st~rkefraktionen. . mitt.: die mikro-ameisens~urebestimmung bei der perjodat- xydatonsbestimmungsmethode yon stiirke recherche des falsifications dans les extraits de vanille. ann. falsifications expertise chim bestimmung des gesamtstckstoffgehaltes yon milch nach der kjwld~t:l-methode. internationaler standard fil vorteile und grenzen des einsatzes yon markiertem phosphat bei untersuchungen am hiihnerei paper chromatography of carotene and carotenoids determinaton of sevin insecticide residues in fruits and vegetables insecticide residues in meat and eggs. determinaton of sevin insecticide and its metabolites in poultry tissues and eggs bcstimmung der jodzahl yon fetten und en mittels n-bromsuccinimid. . mitt.: l~ber eine !~i gliehkeit zur bestimmung der gesamtjodzahl yon el~iostearins~ure und holz . nahrung [ ] nr. , s. / . kxrr:~e~r, m. a. : ~ ber die anwendbarkeit des thiobarbi~urs~iuretestes boi der untersuchung yon milchprodukten application of gas chromatography to the measurement of gas permeability of packaging materials a sensitive method for quantitative microdetermination of lipids comparison of chemical and microbiological methods for the determination of procaine penicillin in prcmixes and mixed feeds electrophoretc analysis of flour proteins from various varieties of wheat katalytische methode zur bestmmung kleinster manganmengen in lebensmitteln am beispiel der milch eine methode zur papierchromatographischen qualits yon silagen comparison of methods of measuring potassium in pork and lamb and prediction of their composition from sodium and potassium electron capture gas chromatography for determination of ddt in butter and some vegetable oils eine methode zum schnelinachweis yon nitriten in yleiseh-und wurstwaren. arch. lebensmittel-hyg determination ofglyodin residues on pears and peaches eleetrophoretic analysis of flour proteins erstes internationales symposium fiber methoden zur analyse yon lebensmitteln in bordeaux-tatence (frankreich) veto . bis . oktober on the problem of luminescence technique of protein definition in milk beitr~ge zur aminos/iuren-bestimmung in biologischem material aktuelle fragen der lebensmitteluntersuehung, insbesondere der histologischen wurstanalyse untersuchungen yon importiertem tiefgefrorenem tiasenfleisch argentinischer herkunft fish hydrolysates. iv.: microbiological evaluation untersuehungon zum naehweis yon emulgatoren in lebensmit~eln. . mitteilung. forte use of a slice-tenderness evaluation device with pork studies on improvements in quantitative paper chromatography of amino acids in foods. i. : research on procedure of development studies on improvements in quantitative paper chromatography of amino acids in foods. ii.: selection of solvent systems determination of the equilibrium relative humidity of foods untersuchungen fiber die methodik der quantitativen bestimmung yon heizolen und flfissigen treibstoffen im wasser determination of ~-lactalbumin in complex systems hydrogen sulphide in cheddar cheese; its estimation and possible contribution to flavour ascorbic acid measurement. polarographic determination of total ascorbic acid in foods trans-fettsguregehalt yon sehweineschmalz nach fiitterung von schweinen mit rindertalghaltigem kraftfutter. (ein beitrag zur quantitativen infrarotspektroskopischen bestimmung yon trans-fetts~uren in fetten the importance of starch on the microscopic identification of cereal grains in feeds insecticide residues. chromatographic identification of some organophosphate insecticides in the presence of plant extracts chemical and biological estimation of the carotene content in fresh and processed italian apricots sialir acid as an index of the u-casein content of bovine skimmilk foodstuffs analysis. nonvolatile acids of blueberries le dosage de la matibre grasse dans les fromages. ]~tude critique de la m thode en usage au laboratoire municipal la d termination de residus d'insecticides et de fongieides par la m thode polarographique fusel oil determination by gas.liquid chromatography * die brabanter mastitis-reaktion, ein neues verfahren zur ermittlung yon sekretionsstsrungen des euters dutch die kannenmilchuntersuchung uber eine enzymatisehe _&pfels~urebestimmung in wein und traubensaft direct microscopic technique to detect viable yeast cells in pasteurized orange drink die enzymatisehe bestimmung der glucose und saecharose und ibre anwendung in der lebensmittelanalyse a modified zirconium-alizarin method for determining fluoride in natural waters paper chromatography of some cholesterol derivatives determination of moisture by nuclear magnetic resonance and oven methods in wheat, flour, doughs, and dried fruits dye binding by soybean and fish meal as an index of quality the absorptiometrie determination of silicon in water: part l: formation, stability, and reduction of cr and fl-molybdosilicie acids column chromatography of soybean whey proteins enzymatic determination of carbon dioxide in lightly carbonated wines. collaborative study nachweis yon biiffelmilch als verf/flschungsmittel in kuhmilch durch serologisehe methoden photometric determination of phosphate in wines pertinent references to analytical lipid methods published recently, ft spectrophotometric estimation of nucleic acid of plant leaves hemmstoffe in der aalieferungsmilch und methoden zu ihrem nachweis determination of potassium in tobacco determination of chlorides in tebacco phospholipase c determination by egg yolk turbidimetry oher die inhaltsstoffe der rol]kastanie und versuche zu ihrer gehaltsbestimmung mierobiologlcal method for assaying nystatin in animal feeds / ] nr. , s. / . --gaschromatographische untersuchungen yon fuselslen aus versehiedenen g~rproduk-ten. . mitt.: methodik der fusel bestimmung lactose activity measurements. evaluation of laetase preparations for use in breadmaking comparison of methods for determination of lysino in cereals direct determination of calcium in plants, soils, and milk by means of a flame photometer colorimetrie determination of urea in feeds kolorimetrische bestimmung des dihydrox-yacetons fluoride, teeth, and the analyst the quantitative micro-determination of biphenyl in citrus fruit kolorimetrisehes verfahren zur gleichzcitigen bestimmung der weinsiiure und milchsiiurc in wein und most estimation of extra-cellular starch of dehydrated potatoes versuehe zur chromatographischen trennung yon kohlenhydra~en und eiweil en aus dem w~ibrigen extrakt yon roggenvollkornmehl quantitative determination of the amino acid content of rumen fluid from twin steers fed soybean oil meal or urea vemnche zur chemischen differenzierung der eiwei•stoffe des weizens und roggens aromastoffe des brotes. versuch einer auswertung chemiseher gesehacksanalysen mit hilfe des schwellenwertes zur trennung yon sacchariden an kohle]celit~-s~ulen. ern~hrungs-fomchung untersuehungen zur bestimmung der lsslichkeit yon milchpulver * selection of a medium for the isolation and enumeration of enterocoeei in dairy products colorimetric determination of amino nitrogen in corn syrups stxogene und versuche zu ihrem nachweis in gefliigelflelsch. dt. lebensmittel ein beitr~g bert. die verwendung der anatysen-q~arzlampe zu friihzeitiger erkennung der l~anzigkeit colorimetrlsche methode zur bestimmung yon -monoglyceriden in eiskrem determination of fusel oil in distilled spirits zu den m glichen fehlerquellcn bei der histometrischen ermittlung des kollagen-, bzw. gelatine-gehaltes bei briihwiirsten studium der uv-spektren der auf hshere tempcraturen erhitzten e measuring of oil-binding characteristics of flour naehweis der konservierungsmittel mit hilfe cl~r papierehromatographie identification of ch]orogenic acid in castor bean and oranges. canad evaluation of forages in the laboratory. iii.: comparison of various methods for predicting silage digestibility feed microscopy essential oils. determination of botanical and geographical origin of spearmint oils by gas chromatographic and ultraviolet analysis fluorometric determination of chlortetracycline in premixes dfinnschicht-chromatographie yon carotin-und carotinoidgemischen measurement of the sub-sieve particle size distribution of flour chemisehe bestimmung der riickst~nde yon parathion, malathion und diazinon auf blumenkohl, kohlrabi, bohnen und gurken determination of cadmium anthranilate in feeds nachweis yon penicillin und anderen antibiotika in milch microbiological evaluation of protein quality with tetrahymenapyriformis w. .: a simplified assay procedure peanut lipoprotein. ii. : analysis in foods by gas chromatography beitrag zum papierchromatographisehen und spektrophotometrisehen i~achweis fettlsslieher synthetiseher farbstoffe in lebensmitteln und kosmetika elektroehemische sauerstoffbestimmung in olefinischen fetich bestimmung der gesamten sehwefligen s~ure in getr~nken the modified whiteside test. recommended procedures for bulk or blended milk deliveries die bestimmung yon mileheiweib in fleiseherzeugnisscn. dr. lebensmittel zur bestimmung des veresterungsgrades yon pektin a quantitative fluorometrie method for the determination of serpasil (reserpine) in feeds at the micro level fluorimetric mierodetermination of carbohydrates zur l~iethodik der klebrigkeitsbestimmung yon brot frage der papierchromatographischen untersuchung von amylopektin und amylose chemical determination of diethylstilbestrol residues in the tissues of treated chickens gas chromatographic identification of components in m~ple sirup fl~vor extract dark discoloration of canned all-green asparagus. ii. development of a new tin plate for its control thermal conductivity and density of chicken breast mnsele and skim beitrag zur bestimmung der fluoreszenz in spriten microbiological determination of alanine in proteins and foods collaborative study of the method for counting microorganisms in maple sirup glass fiber paper strip charring. a rapid and simple method for monitoring column chromatography of lipids verbesserte mcthode zum serienm~bigcn quantitativen nachweis yon insektizidrficksti~nden bei bst und gemfise use of the shear press in determining fibronsness of raw and canned green asparagus betrachtungen fiber verschiedene methodcn zur bestimmnng des bindegewebeanteiles in rohem fleisch und fleischwaren. arch. lebensmittel-hyg the determination of citric acid in milk and milk sera formaldehyde in maple sirup: an adaption of the nash method polarographische bestimmung dcr ascorbinsiiure und des gesamt-vitamin c untersuchungcn fiber die mbglichkeit zur objektiven beurteilung der organoleptischen eigensehaften yon kokosraspeln analysis of the flavor and aroma constituents of florida orange juices by gas chromatography assay for cyzine in finished feeds dfinnsehicht-ehromatographische trennnngen yon synthetischen lebensmitteffarbstoffen auf ceuulose-schichten evaluation of quantitative methods of determining peroxidase in vegetables. i.: the indophenol and the o-phenylenediamine methods ein beitrag zur untersuchung thermisch oxydierter fette. dt. lehensmittel a more accurate method for determination of caffeine in decaffeinated coffee bestimmung yon vitamin c in friichten, fruchtsiiften, gemiise und konserven naeh der methode naeh tillma~s unter ausschaltung reduzierender stoffe lebensmittelrecht und lebensmitteliiberwaehung nutritional laws and nutritional control briihwiirst~ einfaeher qualit~t, die zu einem kaliber unt~r mm in den verkehr gebracht werden, sind i.s. des w nr. lmg irreffihrend aufgemaeht new regulations under the food and drugs act lebcnsmittel-rdseh orb der kenntlichmaehung fremder stoffe probleme des geltenden lebensmittelrechts glasurmittel ftir r stkaffee beschr~nkt zugelassen food technol. [ ] nr. , s. . n. iv. : revised u. k. preservatives regulations. food teehnol ausschub fiir lebeusmittelrechtliche fragen der fachgruppe lebensmittelehemio und gerichtliche chemic in der geseuschaft dcutscher chemiker vi. t~tigkeitsberieht der eidg. kommission fiir volksern~hrung, lebensmittelgesetzgebung und -kontrolle (eek) zu h~nden des eidg. departementes des i_nnern, umfassend die jahre federal food, drug, and cosmetic act, as amended. selected u. s. government publ. nr. ; h. catalog i~o verkauf yon frikadellen mit brotzusatz in gastwirtsehaften tierarzneimittel und aufzuchtmittel in der landwirtschaftlichen praxis. gesundheitliche erw~gungen zum schutze des konsumenten bei der anwendung yon tierarzneimitteln und aufzuchtmit~ln in der landwirtschaftliehen praxis. teil hi der amtsarzt und das neue lebensmitt~lgesetz. off ist der beschlub des bundesgerichtshofes vom . . - stl~ -geeignet, den vertrieb yon hackfleiseh befriedigend zu regeln ? arch. lebensmittel-hyg zur beurteilung des saccharingehaltes yon meerrettiehzubereitungen. dt. lebensmittel zum begriff ,mai]gebend" in w a abs. des lebensmittelgesetzes aspect sanitaire et ldgal actuel des aliments conservds verbrauchererwaxtung und lebensmitte]kontrolle bei fleiseherzeugnissen bei ger~ueherten fleischwaren. sehwarz-w~lder speck, schwarzw~lder sehinkenspeck, sehwarzw~lder sehinken. arch. lebensmittel-ttyg kampf der wasserverseuchung. aktuelle notwendigkeiten -gesetzliehe ~/isgliehkeiten. ~iiinchener reed. wschr. lebensmittelreehtliehe stellung yon carotin und carotinoiden in der schweiz die lebensmittelgesetzgebung sterreiehs, der sehweiz und der bundesrepublik deutschland. eine vergleiehende untersuchung arzneimittel-lebensmittel dr. lebensmittel-rdseh erwiderung des autors (zur stellungnahme yon f. nitzsc~, d~. lebensmittel-rdseh. [ ] nr. , s. ). i)t. lebensmittel das lebensmittelgesetz und seine auswirkung auf die gummiindtlstrie codex alimentarius austriacus absehliel]ende stellungnahme der ort der kenntlichmachtmg fremder stoffe. dr. lebensmittel-rdsch _rreffihrende bezeiehnung und angaben bei limonade aus mineralamem tafelwasser. dr. lebensmittel zur herkunftsbezeichnung yon lebensmitteln. dt. lebensmlttel aktuelles zur lebensmitteliiberwachung einige beispiele fiir die auswirkung der neuen lebensmittelrechtliehen vorschriften auf erniihrungs-und landwirtschaft auf dem wege zum einheitliehen lebensmittel-rech~ zur ~r yon b. rsssl~: welehe anforderungen sind an alkoholhaltige siigwaren zu stellen? dr die silbertmg yon tafelwiissern mokka-kaffee" auch im handel mit kaffeebohnen keine tterkunftsbezeiehnung [ ] nr. , s. . --zuliissigkeit trod grenzen bildlicher darstellungen yon fleiseh und ylelscherzeugnissen auf paclmngen yon suppen in trockener form. db. lebensmit~el-l~lsch bedeutung und beur~eitung yon galtstreptokokken in vorzugsmileh. arch. lebensmittel-hyg auf clipverschliissen zul~ssig ist die infektion mit trichinen aus amtlich untersuehtem schwelnefleiseh im lichte der mathematischen analyse der bestimmungen der fleischbesehau yon schweinefleiseh msglich? arch. lebensmittel-hyg intema~ionales rundgespr~ch fiber lebensmitte]ehemische probleme in wiesbaden und eltville a. rh. z. lebensmittel-untersuch. u. -forschung. [ ] nr. , s. . --kommission zur priifung fremder stoffe bei lebensmitteln (fremdstoff-kommission) der deutsehen forsehungsgemeinsehaft entwicklung des lebensmittelrechts im nationalen und internationalen bereich entwicklung des lebensmittelrechts im nationalen und inter~ationalen bereich entwicklung des lebensmittelrechts im nationalen und internationalen bereich de warenwet en de hierop berustendc besluiten zum entwurf einer harmonisierung der lebensmittelrechtlichen bestimmungen fiber konservierungsstoffe im gebiet der europaischen wirtschaftsgemeinschaft. er lebensmittelrechtliche stellung yon carotinen und carotinoiden in der bundesrepublik deutschland die instrumente der lebensmittelfiberwachung in osterreich sterreichischer standpunkt zur l~rage der f~rbung yon lebensmitteln mit carotinen und carotinolden. wiss. ver ff. dr. ges. ernkhrung lst es zu vcrtreten, dab das fleisch schwacbfinniger rinder auch in gebr~tetem zustand eingefroren wird? z lrch. lebensmittel-iiyg richtlinien fiber die zulassung yon gegeusachverst~ndigen zur untersuchung yon lebensmittel-gegenproben die ]ebensmittelrechtliche bedeutung yon bildiichen darstellmlgen auf verpackungen diverse problems (education, documentation associations, terminology etc the nutritional education of the food technologist proposed formation of a food engineering panel within the food group studium der hauswirtschafts-und ern~hrungswissenschaften. ern~hrungswirt-sehaft l an information service for the american food industry st international congress of food science and technology. symposium on education and training technically trained people for developing countries lft paces the food frontier un committee on food additives international standardization of fruit and vegetables. food technol terminology and methods for feeding and weighing animals food additives and food standards beitriige zur durehffihrung der umweltsradloaktivitiits- berwaehung training dairy personnel in denmark training opportunities for the sanitarian-specialized in-service training r die benennungen honigkueheniihnlicher oebiieke als lobkuehen und lebzelten sowie als biber und bibenzelten. dt. lebensmittel nutrition and dietetics for the medical student problem of keeping dairy plants supplied with the foreman-type employee literaturdokumentation fiir hochschulassistenf~n a system for naming and describing feeds, energy terminology, and the use of such information in calculating diets a brief sketch of veterinary periodical literature in great britain before the foundation of the veterinary record a conference on nutrition teaching in medical schools the food service industry and its relation to the control of foodborne illness research and educational progress in nutrition informationsm gliehkeiten fiir tieriirzte auf dem measuring readability of health education literature the dai~y literature problem the central food technological research institute the education and function of the nutritionist training in food service for nursing homes. l: tools for evaluation training in food service for nursing homes. iii.: observations on management of twelve units pennsylvania takes a look at nutrition in the orthopedic program zur definition der begriffe ,aroma" und l~ber die notwendigkeit einer priifung for beamtete sehlaehthofleiter. arch. lebensmittel-hyg organisation der dokumentation in einem forschungsinstitut (bundes. forsehungsanstalt f'tir lebensmittelfrischhaltung in karlsruhe). dt. lebensmittel-rdsch on changing the name of our assoziation. for a name change pporbunities in nutrition education questionnaires to identify nursing homes most in need of dietary counsel. publ. health irep lebensmittelwissenschafttiche institute in bulgarien zur stellung des psychiaters in der alkoholfrage naas nutrition chemists. vcter on changing the name of our association. against a name changing begriffe) in sprachen: deutseh, russiseh, polnisch, tsehechisch, slowakisch, ungariseh, bulgariseh, serbokroatiseh, rum~nisch, englisch the role of nutrition in the teaching of medicine key: cord- -pgyzluwp authors: nan title: programmed cell death date: journal: experientia doi: . /bf sha: doc_id: cord_uid: pgyzluwp nan it is widely held that all developmental cell death is of a single type (apoptosis) and that neuronal death is primarily for adjusting the number of neurons in a population to the size of their target field through competition between equals for target-derived factors. we shall draw on our research and on that of others to criticize these views and replace them by the following. at least three types of neuronal death occur, only one of which resembles apoptosis; a neuron can choose between several self-destruct mechanisms depending on the cause of its death. the purpose of the death is to regulate connectivity, not neuron number. competitors for trophic factors are unequal, and many losers have made axonal targeting errors. a neuron's survival and differentiation depend on multiple anterograde and retrograde signals. activity affects retrograde signals and some but not all anterograde ones. the pattern of activity is more important than the overall amount. in rodents, the period of naturally occuring cell death of motoneurons is followed by a period of supersensitivity to axonal injury. thus, in newborn rodents lesion of the facial nerve leads to a rapid degeneration of the injured motoneurons. we have tested whether overexpression, in rive, of the bcl- proto-oncogene was capable of preventing death of axotomized motoneurons. to address this question we used transgenic mice whose motoneurons overexpress the bcl- protein. one of the two facial nerves of newborn mice was transected on the nd- rd post-natal day. seven days after the lesion, the morphology of the facial nuclei was analyzed. in control mice, and when compared to the intact nucleus, to % of axotomized motoneurons had disappeared. in contrast, in the transgenic animals, the number of motoneurons on the lesioned side remained unchanged when compared to the eontralateral nucleus. furthermore, their axons remained visible up to the distal lesion site. these experiments show that, in rive, motoneurons overexpressing the bcl- protein survive after axotomy, and suggest that, in rive, bcl- protect neurons from experimentally induced cell death and could be a target for treatment of motoneurons degenerative diseases. messmer s., mattenberger l., sager y., blatter-garin m-c., pometta d., kate a., james r.w. drpt de mrdeeine, drpt. de pharmacologie, div. de neurophysiologie clinique, facult de mrdecine, gen~ve. clusterin is a widely expressed glycoprotein, highly conserved across species. numerous functions have been postulated for this protein. the most important are roles in lipid transport, as elusterin is associated with apolipoprotein ai in hdl, complement regulation and tissue remodelling, in particular during cell death and differentiation. using cultures of rat spinal cord neurones ( % neurons and - % non-neuronal cells), we have studied the expression of clusterin and ape e in glutamate-induced neuronal cell death to examine potential roles in lipid management. up-regulation of the two proteins was observed. clusterin and ape e appear in the conditioned medium respectively h and . h after incubation with glutamate. control studies, in the presence of a noncompetitive nmda receptor agonist showed the secretion of clusterin and ape e to be diminished by > %. no up-regulation of either protein was observed in complementary studies with exclusively non-neuronal cell cultures. the cellular origin of the secreted proteins is presently under investigation. programmed cell death and tissue remodelling are consequences of hormonally induced restructuring of the rat ventral prostate after castration and the rat mammary gland after weaning. we used the "differential display"-method (liang and pardee, , science : ) to detect and isolate edna fragments whose corresponding rnas are regulated either coincidentally, or in an organ specific fashion during mammary gland involution and postcastrational prostate regression. partial sequencing of clones revealed high, but not absolute homology of fragments with sequences, previously characterized in different biological contexts. these five encode functions which could be anticipated to be important for cell growth and/or programmed cell death, we are presently investigating the functions of several of these transcripts in cell culture and in rive. antisense oligos are being employed in vivo to determine whether these genes contribute to the phenotype of programmed cell death. b epitopes derived from the envelope gp glycoprotein (ep ) or from the viral superantigen of mmtv have been incorporated into inert or live vaccines. the inert vaccine consists of purified chimeric proteins which contain the b epitopes alone or fused to multimeric promiscuous t helper epitopes from tetanus toxin. mice were immunized subcutaneously with these chimeric proteins. the live vaccine consists of an avirulent strain of salmonella typhimurium which expresses the mmtv epitopes in the form of chimeric proteins fused to the nucleocapsid protein of hepatitis b virus. this vaccine is given to mice in one oral dose. the level, duration and isotype of the immune response generated by each vaccine have been measured and compared. the level of protection has been investigated by systemically challenging immunized mice with the relzovims. a reduced binding of oxytocin (ot) occurs with aging in some, but not all, areas of the rat brain (arsenijevic et al., experientia , , a ) . the candate putamen showed the most impressive loss of ot receptors. two other regions, the hypothalamic ventromedial nucleus (vmh) and the islands of caueja (icj) had also an important deficit of ot binding sites. on the other hand, these two regions were known to be sensitive to sex steroids. in the present work, we treated from month old rats during one month with testosterone propionate ( #g/kg s.c., once every days) dissolved in oil. three rats of the same age injected with oil only served as controls. we labelled ot receptors throughout the brain of old rats using a i-labelled ligand specific for ot receptors. analysis of autoradiograms by an image analyzer revealed that the testosterone treatment increased ot binding sites in the vmh, in the icj, and, to a lesser extent, in the bed nucleus of the stria terminalis, a region also sensitive to sex steroids, by contrast, in the caudate putamen, the disappearance of ot receptors was not compensated. in conclusion, the decrease of ot receptors occurring in vmh and icj with aging can be reversed by administration of gonadal steroids. in contrast, the loss of ot receptors in the striatum appears to depend on another mecanism. vasopressin (avp) receptors are expressed transiently in the facial nucleus during development (tribollet et el., , dev. brain res., , - ) . avp may therefore play a role in the maturation of neuromuscular connexions in the neonate rat, and possibly in the restanration of these connexions after nerve lesion in the adult. in order to investigate the latter proposition, we have sectionned the facial nerve in adult rats and used quantitative autoradiography to look at avp binding sites in the facial nucleus at various postoperative times. we observed a massive and transient increase of avp binding sites on the operated side. the number of facial avp binding sites reaches a maximum about one week after nerve section, remains stable during - weeks, then begin to decrease towards control level. the induction of avp receptors is markedly delayed if the proximal stump of the nerve is ligated. to assess whether other motor nuclei would also react to axotomy by up-regulating the expression of avp receptors, we have sectionned the hypoglossal nerve and the sciatic nerve. in both cases, the binding of avp receptor ligand increases massively in the respective motor nuclei, with a time-course similar to that found in the facial nucleus. altogether, our data suggest that central avp could be involved in the process of nerve regeneration. cytotoxic t-cell mediated apoptosis schaerer,e, karapetian,o.,adrian,m. and tschopp,j. inst.de biochimie, univ.de lausanne, epalinges. an apoptotic cell death mechanism is used by cytolytic t cells (ctl) to lyse appropriate target cells. ctl harbor cytoplasmic storage compartments, containing the lytic protein perforin and serineproteases (granzymes), whose content is released upon target cell interaction. we show that these granules are multivesieular bodies and that degranulation releases these intragranular vesicles (igv) having granzymes, t-cell receptor and yet undefined proteins associated. isolated igvs and perforin induce dna breakdown in target cells within minutes. microscopic analysis demonstrates that igv specifically interact with target cell via the t-cell receptor and that their contents is taken up by the target cell. already min. after interaction, distinct igv proteins are found in the nucleus of the target cell.one of the molecules has been identified to be granzyme a, previously reported to be involved in apoptosis. we propose that lymphocytes transfer apoptosisinducing proteins to the nucleus of the target cells using vesicles as vehicles for delivery. cytotoxic t cells kill their targets by a mechanism involving membranolysis and dna degradation (apoptosis). recently, two sets of proteins have been proposed as dna breakdown-inducing molecules in t cells: granzyme a, b and tia-i. in this study, we cloned and further characterized the tia-i mouse homologue. aa sequence comparison with the human tia- showed an overall identity of %. devoid of a signal peptide, tia is yet localized to cytotoxic granules, probably targeted via a gly-tyr-motif. as tia-i, its mouse homolcgue contains three rnabinding domains. expression of tia during development shows a very strong signal in the brain and weaker signals in thymus, heart and other organs. during embryonic development several structures that contribute to organogenesis form transiently and are later eliminated by apoptosis. this pattern of tia expression could indicate its involvement in apoptosis. prostate involution occurs after castration in rats and is associated with the death by apoptosis of a large fraction of the epithelial cells. we have isolated several genes from a prostate involution bacteriophage lambda library using differential screening methods. among these clones, one d~monstrated an especially strong signal when used as a probe against northern blots of prostate mlhna obtained before, and at different times after castration. this gene is down-regulated after castration by -fold within days. intramuscular injection of a testosterone depot resulted in complete restoration of expression within hours. upon sequencing it became apparent that this clone has a high degree of homology to a known ndah dehydrogenase encoded in mitochondrial dna. the clone failed to hybridize to any transcripts from rat organs other than prostate. we are now in the process of isolating the htm~n hc~olog to this gene for use as a biomarker in study of benign hyperplasia and developing carcinoma. this gene is a possible indicator for testosterone-independent cell populations or of cells lacking ftl~ctional testosterone receptor. during the first three postnatal weeks the rat lung undergoes the last two developmental stages, the phase of alveolarization and the phase of microvascular maturation. the latter involves a decrease of the connective tissue mass in the alveolar septa and a merging of the two capillary layers to a single one. speculating that programmed cell death may play a role during this remodeling, we searched for the presence of apoptotie cells in rat lungs between days and . lung paraffin sections were treated with y-terminal transferase, digoxigenin-dutp, and anti-digoxigeninfluorescein-f(ab)-fragments, and the number of fluorescent nuclei was compared between sections at different days. while the number of apoptotie ceils was low until the end of the second week and at day , we observed an about eight fold increase of fluorescent nuclei towards the end of the third week. we conclude that programmed cell death is involved in the structural maturation of the lung. brunner, a., wallrapp, ch., pollack, i, twardzik, t. and schneuwly, s. lehrstuhl genetik, biozentrum universit~t w~rzburg, mutants in the giant lens (g/l) gene show a strong disturbance in ommatidial development. in the absence of any gene product, additional phetoreceptors, cone cells and pigment cells develop. opposite effects can be seen in flies in which the gene product of the giant lens gene can be ectopically expressed by heat shock. a second very typical phenotype is the disturbance of photoreceptor axon guidance. molecular analysis of gil shows that it encodes a secreted protein of aa containing three evolutionary conserved cystein-motives very similar to egf-like repeats. we propose that gil functions as a secreted signal, most likely a lateral inhibitor for the development of specific cell fates and that gil, either directly or indirectly, is involved in targeting photoreceptor axons into the brain. the decrease in cellularity during scar establishment is mediated through apoptosis desmouliere, a., redard, m., darby, i., and g. gabbiani department of pathology, cmu, rue michel server, gen~ve dudng the healing of an open wound, granulation tissue formation is characterized by replication and accumulation of fibroblastic cells, many of which acquire morphological and biochemical features of smooth muscle cells and have been named myofibroblasts (sch rch et el., histology for pathologists, t ). as the wound evolves into a scar, there is an important decrease in ceuuladty, including disappearance of myofibroblasts. the question adses as to which process is responsible for myofibroblast disappearance. during a previous investigation on the expression of (z-smooth muscle actin in myofibroblasts, we have obsewed that in late phases of wound healing, many of myofibroblasts show signs of apoptosis end suggested that this type of cell death is responsible for the disappearance of myofibroblasts (darby et al., lab. invest. : , ) . we have tested this hypothesis by means of electron microscopy and morphometry and by in situ end-labeling of fragmented dna (wijsman et al., j. histochem. cytochem. : , t ) . our results show that the number of apoptotic cells increases as the wound closes and suggest that this may be the mechanism for the disappearance of myofibroblasts as well as for the evolution of granulation tissue into a scar. (supported by the swiss national science foundation, grant n~ s - r. jaggl, a. marti and b. jehn. universit~t bern, akef, tiefenaustr. , bern at weaning the mammary gland undergoes a reductive remodelling process (involution) which is associated with the cessation of milk protein gene expression and apoptosis of milk-produclng epithelial cells. this process can be reversed by returning the pups to the mother within day. elevated nuclear protein kinase a (pka) activity was observed from one day post-lactation, paralleled by increased c-los, junb, ]und and to a lesser extent c-]un mrna levels. ap- dna binding activity was transiently induced and the ap- complex was shown to consist principally of cfos/jund. oct- dna binding activity and oct- protein were gradually lost from the gland over the first four days of involution, whereas oct- m_rna levels remained unchanged. comparing nuclear extracts from normal mammary glands with nuclear extracts from glands which had been cleared of all epithelial cells three weeks after birth revealed that pka activation, ap- induction and oct- inactivation are all dependent on the presence of the epithelial compartment. the increased fos/jtm expression and the inactivation of oct- may be consequences of the increased pka activity. when involution is reversed, both, pica activity and ap- dna binding activity (and fos andjun mrna levels) are reduced to basal levels. our data suggests a role for pka and ap- on progranlmed cell death of manlnmry epithelial ceils. bcl- ~ does not require membrane attachment for its survival activity c. borner*, i. martinout, c. mattmann*, m. irmler*, e. sch&rrer*, j.-c. martinou-j-, and j. tschopp*. * institute of biochemistry, university of lausanne, epalinges, institute of molecular biology, glaxo inc., plan los ouates. cl- (z is a mitochondrial or perinuclear-associated oncoprotein that prolongs the life span of a variety of cell types by interfering with programmed cell death. how it exerts this activity is unknown but it is believed that membrane attachment is required. to identify critical regions in bcl- o~ for subcellular localization and survival activity, we created by site-directed mutagenesis, various mutations in regions which are most conserved between the different bcl- species. we show here that membrane attachment is not required for the survival activity of bcl- o< a truncation mutant of bcl- (z lacking the last amino acids (t ) including the hydrophobic domain is soluble, yet fully active in blocking apoptosis of sympathetic neurons induced by ngf deprivation or l fibroblasts induced by tnfc~ treatment. we further provide evidence for a putative functional region in bcl- which lies in the conserved domains and upstream of the hydrophobic cooh terminal tail. the breakdown of nuclear dna is considered to be a hallmark of apoptosis. we previously identified the perinuclear membrane localized dnase i as the endonuclease involved in the formation of oligonucleosomal-sized fragments (dna ladder). it is not clear how the nuclease is activated and has access to the dna. we show that in thymocytes induced to undergo apoptosis, lamin breakdown preceded dna laddering. by transfeeting hela cells with a constitutively active cdc mutant, nuclear envelope breakdown and typical apoptotic features (ehromatin condensation) were observed. moreover, co-transfection with cdc mutant and dnase i led to dna degradation. we propose that apoptosis can be induced by wrongly timed and hence abortive mitosis leading to uncontrolled nuclear membrane disintegration. s - s - platelet-derived growth factor (pdgf) is thought to play an active role in fibrosing diseases. bronchiolitis obliterans-organizing pneumonia (boop) is a condition characterized by intraluminal proliferation of connective tissue inside distal air spaces. to evaluate pdgf expression in boop we performed immunohistoehemistry on lung biopsies from patients and controls free of fibrosis. sedal sections were stained with an antibody against either pdgf or the monoeyte/macrophage marker cd , in both groups the pdgf ~ cells were essentially tissue macrophages. using point counting to measure volume fraction (vv) , pdgf-pesitive cells represented . + . % (mean+sd) of the volume occupied by lung tissue in the boop cases, and , + . % in the controls (! < , ). similarily, . + . % of the lung tissue was occupied by cd e~ macrophages in the boop cases, compared to . :~ . % in the controls (p<o. ). a positive linear correlation was found between the v v of pdgf ~ cells and the v v of cd @ cells on the cases (r=g. p< . o ). we conclude that boop is characterized by a recruitment of tissue macrophages and that a significant proportion of them express pdgf. as pogf can also induce the macrophage chemo-attractant mcp- , we speculate that maerophages are involved in a positive feed back regulation, and may play a pivotal role in the pathogenesis of boop. barrier lesions in hydrostatic pulmonary edema d.wu, h.bachofen, u.gyger and e.r. weibel department of anatomy, university of bern, ch , bern pulmonary edema induced in isolated perfused rabbit lungs by moderate elevation of perfusion pressure ( tort ) results in the formation of characteristic bleb-like extensions of the thin cytoplasmic leaflet of alveolar epithelium ( bachofen et al. am rev resp dis. vo . . pp - , ) . in this study, we show by means of electron microscopy and morphometry that the frequency of the blebs correlate with the distribution of alveolar edema fluid. we find that % of the epithelial surface is involved in the formation of blebs. three-dimensional reconstruction showed that blebs had near-spherical shape and that their opening toward the interstitium is irregular. these finding demonstrate a great plasticity of epithelial cell extensions when subjected to moderate pressure stress. at high pressure the cell linings demonstrate clear breaks. two major roles have been defined for no: cellcell communication mediated by the stimulation of cgmp synthesis, and cytotoxicity by direct interaction of the free radical no with cellular target. to investigate these effects in human epithelia, which constitute a major source of no in man, we introduced the murine inos sequence into sv -t immortalized human bronchial epithelial cells (beas- b). inos activity was higher than pmol citrulline/min/ mg prot in the first passages of inos transfected cells, but it decreased by subculturing. no inos activity was detected in cells transfected with control vector. in inos transfected cells, no stimulated a cgmp synthesis and c-los expression which could be inhibited by addition of lnma. thus, in human epithelial cells no is able to significantly alter signal transduction pathway. partial pneumoneetomy, i.e. the resection of lung tissue, is known to initiate compensatory growth in the remaining lobes. under appropriate conditions this results in a complete restoration of lung parenchymal structure and function. unfortunately the molecular mechanisms controlling the onset of this compensatory growth are poorly understood. the aim of our study was to find and eharacterise genes which are up or down regulated during this time. for isolating such genes, we chose the differential display approach (p.liang and b.pardee , science :p ). therein a 'end fragment of a subset of reverse transcribed mrnas is amplified by the polymerase chain reaction. the fragments can be separated on a dna sequencing gel. genes differentially expressed in pneumonectomised and sham-treated rats show different patterns on these gels. the fragments of such genes can be isolated from the gel and cloned into vectors in order to provide tools to characterise their gene. a set of such genes, some up-regulated and others downregulated following pneumonectomy, have been isolated, partially sequenced and characterised. time periods. our new plasma marker consisting of highly concentrated gold nanospheres was infused via a femoral vein catheter into the right atrium of anaesthetised and ventilated rabbits. five or seconds after start of infusion, the blood flow was stopped and the lung was fixed by instillation. silver enhanced paraffin sections revealed labeled and unlabeled blood vessels. electron-microscopic sections showed various plasma gold particle concentration. morphometric analysis of electron micrographs showed an increase of the portion of marked capillaries from shorter to longer circulation times. based on our results, we conclude the existance of inhomogeneous plasma flow velocities within the pulmonary microvascular bed. (supported by snsf grant - . ) in schizosaccharomyces pombe pairing of meiotic chromosomes is not accompanied by the formation of a tripartite synaptonemal complex (sc), a structure which connects the homologs in most other eukaryotes. meiotic nuclei revealed novel structures ("linear elements") that might be related to the axial elements ($c precursors) of other organisms and function in meiotic chromosome pairing, recombination, and segregation (baler et al. ( ) jcb : ). to get further insight into the behavlour of meiotic chromosomes, we performed fluorescence in situ hybridizations on spread nuclei with probes from different chromosomal regions. interestingly, all centromeres and telomeres become clustered during meiotic prophase. the centl:omeres are paired already before meiosis, whereas the telomeres initiate pairing specifically during meiotic prophase, followed by pairing of interstitial regions. painting of whole chromosomes revealed that the homelogs occupy together a specific territory in the nucleus. an expression vector containing the vai gene read by rna polymerase ill, injected into xenopus zygotes, yields high levels of rna in virtually all embryonic cells. anti sense fina is produced from a segment of the xhoxla gene inserted in opposite orientation into the vai gene. immunological staining of whole mount embryos shows that target mrna level and xhoxla protein expression of xhoxla protein is diminished in about half of the antisense treated individuals. accordingly, nearly half of the embryos develop typical malformations in regions where the xitoxla gene is normally expressed. somites el the anterior trunk are heavily disorganized and mesodermal denvalives surrounding the intestinal tract are reduced or missing. antisense inhibition, through production of rna in situ from expression vectors, thus represents a useful tool to study the functional role of zygotic genes in xenopus development. cloning and characterization of the mouse homolog to s.cerevisiae sep encoding a dna strand exchange and homologous pairing protein vladimir bashkirov and wolf-dietrich heyer. institute of general microbiology, university of bern, baltzer-strasse , bern. homologous pairing and dna strand exchange are the central steps postulated in the current models of genetic recombination. to date no mammalian gene encoding a homologous pairing protein has been cloned. to get insight into the mechanism of recombination in higher eukaryotes we were interested to clone such genes. protein sequence comparison of the s.cerevisiae homologous pairing protein, sepi, and its homolog from s.pombe, p ex~ revealed several conserved stretches of amino acids in their nh -terminal regions. using fully degenerate primers and mouse testes edna as a template for the polymerase-chain-reaction a product with the expected length has been amplified. this bp dna was shown to encode a putative polypeptide with high degree of homology to both proteins from the two yeasts. the mouse pcr-product was used as a hybridization probe for screening a gtlo mouse testis cdna library. after several rounds of successive screening a total of about kb of mouse edna (msep ) was cloned and sequenced. the putative translation product revealed high homology throughout the entire sequence to its s.pombe and s.cerevisiae counterparts, % and % of amino acid identity, respectively. the msep sequence was shown to be unique in the genome and the chromosomal gone is likely to contain many introns. we want to establish a functional full length edna clone of msepi and characterize the gone and protein product. we have earlier developed a cell free system to study recombinational repair of dna double strand breaks and deletions. the method allowed us to purify and characterize a high molecular weight multiprotein complex (rc- ), which catalyzes dna recombination. a dna polymerase and dna ligase as well as other enzymatic activities copurify with rc- .we have commenced an investigation of homologous dna recombination in nuclear extracts which were prepared fzom normal and mutated mouse thymocytes. the scid mutation in mice causes an aberrant v(d)j joining reaction, an elevated sensitivity to ionizing radiation and a defect in recombinational repair. moreover, the normal mouse thymocytes were sorted into the cd /cd double negative (dn), double positive (dp), and single positive (sp) subclasses and their nuclear extracts, and extracts from rag- -/-mice, were tested. only the scid mice and the cd /cd sp thymocyte extracts were inactive in the recombinational repair reaction. this indicates a development stage specific activity and a scid specific defect of recombinational repair in thymocytes. restoration of the recombination activity was observed in thymocyte extracts derived from scid, which express a wansgene for the t-cell receptor i~ chain. a protein could be purified from normal thymus cells to near homogeneity which specifically rescues the recombination activity in scid extracts. this protein, srsp ~cid recombination stimulatory ~otein), migrates as a single band of app. kda in sds polyacrylamide gel electrophoresis. the ade -m mutation in the ade gene of the fission yeast schizosaecharomyces pombe gives rise to a hot spot for meiotic homologous recombination. to address whether transcription plays a role in m activity, the effect of the deletion of the ade promoter in combination with either the m and m mutations on recombination frequency was studied at the tetrad level. deletion in eis of the promoter abolishes m hot spot activity relative to the m control deletion strain. it is possible that deletion of the promoter has eliminated a sequence necessary for m hot spot activity, rather than lack of transcription being responsible for the loss of m activity. the role of transcription in m hot spot activity is currently being examined. we are analysing meiosis in a temperature sensitive top mutant. complementary temperature-shift experiments showed that topoisomerase ii is required for both meiotic divisions; early meiotic events are not impaired. in a meiotic time course, dapi staining revealed that the cells are blocked before the first meiotic division. interestingly, in meiotic spreads we found that the spindle pole body (spb) cycle is not affected by the block: the final arrest point showed nucleus with completely separated spbs. we want to analyse the number of spbs and spindle formation with immunofluorescence. we are planning to analyse the course of meiosis in a top rec double mutant (rec is recombination deficient) to see if topoisomerase ii activity is necessary for separation of recombined chromosomes. analysis of meiosis in a top rec double mutant (rec is recombination deficient and does not form linear elements) could give us a clue about the function of the linear elements. genetic recombination is able to induce cancer and to mediate its further progression. in cells from cancer predisposed individuals, mitotic recombination can lead to loss of heterozygous tumor suppressor genes as is e.g. observed in the hereditary form of retinoblastoma. one mechanism for gene loss is inter-or intrachromosomal recombination involving short duplicated sequences. our aim is to identify xenobiotics that are able to induce mitotic recombination and to elucidate the involved molecular mechanisms. for that purpose we have constructed transgenic d. melanogaster ( ). rn.). cell lines. cells were stably transfeeted with an extrachromosomal dna element providing a putative recombination substrate+ "l%e constructed element contained two ta'uncated fragments of the neomycin resistance gene (nee r) interrupted by a hygremycin resistance marker (hygr). the hyg r insert was flanked on both sides by identical bp fragments of nee r, different d. m. promoters shown to be constitutively active in various d. m. tissues were inserted ' to both e. coil genes. restoration of a functional nee r gone may occur by deletion of the hyg r after recombination between the homologous regions. stably transfected schneider line cells were obtained and preliminary results concerning chemical induction of genetic rearrangements will be shown. fleck, ., sch~r, p. and kohli, j., institute of general microbiology, baltzerstr. , bern to detect mismatch-binding proteins of s. pombe we performed band shift assays with radiolabeled oligonucleotides containing a defined mismatch. we found two distinct mismatch-binding activities. one shows high affinity to most of the mismatches, but is absent for c/c. this protein might belong to the general mismatch-repair system, which is thought to be related to the bacterial muts dependent pathway. the second activity preferentially binds to those mismatches containing a cytosine and therefore represents a novel type of mismatch-binding protein. baur m., sch~r p. and kohli j., institute of general microbiology, baltzerstrasse , ch- bern homologs of the salmonella typhimurium (mutl, s and and streptococcus pneumoniae (hexa, b) genes involved in mismatch repair have been identified in several distantly related organisms. so far no mismatch repair gone is known in schizosaccharomyces pombe. in order to get more insight into the ~chanisals of mismatch repair in s. pombe we started the cloning of a mutl homolog. degenerated oligonucleotide primers based on conserved regions of mutl homologs were used in the polymerase chain reaction (fcr) to amplify a corresponding dna fragment from s. pombe. a dna fragment of about bp was amplified whose deduced amino acid sequence shared a high degree of homology with paiql of saccharomyces cerevisiae and mutl, hexb. with the help of this dna fragment the gone was isolated from a genomic dna library by colony hybridization. sequence analysis of this mlhl gene (mlh = mut~ homolog) shows an orf of the expected size with three putative introns. while the deduced amino acid sequence of the ' region shares strong homology with the procaryotic genes mutl, hexb and the eucaryotic gene pmsi, the amino acid sequenoe of the ' region shows homology only to pmsi and hexb. the central region of the protein seems to be conserved only weakly. parisi s., and kohli j., institute of general microbiology, university of bern, ch- bern rec and rec are supposed to be genes with major functions in meiotic recombination since mutations in these genes reduce the frequency of meiotic intragenic recembination ~ fold. these genes have been isolated and sequenced but no enzymological activity could be assigned to the protein. a first step toward enzymological and biochemical characterization of rec and rec is to make beth protein products available in sufficient quantities and in reasonable pure form. for this purpose both genes will be cloned in a s. /x~nbe over-expression system using the strong and inducible nmtl promoter.in parallel both genes will also be expressed in e. coli as gst fusion proteins in order to produce polyclonal antibodies in rats and rabbits. these antibodies can then be used for protein purification in s. pombe as well as for immunofluorescence experiments. induction of expression in e. coli produces a band of the expected size of kda for rec and of kda for rec . to remove the gst carrier, the rec and rec fusion proteins can successfully be cleaved by thrombin or factor xa respectively. production of antibodies is in progress. d bbeling, u., nobi, r,, berchtold, m.w. and kuenzle, c.c. institut fdr veterinarbiochemie, universit t zurich, wintertharerstrasse , normally, only pre b-and pre t-cells can rearrange their immunoglobulin genes by v(d)j recombination, but when other cell types are transfected with the rag-i and rag- genes they also become able to perform v(d)j recombination. by transiently transfeeting nih t fibroblasts with both intact rag genes we found da recombination rate of . %. no recombination was detected, when we transfected the intact rag- gene with a mutant rag- gene, which lacks c-terminal amino acids, or the intact rag- gene with a rag-i gene containing a deletion in the topoisomerase iz homology region. these experiments show that these two regions of the rag genes are essential for v(d)j recombination. when the l fibroblast cell line, which has been rendered recombination competent by stable transfection with the rag genes was overtransfected with both wild type rag genes, we found in contrary to an expected increase of v(d)j recombination a reduction by a factor of - . . this decrease was not observed with the mutant rag genes.this result indicates, that there exists an optimal intracellular rag concentration for efficient v(d)j recombination. m. fischer, a. sailer, h. blieler, t. riilicke*, a. aguzzi +, p. autenried and c. weissmann; nstitut fiir molekularbiologie l universitfit ziirich, h nggerberg, ziirich, *biologisches zentrallabor and + lnstitut fiir neuropathologie , universitdtsspital ziirich, zfirich, switzerland the infectious agent of transmissible spongiform encephalopathies such as creutzfeldt-jakob disease in man or scruple in sheep is thought to be prpsc, a posttranslationally modified form of the normal host protein prpc we have shown that mice devoid of prpc (prn-polo) are completely resistant to scrapie. here we report that also heterozygous prn-p o/+ mice with reduced levels of prp c show enhanced resistance to scrapie, as manifested by a long delay in the onset and slow progression of clinical disease. conversely, prn-po/o animals overexpressing prp transgeues exhibit shortened incubation times and accelerated progression of the disease. propagation of the scruple agent is completely abolished in prnpolo animals. high titers of infectivity, about logld units/g of brain, are detected in prn-po/+ mice weeks after inoculation (the earliest time point measured), weeks or more before the animals die of scrapie. in contrast, wildtype animals survive no more than weeks after reaching similar titers of infectivity. a practical implication of our findings is that moderate inhibition of prpc synthesis could mitigate disease progression in spongiform encephalopathies. the ruvb protein has been shown, biochemically and genetically, to be involved in the process of homologous recombination in e. coli. one of its functions is to promote branch migration within holliday junctions formed during the process of recombination. to investigate the mechanism by which ruvb promotes branch migration we have used conventional electronmicroscopy, cryo-electron microscopy, scanning transmission electronmicroscopy, and image analysis to study the interaction of ruvb protein with dna. we observed that ruvb forms multimeric rings on dsdna which encircle the dna. we also observed that such rings tend to localize at the holliday junctions. we propose that ruvb rings can actively move along dna using the energy of atp hydrolysis.this movement continues upon encountering a holliday junction, thus forcing branch migration along the dna. gschwind m. and huber g., pharma division, preclinical research, in some families with early onset aizheimer's disease mutations on the -app gene have been identified which cosegregate with the disease. so far all these pathogenic mutations have been identified on exons and in humans framing the sequence of the -amyloid itself.a genomic clone was isolated from mouse strain c black/ containing the coding regions of the last three exons ( ) ( ) ( ) according to the human sequence. sequencing of all three exons including the large ' non coding region showed a % homology to the previously described balb/c mouse cdna. exon-intron boundaries were determined at the same positions as in human and rabbit genome and the flanking regions in the introns are also very similar, not only is the homology between the human and mouse f$-app very high ( . %) but also the genomic organisation of their genes is nearly identical. therefore homologous recombination can be applied to introduce molecular -app changes and to study their functional consequences on the protein level. we systematically investigated the ability of reca protein to push the dna strand exchange reaction through heterologous regions. we observed that reca can push the strand exchange through substantial heteroiogy (up to bp) when the heterologous insert was placed on the so called proximal end of the linear duplex. in the case of the medial beterology the strand exchange reaction could overcome heterologies of up to about bp. heterology at the distal end was most difficult for reca to resolve, and already bp long insert was completely blocking the progress of the strand exchange. these results, together with earlier electron microscopy studies, allow to propose a molecular mechanism by which reca can exchange strands between partially heterohigous dna molecules. institut for veterin~r-virologie, universit~t bern, ch- bern bovine viral diarrhae virus is a positive-stranded rna virus of the pestivirus group. two biotypes, cytopathic and non-cytopathic in cell culture, can be distinguished. all cytopathic strains characterized so far carry insertions in their genomes. they probably result from a strand-switching activity of the viral rna polymerase during viral replication. to study the switching capacity of the viral enzyme, rna isolated from cells infected with two different strains of bovine viral diarrhea virus were analysed for homologous recombination in an rt-pcr assay. performing the assay we found that fragmented rna present during reverse transcription, the reverse transcriptase enzyme itself and viral rna present in the pcr reaction result in the production of artificial recombination during the assay. it was not possible to detect a homologous recombination activity of the viral polymerase above the background recombination frequency of the rt-pcr assay. our results strongly question the use of the rt-pcr assay for the study of recombination of rna viruses. transpositional rearrangements mediated by insertion sequence (is) elements represent an important source of genetic variation within populations . we analyze dna rearrangements at the level of the complete genome by rflp, using is elements as probes for hybridisation to southern blots samples taken at different time points from independent cuttures of e coli b grown by serial transfer for ' generations and stored at - ~ are used to study structure and evolution of genetic diversity within populations. the seven is elements contribute differently to genetic variation and lead to a succession of mutants affecting the genetic structure of the population. we now search for correlations between rflp patterns and the increase of relative fitness over time observed previously naas t., o~ m, fitch w.m and arber w, ( ) the complete dna sequence of the locusta migratoria mitochondrial genome has been derived from four cloned mtdna fragments. the sequence is . kb in length, contains peptide coding sequences, and genes for trnas and two rrnas. the organisation of the genome shows a strong resemblance to drosophila, the gene order and orientation being the same except for the positions two trnas. this contrasts with the only other non-dipteran insect mtdna to have been sequenced, apls mellifera, which shows differences in the positions of ii trnas. this finding is discussed in relation to the at content of the different genomes. the sequences of the three mtdna genomes are also compared directly and the results related to existing knowledge concerning the evolution of insects. udem, new jersey medical school, newark, nj - , usa nonsegmented negative strand rna viruses are so far not amenable to reverse genetics. the genomes must associate with nucleocapsid (n), phosphoprotein and polymerase proteins to form a functional rnp. as a step towards reverse genetics of measles virus (mv), plasmid vectors were constructed allowing synthesis of rnas corresponding precisely to a mv genome in which all coding and intercistronic regions are replaced by the chloram-phenicol acetyl transferase (cat) coding region, transfection of these negative polarity transcripts into mv infected hela or cell lines gave rise to cat activity which could be serially transferred and massively amplified together with progeny helper virus in cell cultures. both expected mv/cat chimeric mrna and replication product were identified in the progeny. preliminary experiments indicate that only rnas in which the total number of nucleotides is a multiple of six replicate and transcribe efficiently, consistent with the data on natural and modified copyback di rna of sendal virus (calain and roux, j. virol. ( ) ). precise end to end encapsidation of rna, each n protein contacting six nucleotides, might be required. the embryonic tissue specificity of the human cytomegalovirus immediate-early (hcmv-ie) promoter/enhancer (- to + ) was examined by l~-galactosidase staining of transgenic mouse embryos carrying hcmv-ie regulated lacz genes. embryos of three transgemc lines were analyzed between . - . dpc. for all lines, hcmv-ie transcription was primarily confined to subsets of neural and vascular cells. however, extents of transgene expression along the embryonic primary axis were slightly and greatly restricted in two of the lines relative to the third line at all stages analyzed. these differences most likely represent integration site-dependent chromatin constraints that affect levels of hcmv-ie-directed lxanscripfion. together, the three lines can be taken to define a graded potential for hcmv-ie transcription along the anteriorposterior axis of the embryo with greatest permissivity in mid-axial structures which include the dorsal neural tube at the level of the hindbraln and upper cervical spinal cord, the inner ear and vestibular acoustic ganglia, branchial arteries, aortic sac, and lung buds. the cell typespecificity and axially graded permissivity for hcmv-ie transcription in the mouse embryo provide a basis for understanding the pathology and relative frequencies of different types of birth defects caused by congenital hcmv infection in humans. stauffer, y., fford, e. and beard, p., swiss institute for experimental cancer research (isrec), ch-i epalinges, switzerland. hpvi is associated with genital carcinoma. like other human papillomaviruses, hpvi cannot be easily propagated in cell culture, because the viral growth cycle is dependent on the differentiation of its host cell, the keratinocyte. moreover, very little viral capsid protein is found in vivo in infected tissues. the hpvi l and l genes code for the major and minor capsid proteins, respectively. to obtain the viral capsid proteins we made constructs with the l and l genes for expression in e. coli (the pqe system) and in recombinant vaccinie virus (a modification of the phgs-i system) infected cells. the l and l proteins expressed in e. cell contained a histidine tag encoded by the vector. this allowed us to purify the proteins on a sickelaffinity column in order to raise antibodies. these will permit us to detect the presence of capsid proteins expressed from recombinant vaccinia viruses. we expect the l and l proteins expressed together from the vaccinia-based vector to selfassemble to form virus-like particles. to papain-like proteinases. the role of these putative catalytic residues in the activity of the proteinase was studied by site-directed mutagenesis. a minimal proteinase has also been constructed by n-and c-terminal deletion to determine the boundaries of the proteolytic domain, sequence analysis of the c-terminal cleavage product should indicate the site of cleavage. cell entry, uncoat~ng and nuclear transport of adenovirus z . u. f. greber and a. helenius. yale university school of medicine, new haven, ct, u.s.a. to enter cells, viruses must achieve three major goals: transfer their genome across the plasma membrane into the cytosol, target the genome to the replication site and decondense it for replication. adenovirus, an icosahedral nonenveloped particle with a double stranded genomic dna and ii to structural proteins~ enters into human kb cells by a sequential disassembly program during receptor-mediated endocytosi~, acid-dependent penetration into the cytosol, and targeting to the nucleus. during internalization, the fibers, primarily responsible for virus binding to the cell surface, are detached. in early endosomes, the capsid-stabilizing proteins ilia and viii are released. fenton base, a vertexassociated protein implicated in penetration across the endosomal membrane, is removed in endosomes, if penetration is blocked with lysosomotroplc reagents. in the absence of inhibitors, a large fraction of penton base stays associated with the cytoplasmic capsid. the viral dna is disconnected from the shell after proteolysis of the internal protein vi~ a capsid and dna binding component. removal of protein ix, a capsid binding factor, further destabilizes the coat, and the remaining 'stripped-down' virus particles associate with nuclear pore complexes to release their dna-genomes into the nucleoplasm. this stepwise dissociation program is thought to provide the high efficiencies of the entry process needed to successfully deliver the viral dna across multiple barriers to the cell nucleus, the site of replication. ch. schmidt and w. arber; biozentrum der universit~t basel, abt. mikrobiologie, klingelbergstr. , ch- basel bacteriophage p , carried as a single copy plasrnid in e. col~, is widely known for its horizontal gene transfer ability (transduction) in enterobscteria. as a temperate phage, it can lysogenize its host and thereby provides it with accessory functions (restriction of foreign dna by ecop , functions expressed by transposons carried on the p genome or integrative suppression). these properties reflect both symbiotic and evolutionary functions of pi. p regulates expression from its genes by two different mechanisms: trans(:ription of early regulatory genes is repressed by the phage encoded repressors c and bof, whereas transcription of its morphogenetic genes from p specific late promoters is induced by the early expressed gpl . by site-directed mutagenesis the late promoter ps, composed of the - inverted repeat aagttactt and the - box, was functionally characterized. p and its derivatives compare well with several other late promoters of pt with regard to their sequence dependent strengths: the strong promoters ps, pdar and lpr b all share the perfect inverted repeat around position - , whereas the weaker promoters lpr , lpr a, lpr b and lpr contain mismatches in their inverted repeats or the inverted repeat is positioned mere upstream relative to the transcription initiation site. homologies. however, the phages formed two immunity groups. the results may suggest a common genetic trait providing a selective advantage to lysogenic aa. by using a dna substrate with defined gap size we found that human immunodeficiency virus type reverse transcriptase (hiv~rt) was able to perform strand displacement dna synthesis. this activity was not affected first by calf thymus proliferating cell nuclear antigen and replication factor c and second by escherichia coli single-stranded dna binding protein~ which together allow dna polymerase ~ to perform strand displacement dna syrlthesis (podust , v. and h~bscher, u. ( ) nucl. acids res. , . '-azido- ', '-dideoxy-thymidine tripbosphate inhibited displacement completely indicating that dna synthesis is required for this reaction. the hiv-rt p polypeptide alone could perform limited strand displacement dna synthesis, whereas the hiv-rt p polydeptide was completely inactive, likely due to its inability to replicate extensively on a mi dna template, on the other hand the hiv-rt psl polypeptide e~hanced the strand displacement activity of the hiv-rt d subunit at a molar ratio of : mainly by chasing short products into longer ones. furthermore kinetic experiments after complementation of hiv=rt p with kiv-rt psl indicated that hiv-rt psl can restore rate and extent of strand displacement activity by hiv-rt p compared to the hiv-rt heterodimer d /d , suggesting a function of the kda polypeptide, the mouse mammary tumor virus proviral dna contains an open reading frame in the ' long terminal repeat which can code for a kda polypeptide with a putative transmembrane sequence and five n-linked glycosylation sites. this gene is known to code for a superantigen which deletes a specific subset el co + t-lymphocytes in vivo, the superantigen encoded by the exogenous mouse mammary tumor virus of the gr strain acts specifically on v[ bearing t-cells. we produced recombinant vaccinia viruses to express either the complete or a truncated orf protein after infection of primate cells in culture. the complete err gene in mammalian cells leads to the production of a kda protein which is specifically detected with an anti-orf-peptide antiserum. the kda protein can be labeled with d-[ - h]mannose and its synthesis is inhibited by tunicamycin, an n-linked glycosylation inhibitor, indicating that it is a glycoprotein. the truncated orf protein beginning at the second atg of the open reading frame is also modified, but the c-terminal half of orf, starting at the fifth atg (orf ), has the expected size of the non modified polypeptide. pulse-chase experiments indicate that the kda orf g}ycoprotein has a short half-life of about . - hours in this system whereas the orf truncated protein is even less stable; its half-life is about minutes. the cellular localization of the orf protein and the role it could play in vivo are currently under investigation. conclusion was based on a restriction fragment length polymorphism (rflp) with probes of is-elements. the mutants displayed different growth behavior. the evolutionary stability of of these mutants was further tested by competing a mixture of them for ten days. polymorphism was maintained during this competition but two fitter mutants took over % of the population. implications of these results will be discussed in terms of generation and selection of genetic variants for the adaptation of microbial population. mouse mammary tumor virus (mmtv) is produced in the mammary gland of infected females and transmitted by the milk to suckling new-boms. the passage of mmtv from the gut to the mammary gland is still poorly understood, the nature of the tissue tropism of mmtv has not been elucidated, a single receptor present on mammary epithelial and lymphoid cells might serve for the entry of the virus. a cell-type specific entry mechanism could in part be responsible for this phenomenon. alternatively different surlace molecules might be involved in its uptake. we have studied the cell susceptibility to mmtv using a sensitive method of detection of newly acquired exogenous proviral dna sequences, prior to viral integration and transcription. thereby we confirm that mmtv infects mouse, rat and monkey cell lines of different tissue origin in vitro demonstrating the lack of a strict host range specificity in tissue culture as previously described. we have also observed the infection by mmtv of various human cell lines. furthermore various infection susceptibilities are observed for cell lines of a same species. r. cattaneo, p. spielhofer, k. kaelin, m.a. billeter mo/eku/arbio/ogie /, universit~t zorich, hsnggerberg, zorich subacute sclerosing panencephalitis (sspe), a rare, lethal disease is caused by clonally selected defective measles viruses (dmvs) characteristically mutated. this emerged from sequencing of five mv genes from sspe cases and by functional tests of the envelope glycoproteins hemagglutinin (h) and fusion protein if). these expressed h and f variants migrate poorly to the cell surface but maintain some cell fusion activity whereas the envelope associated matrix protein (m) appears dispensable. thus, the spread of dmv genomes through the brain seems to occur by membrane fusion at local cellular contacts, essentially hidden from the massive immune responses. in more than half of sspe cases, the propagated dmvs contain m genes in which up to % of u residues are replaced by cs. such "biased hypermutations" are likely due to the simultaneous conversion of many a residues to inosine in double stranded rna regions accidentally formed, mediated by an ubiquitously occurring adenosine deaminase. similar events at less spectacular degrees have now been observed in functional genes of several negative strand rna viruses. thus, this enzyme might be an important evolutionary driving force for mononegavirates. pull, i., wieland, s., nieder~st, b., keist, r., walter, e., and blum, h.e. department of medicine, university hospital z'tirich infection by hbv-positive organ donors remains a serious problem in transplant surgery. here we present a case of a -year-old woman who received a heart transplant from a hepatitis b surface antigen (hbsag) positive donor. the patient was negative for all hbv markers prior to transplantation. two months after transplantation she became hbsag positive and died from subacute liver failure about seven months later. cloning and sequencing of hbv dna revealed several hbv mutants in the serum of the recipient, each with a bp in-frame deletion in the pre-s region. transfection studies in huh- cells with the predominant pre-s deletion mutant showed a higher replication competence compared to wild-type hbv. sequence analysis of pcr products from serum of the donor showed mainly wild-type hbv dna in the pre-s region without the pre-s deletion. these data demonstrate that mutant viruses accumulate in immunesuppressed patients. specific mutants may play a critical role in the severe or fatal clinical course of hbv-infection in transplanted patients. in non-neuronal cells herpes simplex virus (hsv) establishes a productive lyric infection starting with transcription of its immediate early (ie) genes by corecruitment of a viral protein (vpi ) and a cellular factor (oct-l) onto characteristic oetamer-like sequence motifs ftaatgarat) in the promotors of the ie genes. in neurons, however, hsv establishes latent infections whereby transcription of the ie genes is prevented and the lyric program aborted. our efforts are focused towards determining which neuronal factors are involved in the regulation of hsv ie gene transcription and whether they may be involved in hsv latency or reactivation in neurons. in electrophoretic mobility shift assays (emsa) using mouse brain nuclear extracts we found that neuronal oct factors (noct- , noet- and noct- ) bind to taatgarat sequences from the viral icp and icp promoters. an additional brain protein also binds to these sequences but not to a consensus octamer sequence (atgcaaat) as do neuronal oct factors. emsa assays with mutated taatgarat probes show that the taat sequence is crucial for this binding and we have tentatively named this brain protein taat- . we are currently testing the noct factors in in vivo and in vitro transcription assays to study their involvement in hsv ie promoter regulation. biochemical and functional analysis of a vaccinia virus recombinant containing two copies of the p k gene. schmutz, c., and wittek, r. institut de biologie animate, univei-sit~ de lausanne, ch- lausanne. the most abundant protein of the vaccinia virus envelope is the palmitated k protein. this protein is required for envelopment and subsequent release of virus from infected cells. the amount of extracellular enveloped virus (eev) produced varies considerably depending on the virus strain and host cell line but remains in any case low (< % of the virus production). if p k is a limiting factor for envelopment, its overexpression might be a taeans to increase the production of eev. for this purpose a vaccinia virus recombinant containing a second copy of the p k gene was enginee,'ed. western blot analysis clearly showed increased levels of this protein. titration assays revealed slightly lower yields of both intracellular naked virus (inv) and extracellular enveloped virus (eeu in ceus infected with recombinant virus. unexpectedly, with recombinant virus, the ratio of eev versus inv was significantly lower when compared to wild-type virus infection, despite the higher level of p k expression. we are currently testing whether this is a consequence of a lower production, or a reduced infectivity of eev particles in cells infected with recombinant virus. hanspeter stalder, christian hertig and ernst peterhans institut f~r veterin~r-virologie, universit-st bern, ch- bern bovine viral diarrhoea virus (bvdv) causes acute transient infection in animals exposed to virus postnataly and persistent infection in animals infected in utero in the early phase of gestation. the latter is associated with specific immunotolerance to the infecting viral strain. animals infected in utero may be born normal and remain healthy despite the persisting infection with a noncytopathic biotype of bvdv. mutation to the cytopathic biotype, or superinfection with an antigenically similar cytopathic biotype, results in lethal mucosal disease. we have pcr amplified and sequenced parts of the region coding for the surface glycoprotein e and p , a nonstructural protein associated with enzymatic activities. over a time of one year, virus obtained from an immunotolerant persistently infected animal remained identical in its nucleotide sequence in the analyzed regions. contrasting with this remarkable evolutionary stasis over some - virus generations is the heterogeneity of viral isolates obtained from different persistently infected animals. we propose that the muitilineage distribution of bvdv is the result of antigenic drift in the population of acutely infected animals, combined with evolutionary stasis in immunotolerant animals. in to % of naturally infected goats, the sevedty of disease in infected animals correlates with the antibody titers to the viral envelope protein (enw) and especially to the gp transmembrane podion (tm). in order to analyze linear b epitopes of env, we produced a random library of caev env polypeptides fused to ~-galactosidase and expressed in e. coli. the complete env gene obtained from an infectious clone of caev-co was subcloned in a puc plasmid and dnase digested, random fragments of bp were inseded in ~,gtl dna and expressed in e. coil y . as the first step of b epitope mapping and in order to identify immunodominant group epitopes we used high titer sera from naturally infected swiss goats to screen the library. six immunoreactive clones were obtained and subsequently sequenced. all six mapped to the tm region of env. a fine mapping of these immunoreactive regions was performed using pepscan technology and elisa with synthetic peptides. four epitopes could be identified including the eysteine loop corresponding to an immunodominant epitope in other lentiviruses. these peptides were used in vitro to test the reactivity of sera from naturally and experimentally caev-infected goats and in rive to modulate the immune response of goats to tm. the minute virus of mice (mvm) has a kb linear single stranded dna genome. at both termini, short selfcomplementary sequences are folded into stable hairpin structures. the ' (right) hairpin is a single stem structure with only three unpaired bases within the stem. these unpaired form a bubble structure. we examined the requirement in mvm lytic infection for the bubble structure in the ' terminal hairpin of mvmp. mvmp rf dna containing the entire ' extremity and extending to the hha i site downstream of the axis of symmetry of the ' extremity was cloned into pgem-szf(+). this construct contains more than half of the ' palindrome, however it lacks the sequence information required to form a bubble. murine fibroblast a cells were transfected with this dna and the ' terminal sequence of resulting virus was analysed. a bubble was seen to be generated, however the sequence of nucleotides contributing to the bubble was of n n-viral, plasmid origin. the nucleotides in question were removed from the original plasmid construct and the experiment repeated. virus was obtained as before. sequence analysis of the ' end indicated that both flip and flop forms were present. the corresponding ' hairpin deduced from these two forms contains no unpaired bases at the position where the bubble is normally located in wild type mvmp. the immediate early lie) gene promoters of herpes simplex virus type- share a similar sequence which in some cases overlaps with a binding site for octamer binding proteins. this sequence confers responsiveness to a viral transaetivator, vp . on these promoters, vp combines with multiple cellular proteins to form an activating complex. we set up a reconstituted in vitro system consisting of hela nuclear extract and recombinant oct- /pou domain protein (without the activation domain of oct- ) and vp where we showed that the activation of ie genes is not mediated by oct-i itself. rather, oct- serves to recruit vp that has no intrinsic dna binding capacity. vp , however, possesses a stong acidic c terminal activation domain of - amino acids which is indispensable for the in vivo function of the protein. as this and other acidic activation domains possess only a relatively weak activating capability when we test them in in vitro transcription assays we are interested in investigating the role of this acidic activation domain in vitro i.e. under condition of cell free extracts and on naked template dna. we are using recombinant oct- /pou domain protein and recombinant vp that has been truncated at the c terminus. additionally, we are also testing different truncations of vp in vitro that have been produced in cos cells. furthermore, we are also trying to see whether these different truncations already affect the assembly of the actvating multiprotein complex. we have studied the kinetics of synthesis of transcripts initiated from the vaecinia virus . kd gene early promoter. unexpectedly, after a first burst of rna synthesis early in infection, the promoter showed a second peak of activity in the late phase.reactivation of transcription was neither dependent on the presence of the sequences upstream of the early promoter, nor on the location of the promoter in the genome. reactivation seems to be dependent on virus assembly since it is prevented by rifampicin, that specifically inhibits an early step of viral morpho-g~nesis. analysis of several other early genes showed that reactivation is not unique to the . kd early promoter. analyzing, by in situ hybridization using digoxigenin-labelled riboprobes, the expression of the viral genome relative to that of an array of cytokines including tnf-(z; ifn-% il-i~, il- , il- and gm-csf. initial experiments suggest that viral genome expression is restricted in the inflammatory infiltrates whereas certain cytokines, particularly tnf-oq are strongly expressed in the synovial membranes. overall, the results point to a prominent role of macrophage activation in the maintenance of chronic inflammation and possibly also in the development of arthritic lesions. the core protein of the hepatitis b virus (hbv) interacts with the viral rna pregenome and the reverse-transcriptase.jdna-polymerase to form a core particle which allows for replication of the viral genome. deletion analysis of the core protein revealed, that the arg-rich carboxyterminal domain is required for rna encapsidation and productive viral dna synthesis. we demonstrate, that the duck hepatitis b virus (dhbv) core protein can be derided into two domains comprising amino acids - (n-ter) and - (c-ter), respectively. the co-expression of these two core subdomains leads to the formation of replication competent core particles. furthermore, we demonstrate that the carboxy-terminal domain of dhbv core (c-ter) can be functionally replaced by the corresponding domain of the human hbv. the complete hbv core protein, however, does not form replication competent core particles with the dhbv pregenome and rt. these results imply that the carboxy-terminal part of the hbv core proteins define an exchangeable, functionally conserved domain. influenza ha mediates the adhesion and penetration of host cell membranes. acylation of three conserved cysteine residues in the membrane spanning region of several ha subtypes has been shown. the biological significance of this hydrophobic modification remains to be elucidated. a possible role for the membrane fusing activity has been controversially discussed in the past. removal of covalently bound fatty acid from protein by incubation with hydroxylamine is a commonly applied method. as we shall show for a number of different envelope viruses with acylated glycoproteins, this treatment leads to quite different consequences in a functional seiase depending on the virus used. as an alternative approach to study the biological significance of palmitoylation we expressed wild-type influenza ha and an hamutant lacking the fatty acid binding sites using the baculovirns system. both wild type and fa--mutant ha, after expression in insect cells bind to red blood cells, induce hemolysis, and fuse efficiently with fluoreseently labeled erythrocyte ghosts. hydroxylumine treatment of both recombinant wt-and mutant-ha leads to a loss of hemolysis and of fnsion activity. at least in the ease of fa--i-ia its inhibitory action must be related to some other effect than fatty acid cleavage. these results indicate that hydroxylamine treatment may not always be suitable for the generation of fatty acid free glycoproteius or virus particles for functional studies. gesemann and j.g. nicholls, biocenter univ. basel, basel, switzerland depolarization of leech neurons leads to growth cone collapse and neurite retraction. this response to depolarization is influenced by the substrata on which the cells are growing and is mediated by the influx of calcium (s. grumbacher-reinert and nicholls, , j. exp. biol. : - ; neely, , i. neurosci. : - . the morphologieal changes induced by depolarization of leech neurons growing on leech extracellular matrix extract is accompanied by a loss of microfilaments in the growth cones. leech neurons growing on a different substrate, the plant lectin coneanavalin a (coma), did not retract their neudtes or lose rnierofilaments, but continued to grow after depolarization. we attribute this to the reduced calcium influx during depolarization of neurons on cona (ross et al., , pnas : - ) . increasing the intraeellular calcium concentration by incubating neurons with the calcium-ionophore a , led to cessation of motility and disruption of microfilaments but not microtubutes in growth cones on coma. to investigate the mechanism by which calcium affects the organization of microfilaments we stained neurons with antibodies against gelsolin, a protein that severs microfilaments when it is activated with calcium. we have observed that these antibodies colocalize with microfilaments in leech growth cones, we are now analyzing the nature of this antigen that cross-reacts with the anti-gelsolin antibodies with the goal of establishing its potential role in the calcium-induced disruption of microfilaments in leech neuronal growth cones. this work was supported by a grant from the swiss nationalfond no. . to j.g. nicholls. activation of metabotropic glutamate receptors (mglurs) was studied in voltage-clamped ca cells in rat hippocampal slice cultures using the whole-cell pateh-clamp configuration. in saline containing mm k +, s, r-acpd ( ~tm), a mglur agonist induced an inward current associated with an increase in membrane conductance. the reversal potential, assessed with a voltage ramp protocol from - to - mv and calculated by linear regression, was - . • . mv, and was shifted to - . • . mv when the concentration of external monovalent cations was halved. these results indicate that the conductance underlying this current is selective for cations (mainly k + and na+). the steady-state i/v curve for the s, r-acpd-induced inward current showed a slight inward rectification. in most of the cells, this inward current was followed (or overlapped) by an outward current concomitant with a decrease in a k + conductance (ere v = - . ~ . mv in [k+]o = mm and shifted to - . :t: . mv in [i +]o = ram). this k + current was voltage-independent in the membrane potential range of- to - mv. similar results were obtained with methacholine, a cholinergic muscarinic agonist. the non-specific cationic current, seen only in the presence of elevated extracellular k + concentration, could play an important role during intense neuronal activity. the c-myc protein has been implicated in the regulation of cell growth and differentiation, c-myc specifically interacts with max which in turn forms heterodimers with mad and mxil and homodimera with itself. we present evidence that this network o! proteins is regulated both at the level of relative protein concentration as well as by post-translational mechanisms. in the hematopoietic cell lines u- , ml- and hl- o e-myc mrna and protein are rapidly downregulated after induction o| differentiation whereas max expression is not significantly altered. in contrast the expression of mad is induced with properties similar to immediate early genes, taxi is induced to a lesser degree and with slower kinetics, in an effort to study the regulation of o-myc we have mapped all the major in vivo phosphorylation sites in both c-mye and max. two sites in the n-terminal transactivation domain are important for the regulation of the transforming potential el c-myc both in established cell lines as well as in primary rat embryo tibroblasts. however, no difference in the transactivating potential of corresponding mutants was observed. phosphorylatien of max at sites close to the basic region increases both on-and off-rates of either myc-max hetero-as well as max homodimers to dna. brain research institute, university of z~rich, ch- z~rich, switzerland gaba b and adenosine receptors act via pertussis toxinsensitive g-proteins of the gi/g o class to mediate longlasting inhibition in the cns. this class of g-protein is negatively coupled to the enzyme adenylyl eyclase. our aim was to determine whether the inhibition of adenylyl nyclase mediated by these receptors has functional consequences for electrical signaling in hippocampal neurons. the calciumdependent k + current, i~, underlying adaptation of cell firing, was used as an electrophysiological bioassay for adenylyl cyclase activity, as it is very sensitive to intracellular levels of camp. accordingly, the ~-adrenergic agoniat iaoproterenol ( - nm), that activates g,-linked receptors, reduced the amplitude of i~ by . • . % in voltage-clamped ca pyramidal cells in ttx (n= ). reduction of imm by isoproterenol was curtailed to . • . %, (p < . ) in the presence of baclofen (i ~m), acting at gaba b receptors, or adenosine ( nm). thus, the inhibition of adenylyl cyclase activity due to activation of gaba a and adenosine receptors can modulate responses to other neuretransmitters by an interaction occurring at the level of intracellular signal transduction. furthermore, stimulation of these receptors will tend to enhance i~, suppressing repetitive cell firing. this represents a further mechanism which will result in prolonged inhibition of hippocampal neurons. a study of the possible modulation by nitric oxide (no) of endogenous dopamine (da) release was performed in bovine retina in vitro. isolated half retinas were incubated in kkg at ~ for successive -rain incubations, and da was measured in the incubation medium by hplc with ec detection. when retinas were incubated in the presence of the no-generating compound hydroxylamine ( ttm), a decrease in either basal or k +-( ram) stimulated da release was observed during the nd -rain period. tested under similar conditions, dibutyryl cgmp ( mm) was ineffective. the no synthase inhibitor l-ng-nitro-arginine methyl ester (l-name, gm) increased basal da release during the st and nd incubation, whereas it did not affects the k+-stimulated da release. in addition, its effect were potentiated in calcium-free medium. finally, we have also shown in cell-free experiments that retinal no synthase activity was totally calcium-dependent and blocked by l-name. taken together, these results suggest that endogenous no regulates da release via a cgmp independent mechanism. division of endocrinology, university hospital, ch- geneva while the stimulation of aldosterone biosynthesis by angiotensin ii (angii) in bovine glomerulosa cells clearly depends upon a sustained influx of ca +, the pathway for ca § entry is not yet accurately defined. at least two mechanisms seem to be involved: ) the opening of voltage-operated ca z § channels, and ) the stimulation of a "capacitative" ca + entry regulated by intraocllular ca § stores; however the relative contribution of these pathways to the stimulation of steroidogenesis needs to be determined. two pharmacological tools were used: nieardipine, a blocker of to and l-type ca + channels, and thapsigargin, which releases ca ~+ and therefore induces a capacitative ca ~+ influx. nicardipine ( lam) completely blocked the cytosolic ca + response to k +, which exclusively activates voltage-operated ca . channels, but only partially ( %) the response to angli. in the presence of nicardipine, angll was unable to stimulate a ca + influx aiter depletion of ca § stores with thapsigargin, demonstrating that angli principally stimulates a capacitative ca + entry pathway. in addition, nicardipine completely blocked the steroidogenic response to k +, but only % of the response to angll. thapsigargin-induced aldosterone formation, as the response to angll, was markedly potentiated by low concentrations of k +. in conclusion, this study shows that in bovine glomerulosa cells, angli activates a sustained ca + influx, principally through a capacitativc pathway, leading to aldostcrone formation. this finding could partially explain the poor efficiency of dihydropyridine ca § antagonists for preventing aldosterone secretion. harald holder and john m. lucocq*. institute of anatomy, university of berne, ch berne *depanment of anatomy and physiology, university of dundee, uk dundee dd hn. subjection of hela cells to ~ for minutes leads to a complex activation pattern of map-kinases as revealed with renaturadon gels. exponentially growing hela cells show activallon of a myelin basic protein (mbp) phospborylating kinase migrating with an apparent molecular mass of around kda upon heat shock induction. in contrast, in cells arrested in go by serum starvation for h, essentially three different proteins phosphorylafing mbp are activated subsequent to heat shock treatment. these kinases migrate with apparent molecular masses of around kda, kda and kda. western blot analysis with antibodies recognizing the human boraologues of erkl and erk revealed that considerable pools of erkl and erk show retarded elec/rophnretic migration behavioar indicating that they are phosphorylated and probably activated in heat subjected and growth-arrested hela cells. down-regulation of protein kinase c (pkc) by a prolonged treatment with -o-tetradecanoyl-phothol- -acetate (tpa) did neither change the major activation pattern observed upon separation of cell lysates on renaturation gels nor that one observed on western blots decorated with anti-erkl or anti-erk antibodies. these results suggest that heat shock induced activation of at least erkl, erk and the kda mbp-kinase is not mediated via activation of pkc. this work was supported by swiss nsf grant no. - . . effects of dominant negative glucocorticoid receptor mutants in cell cultures and transgenic animals stefano brenz verca, stefan schneider, sandro rusconi, institut fiir molekularbiologie h der universitat zf~rich, winterthurerstr. , switzerland the glueocorticoid receptor (gr) is involved in a large number of developmental and biochemical processes. recently, we obtained a trans-dominant negative gr mutant (tdn) -called gr[aia] -by a frame shift of the cag-repeat of the rat gr (see poster by hug et al.) . the gr[ala]-mutant shows normal llgand-binding and dna-binding but cannot stimulate transcription (lanz et al., steroids; in press) . modulatable versions of the tdn-mutant have also been created by substituting the hbd of the grmutant with the hbd of sex-hormone receptors. these constructs have only been tested so far in transient cotransfection assays. therefore, we are currently trying to express them permanently in cell lines (rat hepatoma cell line fto- b) in order to verify whether they can affect the transcription level of a known endogenous gr-dependent target-gene, like the tyrosine aminotransferase (tat) gene or a permanently transformed mtv-iacz reporter. so far no rapid test-system has been described, that allows the quantitative analysis of such effects in transient expression assays. we are about to set up such a rapid and generally applicable transient test-system. in the future we plan to express dominant negative gr mutants in a tissuespecific manner in transgenic mice. it will be particularly interesting to investigate their effects on the immune system or on liver functions. these studies investigated growth cone responses m brief local encounters with surface molecules implicated in neuronal pathfinding using chick drg neurons. we tested two interrelated hypotheses: ) discrete points of information (guideposts) override sustained surface information and dominate growth cone behavior and ) guidance molecules provide such information by activating second messengers instead of simply increase adhesivity. the potential guidance molecule lamialn was covalentiy coupled to polystyrene beads in order to mimic guidepost cells. encounters with laminin model guideposts significantly altered the behavior and morphology of growth cones advancing on fibronestin or polylysine. growth cones steered towards theses beads in a saltatory medea once a long lasting contact with a single filopodium had been established. subsequent to a pause at the bead, growth cones advanced with a significantly increased growth rate for the next llmin. the average increase in rate stimulated by bead contact was times the basal rate on polylysine and . times that on fibronectin. positioning of multiple model guideposts along a path and within filopodial reach maintained this bead stimulated rate of outgrowth and directed growth cone advance. the laminin induced pattern of events was totally blocked in the presence of either h or sphingosine, two pkc inhibitors, or neomycin, a plc inhibitor. neither of these inhibitors affected neurite outgrowth on the substrata alone. our results demonstrate the necessity of filopodial contacts, the importance of spatially restricted stimuli and the activation of transducing pathways as key mechanisms in neuronal pathfinding. mechanisms of glucocorticoid receptor desactlvation by homopolymeric alanines martin hug, manuela h&fferer and sandro rusconi, institut fiir molekularbiologie der universitat uz zart'ch, winterthurerstrasse , ziirich, switzerland the rat glucocorticoid receptor (gr) edna contains in its y-portion a (cag-repeat that encodes a siring of gin residues interrupted by arg. a mutant with the frame-shifted repeat which codes now for poly-ala residues (called gr[aia]) is completely trans-inactive, is more cytoplasmitally located in absence of ligund and exhibits dominant-negative properties in presence of ligand (lanz et al., steroids, in press) . we generated gr recombinants with increasing numbers of cag-triplets in three possible reading frames (oligo-gln, -ser or -ala). the gr-oligo[ala]~-~. mutants showed an activity comparable to wild type gr, whereas the groligo[ala]>_ were inactive. the severity of the effect of oligo a.la su'ings seems to be dependent on the position of the repeat along the primary sequence (e.g. progressive decrease of the effects by moving toward more earboxy-position). we have also examined the effects of the oligo[ala] , , , , on gal chimeric factors and found that the inhibition by ala repeats is probably factor-specific. our results made us speculate that possible differences in post-translational modifications may be at the root of the negative effects by the ala repeats. we believe that these effects are due to transient association of the nascent gr[aia] mutant proteins with intraceuular membranes. these hypotheses are currently tested. the activation of steroidogenesls in calcium-clamped bovine glomerulosa cells and its modulation by angiotensin ii and camp, python cp, laban op, rossier mf, vallotton mb and capponi am division of endocrinology, university hospital, geneva, switzedand. the ca +-messenger system plays a crucial role in the regulation of steroid secretion in adrenal zona glomerulosa cells, as it is known to mediate the action of angiotensin ii and potassium ion. in the present study, we used intact isolated glomerulosa cells in which the cytosolic free ca + concentration ([ca +]c) was clamped at various levels with the ca +-ionophore, ionomycin, in order to locate the sites of action of ca +, to determine their sensitivity towards ca + and the modulation by other physiological factors. by measuring simultaneously steroid synthesis and [ca +]c, we show that ca +-clamping ( - nm) induced a concentration-dependent increase in the production of both pregnenolone ( ___ % of the basal level) and aldosterone ( + % of the basal level, ec = nm). by contrast, ca + did not influence the conversion of t-deoxycorticosterone into aldosterone at concentrations up to rim, although at higher concentrations we observed a modest but significant inhibition. in addition, ca +-clamping did not affect the formation of pregnenolone from a freely diffusible analogue of cholesterol, hydroxycholesterol, indicating that ca + acts at a step upstream of cholesterol side-chain cleavage. both angiotensin ii and a membrane-permeant surrogate of camp, dibutyryl cyclic amp, potentiated the steroidogenic response to increases in [ca +]c . in summary, these studies reveal an exquisite sensitivity of the glomerulosa cell steroidogenic pathway toward very small physiological changes in [ca +]c. radtke, rainer heuchel, oleg georgiev, gerlinde stark#, michel aguet# & walter schaffner institut fiir molekularbiologie , universilat ziirlch, z' rich # lnstitut rir molehdarbiologie i, universitiit ziirich, ztirich we have previously described and cloned a factor (mtf- ) that binds specifically to metal-responsive dna sequence elements in the enhancer/promoter region of metallothionein genes. mtf- is a protein of . kda that contains six zinc fingers and multiple domains for transcriptional activation. here we report the disruption of both alleles of the mtf- gene in mouse embryonic stem ceils by homologous recombination. the resulting null mutant cell line fails to produce detectable mounts of mtf- . moreover, due to the loss of mtf- the endogenous metalinthionein-i gene is silent, showing that mtf-i is required for both basal and metal-induced transcription. accordingly, reporter genes containing either synthetic or natural metal-reslxmsive promoters were not transcribed after transfection into the null mutant ceils. cotramsfection of the mtf-i expression vector rescued both basal and heavy metal-induced transcription. these results demonstrate that mtf- is essential for normal metallothionein gene regulation. rat pheochromocytoma cells (pc ) were differentiated with ngf for - days, a time sufficient for the formation of growth cones and elongation of neurites. addition of the phosphatase inhibitor calyculin a (cl-a) ( . . . nm) led to a concentrationdependent collapse of growth cones and retraction of the neurites within minutes. after the retraction, the cell bodies showed the appearance of a grape-like domain opposite to the cell nucleus. they recovered to normal shape within about - minutes if the inhibitor was washed out. the neurite retraction was further analysed using both low-light level fluorescence video-microscopy and fura- single cell microfluorimetry. the onset of retraction started in the central part of the growth cone prior to a complete collapse of filopodia. the basal intracellular free calcium concentration ([ca +] i) remained constant during neurite retraction. as a measure for the viability of cl-a-treated cells, agonist-isduced responses of [ca +] i were analysed. ca + entry across voltage-activated ca -~ channels was inhibited by % after minute treatment with cl-a, while the atp-induced ca + entry via ca *-permeable cation channels was not significantly reduced. however, the onset of the atp-induced rise in [ca +]i started at the grape-like domain. the speed of the ca + wave across the cell body was slower than in control cells. the addition of . gm okadaie acid, a different type of phosphatase inhibitor, caused no cell shape change or neurite retraction within hours. taken the specificity of the two phesphatase inhibiters into account, the observed effects seem to be induced by inhibition of a ppl-class phosphatase. myosin in nematocytes of hydra michel nakano & robert p. stidwill dept. zoology, university of ztirich, switzerland the functions of nonmuscle myosins during cell locomotion are not clear. several models have been presented reaching from very active participation of the molecules in different parts of cells to only minor or no roles at all. there is increasing evidence that the functions of myosin ii (capable of forming bipolar filaments) are quite distinct from the ones of myosin i and generally that the different members of the growing family of myosin-like proteins may not all have identieal functions. we have attempted to demonstrate the occurrence and determine pattern of myosins in tissue of hydra and especially in migrating nematoeytes mainly for two reasons. first, nematocytes are fairly rapidly moving cells which can be studied in vitro and, second, the question is of interest from a evolutionary point of view. we have utilized several antibodies against vertebrate and invertebrate myosins for immunofluoreseenee (confocal laser scanning microscopy) and western blotting studies. in addition to these results findings on the screening of a ~.zap c-dna library are presented. the jaki protein tyrosine kinase is a member of a family of intracellular kinases characterized by having two kinase catalytic domains: a bona fide tyrosine kinase domain and an amino terminally positioned kinase-related domain. recently it has been shown that jaki and the other members of this family (jak , tyk ) are intimately involved in cytokine and growth factor signalling. the presence of two putative nuclear localizing sequences in the amino terminus of the protein prompted us to examine the subcellular localization of this protein. immunohistochemical and biochemical analysis revealed that a part of jaki was localized to the nucleolus. metabolic labelling showed that (a) the jaki protein partitioned rapidly into the nucleus and (b) the nuclear jaki became smaller with time. the size difference was not due to either co-translational glycosylation or phospherylation. these results suggest that part of the jaki protein is rapidly translocated to the nucleoli where it becomes processed. revealed that this clone is expressed predominantly in spleen, mammary gland and thymus as two transcripts, a major species of approximately kb and a minor species of approximately kb. nucleotide sequence analysis revealed a % homology to the porcine syk gene. the syk gene is expressed as a major . kb transcript and has been reported to be lymphoid specific. due to the discrepancy of transcript size and number, we suggest that our cdna clone represents a novel member of this family of ptks. we are currently isolating a full length sequence from a mouse spleen cdna library. in addition, we want to characterize which cell(s) in the mammary gland are responsible for the observed expression. expression and function of g-protein isoforms were studied in differenflaring ( c ) and nondifferentiaflng (( ) subclones of red- rauscher murine erythroleukemia cells. erythroid differentiaflon induced by the combined action of erythropoietin (epo) and dmso was associated with a selective loss (> %) of immunoreactive gai . simultaneously, a iruncated form of g~ accumulated in the cytosol, while the membrane concentrations of gai , gets and gaq/ remained unchanged. nondifferenfiating ( cells contained sigrtificanfly higher levels of most get subunit isoforms that remained unchanged upon epo/dmso treamaent. changes in adenylyl cyclase activity and in intracellular ca ion levels ([ca]i) resulting from activation of g-protein-coupled receptors were monitored for differentiation-dependent effects on transmembrane signaling. adp-mediated changes of [ca]i in native and differenflaflng c cells were consistent with p t receptor coupling to gai . thrombin receptors seemed to couple preferentially to gai in g cells and to gaq in c cells. our results document substantial alterations in g-protein-mediated signaling during erythroid differentiation. to investigate the involvement of protein tyrosine kinases (ptks) in the growth control of the mammary gland, we have used pcr based cloning to identify ptks expressed during normal mammary gland development. this approach led to the isolation of three members of the eph-related family of receptor ptks: myk-;, a novel member expressed predominantly in lung, heart and mammary gland; m-eck, the murine homologue of the human eck gene and mek , the murine homologue of the chicken cek . all three ptks were expressed in a distinct and narrow window of mammary gland development: puberty and estrus cycle. no expression was found either in late pregnancy or in the end differentiated state of lactation. vet-expression was found in the undifferentiated and invasive tumors of transgenic mice expressing the h-ras oncogene. in contrast, no elevated expression of all three genes was detected in the well differentiated, non-invasive mammary tumors characteristic of c-myc expressing transgenic mice. these results suggest that members of this family of ptks are involved in the control of proliferation of the mammary gland but are incompatible with its differentiation. np-tcii is a lymphocyte specific transcription factor (lattion a.-l. et el., mol. cell. biol., , : - a nucleotide receptor that mediates phosphoinositide and phosphatidylcholine hydrolysis by phospholipases c and d, respectively. here we report that atp and utp potently stimulate mesangial cell proliferation. both nucleotides stimulate phosphorylation and activation of map kinase and the up-stream map kinase kinase. activation of map kinase by both nucleotides is dose-dependently attenuated by the p receptor antagonist suramin. stimulation of protein kinase c (pkc) by phorbol ester increased map kinase phosphorylation and activation, and downregulation of pkc partially inhibited atp-and utp-induced map-kinase activation. inhibitors of pkc which display a selectivity for the ca +-dependent isoenzymes (c~, [ , ) , as compared to the ca +-independent isoenzymes ( , e, ~, rl) such as staurosporine and the specific pkc inhibitor cgp did not inhibit nucleotide-stimulated map kinase phosphorylation, thus implicating the involvement of a ca +-independent pkc isoform. in conclusion, these data suggest, that atp and utp trigger the activation of the map kinase signalling cascade in mesangial cells and this may be responsible for the potent mitogenic acitivity of both nucleotides. subcellular ion-gradients in ca signaling p. lipp & e. niggli, department of physiology, univexsity of bern recent experimental evidence has indicated an important role for submicroscopic concentration gradients during ca-signaling in various cell types. in heart muscle cells influx of ca via l-type ca channels triggers ca release from the sarcoplasmic reticulum (sr) and links electrical excitation to mechanical contraction (ec-coupling). we used a ratiometric confocal method (fluo- and fura-red) to follow the intraceuular ca-concentration while ha-and ca-currents (inn, ica) were elicited in the whole cell mode of the patch-clamp technique. both, icand in, were able to trigger ca release from the sr. kinetic differences between ini and ic -induced ca-transients, li-substitution experiments and the existence of a residual inn-induced ca transient in the absence of sr function suggested that these ca signals were mediated by the ha-ca exchange running in the ca influx mode. control experiments showed that ins-induced ca transients did not result from spurious activation l-type ca channels. the significant increase of the na concentration required to activate the ha-ca exchange can only occur provided cytosolic diffusion of na is restricted in the vicinity of the exchangers. this limited diffusion results in significant ha-gradients in the subsarcolemmal space. in conclusion, i~iinduced ca influx may represent a safety factor for ec-coupling while ic, ensures sustained ca-release and is the source for refilling the sr with ca. supported by snf. the regenerative ca-induced ca release (cicr) mechanism is an important amplifier in cardiac signal transducilon. the cicr contributes to the ca transient responsible for mechanical activity, but also generates ca waves propagating within the cytosol. we investigated subcellular ca signals in neonatal rat and adult guinea-pig ventricular myocytes using ratiometric confocal microscopy with a mixture of two fluorescent ca indicators. individual resting myocytes exhibited spontaneous ca release events from the sarcoplasmic reticulurn (sr) that were attributable to three distinct patterns: i) local ca release events without propagation; ll) planar ca waves propagating across the cell; iii) spiral ca waves spinning around a nucleus or shifdng along a subcellular region without entering it. transitions between these patterns were frequently observed, suggesting that a particular release pattern is not the property of an individual cell but reflects the functional state of the sr. the existence of focal release and refractory zones within a single cell indicates that the sr is not uniform on the subcellular level. the sr seems to he a network of elements with distinct functional properties. a suhcellular variability in the positive feedback of the cicr mechanism can account for the observed features of ca signaling. the degree of positive feedback exhibited by a single sr element may depend on its ca content. to characterise the intracellular signalling and regulatory properties of the cloned parathyroid hormone (pth) receptor we have stably transfected two renal epithelial cell lines with cdna's encoding the pth receptor (provided by dr g. segre). the proximal tubular, porcine (llc pk [clone ]), and the distal tubular, madine darby canine (mdck) cell lines were transfected since the culture of either cell line on permeant filter supports leads to the development of polarised epithelial cell layers that mimic the epithelial organisation in vivo. transfection of the cloned pth receptor into each of these cell lines allowed us to examine, independently, the functional properties of the receptors expressed at the apical and/or the basolateral cell surface. while the basolateral application of pth produces a large increase in camp production in both cell lines, the relative efficacy of an apical application of pth differs between the cell types. the transfected llc pk cells exhibit a greater response to apical pth compared to the transfected mdck cells. pth-mediated regulation of intrinsic phosphate transport was also examined in polarised, transfected mdck cells. neither apical or basolateral application of pth affects intrinsic apical or basolateral ha-dependent or phosphate transport activities. similar experiments are being performed with the transfected llc pk cells. a. and preiss, a. biozentrum der universit&t basel, ch basel hairless, a recessive larval lethal mutation, causes dominant defects in sensory organs of adult drosophila flies.manifold genetic interactions indicate that hairless antagonizes neurogenic gene function suggesting an involvment also in neurogenesis. in order to gain insight into its function we have cloned the hairless gene which encodes an extremely basic, very serine rich protein of ii kd calculated molecular weight. no significant homologies to proteins of known function could be identified. therefore, we are concentrating by various means on a structure -function analysis of the hairless protein. firstly, we are testing a series of hairless deletion constructs for rescue capacity and generation of anti-hairless phenotypes after overexpression compared with the wild type gene. secondly, we are analysing hairless protein distribution throughout development, also at the subcellular level. heat induced hairless protein runs at ~ kd, possibly due to phosphorylation which might be intrinsic to hairless function, a hypothesis we are currently scrutinizing. thy- and cd surface glycoproteins are both capable of delivering proliferative signals to t cells upon cross-linking with appropriate antibodies. the tyrosine phosphatase cd is a key regulator of t cell proliferation as it controls the activity of src family kinases p /~k and p fy n. associations of thy- with cd , and of cd with cd have been reported after chemical cross-linking of intact cells and it is likely that such associations constitute part of the structural basis for the t-cell stimulating capacity of thy- and cd accessory molecules. we report that thy- , cd and cd can be specifically crosslinked at the cell-surface and isolated as multimolecular complexes containing a kda protein that is recognized by an anti-bip (grp ) antibody. in vitro kinase assays show the selective association of p ick and p fy n with thy- and cd contained in multimolecular complexes. the multimolecular structure we describe could representa transducing unit incorporating surface receptors and regulators of tyrosine phosphorylation that are stabilized through interaction with the bip protein in the plasma membrane. structure-function-analysis of hairless, a gene involved in drosophila nervous system development maier, d., functional coupling of ppars and trs ijpenberg, a., wahli, w., desvergne, b., institut de biologie animale, lausanne we are interested in a possible functional coupling of thyroid hormone (tr) and peroxisome proliferator-activated receptors (pfar). to this end, we performed cotransfection experiments in nih t cells using cat reporter constructs containing the promoter of either the t responsive malic enzyme (me) gene, or the ppar regulated acyl coa oxidase (aco) gene. the me reporter construct showed very moderate response to the activated mouse ppar (mppar), whereas combination of try and unactivated mfpar potentialized the t induction. a putative ppar response element was identified within this promoter and will be further investigated. the aco reporter constructs were not responsive to trs. in constrast, a tr and t dependent inhibition of the fpar mediated activation was observed. the mechanism of functional coupling of these receptors, which may possibly involve the -cisretinoic acid receptor rxr, will be further investigated by molecular analysis. unliganded steroid receptors form a complex with hsp via their hormone binding domain (hbd). the receptor activation involves a hormone-induced release of hsp . we genetically addressed the role of hsp in budding yeast by analyzing three kinds of hsp (the essential yeast homologue of hsp ) mutants. these mutations affect either the quantity of the protein (low versus normal levels), its integrity (deletion analysis) or its nature (hsp homologues from other species). hsp mutant are examined for their ability to complement a hsp deletion mutant. moreover, they are tested for functional interaction with a hbd. interestingly, a viable deletion of a charged region, suspected to be involved in hsp -hbd interaction, only slightly interferes with hormonal activation of both estrogen or glucocorticoid receptors. furthermore, a short deletion in the last third of hsp leads to the production of a dominant negative mutant which prevents ceil growth at elevated temperature. the ability of various hsp homologues to palliate or not the absence of the yeast protein allows us to define, using chimeras, the regions of the protein which are necessary and sufficient to support cell life. zhang,h.,reynaud, s. and spohr,g. d~partement de biologie cellulaire, sciences iii, , quai ernest-ansermet, ch- gen&ve id cdnas expressed during early xenopus development have been isolated and sequenced. the encoded protein is homologous to the proteins described in higher vertebrates. northern blot analysis reveals that transcription starts soon after mid blastula and decreases to some extent after tailbud-stage. lower levels of transcription are detected also in adult frog tissues such as liver and heart. microinjection into oocytes of in vitro-synthesized myodor/and id-mrna, along with a cat reporter gene whose expression is under the controll of an a -actin promoter supplemented with four e-boxes shows that myo d function is repressed by id. to investigate the importance of negative regulatory sequences we have isolated and characterised the id promoter. as a strategy for identifying cis-regulatory elements, chimeric genes consisting of different ' elements of the promoter were attached to the cat coding sequences and microinjected into embryos. we are studying the tissue distribution of the rat peroxisome proliferators activated receptor (ppar~), a member of the nuclear hormone receptors superfamily, during development and in adults. high levels of ppar~ mrnas are detected in adult liver, kidney and heart. muscle, stomach, and intestine show moderate amounts of the ppar~, whereas brain, testis and lung present a very low expression. during postnatal development, we observe a continuous increase of the ppar~ expression in the kidney, in contrast to a steady level in the liver. anjard, c., groux, b ., gamboni, s. and reymond, c.d., institut d'histologie, unil, rue du bugnon , lausanne. the camp dependent protein kinase (capk) from dictyostelium is composed of a single catalytic (c) and a single regulatory (r) subunit. the c subunit we characterised, pkac is about twice the size of its mammalian counterpart. we showed that it possesses all properties of a catalytic subunit. it associates with the r subunit in absence of camp, it copurifies with capk activity and an increased activity is observed in cells over-expressing pkac. furthermore, we dissected its role during development and cell differentiation. overexpressing pkac under cell type specific promoters allowed to show the requirement for its activity during spore differentiation. pkac seems to play a more complex role in prestalk cells. we are now dissecting the functional regions of the pkac protein, making use of the known tertiary structure of the c subunits of mammalian enzymes. dna binding properties and stimulation of gene transcription of baculovirus expressed xppar~. hihi, a.k, mermod, n., medin, j., ozato, k. and wahli, w., inst. de biol. animale, uni lausanne, ch- lausanne. lab. of mol. growth reg., nih, bethesda, md, usa. the peroxisome proliferators activated receptors (ppars) are members of the steroid/thyroid nuclear receptor superfamily. so far, they have been found in amphibians, rodents and humans. in xenopus laevis, subtypes (xppara,~.y} have been isolated. in order to study the mode of activity of the ~ form, the xppar~ gene product was overexpressed using a baculovirus expression system. the nuclear protein obtained has the correct molecular weight of kd. gel shift assays showed that nuclear extracts containing the recombinant protein are able to specifically bind the 'ppar response element' which is a direct repeat of the core aggtca motif. this dna binding activity is potentiated by another nuclear receptor, the mouse rxr~ and is inhibited by phosphatase, suggesting a role for phosphorylation in ppar binding to dna. a functional approach, using an in vitro transcription system, was chosen to define ppar and rxr action on transcription stimulation, as well as the role of their respective activators. arachidonic acid ( . - ram) induces various patterns of ca +i signal in bronchial smooth muscle cells, as revealed by single cell dynamic video imaging of fura- loaded cells. most frequently, ca +i oscillations were observed, although other patterns (a single ca +i transient; a single peak followed by a sustained elevated level, or a sustained ca *i elevation solely) were occasionally seen. the amplitude of ca *i oscillations was related to the concentration of arachidonic acid. the frequency histogramm was noticeably shifted to higher frequencies by nordihydroguaiaretic acid (ndga, . rtm, a lipoxygenase inhibitor), whereas it was markedly shifted to lower frequencies by indomethacin ( pm, a cyclooxygenase inhibitor) and by , , , -eicosatetraynoic acid (etya, ~tm, a lipoxygenase and cyclooxygenase inhibitor). these observations suggest that: ( ) arachidonic acid per se elicits ca + i oscillations in these ceils; ( ) cyelooxygenase activity products modulate the frequency of these oscillations. promoter analyses of a growth factor regulated gene t. triib, m. kalousek, r. kessler, and r. klemenz dept. of pathology, university hospital, ziirich stimulation of quiescent cells to enter the cell cycle results in altered gene expression. some of the first genes to be induced (immediate early (i.e.) genes) encode transcription factors which are thought to pass on the mitotic signal to the delayed early (d.e.) genes. we have analysed the promoter elements required for growth factor mediated induction of the d.e. mouse gene t which encodes a secreted glycoprotein of the immunoglobulin superfamily. a bp region located between . and . kb upstream of the transcription start site is sufficient for growth factor mediated inducibility. it contains an ap- binding site which is absolutely required for t gene expression. it is surrounded by e-boxes. at least of them are essential for efficient gene induction. thus the i.e. proteins encoded by the fos and jun gene family which form the ap- complex can activate the d.e. gene t in collaboration with helixloop-helix transcription factors binding to the e-boxes. the increase of radiolabelled inositol phosphates ([ h]ips) production in agonist-stimulated human airway smooth muscle cells (asmc) was determined by hplc. in order to observe the role of calcium, pkc and pla on plc activity during agonist stimulation, the effects of pharmacological agents (able to alter calcium, pkc and pla ) were tested on ip production elicited by a sec stimulation with histamine. the inhibitor of pkc staurosporine increased, while pma (an activator) decreased the histamine-stimulated production of [ hi , , -ip and of its degradation products. an inhibition was also observed with the two pla inhibitors, quinacrine and p-bromophenacyl bromide. in calciumfree buffer, no difference in the ip increase was shown, but an inhibition was observed when thapsigargin, an inhibitor of the ca +, mg +-atpasc of the sarcoplasmic reticulum, was added in the same conditions. the calcium ionophore ionomycin increased the lp production in presence of external calcium, whereas it completely blocked the stimulation in calcium-free buffer. the results suggest that plc activity, in human asmc, is modulated by a positive feedback of calcium and by a negative feedback of pkc and pla . regulation of hormone-stimulated calcium signals m. boolman, afrc, laboratory of molecular signalling, dept. zoology, cambridge, uk stimulation of cells with hormones that activate inositol , , -trisphosphate (insp~) formation, leads to an increase in the intracellular ca . concentration ([ca +]~). these hormone-stimulated [ca § signals have a complex temporal and spatial regulation, with the [ca *]~ increase often taking the form of a series of repetitive spikes or oscillations, arising from a steady basal level. the spatial counterpart of a [ca +]~ spike is a wave, where [ca § is first elevated in a localised region of a cell, and then spreads throughout the cell in a regenerative manner. the [ca~*]~ spike frequency is modulated by several environmental factors including hormone concentration, extracellular calcium and thiol reagents. current evidence suggests that a cell can interpret these complex [ca +]~ changes to regulate a diverse range of cellular activities. although many of the biochemical steps between hormonereceptor activation and the [ca § response are known, the precise mechanism generating these complex [ca +]~ signals is unclear. in order to understand this mechanism more clearly, we have investigated the regulation of insp~-mediated ca ~ § release from intracellular stores in intact cells. our current data suggests that under certain conditions, the insp -sensitive intracellular stores behave as functionally discrete units, and that during a ca § spike these stores become functionally coupled by the diffusion of ca z+ from one store to the next, triggering a regenerative ca + release. phenylarsenoxide as a tool for identifying proteins involved in the secretory process wiedemann. c., schaefer, t.,*gitler, c., burger, m. m. friedrich-miescher institut, p.o.box , ch- basel "department of membrane research and biophysics, weizmann institute of science, rehovot , israel phenylarsenoxide (pao) at btm blocks exocytosis in chromaffm cells of the bovine adrenal medulla. this inhibition can be reversed by small dithiois, such as dithiothrcitol or , ~limercaptopropanol. because trivalent arsenicals interact specifically with dithiol containing proteins, we conclude that dithiol-proteins do play an important role in regulated secretion in chromalym cells. to identify dithiol-proteins putatively involved in the secretory process, we compare pao-binding proteins from chromaffm and pci cells and from fibroblasts as well as from sulx:ellular fractions by -dimensional gelelectrophoresis. the proteins are identified by radioactive pao bound to the dithiol in a complex that witltslands sds-page. affinity chromatography on pao-scpharose with various protein fractions is used to purify the dithiol-proteins of interest. enzymatic activity, e.g. kinase or phosphatase activity, can be measured in the eluates of such a column to give further hints at the possible function of dithiol-proteins. rac-pks (~elated to pka_ and pkcc protein kinnses) represent a serine/threonine kinase family whose catalytic domain is closely related to those of protein kinases a and c. they contain a ph (pleckstrin homology) domain n-terminal to the catalytic domain. in addition, rac-pk~ was shown to be a proto-oncogene. in order to investigate the role of rac-pk in cellular signallimg we undertook genetic and biochemical analysis of the drosophila homolog (drac-pk). the drac-pk gene encodes multiple classes of transcripts. antisera raised against the drac-pk recombinant protein specifically recognize distinct molecular weight forms ( , and kda). all forms are expressed throughout development and are both maternally and zygotically regulated. the drac-pk gene is localized at b -i region of the third chromosome, betwee~ the pannie~ and the stubble genes. universit de lausanne, rue du bugnon , lausanne when spinal meninges homogenates were incubated with reduced glutathione (gsh), a cofactor of prostaglandin (pg) biosynthesis, [ c] arachidonate (aa) was bioconverted into a major and a minor product which comigrated on tlc with pge and pgd respectively. in absence of gsh: ) pgd could not be detected; ) the level of pge was dramatically reduced but surprisingly counterbalanced by accumulation of an unusual [i c] aa metabolite which exhibits particular traits of migration on tlc and of retention time on hplc. this aa metabolite: ) does not correspond to a degradation product of pge ; ) crossreacts with pge antibody; ) fits the conditions required for a hydroperoxy pge (chemical degradation into pge by hydroperoxyde reducing reagents such as snc or gsh-hemin). it is therefore inferred that depletion of gsh favours accumulation of hydroperoxyprostaglandins which would participate to oxidative injury. the ecdysteroid receptor (ecr) and ultraspiracle (usp) from both, chironomus tentans and drosoplu'la melanogaster, were expressed in e. coil as fusion proteins with glutathione-s-trsusferase. the identity of the expressed recombinant proteins was confirmed by western blot analysis. the drosophila ecr binds to its cognate hormone response element as a heterodimer with usp as a partner. we now want to compare the capabilities of the two receptor partners from both insect species in regard to dna binding and heterodimerization by means of gel mobility shift assays. the use of naturally occurring andartificial hormone response elements will allow to define a hormone response element consensus sequence for ecr-usp heterodimers and, if existing, for ecr and usp homodimers, respectively. for other members of this receptor family it was shown that the dna binding domain and adjacent sequences alone sufficiently defines response element specificity for both homo-and heterodimers. the dna binding domains of ecr and usp from the two species show a high similarity ( % and % identity, respectively). the corresponding sequences were selected for overexpression in e. coli. in combination with immtmoprecipimtion of receptor-dna complexes and pcr amplification steps, the expressed proteins will be used for the detection of naturally occurring hormone response elements within genomic dna. university of fribourg, ch- fribourg. various agonist-induced cell responses in neutrophils and fibroblasts, such as chemotaxis and cytoskeletal rearrangements, have been shown to parallel the synthesis of ptdins( , , )p . but the importance of this rise is not clear. we show here that wortmannin blocks pdgf-induced production of ptdins( , , )p in human foreskin fibroblasts with an ic of about nm. a similar inhibition was observed in in vitro assays (ics =lnm) with pi -kinase ii~nunoprecipitated by antibodies directed against the kd subunit (p ). on the other hand, wortmannin did not affect pdgf-mediated phosphorylation of p , indicating the correct interaction of p with the pdgf-~ receptor. the pl /p complex of the pi -kinase remained intact in the presence of ~m concentrations of wortmannln. these results are consistent with a direct, specific inhibition of pi kinase by wortmannin at concentrations relevant for its previously reported effects on cellular responses. when stimulated with pdgf, human foreskin fibroblasts form circular membrane ruffles rich in filamentous actin. the fact that wortmannin inhibits these pdgf-mediated aotin rearrangements suggests the need of pi -kinase activity as a signal for this cell response. caicineurin is a calcium/calmodulin-regulated protein phosphatase which is comprised of a catalytic subunit a and regulatory subunit b. although calcineurin was discovered in brain, the calmodulin stimulated protein phosphatase which has caicineurin like structure has been described in other tissues. the protein structure show that the calcineurin b is very conserved in different tissues and species, using anti-calcineurin b monocicnal antiserum, the tissue extracts from rat have been explored. immunoreactivity was obverved in all tissues, although its intensity was different. the highest intensity was found in brain. spleen, heart and thymus had an intermediate level intensity. the results were supported by the quantitative measurement of calcineurin. the caicineurin concentration was to fold higher in brain than that in other tissues. furthermore, the distribution of the calcineurin activity in rat tissue was studied using dephosphorylaticn assay of calctneurin. the highest caicineurin activity was found in brain too, but was only to fold more than that in other tissues. the specific activity of calcineurin was different in tissues. these results indicate the caicineudn activity might be regulated in tissue specific fashion. burst and cytoskeleton arcaro a. and wymann m.p., institute of biochemistry, university of fribourg, ch- fribourg chemoattractants like n-formyl-met-leu-phe (fmlp) stimulate in neutrophils a rapid and transient increase in the levels of ptdins( , , )p (pip ) which is due to the activation of ptdlns( )-kinase. pip has been proposed to be a second messenger molecule, but its cellular targets are presently unknown.here we report that pretreatment of human neutrophils with wortmannin inhibits the fmlp-etimulated production of pip in a dose-dependent manner (ic = nm). furthermore, ptdins( )-kinase activity immunoprecipitated from resting cells is totally abolished by i nm wortmannin (ic = nm). these results show a potent and direct effect of wortmannin on ptdins( )-kinase. wortmannin also inhibits the respiratory burst of neutrophils at nanomolar concentrations, which suggests that pip is involved in the signalling pathway controlling activation of the nadphoxidase.when pretreated with wortmannin and stimulated with fmlp, neutrophils display oscillatory changes in factin content that correlate with oscillations in cell ~hape. this, and the inhibition of ptdins( )-kinase, suggest a modulatory role for pip in cytoskeletal rearrangements.we are currently investigating the effects of pip on the functions of gelsolinl a protein that is proposed to mediate actin polymerization and can be regulated by polyphosphoinositides and ca +. matthias gstaiger, walter schaffner and christopher hovens institut f'tir molekularbiologie ill der universitat z'tirich, ch ziirich the octamer motif atgcaaat plays a central role in mediating the activity of a number of both lymphoid specific and ubiquitous promoters and in the immunoglubulin heavy chain intronic enhancer. the activity of thils motif in lymphoid cells is presumably mediated by the lymphoid cellspecific factor oct- a. when ectopically expressed in non-lymphoid ceils, oct- a is not sufficient to mediate enhancer activity of a multimerized bp enhancer core element derived from the immunoglobulin heavy chain intronie enhancer, a segment, which is however highly active in b-cells. this and other findings support the supposition that additional b-cell specific factor(s) can account for the specificity and extent of the octdependent transcription observed in lymphoid cells. to further our understanding of the molecular mechanisms involved in mediating lymphoid-specific expression, we have employed the yeast two hybrid system to identify human proteins capable of physically associating with human oet- a. to this end, a oct- a expressing yeast strain has been constructed which bears two integrated reporter genes, his and lacz under the control of the octamer motif. transformation of this strain with a cdna library has allowed us to isolate clones interacting with oct- a. we are currently analysing these candidates to determine the veracity of these interactions in higher enkaryotic cell background. in general, untransformed nuclear hormone receptors are predominantly confined to either the nucleus or the cytoplasm of a cell. receptors of the latter class are translocated to the nucleus upon interaction with ligand. our studies of the use of the ecdysteroid receptor complex from insects, consisting of a heterodimer formed between ecr and usp, as a tool for target gene activation in vertebrate cells have revealed a novel mechanism of nuclear translocation of ecr that does not depend on hormone action. after transient transfection of respective expression plasmids into hela or cv- cells, the subeellular distribution of the individual receptor types was analyzed by indirect immanofluorescence staining. while ecr alone was detected in both cellular compartments, nucleus and cytoplasm, usp was predominantly found in the nucleus of either host cell. cooxpression of both ecr and usp resulted in an efficient transloeation of ecr into the nucleus. usp appears to trap ecr in the nucleus, presumably by the formation of a heterodimeric complex. thus, heterodimer formation of ecr and usp not only appears to be required for high affinity binding to cognate hormone response dements (and subsequently for transactivation of an adjacent target gene) and for binding to ecdysteroid but in addition for an enhanced translocafion of ecr into the nucleus. activation of rodent macrophages with cytokines or bacteria is accompanied by the induction of a ca +-independent nitric oxide (no) synthase which plays a key role in antimicrobial and antitumoral activity. firm evidence for expression of a similar enzyme in other mammals or in man has been lacking. we now show that bovine bone marrow-derived macrophages produce nitrite in an l-arginine-dependent manner upon stimulation with heat-killed grampositive or gram-negative bacteria. homologous interferon- and tumor necrosis factor-~ induced little no -production, but primed macrophages for enhanced n -production induced by salmonella dublin or lps. this is one of the first demonstrations of no -production by nonrodent macrophages and encourages a search for an involvement of reactive nitrogen species in the killing of parasites or tumor cells by macrophages from mammals other than rodents. lectins are used in many analytical methods to study the carbohydrate structure of glycoproteins. we report here a technique which combines the high resolution of two-dimensional gel electrophoresis ( -dpage) to separate plasma proteins, the specificity of lectins to bind carbohydrates and the sensitivity of chemiluminescence. this method is compared with that of a commonly used colorimetrio reaction. since the reference plasma protein map obtained by -d page is available (i), the technique described here allows a quick and specific identification of plasma glycoproteins. therefore any ma j or glycosylation modification which may happen in some diseases can be detected. (i) electrophoresis ; : - . this work was supported in part by fcar (quebec, canada). in our laboratory we use genetic and biochemical approaches to study translation initiation, in particular the initiation factor eif- a in yeast. this factor from higher eukaryofic cells is known as an rna dependent atpase and functions as a rna helicase together with another initiation factor, elf- b. it belongs to a family of putative rna helicases, the d-e-a-d proteins. to find new genes involved in translation initiation, suppressors of a temperature sensitive eif- a mutant were isolated. one of the suppressors (stm ) is a single copy gene which encodes a protein resembling the human translation initiation factor eif- b. disruption of the gene is not lethal to the cell, but affects growth. polysome analysis of strains with a deleted stm gene have shown that this new gene is also involved in translation initiation. it carries six repeated sequences of amino acids of unknown function and has -like the human eif- b -a rna binding domain. the second suppressor (stm ) is also a novel gene. it encodes a large protein which shows no homology to other proteins present in the data libraries. it contains high portion of charged amino acids and is rich in glutamines and serines. its function in translation initiation is currently under investigation. tobacco translationini~ationfactore~ aisencoded by a large anddiverse genefamily karl brander, therese mandel, isabelle lutziger, george owttrim and cris kuhlemeier, institute of plant physiology, university of berne, altenbergrain , ch- berne using heterologous probes we isolated cdnas coding for translation initiation factor eif- a from tobacco, eif- a is encoded by a large multigene family of which at least nine members are expressed in leaves and most if not all other organs of the tobacco plant. while screening genomic libraries we found several additional genes. two of them code for genes highly homologous with eif- a throughout the coding region, but with changes in the gkt, ptrela and dead-box motifs. a third gene is expressed exclusively in the developing male gametophyte. this eif- a-related gene may have a role in the well-documented regulation of translation in pollen. in order to study the function of these genes we have begun altering their expression in transgenic tobacco plants. seauence reauirements for a non-aug protein initiaf~jon non-aug initiation codons are still infrequent and were initially observed in animal viruses, but a number of cellular examples have now also been reported. several years ago we reported the use of an acg in the p/c mrna of sendal virus (sen) to initiate the non-structurar c' protein. we recently described a c' protein in the human parainfluenza virus type (hpiv ), which is initiated from a gug. in both cases, the c' protein is an n-terminally ebngated form of the c protein which is initiated at the second aug, in the + reading frame relative to the first aug. this aug is used to initiate the p protein, an essential component of the viral rna polymerase. unlike the acg in sen, the gug in hplvt is clearly not a weak start codon since c' is the most abundant protein expressed from this mrna both in rive and in vitro. it appears that not any upstream gug in an optimal context will function as a start codon. for instance, the p gene of hpiv cannot express a c' protein (the orf is closed by stop codons) but it does contain a gug in an optimal context upstream of the p protein aug, which could encode a p' protein. nevertheless, we have been unable to demonstrate that this gug functions as a start cedon. with the use of chimeric mrnas between hpivl and , we have shown that positions + and + (the first g of the gug triplet being + ) are the major determinants of the efficiency of gug as an initiaton codon for protein synthesis. we are currently investigating the possibility that these nucleotides may increase the initiation rate of weak non-augs such as the acg of sen. biological activity of the heterodimeric subunit srp / of the signal recognition particle is retained in fusion proteins fabrice bovia & katharina strub, d partement de biologie cellulaire, universit de gen~ve, ch- gen~ve the targeting of secretory proteins to the membrane of the endoplasmie reticulum is mediated by a cytoplasmic ribonucleoparticle, the signal recognition particle. it is composed of proteins and f rna molecule (srp rna). the smallest proteins srp and srp are required for the translational control function of srp. it effects a specific arrest in the biosynthesis of er-targeted proteins. these polypeptides bind to the srp rna exclusively as a heterodimer (srp / ). we have constructed single-chain variants of this dimer using a short peptide linker of aa to connect the c-terminus of the kd subunit to the n-terminus of the kd subunit ( - ) and vice versa ( - ). we found that both proteins bind srp rna with a similar efficiency as the wild type protein. glutaraldehyde cross-linking experiments and sedimentation analysis indicate that the fusion proteins fold into a similar structure as srp / and bind srp rna as a monomer. furthermore, they can functionally replace srp / in the elongation arrest and translocation assay. since the fusion proteins are circular permutations of each other, we can conclude from this results that n-and c-termini of both proteins have no essential role in folding, rna-binding and in mediating their biological activity. furthermore, the possibility to express an oligomeric protein as a single polypeptide facilitates the analysis of its functions as well as its structure. a s - studer, r. and hunziker, p. e., iochemisches institut der universit~t z rich, ch- z rich metallothioneins (mts) are small cys-rich proteins which are inducible by a variety of agents such as bivalent transition-metal ions, tumor promoters, cytokines and hormones. the biological role of mts is still unclear. initially postulated to serve in the sequestration of the toxic heavy metals such as cadmium and mercury, they are now believed to play a major role in the cellular metabolism of essential metals such as zinc and copper. to explore this function further we have now established a method to quantify basal mt production in several human cell lines. thus, cellular isomt contents were monitored by h.pj.c, and the rate of synthesis was followed by the incorporation of ~ss-cysteine. the results show that maximum mt synthesis and accumulation occurs when the cells enter the exponential growth phase. with increasing celldensity the mt content decreases progressively until confluence is reached. in correlation with preliminary measurements our results suggest a close relationship between the mt content, the cell growth rate and the intracellular abundance of zinc. vincent bernard, adrian etter, heinz tobler and fritz mtiller. institute of zoology, university of fribourg, fribourg (switzerland). the nematode ascaris lumbricoides undergoes chromatin diminution which causes a loss of dna in all somatic ceils. we have identified a ribosomal protein (rp) gene (coding for alep = ascaris lumbrieoides eliminated protein ) which is located within the germqine specific material. alep shows strong homologies with the ribosomal protein s (rps ). the transcription of its gene takes place only in the germ-line. d-gel electrophoresis on germ-line and somatic purified ribosome fractions shows that the alep protein (rps ) is present only in the germ-line ribosome. does the alep germ-line specific protein have a homologue in the somatic ribosomes? we have cloned a homologue of alep from ascaris somatic tissue named rps ', we are currently studying the expression pattern of this rps '. these results indicate the existence of a developmentally regulated ribosome heterogeneity between the germ-line and somatic ceils. what is the function of rps ? since ascaris is not suitable for genetic analyses, we isolated rps from the nematode caenorhabditis elegans. the c. elegans rps cdna shows only % homology at the nucleic acid level. rps is located on the c. elegans chromosome iv. the identification of a rps mutant in c. elegans will indicate its function. favre, d, l, pavlovic, j and michel, m.r. t institute of medical microbiology, university of beme, ch- berne. institute for immunology and virology, ch- ziirich we have successfully purified the recombinant c (recc) protein from spodoptera frngiperda (sf ) insect cells which were infected with acnpv-c (a). the recc protein retained the biological activities of native c protein obtained from wild type sfv (b). our results suggest that the c protein is responsible for the cellular shut-off of host protein synthesis by inhibiting the initiation of translation. we are currently investigating the molecular mechanisms involved in this shut-off. tif is a new eukaryotic initiation factor which shows % identity with the sequence of mammalian translation initiation factor eif- b. the tif gene is not essential for growth; however, its disruption results in a slow growth and coldsensitive phenotype. in vitro translation of total yeast rna in an extract from a tif gene-disrupted strain is reduced as compared to a wild-type extract. the translational defect is more pronounced at lower temperatures and can be corrected by the addition of purified yeast tif ,or mammalian eif- b. in vivo translation of ~galactosidase reporter mrnas with varying degree of rna secondary structure in the ' leader region in a tif gene-disrupted strain show preferential inhibition of translation of mrna with more stable secondary structure. chloroplast protein synthesis requires the import of elongation factors ef-tu (tuf-gene) and ef-g (fusgene). it seems that the expression of both genes is light regulated. we recently cloned and sequenced a chloroplast specific nuclear fus gene in soybean (biochim.biophys acta : - , . further analysis revealed that a second very similar (sequence, anatomy) nuclear fus- gene exists which, however, shows strong sequence diversion in the 'trailer region. several cdna clones were partially sequenced but sofar only transcripts from the fusi gene were retrieved. we currently study the promoter regions of either gene to test possible differential expression of fus genes. comparative mapping and sequencing studies of the two genes in the "-region indicate that a major dna insertion occurred distal to the coding part of both fus genes what may also influence gene expression. the rna-binding domains in the kd and kd subunits of the signal recognition part~clehavea putative~-h~licalstructure. nazarena buiand katharina strub dpt de biologie cellulalre, science ill, universit gen~ve the signal recognition particle (sr ), a cytoplasmic ribonucleoprotein is important for targeting nascent secretory proteins to the endoplasmic reticulum. srp and srpl are constituents of srpandare, together with the alu sequences of srp rna, required for the elongation arrest activity of the particle. srp and srpi form a heterodimer in the absence of the rna and bind the rnaexclusively as a heterodimer. the primary sequence of srp and srpi revealed that both proteins are highly basic and lack structural similarities to other characterized rna-binding proteins. we have therefore been interested in characterizing the molecular nature of rna-protein interactions. the analysis of n-and c-terminal deletion mutants of the proteins have shown that both proteins contribute to the formation of an rnabinding pocket, the rna-binding and dimerization domains in both proteins, are found in regions that have a predicted ~~helical structure. in sp~pi , this u-helical region is located betwen aa and i at the c-terminus; it contains the rna binding and dimerization domains adjacent to each other. our results support the model that the hydrophobic face of the putative u-helix is implicatedinprotein-protein interactions and the positively charged face in rna-protein interaction. in sp~pg, the two rna-binding domains found n-and c~ terminally, also have a predicted amphipatic s-helical structure. it has been proposed that the decline in protein synthesis observed in aging organisms may result from a decrease in the protein synthesis elongation factor ef-la. john shepherd's finding that transgenic drosophila melanogaster carrying an additional copy of the ef-la gene have an extended lifespan further indicated that the ef-la gene may play an important role in determining longevity (shepherd et al., ) . in order to test this hypothesis, we have quantitated ef-la mrna, ef-la protein, as well as catalytic ef-la activity in john's flies. young and aging ef-hx transgenic (ef) and control lines (( ) were examined. because the the additional ef-lc~ copy is under the control of the hsp promoter, flies were aged either at ~ or at ~ the results show that transgenic ef flies do not express more ef-lct than the control flies, neither at the rna nor at the protein level. the question arises, whether the lransgene can be induced at all. this was tested by doing rnase protection assays. transgenlc message can be detected only in ef flies heat shocked at ~ but not in ef flies grown at ~ nor in control flies at ~ from this experiment we conclude that the transgene is indeed inducible, but is not expressed in detectable amounts at ~ we conclude that the difference in the lifespan does not result from the overexpression of the ef-la transgene. phenobarbital (pb) induces the expression of liver enzymes involved in the metabolism of drugs and other foreign compounds (xenobiotics). these enzymes include several cytochromes p , nadph:p reductase, aldeyde dehydrogenase, epoxide hydrolase, udp-glucoronyltransferases and glutathione-s-transferases. many other proteins not yet recognized may however be induced by pb. our first approach therefore is aimed at identifying gene products which respond to pb treatment. as model systems we are using either chicken embryos (induction in ovo) or primary cultures of chicken embryo hepatocytes (induction in culture) (althaus et al., jbc pp , . to differentially display mrna of induced vs non-induced cells we are using a rt-pcr technique with sets of random primers. electrophoretic separation of amplification products allows to identify mrna species which are induced (or decreased) following treatment. data obtained from these experiments and sequence information revealed from extracted pcr products indicate that pb treatment results in the transcriptional avtivation (or repression) of a variety of so far unknown genes. antithrombin -binding heparan sulfates from ovarian granulosa cells hosseini g., de agostini, a., clinique de st rilit , hcug, gen~ve. at the time of ovulation, several serine proteases of the plasminogen activator and coagulation cascades are activated in the ovary. these proteolytic activities are tightly controlled in time and space, and the heparin-activated serpins plaminogen-aetivator-inhibitor- , protease nexin- and antithrombin (at) are present in the follicular environment. we have observed that granulosa cells produce a subset of heparan sulfate proteoglycans that bind and activate at. these at-binding heparan sulfates (ahspgs) endow the vascular endothelium with antithrombotic properties. we are now examining the role of ahspgs in the extravascular compartment formed by the ovarian follicle. using i-at binding assays, we have detected ahspgs on granulosa cell surfaces and culture media, in amounts comparable to those found for endothelial ceils. after h of stimulation by the gonadotropin fsh ( - ng/ml) granulosa cells increased the amounts of ahspgs they released in culture media by - -fold, while keeping their cell surfaceassociated ahspgs constant. analysis of s-labelled hspgs revealed that heparan sulfates constitute about % of the surface-associated and % of the soluble granulosa cell glycosamineglyeans. finally, using i-at autoradiography on rat ovary cryosections, we have localized ahspgs on the granulosa cell layers of large antral follicles, while ahspgs could not be detected in smaller follicles. these data suggest that granulosa cell ahspgs could be critically located to modulate proteolytic activities in the changing environment of the growing follicle. division, cibabasel, switzerland. the development of chelators which can mobilise excess storage iron (fe) and permit its excretion is necessary in the treatment of fe overloaded diseases. although oral administration of such a pharmaceutical is highly desirable, no orally active fe chelator is so far in regular clinical use. searches for orally active and safe fe chelators require appropriate animal models of fe overload in order to evaluate the potential of these drugs. an animal model is particularly useful for testing the capacity of new fe chelating ce~oounds in enhancing the mobilisation of clinically relevant storage iron. iron loading in animals was achieved by dietary supplementation with carbonyl fe, fe fumarate and , , -trimethylhexanoyl ferrocene (tmh-ferrocene). liver fe concentrations were increased - times above normal levels. tmh-ferrocene was the most efficient compound to induce stable liver fe overload. the orally given con'pounds induced a predominantly hepatocellular iron distribution and was found distributed among reticuloendothelial cells only at a later stage. this pattern of liver fe storage is similar to that observed in the early stages of primazy ha ~ochromatosis and late stage of transfusional hae~ochromatosis. eeeently, we and ethers have cloned and characterized novel transcription factors called peroxisome proliferator-actirated receptors (ppak). they belong to the superfamily of nuclear hormone receptors as the steroid, thyroid and retinoid hormone receptors. ppars are activated, beside xenobiotic peroxisome proliferators, by natural fatty acids and induce, together with the receptor for -cis retinoic acld (rxr), the transcription of genes involved in the ~-and ~-oxidation of fatty acids. ppar and rxr bind as heterodimers to the ppar response element (ppre), consisting of a direct repeat of aggtca-iike sequences with one intervening nucleotide. now, we show by gel retardation analysis and transient transfection assays that ppar/rxr heteredimers also bind to and transactivate gene transcription through estrogen response elements (ere). the ere is a palindrome with aggtca half-sites separated by three nucleotides. the selectivity and specificity of the transactivation through an ere was demonstrated by the testing of various related palindromic and inverted palindromic repeats of aggtca sequences, which were all inactive. the possible activation of estrogen responsive genes by fatty acids via ppars in vivo is discussed especially with regard to a debated role of fatty acids in breast cancer. the purpose of this study was to evaluate whether intermittent doxorubicin (dox) treatment in vitro selects for multidrug resistance in rpmi myeloma cells and, if so, to determine the underlying mechanism(s). cells were exposed to constant doses of dox for days alternating with growth in dox-free medium for days. after > months of treatment, the cells developed an atypical mdr phenotype with -to -fold resistance to topo ii poisons. cross-resistance to vincristine and taxotere was < -fold, sensitivity to cisplatin and melphalan remained normal. efflux of mdr drugs was incresed with no effect of verapamit; subcellular dox distribution was normal. expression of topo ii a was found to be reduced by around and - % at the rna and protein level, respectively. minimum mdrt rna overexpression was detected when using rt-pcr; p-glycoprotein was not detectable. mrp rna expression was increased by - % relative to the wild type cells without detectable p . this data suggests that atypical mdr might play a role in acquired dox resistance in human myeloma. mucosal surfaces cover a vast area in the human body. they satisfy its need to communicate with its environment, e.g. in terms of nutrient uptake and sexual reproduction. however, at the same time they offer a vulnerable gate for invasion by pathogenic microorganisms. for immunoprotection of these regions, large amounts of iga antibodies are produced in the bloodstream and transported across the epithelial barrier to the lumen by means of polymeric ig receptor. there the receptor is cleaved, and its extracellular portion remains associated as secretory component (sc) to the secretory iga complex. in the context of a project aiming to mass production of secretory iga as a passive vaccine, we evaluated a series of eukaryotic expression systems for production of sc. the cdna encoding polymeric ig receptor was engineered in a way that only its extracellular domains corresponding to sc were expressed in yeast, in insect cells infected with recombinant baculovirus, and in mammalian cells infected with recombinant vaccinia virus. furthermore, a c-terminal poly-histidine tag was introduced for simple purification of the products. we will show optimization of expression levels for each of the systems as well as a quantitative and qualitative comparison of the products. recombinant vaccinia viruses (rvv) that express gone products from other organisms has become a popular and widely used laboratory expression system. our current interest is directed toward the production of secretory component (sc), a subunit of secretory iga antibody complex involved in mucosal immunity. toward this goal, we constructed rvv governing sc transcription under the control of two viral-based and the t bacteriophage promoters. expression of the protein was assessed in several mammalian cell lines, such as human tk-, human hela (grown in suspension or as a monolayer) and monkey cv-i. optimization of infection parameters and culture conditions were also performed. institut de biologic animale de runiversit~ de lausanne protection of mucosal surfaces is mediated by secretory iga ant/bodies (alga) constituted of dimeric iga and secretory component (sc) . toward the aim of reconstituting slga complexes in vitro and using them for passive oral immunization, sc has been produced in yeast, insect and mammalian expression systems. the gone encoding sc has been engineered so that a) the c-terminus of the protein carries a xhis tag and b) the newly synthesized polypeptide is released in the culture medium. culture conditions have been established to permit recovery of sc in serum-free medium. purification procedures based on nickel chelate and classical chromatographies have been optimized to yield sc under native conditions. finally, in vitro reassociation with purified dimeric igas obtained from hybddomas has been used to assess biological activity of recombinant sc. using a magnet-assisted subtraction-hybridisation technique (mast), cdna clones were isolated that are expressed in nqrm~l l~nq %issue but n__d_i expressed in the corresoondina nsclc tissue, ii of the edna clones are highly homologous to edna sequences for the following proteins: pulmonary surfactant proteins sp-a and sp-b; receptor for advanced glycosylated endproducts of proteins (rage); calmodulin-like protein; natural killer gone (nkg ); matrix gla protein (mgp); glutamine synthetase; ubiquitin; vascular smooth muscle alpha-actin; cytoskeletal beta-actin; vimentin. the remaining edna clones may represent parts of still unknown genes. the mast edna clones were derived from a patient who developed squamous adenocarcinoma. northern blot analysis of normal/tumour rna from three other nsclc patients showed that the lack of gone expression was independent of nsclc type and stage. enzymatic methylation of cytosines in mammalian dna is believed to play a fundamental role in basic gene regulation. methylation in the vertebrate genome is restricted to cpg dinucleotides at the c- position of cytosine. in vitro studies have shown that methylation can drastically influence the dna structure. cpg islands remain unmethylated in normal cells, whereas several immortalised, transformed cell lines contain partially methylated cpg islands. transcription of genes can be inhibited by methylation of cpg's. this modification contributes e.g. to the stable repression of genes on the inactivated x chromosome of females. the collagen vi genes show typical cpg islands in their promoter region and they were shown to be down regulated at the transcdptional level in src or myc transformed cells. given these facts we dee~ded to investigate the effects of methylation in the collagen vi gwene. e could demonstrate that the promoter activity of (x (vi) chicken collagen gone was strongly diminished by methylation in vitro. furthermore dna methylation completely prevented binding of a novel transcription factor to the promoter, whereas binding of sp was not affected. to show evidence of methylation in vivo, we are currently iocalising the exact positions of methylated cpg's in normal and transformed cells by genomic sequencing. alterations of putative tumour suppressor genes in human non-small cell lung carcinoma (nsclc). r. shipman and c.u. ludwig, molecular oncology, lab (zlf), basel, ch- chromosomal subregion llp displayed non-random allelic loss in nsclcs. llp contains the genetic loci for catalase (cat), the wilms' tumour gene (wti) and the follicle-stimulating hormone ~-chain (fsh~). % of the nsclcs were deleted at cat suggesting that loss of a gene(s) near cat may be involved in the progression of nsclc. yac clones containing the cat locus are being prepared for use in a direct selection procedure for cdnas encoded at this region. although % of the nsclcs were deleted at wti, no mutations were detected in the remaining wti allele. wti expression was subsequently demonstrated in fetal lung, fetal hematopoietic tissue, normal adult lung and nsclc. allelic deletion at p , immuno-histochemical and sequence analysis of the p gene were also examined in our nsclcs. these analyses support the contention that aberrant expression of the p gene is a pivotal event in the progression of nsclc. $ - recently, a number of advances have been made which enable the experimentalist to study the effects of low levels of ionizing radiation. the results suggest a threshold to radiation damage exists and that above a critical level, recovery mechanisms in the cell are induced and the biological response falls. thus low levels of radiation cause greater than expected levels of biological response. this has been demonstrated in studies of mutations, cell survival and cancer induction. because the low dose-rates and the low doses per fraction result in the total doses being small, the magnitude of the biological effects induced is insufficient to warrant alarm. identification of genes associated with the revertion of the malignant phenotype of a rhabdomyosarcoma cell line. michele genini, sabina solinas toldo*, ruedi fries* and beat w. schafer, dept. of pediatrics, university of ziirich, steinwiesstr. , ziirich, and *institut for nutztierwissenschaften, eth ztifich, ztirich. the human rhabdomyosarcoma cell line rd is tumorigenic and differentiates very poorly despite the expression of the myogenic factors myf and myf . therefore it can be regarded as natural mutant. to identify genes which are involved in suppression of tumorigenicity or enhance differentiation we applied two different approaches. since the development of human embryonal rhabdomyosarcoma has been associated with abnormalities of chromosome band p , in the first approach we transferred a normal human chromosome into rd ceils via microcell fusion. the microcell hybrids did not show a higher degree of differentiation. suppression of tumorigenicity was observed in one clone but is probably independent of chromosome , as it was shown by chromosome specific painting. ' in the second approach we applied a subtractive hybridization procedure between primary myoblasts and rd cells to find genes specific for the normal phenotype. these genes will be tested in vivo for induction of differentiation and/or tumorsuppression. deficient synthesis of the gpi anchor is the molecular basis of idiopathic paroxysmal nocturnal hemoglobinuria ("pnh") and a similar phenotype can accompany aplastie anemia cpnh-iike"). the population expressing cd , a gpi-anehored membrane glycoprotein, was quantified by flow cytometry in samples from patient p.g. (pnh) and patient m.m. (pnh-like) and amounted to % (p.g.) and % (m.m.), respectively. t lymphocytes from both patients were isolated, expanded and cloned. from both patients, clones expressing the cd marker (cd +) and cd deficient clones (cd -) were obtained. takeda et al. (cell , , - ) showed that in some (cd -) bcell clones, a deletion of the p g-a gene product is responsible for the expression of the pnh-phenotype. pcr analysis of t cell mrna from extracts of both cd + and cd -clones of patient m.m. (pnh-like) using pig-a specific primers revealed a band pattern from which can he concluded that no deletion is present in the pig-a gene product. this result suggests that cd -t lymphocytes of the pnh-like patient express a normal pig-a gene product; a disorder in regulation of pig-a expression or an unrelated biosynthetic defect may explain the cd phenotype in these t cells. further work is aimed at defining the differences in biosynthetic defects of pnh and pnh-like cd -t cell clones. supported by grant - - of the snsf to egb. w@ have recently employed an experimental approach to turn the function of "nuclear messengers" such as c-fos (and c-jun) on and off at will in polarized me/nmary epithelial cells. to this end we used constructs encoding the c-fos (and c-jun) genes fused to the hormone-binding domain of the human estrogen receptor, designated c-foser (and c-juner), we could show that short-term activation ( mins.) of c-foser by estradiole (e ) led to the disruption of epithelial cell polarity within hours, as characterized by the expression of apical and basolateral marker proteins. during the next - days, however, the cells fully regained their polarized phenotype. conversely, long-term activation ( - hours) caused the irreversible loss of epithelial cell polarity, leading ultimately to an epithelial-fibroblastoid cell transition. these cells underwent dramatic changes in gene expression including the complete loss or reduction of epithelial markers such as e-cadherin/uvomorulin, zo-i and cytokeratins, and the de novo expression of mesenchymal proteins like vimentin, type i collagen and fibronectin. expression of the v-ha-ras oncogene (acting upstream of c-fos) did not markedly influence epithelial cell organization when the cells were grown on plastic. however, when cultured within type i collagen gels in the presence of serum, or when injected into the mammary glands of nude mice, rapid cell growth and a clear epithelial-fibreblastoid cell transition was observed. growth properties determine the organ colonization ability of a metastatic murine melanoma cell line. the ability of metastatic tumor cells to specifically colonize distant organ sites strongly depends on the interaction of the metastatic cells with the local organ environment. we have selected a b routine melanoma cell fine (b -ls ) which has an increased ability to colonize the liver, and compared its behavior with either its parental (b -f ) or inng-specific (b -f ) cell line. we have found that adhesion in vitro and organ lodgement in vivo were not different for ls and f . in contrast, b -ls grew at slower rates than the other lines both in vitro and in vivo after subcutaneous or intra-foot-pad injection. analysis of tyrosine-phosphorylated proteins indicated a higher phosphorylation activity in b -ls cells, which might suggest an altered regulation of some kinase(s) in this cell line. intercellular contact with hepatocytes in vitro, or the liver in vivo could restore an efficient growth of b -ls , enabling thus these cells to colonize this organ much better than others. hepatocytes or liver plasma membrane (p.m.) extracts conserved the growth stimulatory activity towards b -ls ceils. chromatographic fractionations of these extracts showed that a major growth sfimulatory protein in this fiver p.m. fraction turns out to be ttansferrin, which in the liver appears to exist in at least two different mature forms. protein import into mitochondria requires atp in two locations: outside the organelle, and in the matrix space. depletion of matrix atp arrests translocation of precursors across the inner membrane, but does not prevent precursors from crossing the outer membrane. as a result, proteins that reach the inner membrane or intermembrane space without passing through the matrix are often sorted normally in atp-depleted mitochondria. external atp is used to maintain precursors in a translocationcompetent state. however, some precursors can fold and still remain translocation-competent, and import of these precursors is independent of external atp. with the folded cytochrome b precursor, it is matrix atp rather than external atp that drives translocation across the outer membrane. thus matrix atp generates a pulling force that can drive both the translocation and the unfolding of precursor proteins. mitochondrial hsp probably functions as the atp-dependent import motor. arsenijcvic d, dulloo ag & girardicr l, dpt of physiology, cmu. geneva, ch. the relationship between body weight, daily energy expenditure (assessed by indirect calorimetry), and immune status (determined by lymphocyte proliferation tests) was investigated in swiss webster mice maintaining a stable body weight several weeks after infection with toxoplasma gondii(me ). following an initial period of weight loss (infection-induced cachexia), the surviving mice could be differentiated into two main groups: (i) those showing a partial regain in body weight (the infected gainers) and eventually stabilizing al a mean body weight % below a non-infected control group, and (if) those showing no weight regain (the infected non-gainers) and eventually stabilizing at % below control levels. immune function was found to be markedly suppressed in the infected non-gainers, whereas it was normal (and similar to control levels) in the group of infecled gainers. daily energy expenditure (and food intake), after normalisation for differences in body weight, was found to be the same in both infected gainers and non-gainers, but lower by % when compared to controls (p< . ); thereby indicating that the infected groups were able to increase their overall efficiency of food utilization in response to the low food inlake. in conclusion, this study conducted during a weight stable phase of chronic infection shows a clear-cut association between the inability to regain body weight and impaired immune function. in contrast, no relationship was found between metabolic rate and body weight nor between metabolic tale and immune function, but the chronically infected mice exhibited the same phenomenon of enhanced metabolic efficiency characteristic of chronic underfeeding per se. identification and functional analysis of chaperonin , the groes homolog of yeast mitochondria. biozentrum, university of basel, klingelbergstrasse , ch- basel, switzerland. phone ++ - - , fax ++ - - . the mitochondrial chaperonin system consists of chaperonin (cpn ; also termed hsp ), which is homologous to e. cull gruel, and chaperonin (cpnlo), which is homologous to e. coil groes. we have used a functional assay to identify cpnl of yeast mitochondria. when dimeric rubisco is denatured and allowed to bind to yeast cpn , subsequent refolding of the enzyme is strictly dependent upon yeast cpnl . in the presence of mgatp, yeast cpn and yeast epnl form a stable complex that can be isolated by gel filtration. the atpase activity of hsp is not inhibited by complex formation with cpnl . a different result is obtained with the e, coil system, where groes is able to completely inhibit the atpase activity of gruel ( ). to test the in vivo role of cpnl , we have cloned, sequenced and disrupted the corresponding nuclear gene cpnio. this gene encodes a protein of , da that is imported into the mitochondrial matrix without detectable cleavage. haploid cells lacking a functional copy of cpnio fail to grow at temperatures between ~ c and ~ c ( ). ( ) rospert, s. et al. ( ) proc. natl. acad. sci. , in press. ( ) rospert, s. et al. ( ) febs lett., in press. characterization of yeast mitochondrial heat shock protein , l. bolliger, b. s. glick and g. schatz, biocenter, university of basel, basel. mitochondrial hsp (mhsp ) is thought to use the energy of atp hydrolysis to pull precursor proteins across the mitochondrial membranes. to facilitate the purification of yeast mhsp , we used the cloned gene (a gift of dr. n. morishima) to modify mhsp with a six-histidine tag at its c-terminus. this construct supports growth of yeast cells lacking wild-type mhsp . we developed a purification procedure that allows us to recover this tagged protein with a purity of about %. experiments are in progress to characterize the nucleotidedependent interaction of purified mhsp with unfolded proteins. in addition, we are looking for partner proteins that may modulate the action of mhspt . we have copurified a protein of about kd together with mhsp . the kd protein could be released from mhsp by atp or adp. when tryptic fragments of this protein were subjected to microsequencing, one fragment showed strong homology to the grpe protein of e.coli. the transmembrane electrical potential of mitochondria (awmit) can be assessed in single hepatocytes by using epifluorescence microscopy. for this end the rhodamine fluorescence of a mitochondria-rich (fr) and a mitochondria-poor region (fp) are measured. the ratio of these signals depends on the awmit according to a modified nernst equation (reber b .f.x., somogyi r. & stucki j.w. ( ) , bba, , - ) . to calibrate this method the cell membrane was permeabifized for monovalent cations with nystatin and gramicidin. then the cytosolie k + was replaced by na + from a k+-free bath solution. subsequent addition of valinomycin made the mitochondrial membrane permeable for k + ions. by the addition of different k + concentrations to the bath, the awmit could be clamped to defined values. the relation between the ratio fr/fp and the awmit could be well fitted to our modified nernst equation. however, the calibration curve flattens out at potentials higher than about inv. therefore the detection limit of changes of a~mit within the physiological range is about - mv. maximal stimulation of the na+/k + atpase by addition of nystatin to the na + containing bath medium caused a drop of awmit of about inv. subsequent addition of ouabain resulted in an almost complete reversal of this drop. this shows that changes in atp consumption can be assessed via changes of audmit. schneiter ph., di vetta v., j quier e. and tappy l. institut de physiologie de l'universitg bugnon , lausanne. the metabolic effects of an exercise performed in the fasting or fed state were studied in subjects over a hour period in a respiratory chamber. a mixed meal containing c labeled carbohydrates was ingested at : am and an exercise session (walking at km/h rain, slope %) was performed at : am (fasting) or am (postprandial). net carbohydrate (net cho ox) and lipid oxidation (indirect calorimetry) and oxidation of exogenous carbohydrates (exo cho ox, breath co ) were measured. glycogen breakdown was calculated as net cho ox -cho exo ox, and glycogen synthesis as cho in -cho exo ox. energy expenditure over hours was similar but net cho ox was % lower, lipid ox % higher and exo cho ox % lower when exercise was performed in fasting vs fed state; moreover, glycogen synthesis was increased by % (p< . ), while glycogen breakdown was increased by % (p= . ). in conclusion, a min exercise in the fasting vs fed state a) increases utilization of endogenous lipids, b) enhances muscle glycogen turnover over a hour period. hormonal regulation of e-abl tyroslne kinase by fusion to a steroid binding domain t. mattioni, o. chini hooft van huijsduijnen, p.k. jackson and d. picard d partement de bielogie cellulaire, universit de geneve, ch- geneve the activities of a wide variety of proteins can be subjected to hormonal control by fusion to the hormone binding domain of steroid receptors. we show that this strategy can also be applied to a tyrosine kinase. in particular, transforming derivatives of c-abl become hormone-inducible oncogenes by fusion to a steroid binding domain. it is noteworthy that the hormone-inducible transformation of nih t cells is completely reversible upon removal of the specific ugand. reversion experiments reveal another facet of abl: its ability to inhibit cell proliferation. hours after hormone depletion, cells are not only morphologically reverted, but the cell cycle appears to be blocked in g . a cytostatic effect of abl overexpression has been reported by several groups but, until today, it could only be examined by comparing different abl derivatives or the same derivatives in different cellular contexts. in contrast, our hormone-regulable system has the advantage that the two distinct functions of abl (transformation versus growth inhibition) can be studied using the same derivative expresed in the same cell line. we are using the hormone inducible system to investigate the mechanisms regulating the transtorming and the cytostatic effects of abl in a variety of cell lines. jiewu yang and herbert zuber institute for molecularbiology and biophysic, eth, z'tirich enzymes from thermophilic organisms show higher thermostability and temperature optima than those of mesophilic organisms. it has been shown that these properties are based on a specific structure of the protein. the primary structure of thermophilic (b. stearothermophilus) and mesophilic(b, megaterium) triosephosphate isomerase(tim) have been determined by cloning and sequencing of the tim genes. comparison of the primary structures of the thermophilic and rnesophilic enzyme showed specific amino acid substitutions, particularly in the c~-helices of the tim barrel, which could play a critical role with respect to thermostability. we have consmacted mutants by exchanging (x-helices between tim of b. stearothermophilus and b. megaterium. helix h , h and h of tim from b. megaterium, corresponding to amino acid residues - , - . and - respectively, have been exchanged. the properties (thermostability and temperature optimum) of the hybrid-mutants were compared with the wild type enzymes. in a comparative investigation of structural and functional parameters related to the oxidative substrate metabolism in skeletal muscle cells of dogs and goats, a close relationship was found between intracellular lipid stores and mitochondria. as revealed in serial sections with subsequent em analysis, each lipid droplet was in direct contact to one or several mitochondria. quantitatively, % of the lipid droplet surface in goats and % in dogs were in direct contact to outer mitochondrial membranes or -in other terms - % of outer mitochondrial membranes in goats and % in dogs were in direct contact to lipid deposits. these findings were in good agreement with the physiological data showing a times higher oxidation rate of fatty acids drawn from intracellular stores for dogs than for goats. human muscle adapts to altered load with changes in the expression of enzymes involved in energy metabolism. endurance exercise in humans is known to increase the oxidative capacity of skeletal muscles (hoppeler, int. j. sports med. ( ), , ) . at the ultrastructurai level, a higher mitochondriai content contributes to the higher oxidative capacity of skeletal muscles. little is known about the molecular mechanisms that lead to such adaptations in humans. the expression of mitochondriai components involves complex regulation of both mitochondriai and nuclear encoded genes. we have developed a pcr approach to quantify dna and rna from human skeletal muscle cryostat sections and addressed the question, whether the structural adaptations in endurance trained athletes are accompanied by concomitant changes in respective rna steady state levels. preliminary data indicate a higher concentration of coxiv and coxl ( . and . times, respectively) in trained subjects. no differences were observed for mtdna, despite a two fold higher mitochonddal content. human skeletal muscle contains three main fiber types which are based on the myosin heavy chain composition of the individual fiber, the isoforms of other contractile proteins within a given fiber type can vary leading to a continuum of different phenotypes. we are studying the fiber type specific expression of the mrnas of myosin alkali light clmins (mlc) using in situ hybridization, especially the rarer slow form mlc lsa. differences were found between shoulder muscles and leg muscles: m. deltoideus yielded stronger signals for mlc lsa and mlc lsb in the least glycolytic type i fibers (ia), weak but detectable signals for mlc lsb and mlc lf/f mrna in more glycolytic type i fibers (ib) and strong signals for mlc lf/ f in type ii fibers. in m. vast. lat. mlc lsb was strongly expressed in type ia as well as ib fibers, mlc lsa mrna was only found in some ia fibers. mlc isa has been shown to be expressed at a particular time point during muscle development and regeneration. in a biopsy of a becker dystrophy patient, we found very strong signals in some small fibers displaying the morphology of regeneralmg fibers. thus this probe could be useful to dete~t regenerative processes in pathological muscle samples. the aim of this study was to validate the complementarity of two new non-invasive methods for the assessment of autonomic regulations. reactions to a moderale exercise ( w on a cycloergometer) were compared in cardiac transplant recipients (ctr) who modulate their heart rate (hr) by means of circulating calecholarnines, their heart being donervated, and in normal subjects where such exercise level doesn't stimulate the sympathetic system. the methods used were: ) the spectral analysis of hr variability where the high frequency component (hf, above . t z, related to the respiratory frequency) is a marker of the vagal activity, the low frequency component (lf; at about . hz) depends on both sympathetic and parasympathetic activities and the lf/rf ratio indicates changes in the sympatbo-vagal balance; and ) a pure sympathetic index obtained from the amplitude of the t-wave of the ecg (twa) which is decreased by adrenergic stimulation of the myocardium. our results indicate that in both groups hr was increased by the exercise. in ctr, a net decrease in twa (- _+ %, n= ) was measured. contrasting with this, in normal subjects, no change in twa (+ _+ %, n= ) but a decrease in beth hf (- _+ %, n= ) and lf (- _+ %, n= ) without change in lf/hf ratio (- +_. %, n= ) were observed. these results indicate that ctr subjects increase their hr by an adrenergic stimulation, whereas the normal subjects regulate their hr for such moderate exercise only by redudng their vagal tone. there was a significant relationship (r= . , p< . ) between ps and the ree. the slope of the regression line indicated a net cost of ps of . kcal/g. we conclude that obesity in children is associated with an absolute increase in whole-body protein turnover consistent with the increased ffm and ree. the "glucose paradox" in the anoxic and reoxygenated embryonic myocardium raddatz, e., tran, l., rochat, a.-c., kucera, p. and de ribaupierre, y. institut de physiologie de l'universitd, ch- lausanne. the hearts of -day-old chick embryos explanted in vitro react rapidly, reversibly and reproducibly to anoxia and reoxygenation. this work deals with the effects of exogenous glucose on the mechanical activity of the hearts submitted to strictly controlled normoxia-anoxia-normoxia transitions. the anoxic periods lasted from to seconds. instantaneous heart rate, amplitude of contraction, velocities of contraction and relaxation of the ventricle wall were determined optically. in presence of and mmol/ of glucose ) the arrest of cardiac activity under anoxia was delayed by and %, respectively, ) the duration of the postanoxic cardioplegia was reduced and ) recovery was faster. paradoxally, these concentrations of glucose induced arrhythmias both during anoxia and reoxygenation. the incidences of such arrhythmias increased with the duration of anoxia. the absence of glucose or blockade of glycolysis suppressed arrhythmias. image analysis showed that the observed myocardial dysfunctions originated in the atrium and were sometimes associated with disturbances of propagation. the microinjeetion of okadaie acid into incompetent oocytas leads to the release of the prophase block with entry into m-phase. these g /m-like alterations include chromatin condensation, germinal vesicle breakdown (gvbd), microtubules reorganization and formation of an abnormal (often multipolar) spindle of meiosis i, with chromosomes not aligned on the metaphase plate. the polar body is never extruded. moreover okadaic acid microiujection induces the emergence of phosphorylated cytolasmic foci as detected by the monoclonal antibody mpm- , known to react with mitotic phosphoproteins associated with centrosomes. the kinetics of gvbd following mieroinjection is extremdy retarded ( - hrs) when compared to spontaneous maturation ( ran-lhr) and depends on the oocyte diameter. interestingly, when inc~ompetent oocytes are cultured during hrs and then microinjeeted with okadaic acid meiosis resumes within a short delay ( -rhrs). mpm- epitopes are present on ecntrosomes only after okadaic acid injection. following gvbd one of the step depending on protein synthesis, the expression of tissue type plasminogen activator, is triggered. indeed oa-injected incompetent oocytes produce small amounts of this activator. altogether these results argue for an effect of okadaic acid favoring entry into mphase including microtubule organization, centrosomes phosphorylatiou and the recruitment of one of the masked mrnas (tpa). st~rillt~, ncb~, c~- l g~. ~t rs-inse~, monf~llier, france. meiotic reinitiation of the mouse oocyte is caracterized by a slow entry into metaphas~ i, b ~jinnin with germinal ve~ir break@own (gvbd) and ending with spindle formation. it is accompanied by a cascade of protein kinases and phosphatases increasir~g protein phosphorylation. the activation of histone hi kinase (maturation promoting factor, mpf) and of the hapk have been compared during spontaneous or okedalc acid (oa)-induced rhetoric reinitiation. in spontaneously maturing oocytes, hist ne hi kinase activity increases before gvbd ( x) and is a~sociated with the disappearance of the upper (tyrosine phosphorylated) migrating form of p cdc , following gvbd, histone hi klnase activity culminates ( x) in metaphase i. activation by phesphorylation of p mapk occurs after gvfld. in contrast, no increase of histone hi kinase is detec~cable in oocytes induced to reinitiate meiosis by a transient exposure %o oa, neither before gvbo nor during the following hours of culture. the upper migrating form of p cdc is present for houra. the activation of p mapk b~lins before gvbd. furthermore, when oa is applied on coclrces microinjected with p sucl, neither iocrease of histone hi kinase activity nor p cdc dephosphorylation are observed although gvbd is induced; p mapk is activated. altogether these results suggest that gvbd may or may not be associated with a detectable activation of histene hi kinase, depending on the experimental conditions. activation of ps cdc and p mapk are separable events. we propose that, in the mouse c~yte, oa might be able to aetivaf an alternative pathw~ leading to gvbd that is cdc -independent and that involves p mapk activation ensuing mpf-independent phosphorylations. s - urich, k., basel, switzerland transformation of cells by polyomavirus is mediated by middle-t antigen. this viral protein is able to form complexes with src family tyrosine kinases (e.g. c-src), phosphatidylinositol- -kinase (pi -k) and phosphatase a (pp a). c-src associated with middle-t is constitutively dephosphorylated at tyrosione , a site negatively regulating the activity of this enzyme. this results in high tyrosine kinase activity in interphase and mitotic polyoma-transformed cells. middle-t is transiently hyperphosphorylated during mitosis as reflected by an increase in the apparent lvl, on sds acrylamide gels. two putative phosphorylation sites for a cyclin-dependent kinase present in middle-t, threonine and threonine , are also phosphorylated by purified p ~a~ in vitro. we constructed mutant middle-t genes where individual phosphorylation sites were converted to alanine residues. mutants lacking threonine were still able to associate with all cellular enzymes described above yet defective for gell transformation. interestingly, the defect of this mutation could be compensated by additional changes in the middle-t protein sequence introduced further downstream in a domain suspected to play a role in the targeting of this protein to intracellular membranes. $ - schmidt, s., fa~khauser, c., and viesturs, s. isrec, epalin~es, switzerland little is understood of the mechanisms which regulate cytokinesis, or how it is integrated with mitosis. we have cloned a number of genes from the fission yeast s. po~be which are required for the initiation of septation and are characterising them. defects in the cdc and cdcll genes result in continued nuclear division without cytokinesis, while cdcl mutants undergo multiple rounds of septum formation without cell cleavage. our analysis suggests that phosphorylation will play an important role in the regulation of cytokinesis and its integration with mitosis. a functional cdcl product is required for maintenance of p r activity in mitosis and the primary sequence of the cdc protein indicates that it encodes a protein kinase. data concerning the role of the cdc , cdcll and cdcl genes will be presented. rfc was originally identified in human cell extracts as one of the cellular factors essential for the replication of sv origin-containing dna in vitro. it is a five subunit protein with one large subunit of -i kda and four small subunits of - kda. rfc binds specifically to primertemplate structures at the '-end of the primer. together with proliferating cell nuclear antigen (pcna) it serves as a primer recognition factor for dna polymerase ~ and ~. to show the invobement of rfc in cellular dna replication we are currently cloning s. cerevisiae rfc. the amino acid sequences of a number of tryptic peptides have been obtained. this information was used to amplify parts of the s. cerevisiae genes by per and to screen a genomic library with these probes. three genes were cloned so far. the large subunit, rfc , codes for a kda polypeptide that is % identical to the large subunit of human rfc (lirfc). the sequences for two of the small subunits were also obtained. rfc , coding for a kda polypeptide, is % identical to the hrfc kda subunit. rfc , coding for a kda polypeptide, is % identical to the hrfc kda subunit. there is also considerable similarity between the different subtmits. rfcj, rfc and rfc , as well as the hrfc subunits, have consensus sequences for a atp/gtp binding site. all three genes are essential in s. cerevisiae. p is a major tumor suppressor gene which spefically interferes with the onset of e phase. we have therefore cxa~ined the possibility that p modulates the normal functions of enzymes and proteins directly involved in dna replication. we have shown that human wtp protein binds physically to calf thymus single stranded dna binding protein a, rp-a, we have also found that p blocks dna helicases in a typical strand displacement assay. this may reflect the fact that p is able to reanneal complementary dna single strands. since both wt and some mutant forms of p share this function, they are unlikely to be related to the p tumor suppressor activity. to our surprise, among the helicases which were tested we determined that the p inhibition could be released by rp-a in the case of cellular but not in the case of viral dna helicases. additionally, we found that p can partially inhibit dna polymerase ~ and s holoenzymes on singly primed mi dna. this inhibition, however, was only evident when e. coli ssb was substituted for rp-a. our data thus show that p exerts an inhibitory effect on several enzymes involved in dna replication and dna repair. the p -rp-a interaction may function to protect replication enzymes from inhibition by p under certain physiological conditions. catalytic subunit showed that pol ~ is the most conserved replicative pol (cullmarm g., hindges r., berehtold m.w. and htibscher u., gene, in press). we synthesized pcr primers and cloned three fragments spanning the whole catalytic subunit. the resulting pcr products, called n-term, middle and c-term have a size of , and bp, respectively. the primer for the n-term and middle fragments contained a histidin tag in order to purify the recombinant proteins by using a ni-nta column. these fragments were cloned into the expression vector pgemex . because of the natural termination codon in the c-term fragment, the histidin tag was placed directly in the vector in front of the fusion protein, generating a new vector, prhh s. all three fragments were expressed in e.coli. the fusion proteins could be purified in a single step and were used to raise polyclonal antibodies. finally, the entire pol sequence was joined together and could be expressed. data on the characterisation of the antibodies and the proteins will be shown. hsp from calf thymus can influence dna polymerase under heat shock conditions. we have generated antibodies against dnak protein, the hap homolog from escherichia coil, and used them for detecting of hsp protein during its purification from calf thymus tissue. the apparently purified protein has a molecular weight of kda, and has been recognized by antibodies against dnak and eucaryotic hsp / , it possesses a very week atpase activity, which can be stimulated by different poly-and oligopeptide substrates. results will be presented showing the influence of hsp on dna polymerase r from calf thymus under heat shock conditions. identification of a vertebrate cdc mutant which is unable to complete the g /s transition. nicole sr and viestars simanis. chemin boveresses , epalinges,.isrec. s. pombe strains have been constructed which should permit the generic and molecular analysis of the events which occur in late gi when cells become committed to the mitotic cell division cycle and the initiation of dna synthesis. a number of mutants of the chicken cdc gene were eonsuueted during the course of analysis of phosphorylation sites some of which lead to the interesting phenotype of cold sensitivity when expressed in a cdc null background, when the only source of p cde is the chicken homologue of the gene, expressed from a muldcopy plasmid. in order to create a genetically tractable strain, the wild-type chicken cdc gone and one mutant into the s. pombe genome to create a strain in which cell cycle progression is dependent upon the chicken cdc gene. analysis of this strain indicates gnat the cells block predominantly before replication of dna. in order to determine whether the cells were arrested before or after the execution of the start control their ability to conjugate at the arrest point was assessed. consistent with a block before the traverse of start and contmim~ent to s-phase, cells were able to conjugate with high efficiency. further support for the view that this mutant is defective only for the g -s transition is provided by the observation that it is able to complement a cdc a l mutation in trans. this mutant is defective only for ( function and not traverse of start. tl~e double mutant is viable at the restrictive temperature of either of tile parents alld is no longer cold sensitive. further work will concentrate on cloning genes involved in the traverse of gi into s phase in fission yeast. (masson and kreis, , j. cell biol. : ) where it is localized on a subset of microtubulea. when caco- cells are grown to confluency and become polarized, e-map- expression levels increase concomitant with a higher microtubule stability. transfection of fibroblastic cells (veto), which do not contain any detectable e-map- , with cdna encoding this protein, results in stabilization of microtubules to nocodazole treatment. these data suggest that e-map- may be involved in the stabilization and reorganization of microtubules in polarizing epithelial cells. since e-map- is a stabilizing protein, its interaction with microtubulen should presumably be modified at the onset of mitosis to allow disassembly of the interphase microtubule network and formation of the mitotic spindle. indeed, immunolabeling for e-map-t varies during mitosis. in early prophase, no e-map- can be detected on the microtubules of the forming mitotic spindle. the protein is observed on the spindle microtubules of metaphase cells and staining becomes bright on the new interphase microtubules of telophase cells. analysis of the protein in cells blocked in mitosis by low concentrations of nocodazole indicates that e-map- is hyperphosphorylated. this hyperphosphorylated form of e-map- is no longer able to co-sediment with microtubules in in vitro microtubule-binding assays. phosphopeptide map and phosphoamino acid analysis show the existence of novel phosphorylation sites in e-map- during mitosis compared to the protein during interphase, we are currently characterizing the kinases involved in the modulation of e-map- activity. little is known about the specific functions of calcium-binding proteins in epithelial cells ~md in particular in tumoral cells of epithelial origin. calretinin (cr) and parvalbumin (pv) are supposed to be involved in cell proliferation. we observed the endogenous expression of cr in several cell lines from human colon adenoearcinomas, and we obtained stably transfected cells for pv in a human ovarian adenocarcinoma cell line. we studied the cell cycle in correlation with the endogenous or ectopic expression of cr and pv, respeetiveiy. g and mitotic celts are strongly labelled with the cr-antiserum , while pv immunoreactivity is dominant in interphasic, but not in mitotic cells. in cr expressing cells, the cell cycle seems to be normal and the rate of proliferation to be proportional to the percentage of positive cells. the cell cycle is inhibited at mitosis after the addition of cr antisense oligo nucleotides to the culture medium. the cell cycle is blocked in gi/s and g in the cells eetopically expressing pv. the results support the hypothesis that these two calcium.binding proteins, cr and pv, could intervene at different moments and probably with different mechanisms on the mechanism of the cell cycle in the tumoral cells studied. production of polyclonal antibodies against calretinin cr- k gander, j.-ch., gotzos, v., cello, m.r. and schwaller, . institute of histology and gen. embryol., university; -fribourg a mrna for an alternatively spliced form of calretinin, ealretinin- k (cr- k) was detected in widr cells (a cell line from a human adenocarcinoma). we therefore decided to produce antibodies specific for this protein. it is directed against the amino-acids localized at the c-terminus of the truncated protein which are unique for this protein and not present in full-length calretinin. two different approaches have been chosen to obtain a specific antiserum. we have produced a chimeric protein containing the cterminal region of cr- k and the (h) (nanp) polypeptide as carrier. the fusion protein was expressed in e. coil and purified on a nickel chelate column. as a second method, a synthetic peptide (corresponding to the last amino acids of cr- k) was crosslinked with the carrier protein klh (keyhole limpet hemocyanin). the antigens were used to immunize two rabbits. after the first boost, the sera were tested. we have observed that both antisera recognize besides the protein used for immunization also cr- k (expressed in e. coil) in a western blot specifically. no other protein bands of either e.coli extracts or from eytosolic extracts of widr cells crossreact with the antibodies. both antisera detected the protein cr- k in immunohistochemieal stainings of widr cells confirming the presence of cr- k in widr cells. the tctp (p ) is a growth related tumour protein which is expressed under translational control. homologous acidic proteins have been found in higher plants and in saccharomyces cerevisiae (ykl ) . the striking conservation of this polypeptide between these very divergent organisms suggests some important but still, unknown cellular function. to address this question, a null mutation was constructed in yeast, by deleting almost all the ykl structural gone. the deleted strain shows no detectable phenotype. therefore, we conclude that, in yeast, this gene is dispensable. the protein ykl has been overproduced in e. coil and injected to rabbits to raise antibodies. we are now characterizing the ykl expression at rna and protein level. ellenrieder,c., forrer,p., kosovsky,j., rischle, m., nueller, r. and jaussi, r., institut f~r medizinische radiobiologie, paul scherrer institut & universitgt zh, ch- villigen radiation therapy of tumors could be improved by influencing cellular radiation sensitivity. yeast strains with defective cell cycle checkpoint genes (e.g. radl, rad , radg) show increased radiation sensitivity. cross-hybridization of a probe from the tad gene on human mrna northern blots yielded a single prominent signal. experiments to clone the corresponding kb cdna are underway. we have cloned a hamster cdna encoding the homologue of human cdk . probably, this cdna encodes the p protein from hamster whose phosphorylation responds to radiation checkpoint signals and who does not bind to p sucl beads. treatment of cells with cdk antisense oligonucleotides is hoped to modulate the radiation sensitivity. proliferation of smooth muscle cells (smcs) is a major event in vascular development and atheromatous plaque formation. in order to characterize smc replicative potential, newborn rats have been injected with h-thymidine ( h-tdr) during their first week of life when most of smcs were proliferating; then, h-tdr labelling was evaluated in adult rats. depending on the number of mitosis that smcs had accomplished after the first week of life, four different smc subpopulations could be defined indicating that rat aortic smcs are heterogeneous in their replicative activity. -bromo- '-deoxyuridine (brdu) incorporation after balloon induced endothelial denudation of rat aorta in adult rats showed that smcs entering into the cell cycle were mainly devoid of h-tdr labelling, this category could derive from two distinct smc subpopulations: smcs which were arrested in their proliferation before or just after birth or smcs which had actively replicated during development. thus, one (or two) subpopulation(s) of rat aortic smcs, characterized by a particular replicative activity during development, is (are) selectively activated after balloon induced endothelial denudation. the situation in vivo of dermal fibroblasts embedded within the connective tissue can be emulated in vitro by growing the cells in collagen gels. we have investigated how fibroblasts cultivated within a matrix react upon wounding of this dermal equivalent. human skin-derived fibroblasts (kd-cells) growing within attached collagen matrices were monitored over a period of days. fluorescently labeled cytoskeletal elements (tubulin, vimentin, acfin) and fibronectin served as phenotypica/ markers. the d-arrangement of fibroblast microtubules, intermediate filaments and microfilaments, as well as of fibronectin was visualized by clsm and optical sectioning. cells at the wound margin, adjacent to the wound, and in non-wounded controls, up to a depth of about gm were analyzed. upon wounding, i.e. upon excision of a mm "punch biopsy" from the center of the collagen matrix, fibroblasts in the wound region oriented towards the matrix defect. layer-like structures of increased cell density evolved over time at the wound margin. the tissue defect was covered by fibroblasts, reminescent of the situation in vivo. global yeast genome expression analyzed by -d page after tctp homolog gene (ykl ) disruption. sanchez a translationally controlled tumor protein(tctp) was identified and microsequenced from a human liver sample. previous publications described the presence of a gene related to tctp in plants, mouse erythroleukemia and ascetic tumor, human mammary carcinoma and more recently in the yeast. tctp has no known function but its high degree of homology from plants to human underlines its probable crucial role in cell function. in order to study multifactorial chan~es in protein expression as a function of the ykl gene disruption m the yeast, we used high resolution two-dimensional gel electrophoresis combined with an efficient computer system (melanie). two groups of gels (ykl +(n= ) and ykl -(n=l )) were analyzed, compared and classified using correspondence analysis. there was no significant difference between the two populations of gels according to either qualitative and quantitative spots expression except for two spots which completely disappeared. the first one corresponded to ykl gsne product. the second one corresponded to a kda peptide with an isoelectric point of . . this ykl associated peptide still needs to be characterized and linked to the yeast genome. demyelinating and neurodegenerative diseases are associated with altered cellular responses including processes mediated by receptor protein tyrosine kinases. using a pcr cdna cloning approach we have identified a novel member of the eck/elk/eph genefamily in myelinating serum-free brain cell cultures. alternatively spliced cdnas encoding complete open reading frames for putative transmembrane proteins have been isolated from both rat and human brain. a recombinant protein derived from the rat cdna was demonstrated to have protein tyrosine kinase activity. an affinity purified antiserum to this protein was used to identify a glycoprotein of kd molecular weight in brain cultures, and developping and adult brain tissue. the protein expression is most prominent during embryonal development and during the first three weeks of postnatal life. by in-situ mrna hybridisation the transcripts were found predominantly in restricted neuronal populations. in order to investigate systematically the effects of geometrically defhied abrupt tissue expansion on the characteristics of impulse propagation, we constructed expanding cellular structures in vitro using patterned monolayer cultures of neonatal rat heart cells (photolithographically patterned substrates.) the preparations, which consisted of narrow cell strands ( - tim wide) inserting into large reetangular cell areas (side lengths > ~m), were stained with the voltage-sensitive dye di-g-anepps and were imaged onto a x photodiode matrix array (maximal spatial resolution .tin x am in the object plane). while impulse propagation was uniform in linear cell strands (average conduction velocity . m/s), a transient decrease in conduction velocity hi the transitional region was invariably observed in the suddenly expanded structures. this decrease was accompanied by marked distortions of the local action potential shapes due to bidirectional electrotonic interactions across the transition: upstream from the expansion, the repolarization phase was interrupted, a few ms after its inception, by a small secondary depolarization ("spike and dome" configuration), while, downstream, the action potential upstrokes were preceded by prominent prepotentials. in those cases where conduction failed at the tlansition, action potential durations upstream were dramatically abbreviated. these findings showed that phenomena similar to those recorded in intact purkinje-fiber-ventricular preparations can be observed in cell ensembles in vitro having comparable geometlins but consisting of cells with uniform active membrane properties. supported by swiss nsf grant a- (sr) and usphs grant ns ms). it is a noncollagenous glycoprotein with a molecular mass of kda consisting of three identical subunits. cmp is selectively extracted with edta-containing buffer what indicates a divalent cation dependent anchorage of the protein in cartilage matrix. electron microscopy revealed the presence of three compact globular subdomains and sequence analysis indicated the presence of a coiled-coil c~-helical assembly domain formed at the c-terminal end of each subunit. the trimeric structure was retained after complete reduction under native conditions which reveals that the subunit structure of cmp is not only stabilized by disulfide bridges but also by the coiled-coil assembly domain. tissue distribution studies in mouse revealed that cmp is selectively expressed in some but not all cartilages. adult rat cardiomyoeytes (arc) in culture degenerate the myofibrillar apparatus and after attachment to the substrate they grow and reassemble new rnyofibrils. the appearance of new, well organized rnyofibrils can be observerd in several distinct regions. they appear close to the membrane proximal to the culture substrate and in the perinuclear region close to the substrate. previous studies have shown that embryonic cultured cells assemble new myofibrils which insert in adherens junctions at sites of cell-cell contacts and in sub-sarcolemmal adhesions plaques (saps) at sites of cell-substrate contacts. this saps are thought to function as the nucleation sites for myofibril formation. we have investigated the formation of this saps in cultured adult rat cardiomyocytes. using confocal light microscopy we can show that the interaction between rnyofibrils and membrans occurs close to the membrane proxirnal to the substrate and that this saps are flanking both nascent myofibrils and sfls, the latter structures being believed to serve as scaffold for myofibrillogenesis, additionally we have used electron microscopy to investigate the formation of this structures at higher resolution. $ - muscle satellite cells (sc) are quiescent myoblasts, ready to be activated and to regenerate new muscle fibers in case of muscle damage. in vitro, single human muscle sc, cultivated as clones, give rise in proliferating conditions to two subpopttiations of cells, cells expressing both ~-striated muscle actin and desmin (c~-sr+dsm+), and cells expressing desmin alone (dsm+). in culture conditions promoting differentiation, a clone .typically gives rise to a fusing progeny, yielding c~-sr+dsm + myotubes, and to non fusing myoblasts (nfmb). the latter are dsm +, a phenotype similar to quiescent sc found in situ. in order to determine whether nfmb are the in vitro equivalent of in situ quiescent sc, this first generation of nfmb were selectively collected and subcultured. in proliferating conditions, nfmb were able to resume proliferation and to give rise to a progeny with both c~-sr+dsm + and dsm + cells. when cultured in differentiating conditions, nfmb formed ct-sr+dsm + myntubes, and a new generation of dsm + nfmb. when nfmb of the second generation were again selectively collected and subculture& they resumed proliferation and ~re again able to yield both myetubes and a third generation of nfmb. nfmb of the first generation were also cultivated as clones, and to % of them were able to resume extensive proliferation, yielding in their progeny both c~-sr+dsm + and dsm + cells, which differentiated into myotubes as well as nfmb. these results strongly suggest that sc have the stem cell property, of selfrenewal, yielding in their progeny both cells ready to differentiate into muscle and ceils similar to themselves. extra cellular matrix (ec~ regulates the expression of b-casein in cultured mouse mammary epithelial cells. we have developed a functional ~ epithelial cell strain, which expresses high level of milk proteins, forms alveolar-like structures when plated onto a reconstituted basement membrane and secretes casein unidirectior~lly into a it~aen. we have further shown that fflv~dependent regulation of b-casein occurs mainly at the transcriptional level and that ' sequences play an inloortant role in this regulation. we have located a bp transcriptional enhancer (bcei) within the ' flanking region of the b-casein gene. using functional assays, we show that bcei contains responsive elements for ~ and prolactin-dependent regulation. bcei placed upstream of a truncated inactive b-casein promoter reconstitutes a promoter even more potent than the intact prcmoter, which contains bcei in its natural context more than . kb upstream. this small fusion promoter reconstitutes the nonaal regulation pattern. we show that bcei mediates f/ dependent regulation even when linked to a het~rologous viral promoter. purified satellite cells (sc) were obtained from human skeletal muscle biopsies following cell sorting by flow eytometry. a chemiluminescent assay for acetylnholine (ach) revealed the presence of , nmoles/weu of . sc. in elonal cultures of proliferating sc, this intracellular level of ach was .l nmoles/well. when cells were cultured in the presence of an esterase inhibitor ( ixm phospholine), the ach amount was enhanced fold. conversely, cultivating the cells in presence of a potent inhibitor of choline acetyl transferase ( gm bromoach), gave a % decrease of ach content finally, the release of ach was detected in the supernatant of cultured sc, following a min incubation, and the measured level of ach was pmoles/well/min. the presence of an ach-like compound in myogenic cells in both freshly isolated and in proliferating sc suggests that sc may contain ach in situ. in additiou, the fact that ach was present in the extracellular medium suggests that ach can be released spontaneously by the cultured sc. type xii collagen is an extracellular matrix protein associated with collagen fibrils in vivo. as observed for several other extracellular matrix proteins, the synthesis of type xii collagen by chick fibroblasts cultured in . % fcs is stimulated by tgf-i~i. two splice variants of type xii collagen are known, with subunits of either kda or kda. by using antibodies specific for the large ( kda) form of type xii collagen, we could show a more restricted distribution of the large variant compared to the smaller form in the embryo. while only the large variant carries chondroitin sulfate, both variants are identical in their collagenous domain. it is thought that via this domain, type xii collagen binds to collagen type i fibrils. we demonstrate here that type xii collagen can bind to collagen i fibrils also in vitro. by neutralizing acid soluble collagen type i, we could coprecipitate type xii collagen together with newly formed collagen type i fibrils. this interaction occurs in physiological saline but is highly salt dependent. in further studies we investigated wether s-methionine labeled type xii collagen treated with chondroitinase abc, ct-chymotrypsin, or collagenase can still be precipitated together with type i fibrils. in addition, we found that type xii collagen also affects the rate of type i collagen fibril formation. h.u. keller department of pathology, university of bern, ch- bern locomoting blebbing cells colchicine ( -sm) can induce locomotion associated with blebbing in walker carcinosarcoma cells. blebs expand at a rate (about ~m/sec) which is much faster than known rates of actin elongation. this suggest that the membrane is directly pushed forward by forces other than actin elongation, possibly by hydrostatic pressure. this interpretation is suggested by the observation that there is significant f-actin staining all along the cell membrane but not with the cytoplasmatic content the blebs. blebbing is suppressed by . m sorbitol. the finding that cytochalasin d suppresses blebbing shows that actin polymerization is nevertheless indirectly instrumental in bleb formation, possibly by generating a high intracellular pressure. a tentative model explaining locomotion of blebbing cells is presented. osteopetrosis encompasses a family of diseases with different causes and the common phenotype of impaired bone resorption. the osteopetrotic mouse mutant oo/oo is deficient in csf- , the growth factor for the cells of the mononuclear phagocytic system. the phenotype of oo/ou mice is characterized by a low number of peritoneal macrophages and peripheral monocytes, and a virtual absence of osleoclasts, leading to the osteopetrotic phenotype. injections of csf- into op/oo mice reversed the osteopetrotic phenotype, proving the requirement for csf- during the process of osteoclastogenesis. by in situ hybridization, expression of csf- was demonstrated during bone development. osteoclast precursors and mature osteoclasts were identified as putative target cells for the growth factor, since these cells express csf- receptors, which is encoded by the proto-oncogene c-fm~. subsequently, specific binding of csf- to osteoclasts was shown. expression of c-fms parallels the expression of csf- in bone both in time and place, suggesting a local action of this growth factor in osteoclast formation, but also in the modulation of osteoclastic activity. different transcripts, raised by alternative splicing of a commmon nuclear rna precursor, have been found to encode a secreted and a membrane associated forms of csf-i. the secreted form can be modified by attachment of a glycosaminoglycan side chain, which serves as an anchor to integrate the peptide into the extracellular matrix. investigation of the role the different csf- forms play during recruitment of osteoclasts and regulation of their activity will provide further insights into the mechanisms governing these processes. comparative sequence alignments of known voltage-gated k + channels revealed two conserved cysteine residues in the putative transmembrane segments $ and $ . if the cysteines were connected by a disttifide bridge, it would put a structural consllaint on possible channel models by placing $ next to $ . we used site-directed mutagenesis to study systematically the potential roles of these invariant cysteines. fourteen of substitutions in $ (c ), and of substitutions in $ (c ) maintained channel function in xenopus oocytes microinjected with mutant crna. therefore, the conserved cysteines are not essential for k + channel expression in xenopus oocytes. however, electrophysiological characterization of the cysteine replacement mutants revealed distinct roles for the two cysteines. inactivation, deactivation, and ion permeation did not considerably change in $ mutants. in $ , in contrast, cysteine replacement by leucine, asparagine, glycine and valine accelerated inactivation and deactivation kinetics substantially, whereas serine and threonine showed opposite effects. furthermore, the voltage dependence of deactivation was differently affected. in summary, it appears that the side-chain at position in $ of kv . is involved in channel gating by participating in the transitions from the open to the closed and inactivated state of the channel. (supported by grants from the nih and mda to rhj, and from the snf and the swiss foundation for medical-biological grants to rdz.) the segment between $ and $ of voltage-gatedk + channels, called h or p region, has been implicated to form part of the ion conduction pathway. little is known about the conforroation of this region although various models have been proposed including a j barrel-like structure formed by four adjacent antiparanel j sheets. to gain insight into the secondary structure of this region, we used cysteine substitution mutagenesis and sulfhydryl-specific, membrane-impermeant reagents to probe the accessibility of amino acid side chains in and around the p region of kv . (drki). twemy-eighi positions from k to t were each mutated to a cysteine. after expression of mutant k + channels in xenopus oocytes, cysteine side chain accessibilities were probed by superfusion with ch so sch ch nme + (mtset), mtset can form a mixed disulfide with an accessible cysteine, and this may lead to current reduction if the covalently modified side chain is in or close to the ion conduction pathway. thus far, we have identified four mutations that showed k + current reduction after superfusion with mm mtset. the mutants p c, i c, y c, and k c showed %, %, %, and % reduction in current amplitude, respectively. in coutrast, mutants $ c, a c, t c behaved like wild type kv . with less than % current reduction. our results suggest that the side chains of p , , y , and k are directly accessible from the extracellular environment. taken together, these residues may, therefore, face the lumen of the ion channel pore. furthermore, the degrees of inhibition are in agreement with a model in which the ion conduction pathway narrows from residue k to y to i . (supported by grants from nih and mda to rhj, and from the snf and the swiss foundation for medical-biological grants to rdz.) motejlek k., h,,iuselmann r., and liig:her b.; pharmakologisches institut der universitiit ziirich, winterthmtr. , ch- zlirich comparison of ' flanking sequences of three gabaa-receptor subunit genes (atl, ~, ) revealed a novel conserved purine sequence dement with the consensus sequence gagaggggagaoga gagag(gg/aa)g. this element is present once each in the (zl and subunit gene promoters and in seven tandem copies in the gene promoter. a novel dna binding activity (bsf ) was identified that binds to various versions of this purine element. bsf was found ordy in brain but not in any other tissues tested. furthermore, the factor is distinct of beta, another brain-specific dna binding .protein with a purine-rich dna recognition sequence. the expression of bsf during differentiation of eerebeuar granule cells in vitro correlates with the expression of gabaa-receptor subunit genes. this correlation suggests a role for bsf as a transcriptional activator of neuronal genes. therefore, bsf may be important for cell ty~specific regulation of gabaa-receptor gone expression. we also found that bsf binds to wheat germ agglutinin, suggesting that this protein is glycosilated. taking advantage of this property, we are in the course of purifying bsf , the question, whether bsf is indeed a transcription factor is being investigated in vivo and in vitro. to produce a simple method of estimating the free magnesium concentration ([mgzq) in solution, we have manufactured dip cast mg + macroelectrodes using the neutral mg carrier eth .we have used the elecmides to measure the dissociation constant (k~) for mgatp in a background solutions mimicking the intracellniar milieu. for the mcasmement of the big atp dissociation constant, the background solution contained mmol/l n%atp. this solution was titrated with mgci and the changes in [mg ~] above . retool/ monitored with the maeroelectrode. during the titration the ph was maintained constant. fitting such titration curves with the standard hyperbolic binding equation, after correction for zero drift, gave the following mean+sd values for the ka (pmol/ at ~ ph . , . -+- , (n= ); ph , . :k- . (n= ) and ph . , .g~ . (n= ). elevation of the [naq to m~l/ and reducing the [kq to ( mmol/l was without effect at ph . (n= ). reducing the temperature to ~ increased the k a at . to . :k . (n= ). mg =+ macroelcctrodes provide an easy way of measuring [mg z § in solution. [mg ~] can also be measured in a background solution containing i mn~i/l ca, although the electrode response is reduced. interference by protein to date prevents their use in plasma. similarily to the block by zn + and ca +, protons only partially block the channel. the ability for all mutated channels to. conduct na + current, and the partial block induced hy zn +, ca + or h + indicate that y is not located deep in the pore but rather in the extracellular mouth of the channel. progesterone modulates the activity of glycine receptor expressed in colliculli neurons of neonatal rats. maury, k. & bertrand, d. dpt of physiology, cmu, geneva . steroids are potent narcotics and have been shown to act on ligand-gated channels activated by gaba or nicotine. however, little is known about their possible actions on other receptor types. for instance, while it was demonstrated that progesterone inhibits glycine receptors expressed by spinal cord neurons isolated from chick embryos, nothing has yet been reported for brain receptors. in this study, we have determined the properties of glycine channels expressed by brain neurons of neonatal rats using the whole-cell voltage-clamp technique. in inferior colliculi neurons, glycine elicited little-desensitizing inward currents which reversed around - mv. these currents were blocked by strychnine in the nanomolar range. surprisingly, however, we found that progesterone, in the micromolar range, potentiated the glycine evoked currents. these results, contrasting those obtained in chick spinal cord neurons, suggest that progesterone enhances or antagonizes glycine currents depending on the subtype of the receptor. the mammalian auditory organ (organ of corti) relies on two groups of sensory cell for its normal hearing: inner hair cell & outer hair cell. ihcs are mainly innervated by afferent fibers while the ohcs essentially receive efferent fibers. the ohcs possess unique electromofile properties which are thought to enhance the motion the basilar membrane and to refine the mapping of the sound frequency. we found, using whole ceil recordings, that - % of the fleshly dissociated ohcs displays fast inward currents. the magnitude of peak currents which can be as large as na vary from cell to cell. further experiments have revealed that this current is sensitive to ttx and strongly reduced when extracellular sodium is replaced by choline. its kinetic is relatively slow compared to that of sodium current of typelspiral ganglion cell, and has a more negative inactivation. other voltage activated currents and ligand-gated currents are under inverstigation. these results will provide further insights in the hearing transduction mechanism. the formation and the properties of homotypic and heterotypic gap junctions were studied using two types of insect cells, c / and sf . two single cells were pushed against each other and the subsequent de novo formation of gap junction channels was assessed by means of the dual voltage-clamp method (bukauskas and weingart: pfl~g. arch. : , it was symmetrical for homotypic junctions. the vj-sensitivity was less pronounced in sf cells. hence, the relationship for heterotypic junctions was asymmetrical. all pairs examined revealed a s-shaped relationship between gj(ss) and v, which was virtually superimposable (gj(ss) declined upon depolarization). each channel contains a vm-gate and two ~-gates. the vj-gates are operated independently. they close when their intracellular aspect is made positive. shaped curve compatible with a two-state boltzmann process. half maximal inactivation was reached at - mv and + mv, implying an asymmetrical gating behavior. in case of an asymmetrical protocol (~ and vewas stepped simultaneous, but in opposite direction), the q-dependent asymmetry disappeared (half maximal inactivation at - mv and + mv). the asymmetry in gj-gating, attributable to the pulse protocol, was also reflected in the time constants of ij inactivation (r cerebellar granule cell cultures were used to study the regulation of the nmda receptor expression. we have shown previously that chronic membrane depolarisation ( him k +) or treatment with nmda {i ~m) promotes the functional expression of nmda receptors, as assessed by nmda-evoked ca + influx. we have now developed a rnase protection procedure for the quantitative determination of the mrna levels of the nmda receptor subunits known so far (nr-i, a, b, c, d). the growth conditions mentioned above failed to alter the mrna levels of the different subunits. at the protein level, nmda receptors were evaluated quantitatively by the labeling pattern obtained by the new photoaffinity ligand i-cgp a. the intensity of the phctolabeled bands, thought to represent nr-i and nr- a subunits, was increased by treatment with high k + or nmda compared with control cultures. this result suggest an increase in receptor protein by high k + or nmda treatment. thus, posttranscriptional mechanisms seem to play an essential role in the modulation of the nmda receptor activity. the gene encoding the pore-forming subunit of the rat epithelial na + channel (~renac) was cloned recently and shown to belong to a novel gene family coding for cation channels (nature , p - ( ) . transport of na + ions through these channels is the rate-limiting step of na + absorption and thereby controls osmotic balance of body fluids and secretions. in order to study the implication of ~renac in regulating sodium balance, blood volume and blood pressure and therefore its involvement in hypertension, we expressed ~renac under the control of the human cmv promoter in transgenic mice. several lines of mice showing expression in different tissues were obtained. progress in the analysis of these mice will be discussed. activation of metabotropic glutamate receptors increases the excitability of neurones in the lateral septum of the rat. to elucidate the mechanism(s) of this action, we used whole-cell patch-clamp recordings from coronal brain slices of young animals. in voltageclamped cells, the selective agonlst (is, r)-acpd, at - /~m, had the following effects, a) it evoked a sustained inward current, which was resistant to ttx and to cadmi~am and which persisted in neurones loaded with bapta, a calcium chelator. this current displayed inward rectification and was reduced, or suppressed, when extracellular sodium was partially replaced with n-methyl-d-glucamine. b) it elicited a ttx-insensitive, voltage-dependent inward current, which activated at - /- mv and which reversed at aound mv. this current was suppressed in a low-calcium/high-magnesium perfusion solution and was undetectable in the presence of intracelhilar bapta. we suggest that acpd exerts a dual action on lateral septal neurones. it causes a steady depolarization by generating a sodium-dependent current and it triggers transient plateau potentials by inducing a calcium-dependent cationic current. interaction between these currents results in a powerful, self-reinforcing neuronal excitation. we have investigated the effects of protein kinase c (pkc) activators ( [~-pma, oag) and of phosphatase inhibitors (okadalc acid, calyculin a) on voltage-gated ca + and k + channels in ngf-differentiated pc ceils. whole-cell ba + and k + currents were recorded with the patch-clamp technique. by using specific ca + channel blockers (co-conotoxin (cgtx), isradipine) we found types of ba + currents (iba): a) a ~cgtx-sensitive iba; b) an isradipine-sensitive iba; and c) a t -cgtx plus isradipineresistant iba. [~-pma and oag specifically down-modulated the isradipine-sensitive iba. the inhibition of iba was prevented by staurosporine and pkc ( - ) ( pk inhibitors). the delayed rectifier k + current was unaffected by pkc activators. applied externally, okadaic acid and calyculin a inhibited the total iba by affecting several components of the ba + currents. however, the ~-cgtx plus isradipine-resistant iba was unaffected by okadaic acid. in conclusion, our results suggest a differential modulation of voltage-gated ca + and k + channels by the pkc signalling pathway in ngf-differentiated pc cells. agrin, a protein isolated from basal lamina extracts of the electric organ of the marine ray, is thought to mediate the motor neuron-induced aggregation of acetylcholine receptors (achrs) in muscle fibers at their neuromuscular junction. recent cloning in the marine ray, rat and chick has revealed that agrin can be alternatively spliced at two sites in its c-terminal half. site a encodes either or amino adds and site b encodes either , , or ( + ) amino acids. studies of agrin mrna expression indicate that early in synaptogenesis, chick motor neurons contain high levels of a b . in contrast, non-neuronal cells and muscle calls contain a b and aob mrna. in the adult ray, electric lobe motor neurons that innervate the electric organ contain a b , whereas agrin mrna in the electric organ again lacks both sites (mcmahan et al. ( ) , curr. up, cell biol. : - ). to investigate the functional properties of agrin isoforms in more detail, we have now compared their specific activity to aggregate achrs on cultured chick myotubes. heterologous expression of the c-terminal half in cos- and cells revealed that chick agrin isoforms a b and a b were highly active, while a bll was up to fold lower in activity. no activity could be detected for the a b and aob isoforms. in agreement with these findings the a b isoform of the marine ray also showed high achr-clustering activity. therefore, we conclude that motor neurons throughout development synthesize highly active agrin isoforms while the postsynaptic target ceils do not play a primary inductive role in the formation of synapses. we have used whole-cell patch-clamp recordings and hypothalamic slices in order to characterize the effect of n-methyl-d-aspartate (nmda) on suprachiasmatic neurones. in ceils clamped at or near their resting membrane potential, nmda ( - #m) generated an inward current of - pa, which was insensitive to ttx and which reversed at about inv. the nmda current-voltage (i-v) relation contained a region of negative slope conductance. the nmda current was reduced or suppressed by d(-)- -amino- -phosphonopentanoic acid (d-aps) or by mk- . it was potentiated by reducing the extracellular magnesium concentration from to . ram, or by adding glycine ( /~m) to the perfusion solution. in a majority of neurones, lowering the extracellular calcium concentration from to . mm caused a . to -fold enhancement of the nmda current. i-v relations indicate that in the low-calcium solution, the region of negative slope conductance was attenuated. this effect was due to extraceuular calcium, since it persisted in neurones loaded with the calcium chelator bapta. we conclude that nmda channels present in suprachiasmatic neurones may be modulated by extraeellular calcium. institut, and abt. pharmakologie*, biozentrum, basel. during innervation of skeletal muscle, the myonuclei underlying the synapse begin to express acetylcholine receptor (achr} ~-subunit gene and they remain activated after the nerve is removed. our experiments show that this is due to a factor in the synaptic portion of the muscle fibre basal lamina (bl). one candidate for this factor is agrin, which regulates ache clustering in the synaptic membrane: i) unlike in untreated muscle, the level of s at synapses was strongly reduced in cultured rat muscle fibres after proteolysis of synaptic bl. ) conversely, when rat myotubes were cultured on bl isolated from adult muscle, they preferentially expressed achr accumulations and ~-mrna at sites where they contacted the synaptic portions of the isolated bl. ) when various substrates were impregnated locally with agrin a big, an isoform expressed by motor neurones in fetal spinal cord, it locally induced in the myotubes the expression of s-mrna. art increase of functional nicotinic acetylcholine receptors precedes the fusion of cultured human muscle satellite cells. r. m. kranse, c.-r. bader and l. berrtheim. department of physiology and division of clinical neurophysiohigy, university medical center, geneva , switzerland nicotinic acetylcholine receptors (nactlr) are expressed on embryonic myoblast and acb may play a role in mechanism of fusion. our study focuses on the appearance of functional nachr in freshly isolated and cultured satellite cells (sc) from normal human muscle biopsies. sc cart be conditioned to either proliferate or fuse to form myotubes depending upon culture media. presence of ach-activated current was investigated using the whole-cell and single.channel patch-clamp technique. in freshly isolated sc (whose properties should be close to the quiescent in vivo sc/no nachr were observed (n= ). in the proliferating state, % (n= ) of the satellite ceils (which are also called myoblasts) displayed a small ach-activated current ( pa/pf) and, as expected, after fusion of salellite cells into myotabes, % of the ceils displayed an ach-activated current (n= ; pa/pf). to examine, whether the increased expression of nachr precedes sc fusion, sc were cultured in a medium which promotes fusion, but were prevented from fusing by keeping them at a low density. in this culture condition, % of the sc (n= ) displayed an ach-activated current and, in addition, these cells expressed a times higher ach-current density. ( pa/pf) than the proliferating sc. we also observed that, in high density cultures, a small population of sc do not fuse even atter months in the medium promoting fusion. these non-fusing myoblasts (nfmb) had no nachr. our results suggest that the appearance of nachr may be related to the process of sc fusion as i) quiescent sc in vivo do not express nachll ii) nachr expression increases in conditions promoting fusion and iii) no nachr were observed in nfmb. the latter cells may be equivalent of quiescent sc in vivo. $ - we have investigated the sensitivity of neuronal nicotinic acetylcholine receptors (nachrs) of known subunit composition to various cholinergic antagonists. neuronal nachrs were expressed in xenopus oocytes after injection of pairwise combinations of (x or c~ with either ~ or ~ crnas. the two-electrodes voltage-clamp technique was used to measure currents induced by rapid application of low concentrations of acetylcholine (ach) together with increasing concentrations of antagonists. the response of ct ~ neuronal nachrs to ach was halfmaximally inhibited by following antagonist concentrations (ic ): hexamethonium ( . i.tm), mecamylamine ( . p.m), pentolinium ( . i.tm) and trimetaphan ( . ~tm). with (x nachrs the ic s of the same antagonists were about times higher. finally, (+)-tubocurarine was a conventional competitive inhibitor of ach in ~ and t~ ~ nachrs. in contrast, low concentrations of (+)-tubocurarine increased the response to low ach concentrations in a ( and t~ ~ nachrs. these results further underline the importance of [l subunits in determining the functional properties of neuronal nachrs. supported by the hochschnlstiftung and nf grant . . to abc. previously, the role of the transglial tubular system was shown for the squid giant axon: in na + free (tris) solutions the series resistance increased in correlation with a decrease of the tubular opening density (tod). in the crayfish giant axon, which show similar tubules, we correlate the change in tod, measured in freeze-fracture preparations, witl] the conduction velocity. the control to'd was estimated to be per #ms. after and rain tris-inkubation the tod decreased to , and , , respectively. this reaction was reversible when these axons were reincubated in na~-c ntaining solution. after rain reincubatiou the tod was . per tam'z. to determine the influence of decreased tod on the impulse conduction velocity, the nerve was incubated for min in tris solution followed by reincubation. impulse propagation then recovered in two phases. during an initial fast phase with a short time constant of - rain na + diffused back into the periaxonai space and allowed impulses to occur with a low conduction velocity. in the second phase with a long time constant of min, conduction velocity recovered slowly to normal values. this time course was comparable to the recovery of tod, as estimated from recent freeze-fracture preparations after short reincubation. these results indicate that the normal tod is a necessary condition for the normal excitability of the axon and determines its conduction velocity. most spinal cord neurons respond to the inhibitory action of gaba and glycine, suggesting co-expression of gaba a-and glycine receptors n individual ceils. while glycine-receptors are exclusively found in post-synaptic densities, the cellular localization of gaba areceptors has not yet been characterized. in this study, the distribution of gabaa-receptors in the spinal cord was analyzed with an antiserum recognizing the (xl-subunit. double-and tripleimmunofluorescence staining were employed to identify synaptic receptors apposed to gabaergic terminals (immunoreactive for glutamic acid decarboxylase) and to assess their co-localization with glycine-receptors (visualized with an antibody to the kda protein gephyrin). staining for the gabaa-receptor ctl-subunit decorated the soma and dendrites of numerous neurons in laminae iii-viii and x, revealing their morphology in clear detail. by contrast, laminae i and ix contained little immunoreactivity for these gabaa-receptors. most gabaa-receptor-positive cells also exhibited a prominent glycine-receptor immunoreactivity. both types of receptors had very similar distribution patterns and were frequently co-localized in sites apposed to gabaergic boutons. these results indicate that gaba aand glycine-receptors may co-exist within single post-synaptic densities, suggesting a possible synergism between gaba and glycine neurotransmission in spinal cord neurons. prenatal benzodiazepine (bdz) exposure changes both behavioral and neuiochemical parameters of developing iats. these changes are not a consequence of remaining bdz in brain tissue, but rather indicate alterations in bdz receptol binding and function. since the bdz binding site is located on the gaba~ receptor complex, it is possible that prenatal bdz treatment influences the expression of gaba~ receptor subunits. to evaluate effects of pienatal bdz exposure on mrnas expression specific for several gaba~. receptor subunits (~i, ~ , ~ & y ), we treated plegnant rats with diazepam (l. mg/kg/d; s.c.) from gestational day to . we then analyzed specific mrna levels in offspring at four diffeient developmental stages (gd , pns, pni & adult) by in situ hybridization. pleliminary data flom optical density measurements indicate a deciease in expression of mrna in particular for ~-subunit of gab~ receptors in different brain reglons of plenatally diazepam-treated zats. processing of sensory information within the auditory system has been well characterized using histological and eleetrophysiologlcal techniques. however, relatively little is known about the neurotransmitters involved. the present study was performed to elucidate the possible role of excitatory (eaa] and inhibitory (i.e., gaba) amino acids in neurotransmission within the primary auditory cortex (all the main afferent to the ai, the ipsilatera} medial geniculate body (mgb), was electrically stimulated and evoked responses were recorded intracellularly in the ai region in halothane anesthetized cats. a seven-barrelled iontophoresis pipette glued alongside the recording electrode allowed localized application of eaaergic and gabaergic compounds onto the recorded neuron. in most ai neurons, mgb stimulation evoked a short latency, short duration epsp followed by a long-lasting ipsp. pattern, duration, and amplitude of synaptic potentia{s were highly variable and strongly dependent on stimulation intensity, reduction of gabaa receptor-mediated inhibition by iontophoretic application of either bicuculline or sr markedly enhanced mgb stimulation-evoked epsps. only the late component of this enhanced epsp was reversibly blocked by the competitive nmda receptor antagonistl ap at currents which selectively blocked neuronal excitation induced by iontophoretic application of nmda, our results demonstrate that in the eat ) nmda receptors in ai are activated following mgb stimulation and that ) a dominant gaba~ receptor mediated inhibition usually masks this nmda receptor activation under our experimental conditions, a highly purified and specific cell wall degrading endo-l, -u-l-arabinanase was isolated from an a. niger pectinase preparation by low and medium (fplc) pressure column chromatographic separation methods. the isolated enzyme was most active on linear , -~-l-arabinans (- i.u./mg), whereas branched arabinans from sugar beets were degraded to a lesser extent (- i.u./mg). the enzyme was shown to be electrophoretically pure after silver staining (sds-page) and its identity was confirmed through specific binding to an antiserum directed against endo-l, -~-l-arabinanase. the major physico-chemical characteristics of the enzyme were the following: mr ' , iep ~ . , ph optimum . , temperature optimum ~ ph stability . - . , temperature stability s oc, km = . mg/ml, vmax = . . - ~unol/min. zn and hg showed potent inhibitory effects. . ) is a specific enzyme of the glyoxylate cycle, which plays a key role in the initiation of gluconeogenesis in germination oilseeds, this pathway is localized in glyoxysomes, single membrane organelles which are converted into peroxisomes when lipid reserves are depleted. the glyoxylate cycle enzymes have been the subjects of numerous conflicting reports typically addressing the problems of their synthesis (on free or bound ribosomes), targetting and suborganelar localization (membrane bound or matrix proteins). recent biochemical studies have shown that ms from germinating soybean cotyledons is an interconvertible enzyme (membrane bound, aggregated or soluble fomls) which is significantly affected by its ionic environment (henry et al., , plant science : - ) . microscopy studies have now been carried out using immunofluorescence staining or immunogold-silver staining for light microscopy, and immunogold labelling for electron microscopy. all results consistently characterize ms as a glyoxysomal mawix enzyme. chorismate mutase (ec . . . ) catalyzes the first step in that branch of the shikimate pathway which leads to the aromatic amino acids phenylalanine and tyrosine. we have isolated a cdna for this enzyme from the higher plant arabidopsis thaliana by complementing a yeast strain (aro ) with a cdna library from a. this is the first chorismate mutase cdna isolated from a plant. the a. thaliana chorismate mutase expressed in yeast revealed allosteric control by the three aromatic amino acids as previously described for plastidic chorismate mutase isozymes. an attachment of rubisco to chloroplast membranes under cu++-stress has been described for wheat (mehta et al., j. biol. chem. , - . the displacement to the membranes has been discussed as a possible step in the degradation of this predominant stromal protein, e.g. during leaf senescence. in our recent experiments it became evident that such interactions are not restricted to rubisco. several other stroreal, and even a peroxisomal enzyme (glycolate oxidase), were also detected in the membrane fraction after to hours of oxidative stress induced in wheat seedlings by a treatment with mm cuso . the velocity and the degree of membrane attachment varied for different enzymes. based on these results it was interesting to check the solubility of rubiseo and its previously described kd fragment which accumulates during the incubation of bean leaf discs under oxygen deficiency (hildbrand and feller, experientia , a ) . neither rubisco nor the breakdown product were found to accumulate in the membrane fraction. thus, at present, the steps involved in rubiseo catabolism cannot be generalized. different mechanisms depending on the metabolic situation and on environmental conditions should be considered. overexpression of the four parsley phenylalanine ammonia-lyase (pal) isoenzymes in e. coli. characterization of the enzymes and kinetic analysis of the inhibition by -aminoindan- -phosphonic acid. christoph appert, j/irg schmid, jerzy zorn* and nikolaus amrhein institute of plant sciences, eth-z/jrich, zijrich. *technical university wroclaw, - wroclaw, poland. all four phenylalanine ammonia-lyase (ec . . . ) isoenzymes of parsley (petroselinum crispum nym.) were expressed as glutathione s-transferase fusion proteins in e. coil after affinity purification, the glutathione stransferase moieties were cleaved off with factor xa and the phenylalanine ammonia-lyases were characterized. the proteins form enzymatically active tetramers, even as fusion proteins. the four isoenzymes have comparable km values for l-phenylalanine ( gm to . /.tm), similar temperature ( ~ and ph optima ( . ). the aminooxy-and phosphonic-analogues of l-phenylalanine are competitive inhibitors of the enzymes. -aminoindan- -phosphonic acid (j. zo{a & n. amrhein, liebigs ann. chem. , ) was found to be a potent slow-binding inhibitor of these and other phenylalanine ammonnia-lyases, both in vivo and in vitro. chlorophyll a fluorescence has been used to compare the drought resistance of two varieties of solanum tuherosum (avr/i)c- st- and sibtema). fast fluorescence rise kinetics were measured during the first second of illumination with a time resolution of l ms and z bits in fluorescence intensity. the rise kinetics shows the typical steps called fo -j -i -p. with this values different indexes have been defind with the goal to compare the behaviour of these two varieties of potato. salicylic acid (sa) was proposed as a putative signalling molecule for the induction of systemic acquired resistance (sar) in infected plants. we studied its biosynthesis as follows. cotyledons of cucumber plants were injected with a solution containing pseudomonas lachr~ans and fed with radioactive sa precursors by injection of c-benzoic acid ( c-ba) or cphenylalanine ( c-phe). alternatively, leaf of cucumber plants were inoculated with tobacco necrosis virus (tnv) and leaf disks were then vacuum-infiltrated using c-ba or c-phe. free and bound c-phenolies were quantified by hplc. incorporation of c into sa was found in the two plant-pathogen systems. i~c-ba gave mostly c-sa and an unknown polar compound. c-phe gave also some c-sa, in addition to c-labelled lignin precursors like ferulic and p-coumaric acids. the biosynthetic pathway of sa from phe through cinnamic acid and ba will be discussed. after infection with a necrotizing pathogen, systemic acquired resistance (sar) is often observed in plants. in cucumber, salicylic acid (sa) increases in the lower infected leaf as well as in the upper uninfected leaves. sa was also found in the phloem sap of infected cucumber plants and was proposed as a signalling molecule for the induction of sar. we intend to clarify whether sa is translocated from the site of infection to the upper leaf, or wether it is made in the phloem tissue upon the action of a primary systemic signal. one cotyledon of cucumber plants was first infected with pseudomonas lachrymans and then fed with c-sa, c-benzoic acid (ba) or c-phenylalanine. after different times, cotyledons and first leaves were collected and the radioactive ~henolics were analyzed by hplc. in some cases, c-sa was found in the first leaf. in addition, two unknown radioactive compounds were detected in leaf after treatment with c-sa and c-ba respectively. we will discuss signalling processes for the induction of sar in cucumber. one modern approach of creating virus resistant grapevine plants is the introduction of resistance genes into existing grapevine varieties by genetic engineering. the goal of this work is to use the coat protein (cp) mediated strategy to induce resistance to nepovirus in vitis spp. as a first step, several chimeric nepovirus cp genes, were constructed by addition of various promoter regions upstream of the gflv and armv cp regions. their ability of conferring resistance to nepovirus infection in transgenic nicotiana benthamiana and n. tabacum plants will be presented. the best constructions will be used to transform several grapevine varieties in order to create nepovirus resistant grapevine plants. $ - the bctalains axe a class of natural pigments found only in plants of the order caryophyllales and in some fungi. the first step in betalain biosynthesis is the conversion of tyrosine to dopa. subsequently , dopa is transformed to betalamic acid, the bctalain chromophorr through the action of dopa- , -dioxygenase. we have purified and characterised a copper-containing enzyme of - kda from amanita muscaria pileus that catalyses the hydroxylation of tyrosine to dopa. considering that the enzymatic activity was restricted to the coloured parts of the mushroom, we postulate that it is involved in betalain biosynthesis. the enzyme featured a broad substrate specificity, and also oxidised the diphenols to their corresponding ortho-quinones, a reaction typical for tyrosinases. the implications of our findings on betalain biosynthesis axe discussed. chlorophyll a fluorescence was measured under steady state conditions of pea and tomato leaves adapted to low light intensity ( wm- s -i) at different temperatures (havaux and strasser, z. naturforsch. c, - , ). when leaves were exposed for a short period (i min) to heat ( to ~ in darkness, the level of variable fluorescence decreased. when the heat stress was imposed in presence of low light, the variable fluorescence was much less affected and virtually no effect of heat treatment was observed until ~ this protecting mechanism by low light was absent in algae and mostly absent in submerged water plants but fully present in free floating plants on the water surface. we conclude that a mechanism has been developed for higher land plants during evolution which protects the plants against heat damage on warmer days. caryophyllales, e.g. portulaca grandiflora, and in a few fungal species, e.g. amanita muscaria. we analysed the similarity of a key enzyme of the pathway, dopa- , -dioxygenase, in plants and fungi both at the protein and the dna level. antibodies against purified dopa- , -dioxygenase from the fungus a. muscaria crossreacted with protein from p. grandiflora petals and two cdna clones were obtained from this plant. their similarity with fungal sequences and with other dioxygenases is discussed. kinetics of prollne hydroxylauonj intrucellnlur transport and c-terminal processing of the tobacco vacuolar cbltlnase. ernst frcydl, thomas boiler and jean-marc neuhaus botenisehes instimt, abt. pflanzenphysiologie, hehoistxasse , ch- basel the vacuolar chitinase a of tobacco (nicotlana tabacum) is syndietlzed as a preproprotein with ma n-teaminal signal peptide which causes its synthesis in the er mad a c-termlnal extension, which has been idcmified as the vacuolar targeting peptide (vtp) ( ) and whleh is cleaved off from the maybe chitinase. it has recently been shown that mature chltin:~e a contains several hydi'oxyprolines within the short peptide spacer that links the n-terminal cysteine-rieh chitln-bindlng domain to the catalytic domain ( ). we received specific antibodies against mature chitinasr (a kind gift from dr. f.meins. f/vii) and raised antihodiea against a synthetic vtp. we performed pulse-chase experiments and cell [ractlonation with ) stably transformed tobacco plants expressing chitinase a or mutants lacking either the chitin-binding domain and spacer (chitah) or the vacuolar targeting poptid (chitavtp) and ) transient expression of the same conswacts in nicotiana plumbaginifolia protoplasts. in both systems, proline hydroxylatino in chltinase a was detectable after vain. and complete after - rain. the ehltinase intermediate forms were detected in the mlcrosomal and soluble fractions for up to - rain. the mature chitinase continuously accumulated only in the soluble fraction. in both systems chitah showed the same kinetics of intraceilular transport and processing. whereas the transport of cbitavtp seemed more rapid.these results indicate that in vivo cbitinase a is synthetized as a proprotein that is than modified by proline hydroxylation. this second intermedinte form is then transported to the oolgi apparatus and sorted to the vacuole. c-terminal processing occurs late in this pathway or in the vacuole. infection of bean roots with the soil-borne phytopathogenic fungus fusarium solani f.sp. phaseoli leads to a rapid increase of chitinase activity (~five-fold). part of this increase in enzyme activity is due to transcriptional activation of the basic class i chitinase isoenzymes. semi-native polyacrylamide gels stained for chitinase activity using the substrate glycol-chitin revealed the appearence of three additional chitinase isoenzymes that are absent from uninfected control roots. conventional protein purification techniques showed that fusarium solani infected bean roots contain two basic and two acidic chitinase isoenzyrnes. their physiochemical properties and possible biological function will be discussed. proteasomes are multicatalytic protease complexes that function as a major nonlysosomal proteolytic system. they exhibit multiple endopeptidase activities that promote the intracellular turnover of abnormal polypeptides and short-lived regulatory proteins. moss proteasome has been purified from protonemata by successive chromatography on macroprep q, bio-gel a- . , bio-gel a- and ha. the molecular mass was estimated to be , kd by gel filtration. gel electrophoresis of proteasome under nondenaturing condition gave a single band and two-dimensional gel electrophoresis identified the moss proteasome to be composed of different subunits with molecular weight in the range - kd. electron microscopy in aqueous solution showed that it is a cylindrical particle composed of four stacked rings with a diameter of nm and a height of rim. end-on projection established a -fold symmetry of the outer disks. substrate specificity of proteasome indicates that it contains endopeptidase activity against substrates bearing hydrophobic, basic, acidic and glycine residues immediately preceding the cleavage site. antibodies raised against moss proteasome reacted with proteasomes of other eukaryotes, including human. $ - hunger r.e., hess m.w., laissue j,a,, mueller c. the nod (non obese diabetic) mouse, a widely used animal model for insulin dependent diabetes mellitus, shows in addition to mononuclear cell infiltration of the islets of langerhans, massive cellular infiltrates of the submandibular and lacrimal glands with considerable tissue damage. several lines of evidence suggest that both insulitis and sialadenitis represent autoimmune disorders, to investigate a possible role of tumor necrosis faetor-a (tnf-c and activated cytotoxic cells (nk cells, t cells) in the inflammatory process, tissue sections of nod mice were hybridized with radiolabeled rna probes specific for the detection of mrna for tnf-r and the serine proteases granzyme a and b (gra and gr ) and perforin which are expressed in activated cytotoxic cells. tnf-e expressing cells were mainly located in infiltrates and are absent in non affected glands, whereas gra, grb and perforin expressing cells were distributed over the whole section with highest densities in the zones adjacent to parenchyma. the finding that activated cytotoxic cells are present in early stage of disease development indicates that cell mediated cytotoxicity may play a crucial role in initial tissue destruction. the role of tnf-a in autoimmune diseases is not known yet. the appearance, however, of cells expressing this gene in the infiltrate indicates a possible role of this cytokine in the progression of the inflammatory reaction, e.g. by increasing the lymphocyte traffic to this organ. we further demonstrated that the induction of inos was at the transcriptional level. in order to understand the underlying mechanisms we characterized the promoter region of the rat nos gene. a enomic rat library was screened using a cdna-fragment coding for the if-end of the inos cdna (nucleotides - ). a , kb hinc-ii fragment containing the "-flanking region of the inos gene was characterized by dna-sequencing. the transcriptional start site was determined by primer extension. sequencing analysis revealed a multitude of possible cis-acting elements homologues to consensus sequences for the binding of different transcription factors including ifn~' response element (ire), nuclear factor-kb (nf-~b), tumor necrosis factor response element (tnf-re), f-activated sites (gas) and one x-box. nf-kb, a transcription factor involved in signaling and immediate early gene activation during inflammatory processes was induced by treatment of mesangial cells with nm of il- . this was shown by the appearence in nuclear extracts of the dna binding activity of nf-~b using electrophoretic mobility shift assays (emsas) with a p-labeled ~r dna probe. pyrrolidinedithiocarbamate (pdtq), an inhibitor of nf-kb, suppressed the expression of inos mrna in mesangial cells without affecting the dibutyryl-camp-triggered increases in nos mrna levels. this data suggest that the il-i ~ signalling pathway responsible for nos expression requires nf-k activation and is definetly different from the signalling cascade directed by camp. recent findings indicate that the multifunctional cytokine il- , which plays a key role in irm-nune and inflammatory responses, also exerts specific effects in the central nervous system (cns). for example, il- promotes neuronal survival, induces differentiation and modulates neurotrophin production. using the very sensitive technique of reverse transcription combined with polymerase chain reaction the developmental profile of il- and its receptor mrnas was analyzed in various rat brain regions. our results indicate that both genes are expressed in a region-specific manner and are developmentally regulated. these findings support the hypothesis that il- is involved in differentiation and maintenance of neuronal subpopulation in the cns. interleuldn (il- ) expression is strictly dependent on a signal transmitted by the t cell receptor upon antigen stimulation. this signal can be mimicked in vitro with phorbol esters and calcium ionophores. we have found that stimulation with ca-ionophore alone induces expression of il- mrna, but does not induce secretion of il- . in polysome gradient fractionation of cells stimulated with phorbol esters and ionophore, the fractions containing il- rrtrna are found at the bottom of the gradient, bound to polysomes. however, in ionophorestimulated cells the fractions containing il- mrna are clearly shifted to the top of the gradient, and therefore are not lzanslated. this effect is specific for il- , as other mrna's like ~ -microglobulin are not regulated. this data suggests that il- gene is translationally regulated. in addition, in vitro experiments have shown that the lack of translation of il- mrna in ionophore-stimulated cells is due, most likely, to the presence of a translational regulator that specifically inhibits translation of il- mrna. assuming the presence of a translational regulator that specifically represses il- mrna translation, we can predict that, if the represser is in excess, even upon a second stimulus that is by itself able to induce secretion of il- , there will be no translation of il- mrna. the resuhs of such an experiment confirmed our prediction. polysome gradients showed that after ionophore stimulation, il- mrna gets "stacked" with one ribosome, and no further ribosome loading takes place. if malaria is highly endemic, every febrile child is a presumptive malaria case, and there is no differential diagnosis based on clinical, immunological or parasitological grounds. we evaluated the diagnostic usefulness of interleukin- receptor (il- r), tumor necrosis factor-receptors (tnf-r ) and (tnf-r ) . sera came from tanzanian pediatric fever patients and controls, all enrolled in the kilombero malaria project. we found that all receptors marked pediatric fever episodes, whereas stability of observed levels was highest for tnf-r and lowest for tnf-r . tnf-r levels reflected severity of the fever attack. regardless of the presence of a febrile illness, tnf-r levels were strongly associated with parasite density. immunological markers can be applied in rapid pre-and post-intervention morbidity assessments, validation of health interviews and monitoring of convalescence. we have recently shown that human eosinophils produce mrna for interlenkin (il)- when stimulated with calcium ionophom (eur. j. immunol. ( ), ). we now present evidence that human eosinophils contain preformed il- protein although they do not express mrna for il- as measured by rt-pcr. il- protein expression was determined using specific anti-human il- monoclonal antibodies by western blotting, facs analysis and immunohistochemistry. moreover, preformed il- was released into supernatant by % pure eosinophils after priming with gm-csf or il- and subsequent -min stimulation with paf, rantes, ionomycin or pma, however not with il- or il- , as measured by elisa. this observation implies that activation of protein kinase c and/or inwacellular calcium mobilization am necessary events for il- release in human eosinophils. the determined amounts of preformed il- protein was higher in patients with asthma compared to normal individuals suggesting a role for eosinophil derived il- in asthma. since the eosinophil is the predominant cell in the asthmatic airways, we determined l- concentrations in bronchoalveolar lavage (bal) fluids from normal individuals and asthmatic patients. indeed, il- concentrations in bals from both groups were similar to those seen in the supematants released by activated eosinophils. the role for i-,- in asthma remains to be determined. to study the effect of canthaxanthin in vitro, the hydrophobic compound was introduced in vivo into chick high density lipoproteins (hdl) by canthaxanthin feeding. the effects of hdl and hdl associated canthaxanthin on two types of cultures have been tested: flat sedimented cell cultures of embryonic chick neuronal retina, retinal figment epithelium (rpe), brain and meninges, and in reaggregate cell cultures of the neuronal retina. at high canthaxanthin concentrations the formation of colored, birefringent entities were induced in the neuronal retina in vitro. in line with human data, parameters of cellular function and cell differentiation remained unaffected by canthaxanthin at these concentrations. by contrast, fidl was found to induce various cell type dependent effects. proliferation of meninges cells was decreased at low hdl concentrations as concluded from.light microscopic examination and indicated by protein content, and lysosomal and mitochondrial activity of the cultures (ic : - mg hdl apoprotein/l medium). these parameters, however, were increased in brain cell cultures, while they remained unaffected in cell cultures of neuronal retina and rpe. concentrations above . g. hdl apoprotein/l caused a reduction of glial cell differentiation. $ - tnf-c~ had close-dependent antiproliferative activity on hormone-dependent human breast cancer cells that represent an early stage of the disease, whereas hormone-independent cells representing a late stage were not affected. however, in the presence of u/ml interferon ?(inf), high concentrations of tnf-c((lnm) were able to inhibit the growth of hormone-independent cells. using specific monoclonal antibodies in ftow cytometry, no significant changes of either the p or the p tnf receptors were observed upon inf treatment of hormone-independent cells. this indicates that the increased responsiveness of these cells for tnf-c~ is not due to a change in available tnf receptors. the affinity of the receptors for tnf-r are one order of magnitude different for the two cell types. inf treatment shifted the ecs of hormone-independent cells towards the ecs of hormone-dependent cells. thus, the basis for the increased tnf-sensitivity of hormone-independent cells in the presence of inf might be the interconversion of tnf receptors from low-to high-affinity. the adhesion of tumor cells to endothelium is the first step in the processus of metastasis, and is frequently dependent of cytokinemediated activation of endothelial cells. cytokines are also involved in nitric oxide (no) production by various cells. these experiments were designed to evaluate no production during tumor cell adhesion to endothelium. rat brain-derived endothelial and rat colon carcinoma cell lines were used during these experiments. no was evaluated with the griess reagent. activation of endothelial cells with cytokines (tnf-d, and ifn-~ ), only slightly increased ( - %) the adhesion of tumors cells to endothelium. activation of endothelial cells with tnf-ol and ifn-~, induced a - fold increase of no production. however, addition of tumor cells to the endothelial monolayer, either directly or in a semi-permeable membrane, decreased the cytokine-induced no production. these results indicate that no production is modulated during the proeessus of adhesion of tumor cells to endothelium. is induced in rat mesangial cells by inflammatory cytokines such as interleukin ] (il- ~) and requires bh as a cotactor. , -diamino- -hydroxypyrimidine (dahp), a selective inhibitor of gtp cyclohydrolase i, the rate-limiting enzyme for bh synthesis, potently suppressed il-i~induced nos activity, measured as nitrite production. inhibition of no synthesis by dahp was reversed by sepiapterin, which provides bh via a salvage pathway. sepiapterin dose-dependently augmented il- i],-stimulated no synthesis, indicating that availability of bh limits the production of no in cytokine-stimulated mesangial cells. n-acetylserotonin, an inhibitor of the bh synthetic enzyme sepiapterin reductase, completely abolished il-lj~-induced no formation whereas methotrexate, which inhibits the pterin salvage pathway, displayed only a moderate inhibitory effect, thus suggesting that mesangial cells predominantly synthesize bh tom gtp. in conclusion, these data demonstrate that bn synthesis is an absolute requirement for cytokine induction of nos in mesangial cells. inhibition of bh synthesis may provide new therapeutic approaches to the treatment of pathological conditions mediated by no. - [ ) can induce a macrophage-type of nitric oxide synthase (inos). northern-blot analyses of inos mrna-levels and nuclear run-on experiments of control and il- ~ stimulated mesangial ceils revealed that the induction of inos was at the transcripuonal level. here we demonstrate that platelet-derived growth factor bb (pdgf-bb) can suppress the il- dependent inducuon of the inos gene. nortllern-blot analyses of cellular rna isolated from mesangial ceils that were coincubated with il- [ ( nm) and pdgf-bb ( ng/ml, ng/ml and ng/ml) revealed a dosedependent suppression of inos mrna-levels. using run-on experiments with nuclei from mesangial ceils after stimulation with il-i~ ( nm) and pdgf-bb ( ng/ml) we demonstrate that the inhibition occurs at the transcriptional level. basic fibroblast growth factor (bfgf), on the other hand, potentiates the il- dependent stimulation of inos. coincubation of mesangial cells with il- ( nm) and bfgf ( ng/m[, ng/m[, ng/m[, ng/m dose-dependently superinduces inos mrna levels as shown by northern-blot analyses of total cellular rna form control and stimulated cells. run-on experiments with nuclei from mesangial cells stimulated with il- ( nm) and bfge ( ng/ml) revealed an enhanced transcriptional activity of the inos gene. thus, pdgf-bb and bfgf differentaity modulate the il-i~ dependent expression of the inos gene and are therefore an excellent model system to study the crosstalk between signal transductjon pathways. we report that ifnyr-/-mice have an increased resistance to lipopolysaccharide-induced toxicity (lps). lps-induced lymphopenia, thrombocytopenia and weight loss seen in wild type mice were attenuated in ifntr-/-mice. ifntr-/mice survived in the d-galactosamine-lps model when conditions were % lethal for wild type mice, correlating with serum tnf levels of up to fold higher in wild type mice. bone marrow and splenic macrophages from ifnyr-/-mice had a to fold decreased lpsbinding capacity, with serum from these mice lowering macrophage lpsbinding by a further %. thus, depressed tnf synthesis, diminished expression of lps receptors and low plasma lps-binding capacity in the mutant mice likely combine to manifest in the resistant phenotype of ifny r-/mice to endotoxin. in a complementary approach we have screened the ' portion (- . kb/+ . kb) of the gene for tissue specific and/or inducible dnasei hypersensitive sites. in chromatin from fibroblasts or kidney epithelial cells this segment of the il ra gene does not contain any dnasei hypersensitive sites. in contrast, in resting t-and b-lymphocytes, as well as in activated t cells, two sites (dhi, - . kb; dh , - . kb) were found. a third site (dh , - . kb) is detectable in t cells that have been activated with concanavalin a and il . this site maps to the same position as cisacting regulatory sequences that are required for the response of the il ra gene to signals from the il receptor. interleukin- (il- ) production in cultured human dermal fibroblasts can be stimulated by interleukin-i (il-i} added to the culture medium. absence of fetal calf serum and of growth factors (insulin, egf, t ) reduced the stimulation by more than %. egf and t and even more choleratoxin increased the il- production in absence of fetal calf serum. the effect was dose dependent. % human ab serum substituted for i % fetal calf serum. pretreatment of the cultures with serum or growth factors prior to stimulation with il had a stimulatory effect (serum, t , egf) or an inhibitory effect (hydrocortisone, choleratoxin). our results suggest that the il-i signal transduction to il- is modulated by serum components and growth factors as well as through c-amp produced by choleratoxin. interleukin- (il- ) is produced by a variety of cells and plays a central role in host defense mechanisms. its function include induction of il- and il- receptor expression, proliferation and differentiation in t-cells. in cocultures of human dermal fibroblasts and allogenic human t-cells a cell number dependent to fold increase of the il- production could be demonstrated after h and h. conditioned medium from tcells and from fibroblasts failed to induce il- secretion in fibroblast and t-cell cultures, respectively. no up-regulation of il- production was found in cocultures of fibroblasts with tcells killed by ethanol and in t-cell culture incubated with recombinant hll-lc~. after separation of the cells il- production in fibroblast and t-cells returned to precoculture levels. our results indicate that cell-cell contact more than soluble factors must be involved in the up-regulation of il- synthesis in cocultures of t-cells and fibroblast. using cocultures of autologes human t-cells and fibroblasts, we are currently investigating whether the cell contact essential for the il- production depend on immunolcical interactions of t-cells and fibroblasts. rapid growth of tumor often results in oxygen and nutritional deprivation leading to extensive necrosis, which is one of the characteristics of glinblastomas (gbl). neoplastic cells surrounding necrotic areas often present a peculiar arrangement of cells called "pseudo-palisading" -as a hallmark of this entity, together with abundant neovascularization. a special biological milieu is probably formed in these palisading areas by certain unknown cytokines and/or growth factors induced by the necrotic process. plate et al. ( ) reported the expression of vascular endothelial growth factor (vegf) in the palisading cells. we previously demonstrated that gbl cells produce il- , which is known to be an angiogenic factor. the aim of this study is first to assess if il- is expressed in the palisading ceils, and second to determine if hypoxia can trigger il- gene expression. in rive observation using in siru hybridization and immunohistuchemistry showed that il- is highly expressed specifically in the palisading cells. we also demonstrated the mrna expression of il- receptor in the endothelial cells adjacent to necrotic area. to simulate the in vivo condition, two in-vitro models are currently developed to determine if il- is induced by hypoxic insult on cells. first, gbl cell lines are cultured in the absence of oxygen and il- expression is assessed by northern blot analysis. preliminary results have demonstrated the induction of il- by hypoxia. secondly gbl cells are grown in spheroids until development of central necrosis. the il- expression will be assessed by in situ hybridization combined with immunohistochemistry to determine if the ceils surrounding necrosis express il- . it has been proposed by taniguchi and his colleagues that activation of the interferon (ifn)-i gene by virus or double-stranded rna involves induction and modification of irf- . overexpression of irf- can induce expression of the ifn-b gene in certain cells. a role of irf- in the activation of ifn-a genes has also been proposed. furthermore, irf- has been claimed to play the role of a tumor suppressor gene. we have generated mice in which both irf- alleles were disrupted and found them to develop normally. no spontaneous tumors have been observed so far. injection of poly(i)-poly(c) resulted in the same levels oflfn in blood of wildtype and null mice, and equal ifn-ct mrna levels in spleen. induction of wild type and irf ~ embryo fibroblasts (mefs) with virus resulted in the same levels of ifn-a and ifn-i] mrna respectively. in the case of polyo)-poly(c) induction, the levels of both fn mrnas were higher in wild type than in mutant cells; this difference was abolished if the cells were first primed with type i ifn. we conclude that irf- does not play an essential role in the induction oflfn genes. mitsuhiro tada, annie-claire diserens, isabelle desbaillets, marie-france hamon, nicolas de tribolet, erwin van meir; chuv, lausanne there is increasing evidence that a variety of cytokines produced by glioblastoma (gbl) cells modulate the tumor growth by affecting the host's immune response and by stimulating neovascularization. cytokines such as il-i, il- , il- , mcp-i, il- and tgf- , affect each other's production and receptor system and seem to form a cytokine network. among them, il- may play a pivotal role, since il-i can induce or increase the production of other cytokines (il- ,il- ,mcp-i,tgf- ) and modulate their receptor expression. to test this hypothesis, we investigated co-expression of cytokines and their receptors in gbl cell lines and gbl tissues using rt-pcr, northern blot analysis, immunnhistnchemistry and elisa quantification. a majority of the gbl cell lines and gbl tissues expressed il-i~, il- , type i and type ii receptors, indicating the presence of an il- autocrine loop. il- stimulated growth of some of the cell lines. a positive correlation of il- and il- expressions with the presence of an il-i autocrine loop in the cell lines was found. immunohistochemical studies showed the in rive co-expression of the il-i family and the secondary cytokines (il- , il- ) in gbls. antisense oligonucleotide to il-lc~ and/or il- partly suppressed this secondary cytokine expression. these results demonstrate that the il- autocrine loop plays a central role in the formation of the cytokine network in gbls. double-stranded rna-dependent protein kinase (pkr) is induced by type i interferon (ifn) and is implicated in the establishment of the antiviral state. upon activation, pkr phosphorylates the eukaryotic initiation factor eif- , thereby throttling protein synthesis. it was suggested that pkr may be essential for the induction of ifn- gene expression by both virus and double-stranded rna and for the activation of at least some ifninducible genes. recently it was proposed that pkr is a tumor suppressor gene because t ceils overexpressing inactive human pkr gave rise to tumors in nude mice (koromilas et al., ; meurs et al., ) . we have produced homozygous pkr knockout (pkr ~ mice and found that they develop normally and have no striking phenotype. induction of ifn-c~ and ifn- gene transcription by virus was unimpaired in fibroblasts derived from pkr ~ mice, as was the induction of several ifn-stimulated genes. so far, no spontaneous tumor formation was observed. pkr o/o embryonal stem ceils showed no increased malignancy in nude mice as compared to their wild type counterparts. we conclude that pkr does not play an essential role in viral induction of the ifn genes and that its tumor suppressing effect, if any, is redundant. tgf- plays an important role as a negative regulator of hepatocyte proliferation in liver regeneration. exposure of rodents to nongenotoxic carcinogens like cyproterone acetate (cpa), thioacetamide (ta) and phenobarbital (pb) causes abnormal hepatocyte proliferation and liver tumors after long term treatment. this suggests that these chemicals have either a direct mitotic activity or impair the negative regulatory system for growth. therefore the inhibitory activity of tgf- on dna-synthesis induced whith cpa was investigated in cultured rat hepatocytes. after a h exposure to cpa, a dose dependent increase in dna synthesis ( h-tdr incorporation) was observed. the maximum effect was attained with pm cpa (up to . fold) which was stronger than with ng/ml egf (up to . fold). tgf- and tgf- inhibited this response dose dependently (idso = . ng/ml) and reduced it to control levels at ng/ml. these findings show that the tgf- mediated pathway for negative growth control in hepatocytes is still responding after cpa treatment. mouse mxl protein is an interferon (ifn) induced gtpase with intrinsic antiviral activity against influenza a viruses. it accumulates in the cell nucleus and inhibits the transcription of influenza virus genes, recently, we have shown that mxl exerts its inhibitory activity by interfering with the function of the viral polymerase subunit p . pb is responsible for recruiting " ends of cellular mrnas as primers and for the elongation of nascent viral transcripts. to elucidate which pb dependent step of viral mrna synthesis is the target of mxl action, we examined the activity of recombinant mxl protein in an in vitro transcription system of influenza virus. first, we expressed wildtype and mutant mxl proteins, carrying a hlstidinetag at their n-terminus, in e. coil and purified them under nondenaturing conditions by ni-chelate affinity chromatography. mxl efficiently inhibited viral transcription in vitro, while mutant mxl proteins, lacking antiviral activity in vivo ,were inactive. moreover, the use of capped synthetic rna primers revealed, that mxl almost completely abrogated the elongation of viral transcripts, whereas the the cap-binding and endonuclease activity of pb was not affected. a $ - neutralization rates of tnf-a from eight animal species by a monoclonal antibody against human tnf: implication for receptor action? we have recently developed a sensitive bioassay for measuring porcine tnf-a based on homologous pk( ) cells. this bioassay is -to -fold more sensitive than the widely used l bioassay, also, human tnf-a and human tnf- were detected with a slightly higher sensitivity in the new test compared to the l bioassay, we now show that also canine, ovine, bovine, equine and caprine tnf-a can be detected with the pk( ) bioassay. we tested a murine monoclonal anti-human tnf-a antibody for its capacity to neutralize tnf-a from eight different animal species. the action of this antibody revealed that neutralization of tnf bioactivity is a complex phenomenon, indicating species-specific differences in the interaction with tnf receptors. to study this differential neutralization, we are cloning the porcine tnf receptors for expression in a heterologous cell system. administration.of high dose r-tnf(~ in combination with ifn in isolation and perfusion of the limbs in melanoma patients has shown to be very promising with a response rate greater that %. this observation prompted us to investigate the possible expression of the tnf receptors in melanoma cells using the monoclonal antibodies utr- specific for the tnf type a ( kda) receptor and htr- specific for the tnf type b ( kda) receptor. flow cytometric analysis of cultured melanoma cells showed the presence at low level of the tnf type a and to a slightly higher level of the type b receptor. similar results were obtained in vivo by immunohistochemistry on fresh tumor raateriai, treatmem of melanoma cells in culture with the-amp induced up to a fold increase in the number of type a tnf receptors with only a minimal change in the number of type b receptors. this increased expression of tnf receptors is conflrmed by direct binding experiments using i-labeled tnftx. the number of cpm's bound on dbc amp treated cells was about . - fold higher than on untreated control calls, likewise incubation of melanoma cell lines with ifny increased the specific binding of subsequently added i-labeled tnfct. from flow cytometric analysis using the two anti-tnf receptor antibodies it became evident that flint was able to increase the expression of both type a and b receptors depending on the cell line used. characterization of a murine tumor necrosis factor (x-lacz reporter construct tumor necrosis factor ot (tnfcq is a prominent mediator of a variety of different pathologies such as endotoxic shock syndrom and rheumatoid arthritis. it also plays a crucial role in host defence against bacterial and viral infection. up to now, only few data about signal pathways leading to tnfo~ induction are available. an easy to handle tnfc~ -lacz reporter assay will represent a useful tool to study signal transduction on the one hand and provide data about compounds with potential tnfo~ inducible activity on the other hand. in order to develop a routine macrophage cell line capable to easily report tnfc~ induction, different tnfe-lacz reporter constructs were assembled. data about functionality of the different constructs in routine macrophages will be presented in the context of tnfot expression. cloning of a novel protein binding to lymphokine, fos and myc mrna with unexpected enzyme activity j. nakagawa, h-p. waldner, s. meyer-monard, and ch. moroni inst. medizinische mikrobiolegie, univ. basel an au-rich sequence in the ' untranslated region of lymphokines, c-los, and c-myc proto-oncogene mrna is responsible for their rapid decay. we have purified a kd protein by an auuua affinity column from human brain. partial amino acid sequence was obtained and the corresponding edna has been cloned. unexpectedly the edna sequence exhibited significant homology to enoyl coa hydratase and bacterially expressed recombinant protein indeed showed the activity. while rat hydratase did not show rna binding activity, the recombinant protein bound to cfos, c-myc, auuua cluster of il- mrna, and adeno virus iv, but not to mutated ad-iv, nor irrelvant transcript. the binding was competed out by poly u. interestingly the protein seems to be processed from a larger precursor. we suspect that the protein plays a role in mrna turnover and perhaps in oncogenesis. institute for medical microbiology, university of basel in t cells, the immunsuppressive agent cea is known to inhibit ca + dependent induction of lymphokine mrna transcription. in contrast, the immunsuppressive drug rapamycin inhibits the lymphokine induced proliferation. we have analysed the effect of these drugs on a v-h-ras induced autocrine mast cell tumor line which expresses il- constitutively. csa and rapa inhibited autocrine growth by different mechanisms,, because added il- could antagonize inhibition by csa, but not by rapa. whereas csa acted by downregulation of il- mrna expression, rapa blocked il- induced proliferation. cea also inhibited il- superinduction by ca-ionophore, whereas rapa did not. nuclear run on assay indicated that the mechanism is posttranscriptional. therefore, we have introduced il- transgenes with and without the au-rich region in the ' utr of the mrna into a tumor line. in contrast to the wild type, the expression of the mutant construct was insensitive to csa. since the mutant lacks sequences known to regulate rapid mrna decay, we suggest that csa affects the regulation of il- mrna stability. some non-hematopoietic cell lines produce both scf and its receptor, the c-kit protein (c-kit). testing the hypothesis that scf may be involved in secondary growth of nb bone marrow metastasis, we found and previously communicated (beck d. et al, proc aacr, , , ) a low or absent expression of c-kit in nb cells. the mrna and surface expressions of scf gene in nb cell lines grown in chemically-defined medium were evaluated by northern blot analysis and flow cytometry. the scf antigenic concentrations in culture supernatants were determined by elisa and growth-inhibition experiments performed by pre-incubating nb cells with anti-scf antibodies prior to h-thymldine uptake assay. the scf mrna was expressed in of tested lines but no membrane-bound antigen could be detected on those cells. detectable levels of scf in the supernatants were found in / lines (range : - pg/ml). blocking experiments resulted in a decrease of dna synthesis in i out of lines only. from these and previous results, we conclude that scf is synthesized and released by many nb cells in vitro but the functional ~ole of it remains undetermined since scf does not appear to be usually involved in autocrine or paracrine growth stimulation of nb cells (supported by swiss fnrs grant - ). expression of the v-h-ras oncogene in the il- dependent pb- c mast cells leads to the generation of two classes of il- autocrine tumors in vivo. autocrine il- expression in class- tumors results from increased il- mrna stability due to the loss of negative trans-acting posttranscriptional control. this defect can be corrected by somatic cell fusion to the nontumorigenic parental pb- c resulting in downregulation of oncogenic il- expression and concomitant tumor suppression. expression of the v-h-ras oncogene remains unaffected in class- hybrids. in contrast, class- tumors are characterized by transcriptional activation of the il- gene due to the insertion of an endogenous retroviral element (intracisternal a-particle) which cannot be overcome by ceil fusion (see hirsch et al, , j.exp.medicine , - ) . although v-h-ras is required for generation of either class of tumors, expression of anti-sense ras constructs inhibited only the proliferation of class-i tumor cells. experiments are underway to characterize the target and the nature of the class- alteration. nitric oxide (no) promotes vasorelaxatlon of renal vessels in vitro. the purpose of the present study was to assess the role of no in regulating renal hemodynamics in vivo. renal blood flow (rbf) and glomerular filtration rate (gfr) were studied in anesthetized mechanicallyventilated newborn rabbits both before and after the administration of a no synthesis inhibitor, ng-nitro-larginine methyl ester (l-name), at a dose of pg/kg followed by jzg/kg x rain. such a dose of l-name did not modify mean arterial pressure or pulse rate. by contrast, the renal vascular resistance increased by + % (p < . ) while rbf decreased by -+ % (p < . ). gfr and urine flow rate remained constant. the overall results suggest that in normal conditions no may play a role in decreasing the renal vascular resistance of the immature kidney, without altering systemic blood pressure. interleukin- (il- ) is a t-cell derived lymphokine which, in addition to its effects on the immune system, is also able to suppress the growth of certain tumor cells. il- reduced the growth rate of four human colon tumor cell lines up to % in a dose-dependent manner. three other cell lines showed no significant alteration of h-thymidine incorporation or colony formation in the presence of u/ml il- . responder and nonresponder tumors were immunopositive for il- receptor (il- r) expression in flowcytometric analysis, using monoclonal antibodies raised against recombinant il- r and bound biotinyated il- . responsive tumors expressed more receptors. - binds with high affinity to a kda transmembrane receptor (il- r), through which the extracellular signal has been shown to be transduced. coimmunoprecipitation and chemical crosslinking studies have enabled us to demonstrate il- r target proteins. tyrosine phosphorylation of various proteins between and kda appears to be initiated during il- mediated signaling events in colon tumor cells. the cloning of human il- r target proteins will contribute to the understanding of the il- signal transduction pathway at the initial stage. the renal effects of endothelin- were investigated in anesthetized and meehanleally-ventilated newborn rabbits. each animal acted as its own control. in newborn rabbits, a bolus injection of nmol.kg " of endothelin- caused an initial fall in mean blood pressure (mbp) followed by a gradual but significant increase in mbp that lasted for rain. the dramatic increase in renal vascular resistance (+ -+ %) induced by endothelin led to a fall in glomerular filtration rate (- -+ %) and renal blood flow (- _+ %). in spite of the reduction of gfr and rbf, urine flow and sodium excretion rates increased significantly (+ _+ % and + _+ %, respectively). in additional newborn rabbits, a bolus injection of lnmol.kgaof endothelin- -a dose that usually induces marked renal and systemic vasoconstriction in adult models -did not affect systemic or renal hemodynamics. in conclusion, endothelin induces renal and systemic vasoconstriction, and affects water and sodium homeostasis during the neonatal period. however, these effects occur under higher doses than those used in adult animals, possibly reflecting receptor immaturity and/or interference of high levels of counteracting hormones. ontogeny of the endocrine pancreas in xenopus laevis c, maake , w. hanke and m. reine~ke , institute of anatomy, university of z rich, switzerland and department of zoology ii, university of kaflsruhe, f.r.g. the pancreatic islets of anurans show insulin (ins), glucagon (gluc), somatostatin (som) and pancreatic potypeptide (pp) containing cells. since only limited information exists on the ontogeny and on the presence of insulin-like growth factor (igf- ), we studied the development of the gastro-entero-pancreatic (gep) system in xenopus laevis using immunohistochemical techniques. singular ins-immunoreactive (-ir) cells were observed in the pancreas as early as on stage . at stage , the first pp-ir cells were found in the gep system followed by som-ir and gluc-ir cells. the first pancreatic islets consisting of ins-ir and gluc-ir cells occurred around stage . between stage and , i.e. after the onset of metamorphosis, the endocrine cells decreased in number and the islets partly disintegrated. starting with stage , the islets reformed and the amount of endocrine cells increased reaching the adult status at stage . around stage , igf-l-immunoreactions appeared. igf- immunoreactivity was found in pp-ir and gluc-ir cells but not in ins-ir and som-ir cells. it is assumed that islet-derived igf- may p~ay an important role during metamorphosis in xenopus. t. cathomen and r. cattaneo, institut for molekularbiologie i, universit~t zorich, hfnggerberg, ch- zorich the signals used for synthesis of the measles virus fusion (f) protein are poorly understood: a long (> bases) untranslated region is followed by three in frame augs at codons , and . f is a type i transmembrane protein with a postulated signal sequence of amino acids. we confirmed that the aminoterminal residues contain a signal peptide by transfering them to another protein. with the other envelope protein hemagglutinin, f protein induces virus-cell and cell-cell fusion. mutagenesis of the three augs indicates that proteins initiated at codon , or are all functional, but show slightly different fusion efficiencies. forms translated from aug or appear larger in electrophoresis than forms initiated at aug , suggesting that two different signal peptide cleavage sites are used. we are currently investigating whether both f protein forms are incorporated in viral particles. the production of protein isoforms with staggered amino-termini is common in cytoplasmic and type ii transmembrane proteins, but signal sequences usually preclude a similar organization of type i transmembrane proteins. beguin p., beggah a.t., rossier b.c., jaisser f. and geering k. institut de pharmacologie et de toxicologie de l'universit , ch- lausanne recent experimental evidence suggests that na,k-atpase might be a candidate for regulatory phosphorylation by protein kinases a and c (pka and pkc). to verify this hypothesis, we have mutated several serine and threonine residues in consensus phosphorylation sequences of the ~subunit of bufo marinus na, k-atpase. the mutants were expressed in x~nopus ooeytes and phosphorylation was studied in oocyte homogenates upon stimulation of oocyte, pka and pkc. our results indicate that a unique phosphorylation site for pka is located at serine in the c-terminus of the ~-subunit but its phosphorylation can only be revealed in the presence of detergent. on the other hand, mutations of several serine and/or threonine residues located in consensus sequences for pkc phosphorylation did not or only partially abollsh phosphorylation by pkc. in particular, mutations close to the catalytic phosphorylation site of the ~-subunit influenced phosphorylation by pkc, suggesting that either this region contains a real phosphorylation site or is part of a conformational domain necessary for phosphorylation by pkc. the heat-stable antigen (hsa or mouse cd ) gene is differentially regulated but has a housekeeping promoter. roland h. wenger*, georges kshler and peter j. nielsen. max planck institut f(jr immunbiologie, st beweg , d- freiburg i. bsg. "present address: physiologisches institut der universit&t zi.)rich, winterthurerstrasse , ch- z rich, expression of the gpi-anchored murine glycoprotein heat-stable antigen (hsa) shows tissue-specific as well as developmental regulation. during the maturation of several hematopoietic lineages, hsa expression is generally high in immature precursor ceils and low or absent in terminally differentiated cells. we present evidence suggesting that this regulation of the hsa gene (cd a) occurs at the transcriptional level. in addition, sequence and methylation analysis of the cd a promoter revealed characteristics of both "housekeeping" and tissue-specific promoters, including a methylation-free, hpall tiny fragment (htf) island, multiple putative sp and ap- consensus binding sites and a tata box. functional analysis of a . kb dna fragment containing these elements fused to the cat reporter gone in transient transfection experiments showed activity in both hsa expressing and non-expressing cell lines with a strength similar to that of the hsv tk promoter. large fragments from the flanking region of the cd a promoter did not influence the ubiquitous nature of this promoter, finally, the cd a, cd b and cd c genes were mapped to mouse chromosomes , and respectively. we have cloned, sequenced and characterized the gone for the m__itochondriat nadh-cytochrome b reductase (mcri) of saccharomyces cerevisiae. surprisingly, this gone encodes two mitochondrial isoforms of the flavoenzyme: a kda integral protein exposed on the outer surface of outer mitochondrial membrane (mcrlp ), and a kda soluble protein of the intermembrane space (mcrlp ). the smaller intermembrane space isoform is generated from the larger form by the action of the inner membrane protease i (impi) on the outer surface of the inner membrane. this is the first demonstration that a single gone encodes proteins which are located in different compartments of the same organelle. with special interest in any with a mitochondrial localization. degenerate primers were designed on the basis of high homology between members of the abc family, and pcr was performed on yeast genomic dna. ten dna fragments bearing significant homology to the abc family were amplified, nine of which were previously unidentified. disruptions of five of the corresponding genes were performed. disruption of one gene led to markedly impaired growth on rich medium and cessation of growth on minimal medium. the gene was cloned and found to encode a protein of amino acids, a "half-transporter" which likely forms a dimer. ibi~tlnofluorescence on yeast expressing the gene cmyc-tagged at the c-terminus reveals co-localization of the protein with porin and with mitochondrial dna, visualized by dapi staining. nycodenz-purified mitochondria are enriched for the protein by immunoblotting. additionally,the c-myc epitope is protease-protected in mitoplasts, indicating an inner membrane sub-localization and a probable orientation with the c-terminus in the matrix. the gene, termed atmi for abc transporter of mitochondria, thus encodes the first member of the large abc transporter superfamily to be localized to this organelle. present work is aimed at revealing the substrate of this transporter. cycle in cardiac myocyte. the molecular study of this protein has been difficult even after its over-expression was made possible by the cloning of the corresponding cdna. the chief problem is the lack of convenient domains which could be used in the purification of the molecule. we have previously reported the efficient expression of the cardiac na+/ca + exchanger in mammalian cell lines using the t rna polymerase-hybrid vaccinia virus system. we have now constructed the recombinant virus for the exchanger with a xhis-tag at the c-terminal. this tag has enabled the purification of the protein through a ni-nta agarose column. the expressed tagged protein induced na-dependent ca uptake which was not different from that in intact cells, i.e., cells in which the exchanger did not have the tag, the most important aspects of the purification produced and those of the reconstitution of the expressed protein will be described. $ - the neural cell adhesion molecule l is involved in the formation and specification of cell contacts in the central and peripheral nervous system during development, and thus may also play a role in synaptic plasticity of the adult central nervous system, using extracellular recordings of evoked field potentials in the rat hippocampal slice, we found that locally applied polyclonal anti-l and fusion proteins containing the ig-domains i-vi of l specifically block the stabilization of ltp in the ca region. baseline synaptic transmission was not affected by the presence of the anti-l antibodies. the anti-ll-induced effect was dependent on the antibody concentration and anti-l antibodies had no effect on previously established ltp. ltp was also reduced by an antiserum against the recombinantly expressed ig-domains i-vi of l , whereas controt antibodies against liver cell membranes, which bind to neuronal membranes in the hippocampus, exhibited no effect on ltp. the contribution of l to stabilization of ltp within the ca synapses suggests that members of the ig-supertamily of cell adhesion molecules are involved in the structural modifications which are associated with synaptic plasticity in the mammalian central nervous system. an increasing number of surface proteins are described as being attached to the membrane by a glycosyl-phosphatidylinositol (gp ) anchor instead of the conventional transembranous hydropbobic domain. they are initially synthesized with a coohterminal polypeptide extension which is cleaved in the e.r. and replaced by the preformed glycolipid. this processing event is directed by a signal, located in the c-terminal region of the protein, which requires a group of three small amino acids positioned - residues upstream of an hydrophobic c terminal sequence. we previously transformed the transmembfanous glycoprotein d of herpes simplex type i into a gpi-anchored molecule by deleting its cytoplasmic domain and thus creating a molecule, gdww , which meets the requirements for anchoring via a gpi structure. we now demonstrate, using gpi-deficient cell lines and biochemical studies, that a hybrid protein consisting of the thy-i ectoplasmic domain and of the c-terminal region of gdww can be alternative[y expressed in a gpi-anchored or in a transmembranous form. service de p diatrie, chuv, ch- oll lausanne proteolipid protein (plp) is a major myelin protein in the central nervous system (cns). a number of x-linked dysmyelinating disorders have been identified as mutations in the pip gene. we have identified a novel plp mutation in paralytic tremor (it) rabbit, characterized by body tremor, spastic limb paresis and hypomyelination in the cns. reduced levels of plp protein and its mrnas were observed in the cns as well as in the pns. plp sequence analysis revealed a single nucleotide change in exon which results in the substitution of a histidine by a glutamine at position . histidine is localized on the boundary between the first transmembrane region and the intracellular part of the protein. therefore, its position can be crucial for the efficient plp interaction with other proteins and lipids, and correct incorporation of the plp molecule into the membrane. histidine is also part of a short fragment of ten amino acids which is conserved in all species studied. the same amino acid is altered in another hypomyelinated mutant, shaking pup, stressing its functional importance. furthermore, the pt mutation affects the plp "premyelin" functions, including oligodendrocyte maturation. equilibrium constants for octamer dissociation as well as dissociation rates in vitro increased in correlation to the number of charged residues eliminated, e.g. mutant - , in which all four charged residues in the n-terminal heptapeptide are substituted with uncharged amino acids, showed a -fold higher equilibrium constant for octamer dissociation and a -fold increased dissociation rate compared to wild type. this strongly suggests that the charged amino acids in the n-termlnal heptapeptide of mib-ck, and therefore an ionic interaction, play an important role in forming the oetameric molecule. gangliosides and neutral glycosphingolipids (gsls) cell surface molecules are involved in a variety of biological processes and are assumed to play an important role in the mechanisms of hiv entry into lymphoid and epithelial cells. the total lipid-bound sialic acid (lbsa) content and the composition of gangliosides and gsls were investigated on pbmn cells from hiv+ve and normal donors. lbsa level was quantified by a fluorimetric assay, while gangliosides and gsls were assayed with high performance thin layer chromatography (hf'tlc) after multistep lipid extraction of x g membrane fractions. in normal pbmn cells the lbsa content accounted for . /~g/ cells or nmol/mg protein whilst in pbmn cells from hiv+ donors the lbsa level decreased to . ,ug/ cells or nmol/mg protein. the qaantitation of neutral gsls and gangliosides was carried out by scanning spots revealed on hptlc plates and the integrated areas computed with a dedicated software. our findings showed that the most relevant ganglioside present in normal pbmnc was gm ( - % of total gangliosides) followed by small amounts of od ( - %) and other acidic glycosphingolipids. neutral gsls included gl , gl , gl and gl -hexosylceramide. hiv+ pbmn cells were characterized by a decrease of neutral glycosphingolipids as well as gm content which represented only % of the ganglioside fraction, indicating an alteration in the glycolipid composition in the plasma membrane of infected cells. the neuropeptide y (npy) is one of the most abundant peptides of the mammalian brain. upon stimulation of cell surface receptors, npy generates diverse physiologic responses. npy acts on the cardiovascular system. it is a vasoconstrictor by itself and in addition, it potentiates the action of vasoactive substances such as angiotensin ii or norepinephrine. npy also inhibits glucose induced insulin secretion and is a potent inducer of appetite. to facilitate the development of npy antagonists we are investigating the interaction of npy with its cell surface receptor.we first constructed a series of mutants in which negatively charged residues present in putative extracellular domains of the y receptor were systematically replaced by alanines. the mutant cdnas were transiently expressed in hela ceils using a vaccinia virus derived expression system and the abi}ity of the encoded proteins to bind npy was evaluated. the capacity of the mutant proteins to be recruited to the cell surface was assessed by confocal microscopy. we identified initially spacially clustered extracellular acidic residues of the human y npy receptor essential for npy binding. subsequently, we mutated additional residues. based on these data a detailed computer model of the interactions between npy and its receptor was built. functional activity to energy metabolism. l. pellerin and p.j. magistretti. institut de physiologie, universit de lausanne, switzerland. astrocytes play a central role in brain energy metabolism. for example glucose, the exclusive brain energy substrate, is avidly taken up by astrocytes (pnas : , ) . application of glutamate (glu), the main excitatory neurotransmitter, stimulates in a concentration-dependent manner h- -deoxyglucose ( - ( ) uptake by astrocytes in primary culture with an ec of ~ ~m. the effect is not receptor-mediated since it can not be reproduced by glutamatergic agonists such as (nmda, kainate, quisqualate, t-acpd) nor prevented by antagonists (apv, cnqx, ap ). instead, it involves glutamate uptake since the effect is blocked by dl-threo-j hydroxyaspartate, a potent inhibitor of the glu transporter. replacement of na + in the medium by choline also prevents the effect, suggesting a na+ driven process. finally ouabain, a selective inhibitor of the na+/k + atpase, completely prevented the effect of glu. these observations suggest that when glutamatergic synapses are active, glutamate, taken up by astrocytes, stimulates -dg uptake by astrocytes whose end-feet are known to surround capillaries, i.e. the source of glucose. they also provide a simple explanation for the mechanism linking functional activity to energy metabolism. cloning and molecular characterization of the mouse astrocyte glycogen synthase. g. pellegri, c. rossier, s. van berchem, p.j. magistre~i and j.-l. martin. institut de physiologie, universit de lausanne, switzerland. in the brain glycogen is predominantly localized in astrocytes, although its presence has been detected in certain large neurons, in ependymal and choroid plexus cells. glycogen is synthesized by the enzyme glycogen synthase (glys) from udp-glucose. out of the different glycogen synthase isozymes, only the muscle and the liver forms of glys have been cloned. we have isolated and characterized from a mouse astrocyte ~.zapii cdna library, a cdna clone encoding the mouse cerebral cortical astrocyte glys. the . kb clone isolated contains an open reading frame of bp encoding a protein of amino acids. the coding region of the mouse astrocyte glys cdna shares % and % identity with the nudeotide sequences of the human muscle and the rabbit muscle giys cdna respectively. the cellular distribution of the glys mrna studied by northern blot shows the presence of a single transcript of kb in cultures of astrocytes and, to a lesser degree, of neurons. the distribution of glycogen phosphorylase, which was also examined by northern blot shows the presence of a . kb transcript both in cultured astrocytes and neurons with however higher levels expressed in astrocytes. the functional molecular size of the vacuolar h+-pyrophosphatase (h +-ppase ec . . . ) from maize (zea mays l.) roots was estimated by radiation inactivation, both for substrate hydrolysis and for proton transport. light microsomal vesicles enriched in tonoplast membranes were prepared from young ( days) maize roots using sucrose step gradients. these vesicles were still competent for ppi-dependent proton transport after freeze-drying and rehydration of the membranes. frozen native or freeze-dried samples were irradiated with ~ for various periods of time. after thawing the samples, the activities of glucose- phosphate dehydrogenase (added as an internal standard) and h+-ppase (hydrolysis of pyrophosphate (ppi) and ppi-dependent proton transport) were tested. by applying target theory, the functional size for hydrolysis was found to be around mr when the native or freeze-dried samples were irradiated. no ppi-dependent proton transport activity was measurable after irradiation of the native samples. on the contrary, the freeze-dried membranes were still pumping protons after irradiation and a target size of around mr was measured for the transport activity. these experiments suggest that the native h+-ppase from maize roots may exist as a dimer (at least) of the mr catalytic subunit. $ - the na,k-pump moves na + out and k + ions into the cells. the domains of the protein involved in ion translocation have not been determined. we have studied the role of the n-terminal region in the na + translocation by measuring the kinetics of charge movements under na/na exchange conditions in wild type (wt) and two n-terminally truncated forms of the bufo ~ subunit with deletions of the (t ) and (t ) first amino acids expressed in xenopus oocytes. we have developped a new technique to perform fast (< i ms) voltage clamp on whole oocyte. the membrane on one side of the oocyte is permeabilized by digitonin allowing to measure current flowing across the other half. we observed presteady state ouabain-sensitive currents similar to that reported by other techniques (j.gen. physiol. : , ) . the estimated forward charge translocation rate was lower in both n-truncated mutants (wt • , t z , t z s-l). the midpoint potential of the charge translocation (best fit to boltzman equation) was - , - , and - mv in the wt, t and t groups, respectively. the highly charged nterminus of the q subunit has a significant role in the forward translocation of na + ions. deletion mutants of human small intestinal lactase-phlorizin hydrolase (lph) were constructed to determine the role of protein subdomains in the intracellular transport of lph. the mutant lph mact ( aa deletions), which contains the membrane anchor (ma) and the cytoplasmic tail (ct), was transported to the cell surface in a fashion similar to wild type lph. by contrast, lph lacking the transmembrane domain persisted as a mannose-rich polypeptidc. this suggests that the membrane anchor and/or cytoplasmic tail are implicated in the er-egress of lph. another mutant, lph mact ( aa deletions), was not efficiently transported to the golgi apparatus. epitope mapping with monoclonai antibodies as well as protease-sens{tivity assays provided an evidence that the tertiary structure of lph mact is very similar to that lph mact. in view of the variations in the proportions of the complex glycosylated molecules of the two mutants we conclude that the domain between lph mact and lphi mact may play an essential role along the secretory pathway of lph. containing tyrosine. hussein y. naim, and michael g. roth; dept, of biocherni~try, ut-southwestem medical center, at dallas, dallas,texas-usa we have investigated an artificial internalization signal created by site-directed mutagenesis of the short cytoplasmic sequence of the influenza virus hemagglutinin (ha). mutation of cysteine to tyrosine converted ha from a protein essentially excluded from coated pits to one that internalized at rate similar to some cellular endocytic receptors. to determine whether or not the ha-y signal is a degenerate form of the internalization signal found in proteins such as the transferrin receptor and the mannose- -phosphate/igfii receptor, we have mutated amino acid positions in the ha-y shown to be important for internalization of the two receptors. we have changed amino acids on either side of y to those that either fit, or break the pattern of aromatic and hydrophobic residues proposed as consensus sequence. our results indicate that the ha-y mutant contains a sub-optimum sequence for a tyrosine-based internalization signal similar to those found in the receptors for transferrin, ldl, and mannose- -phosphate/igfil. however, amino acids with side chains having different chemical properties functioned well in positions that are important for the internalization signal, indicating that the consensus sequence for this signal is more variable than previously proposed. n-linked glycesylation is an essential protein modification occuring in all eukaryotic cells. core-oligosaccharides are transferred by the enzyme n-oligosaccharyl-transferase frc~ the donor glc man gicnac -dolichol to selected asparagine . resldues of t~e nascent polypeptlde chazn. the donor is build up in a stepwise fashion at the er-membraneby the products of the alg-genes (asparagine-linked-~lycosylafion) mutants defective in this pathway are available, but often lack a phenotype suitable for isolation of the corresponding genes. we found that double mutants of alg or alg with a wbpl mutation (defective oligosaecharyl-transferase) are severely growth retarded at oc. the double mutant phenotype allowed the isolation of both alg and alg genes which have been refractory to cloning so far. both genes encode potential trarsmenbrane proteins and are able to complement the biochemical defects of the corresponding alg or alg mutants. the na,k-atpase produces the driving force for the regulated transport of na across epithelial ceils. it is composed of an c~-subunit which carries the catalytic and ouabain binding sites and a b-subunit. to test the role of the usubunit in transport regulation, we transfected a cells with the b. marinus al-subunit (txl-tbm). this subunit confers a -fold higher resistance to ouabain (k i = /~m) compared to the endogenous pump. taking advantage of this difference, stable transfectants were selected with ouabain. expression of the cd-tbm subunit was visualized by western blotting. approximately half of the positive cell lines showed a high electrical resistance similar to untransfected cells, when cultured on filters. na-transport activity, measured as isc, was lower even in the absence of ouabain, but the relative increase in response to aldosterone was maintained. functional pumps containing the exogenous or endogenous al-subunit were quantified by equilibrium ouabain binding. interestingly, cells cultured on plastic dishes had a large fraction of low affinity binding sites (txi-tbm), while most sites were of the high affinity type when the cells were grown on filters. we conclude that the ouabain-resistant na,k-atpase cd-subunit of b. marinus can be expressed in a cells and used as a selective marker. however, the results suggest that the expression of functional ouabain-resistant pumps containing the al-tbm subunit could be influenced by the degree of cellular differentiation. in infected cells fully functional na/pi-cotransport was found in a time dependent manner; up to a -fold stimulation of na/p;-cotransport compared to uninfected cells was observed aft r - days. transport of phosphate by infected cells was highly dependent on the presence of sodium, exhibited a kmtd~ of around . mm and was dependent on extraceuular i~h'. in agreement with expressed transport of phosphate, infected cells produced an immunoreactive polypeptide of kda that represents a non-glycosylated form of the to kda mature na/p;-cotransporter. we conclud$ that the protein napi- /rat when expressed in sf cells exhibits na/p-eotransport with similar kinetic characteristics as observdd in prox ma tubular apical na/pcotransport. therefore the napi- protein as expressed h sf cells can be used for further structural and functional studies. talin interacts in vitro with actin, vinculin, integrins and bilayers of acidic phospholipids, and nucleates actin filament growth at the bilayer surface. it is therefore believed to be a key protein mediating aetin-membrane linkage. we have analyzed functional properties of proteolytic fragments of purified platelet talin. using limited proteolysis two fragments of and kd were generated. preliminary results, obtained using viscometry and f-aetin-nucleating assays, suggest that the kd fragment contains the actin binding site. we also assessed the lipid-binding capacity of the fragments. when a mixture of the two fragments was centrifuged in the presence of large multilamellar liposomes containing phosphatidylserine, only the kd fragment, but not the kd domain, co-sedimented with the liposomes, suggesting the presence of a specific lipid-binding site in the kd domain. interestingly, this fragment contains the n-terminus of talin which shows homologies to the membrane-binding regions of protein . . we are now analyzing membrane-binding properties of talin domains in more detail using hydrophobic photolabelling. recent studies suggest a role of the neural cell adhesion molecules l and ncam in mechanisms of memory formation (doyle e, et al, j. neurochem. : - , scholey a.b. et al, neuroscience : - , ; lijthi a. et al. unpublished data and see usgeb ) . in the present study we analyzed the effect of chronic intraventricular infusion of polyclonal antibodies against l (anti-u) or ncam (anti-ncam) on the performance of male wistar rats during the acquisition and retention of a spatial learning task. briefly, rats had to remember the position of a submerged escape platform in a water tank (morris water-maze). when the escape platform was removed after acquisition training (= retention test), the performance of animals chronically injected with anti-l showed an impaired search pattern when compared with that of salineqnjected animals (p> . , mann-whitney). whereas control animals spent up to % of their time searching for the platform in the correct (= training) quadrant, the time anti-l animals swam in the correct quadrant was closer to chance level ( %). although monitoring penetration of antkncam into the hippocampus was almost as efficient as that of anti-l , the performance of the anti-ncam-group was highly variable and did not differ from the performance of controls. these results agree with recent findings on the involvement of neural cell adhesion molecules in other forms of learning. we have isolated cdnas from mouse and from xenopus encoding the ~-subunit of the gastric h,k-atpase, which is expressed in the parietal cells of the stomach mucosa. the gastric h,k-atpase transports h + into the stomach lumen with the counter transport of k +, all at the expense of atp hydrolysis. when xenopus oocytes are injected with crna encoding ~h,k from either species, as well as a gastric ~h,k subunit, the two subunit proteins are synthesized, assemble, and form functional pumps located in the plasma membrane as demonstrated by rb+ uptake. the sensitivity to the gastric h,k-atpase inhibitor sch was determined, with the ki for beth species found to be in the low ~m range. spodoptera frugiperda ceils are often used to overexpress eucaryotle proteins using baculovirus vectors. the atomic force microscope (afm) is a device which allows the observer to obtain high resolution images of biological samples immersed in a fluid medium; this instrument has therefore the potential to visualize overexpressed proteins on the surface of living cells. however, the interpretation of the images is sometimes difficult and requires a good knowledge of the specimen topography, i.e. the "normal" plasma membrane of the cell used for the overexpressisn. on our poster we present afm images of the plasma membrane of both fixed and living spodoptera frugiperda ceils. in additon, we discuss the possibility of achieving high resolution in the imaging of dynamic changes which affect /iving systems. the deduced protein sequence consists of amino acids with about % amino acid sequence identicy when compared to mammalian ~h,k-subunits. data from other species and from the structurally similar na,k-atpase has shown that the ~-subunit is necessary for the formation of a functional pump, which consists of an ~/~ heterodimer. following co-injection of xenopus oocytes with crna for both the ~-and ~subunits, we have observed by rb + uptake h,k-atpase activity in the plasma membrane. we are presently studying the role of the ~-subunit in the maturation and function of the h,k-atpase. the atomic force microscope (afm) is a new type of microscope which allows visualisation of inorganic and organic samples with a very high resolution. a sharp tip fixed at the end of a cantilever is used as a topographic sensor. by scanning the sample with this device, a computer records the minute deformations of the cantilever and computes the topography of the sample. the instrument allows also the measurement of the interaction between the tip and the sample as a function of the distance. the knowledge of these interactions allows us to compute several mechanical properties like indentation, elasticity and stiffness. on this poster we report the results of these measurements on cells (sfg, c ), bacteria (e. coli, b. subtilis) and proteins (actin). in addition we discuss the use of the afm as a sensor for probing the mechanical properties of biological systems at a nanometric scale. neonatal thymectomy of balb/c mice induces aig. we have cloned the ~-and ~subunits of the balb/c h,k-atpase and expressed the proton pump in crna injected oocytes. both and ~ proteins are made, form functional pumps, and are immunoprecipated using auto-immune sera. the ~-subunit can also be in~unoprecipated independent of the ~-subunit when oocytes are injected with ~ crna alone. balb/c mice will be immunized with oocyte membranes containing either the ~/~ or ~ antigens in order to study the role of each h,k-atpase subunit in triggering aig. the prion protein is expressed in both astrocytes and oligodendrocytes of the neonatal brain and is down-regulated in adulthood. m. moser, colello, r, , pott, u, and b. oesch institut for hirnforschung der universit~t zorich, august forel str. , zorich the infectious agent causing transmissible spongiform encephalopathies consists largely or entirely of prp sc, a modified isoform o| normal prp (prpc). prpc is most abundant in the brain. the development of the disease strictly depends on the presence of prpc. and incubation times are shortened by overexpression of prpc. the normal function of prpe is not known and its cellular iocalisation in the cns is poorly characterised, except for its evident presence in neurons. here we describe the expression of prp c mrna in both oligodendrocytes and astrocytes during neonatal development of hamster and rat. in adult animals, expression is down-regulated in gila but not in neurons. our findings provide a link to the previously described accumulation of prpsc in astrocytes and the apparently faster course of prion disease in neonatal hamster. our results argue for a major involvement of non-neuronal cell types in prion diseases. such structures we raised mab against membrane protein fractions. a ~lab against a vitronectin receptor containing fraction was found, which if applied in vitro to bi fi mouse melanoma cells, influenced growth, adhesion and morphology. ~tnen bi fi cells prior to tail vein injection were treated, different outcomes were observed. short treatment (lh) resulted in % inhibition of lung colonization whereas long-ter~ treatment ( weeks) resulted in - times more lung colonies. a candidate molecule likely involved in these observed effects could be identified as a kda cell surface protein. sequence information from peptides revealed in several cases % homology to mouse centrosomin a, i.e.a kda cytosolic protein involved in the organization of the spindle apparatus. these data suggest that we may have found a protein which may participate in a link between the extra-cellular space and the microtubular system. we are currently trying to clone this kda protein from a bi fi c-dna library. wahlberg, j.m., and spiess, m. dept.biochemistry,biozentrum, university of basel. signals for correct basolateral sorting of subunit hi of the human asialoglycoprotein receptor are located in the cytoplasmic tail, involving a tyrosine at position . removal of this residue results in nonpolarized transport of the protein to the surface in mdckii-celis. a deletion mutant of hi, lacking of the normal residues of the cytoplasmic tail, including the tyrosine, has been found not to be transported to the cell surface, but to be accumulated intracellularly. the mutant protein is complex glycosylated and sialylated, indicative of passage into or through the trans-golgi. immunofluorescence analyses show defined juxtanuclear, tubular structures, which do not colocalize with markers for er, early endosomes or lysosomes, but which show similar staining pattern as markers for late endosomes and tgn. to define the determinants responsible for the intracellular accumulation a series of deletions and fusions have been constructed. the results suggest that the length of the cytoplasmic domain is one important determinant for missorting of hi. salerr~ n., medilansld, j., pellegrinelli, n. and eder-colli, l. departement of pharmacology, cmu, geneva in chollnergic neurons the enzyme choline acetyltransferase (chat) exists as cytosolic( - %) and membrane-bound activity ( - %}. are these two activities encoded by a single mrna? is membranebound enzyme preferentially associated with a given cellular membrane? in order to investigate these questions we isolated a fulllength cdna ( . kb) encoding drosophila chat and used it to transfect xenopus oocytes, rat fibroblasts and human neuroblastoma cell lines. triton x- fractionation of transfected cells showed that hydrophilic and amphiphilic chat activity were produced. the sp act. of hydrophflic enzyme reached , . and . ~mol ach/h/rng protein respectively in oocytes, fibroblasts and neuroblastoma whereas the sp. act. of amphiphilic enzyme was . , . and . hinol ach/h/mg protein, respectively. amphiphilic chat was less sensitive to inhibition by the product acetylcholine and to heat denaturation than was hydrophilic enzyme. similar results were observed for the native drosophila chat. amphiphillc enzyme sedimentation was affected by the type of detergent present in linear density gradients of sucrose. transfected oocytes were subjected to subfractionation. besides a large amount of soluble chat activity, a significant proportion of enzyme was found associated with a fraction enriched in endoplasmic reticulum (er). preliminary results using transfected mammalian cells indicate that apart cytosolic chat, plasma mernbranes+golgi enriched fractions as well as er-enriched fraction contain chat activity. we have characterized a drosophila melanogaster gene encoding a very hydrophobic protein. the amino acid sequence shows considerable homology ( - % identity) to neurotransmitter transporters. the gene is, however, not expressed in neural tissue, but in the male germline. transcription and translation occurs in early spermatocytes. antibody staining data suggest the following pathway of the protein during spermatogenesis: the protein is synthesized in early spermatocytes and is transported into the nucleus during prophase of rt i. at meiosis it associates with the mitochondria and it stays associated while they fuse to the nebenkern. during individualization it is stripped off in the cystic bulge. inspection of the putative protein sequence shows that it possesses alle the necessary targeting signals to allow its transfer from the cytoplasm through the nucleus into the mitochondria: three nuclear targeting signals, a mitochondrial import signal and, moreover, two pest-sequences for rapid protein degradation are present. we shall present a, currently tested, working hypothesis. in hg-treated toad skins, water permeability is high or low depending on whether the major anion of the inner bathing medium is so~ or ci. we report here the results of experiments designed to determine if, in skins bathed with so -ringer, net water flow (j~,) could be diminished by agents added to the outer medium. toad skins were mounted inbetween glass chambers and exposed to a standard osmotic gradient of mosm. jw was continuously monitored by automatic, volumetric techniques. exposure to lmm hgclz or chzcihg (external side) led to the typical anion-induced j,, changes. re-exposure to hg caused a mild ( %), although significant (n= , p< . ), fall in jw. since cuso is a sh-reagent like hg, we looked at the effects of cuso (lmm). there was a % reduction of j, (n= , p< . ), an effect totally reversible by simple washing of the skins. likewise, addition of dithiothreitol (dtt, mm) in the presence of cu, caused a % reversal of the cu effect ; there was no change in jw if dtt was given before cu, or if both agents were added simultaneously. finally, in hg-treated, glutaraldehyde-fixed skins, lmm and ram cuso caused % and % jw decrements, respectively, that were totally reversible. in conclusion, cu causes a dtt-sensitive inhibition of jr, in hgtreated skins. further work is needed to establish if the cu effect is exerted on sh-groups of apical aquapories of toad skin. $ - the amino acid sequences of the pc- e-subunits and other cryptomonad biliprotein ~-subunits pose the most intriguing question about the phylogenetic origin of these unique chromopeptides. cryptomonad pc- ~-subunits seem not to be closely related to the known phycobiliproteins, linker polypeptides (lpps), bpe or any other light-harvesting chl polypeptides from the thylakoid. for most of them the number of identical amino acid residues was %- %. sequence alignment of c-pe associated lpps with t-subunits from cyanobacteria and red algae and the cryptophytan ~-subunits however show that the identity between c-pe associated lpps and cyanobacterial x-subunits is still significant, whereas it reaches the limit of significance between cyanobacteril and eucaryotic x-subunits. nevertheless it has to be assumed that x-subunit derive from cyanobacterial c-pe associated lpps as well as cryptomonad ~-subunits may derive from t-subunits. whereas the the phycobiliprotein ~-and subunits remained structurally conservative during evolution (e.g. - % identity between cyanobacterial and red algal pe-subunits) the lpps and t-subunits show drastical changes in their chromophore binding domains. $ - functional and evolutionary relationships of the components of the primary events in photosynthesis have been traced using amino acid sequence comparison. basically, the question was posed regarding the interrelationship of the apoproteins of different photosynthetic organisms which gather light and/or separate charges across membranes. do their protein-chemical structure and organisation decipher us certain clues and parts of the path by which they have been selected and derived from a possible primordial reaction centre polypeptide? within that context the light-harvesting-and energy transfer systems of purple bacteria offered as ideal model components. a data set of more than apoprotein sequences has pointed to some keystructural elements which have been partially verified as such by limited proteolysis and genetic engineering. a careful inspection of the determined bacterial antenna-structures revealed some remarkable similarities to eukaryotic antenna and reaction centre apoproteins indicating possible basic structural motifs for complexing pigment molecules, like chlorophylls or bacteriochlorophylls in very distant representatives of phototrophs. a number of glycoproteins of the nervous and/or immune system that are involved in cellular recognition and/or adhesion share the common lz/hnk- carbohydrate structure. in a large proportion of patients with peripheral demyelinating neuropathy associated with paraproteinemic gammopathy (ppn), monoclonal igm antibodies (m-igm) react with the lz/hnk- determinant of mag and p . we plan to test the hypothesis that anti-mag m-igm autoantibodies may alter the expression of hnk- bearing myelin proteins such as mag, p , mog, ncam or pmp . by using monoclonal antibodies to myelin proteins on tissue sections, preliminary results indicate that mbp protein content seems to be strongly downregulated, whereas galc and $ protein do not appear to be affected by the loss of myelin in ppn nerves. gfap protein expression appear to be upregulated in demyelinating biopsy specimens from ppn patients with anti-mag m-igm gammopathy. in conclusion, the expression of some l /hnk- negative proteins might be disregulated by the presence of circulating anti-mag m-igm in the serum of ppn patients. we have isolated a human liver na+-taurocholate cotransporting polypeptide (ntcp) by screening a human liver edna library with a rat edna probe derived from ntcp (pnas , , ) . the cdna codes for a protein of amino acids which shows % amino acid homology with the rat ntcp. expression of ntcp in x. laevis oocytes resulted in na+-dependent bile acid uptake which exhibited saturability with an apparent km for taurocholate of ~m. ntcp mediated taurocholate uptake into oocytes was inhibited by all major bile acid derivatives, bumetanide and bromosulphophthalein. southern blot analysis of genomic dna from a panel of human/hamster somatic cell hybrids mapped the human ntcp-gene to chromosome . in conclusion, these studies supplement the previously suggested selective mammalian distribution of the ntcp gene family and provide the possibility for direct molecular characterization of human bile acid transporter genes. diverse cell surface molecules of the nervous system play an important role in specifying ceil interactions during development. with the aid of a polyclonal antibody against l , a .skb edna clone closely related to l (ch-l ) was isolated from a ~. gtl library derived from poly(a)* l~qa of day mouse brain. using this edna as probe the remaining cdi'ia ' sequences were cloned out of a plasmid library constructed from postnatal day - mouse brain poly (a)+ p, na. two independent full length clones of . and . kb encompassing the entire coding region of chl] were isolated. these represent putative alternatively spliced mp, nas. the deduced primary structure of chl reveals that, like the cell adhesion molecules (cams) mouse l , chicken ng-cam, and nr-cam (bravo), chl is composed of six ig-like domains, a transmembrane domain, and a short cytoplasmatic region. in contrast to the other l family members, only four and a half instead of five fibronectin type hi-like repeals are found in chl . the fibronectin type [h-like repeats were expressed in e. col[ and the protein fragment was used for immunization. as observed for li, ng-cam, and nr-cam, chl is susceptible to proteolytic cleavage. bands of kd, kd, kd, and kd could be detected bywestern blot analysis. by in situ hybridization, a predominant expression by neurons was found in the adult mouse brain. western blot analysis showed expression of chl protein in brain and heart, but not lung, kidney and intestine. in liver, a kd immunoreactive band was found. the relationship of chl to molecules known to be involved in cell adhesion and neurite outgrowth, combined with its pattern of expression, suggests a role for this molecule in cell interactions dndng neural development. work from this laboratory revealed that in hg-treated, glutaraldehydefixed toad bladders, the water permeability coefficient (pf) is high in sod-ringer and low in ci-ringer ; in all these studies the osmolality of the serosal medium was mosm. as an attempt to determine the mechanism(s) underlying such pf changes, experiments were carried out with bladders whose serosal surface was exposed to [so-, hypo-or hyperosmotic media ; the outer surface was always exposed to a hyposmotic solution ( costa). net water flows were measured continuously with automatic, volumetric techniques. after exposure to mm chzcihg ( ', outer medium) followed by fixation with . % glutaraldehyde ( ', serosal medium), the bladders were thoroughly washed. in sod-ringer, pf (/~m/sec) was as follows (n= ) we wanted to determine the molecular weight (mw) of ntcp and its topology in the plasma membrane (pm) of rat liver. a rabbit antiserum generated against the c-terminus of ntcp was used to determine its mw on western blots and its subcellular distribution in liver and in hepatocytes by immunofluorescene (if). site directed mutagenesis and deletion experiments were used to determine the n-linked glycosylation sites. ntcp encodes a kd protein in rat liver pm vesicles. the basolateral localization of ntcp in liver was confirmed by if. ntop could only be immunostained in permeabilized hepatocytes suggesting an intracellular localization of the cterminus, cnda constructs showed the ntcp is glycosylated at positions and ii. the data show a selective basolateral expression of ntcp in rat liver and they suggest that the two ends of ntcp are located on opposite sides of the pm. zymogen granule membranes (zgm) are known to contain proteins with nucleoside phosphatase activity, but their exact nature and function are unknown. the activities require ca ++ and mg ++ and are sensitive to atpase inhibitors but not to inhibitors of na/k-atpase activity. the aim of this study was to isolate individual proteins of pig pancreatic zgm, to attribute enzymatic activity to individual proteins and to characterize them with various substrates (gtp, atp, amp etc.), inhibitors (amp-cp, amp-cpp, gtpgs etc.) and lectins (maa, dsa, gna etc.). we have subjected highly purified zgm solubilized in chaps to ief on immobiline dry strips | (pharmacia) with s ph range -- . (ist dim.). these strips were then electrophoresed in a polyacrylamide gradient gel containing . % chaps and tris/hci at ph . ( nd dim.). nucleoside phosphatase activity was detected histochemically in the d gel by incubation with substrate in the presence of lead nitrate. focussed strips were also subjected to sds-page for comparison. several proteins showed strong activity to gtp, atp and itp. the role of these phosphatases are discussed in the context of the role of gtp-binding proteins and the de-/phosphorylating events on the zgm in intracellular targeting and exocytosis. the disease-specific isoform of the priori protein (prp sc ) is an essential part of the infectious particle causing scrapie or bse. prp sc differs from prp of normal animals (prp c ) by its relative protease resistance. the physical nature of this difference is still unknown. here we analyzed the protease-resistance of prp sc quantitatively. prp sc was rendered completely protease-sensitive at alkaline ph or in guanidinium thiocyanate (> . m). denaturation in m guanidinium thiocxanate (gdnscn) completely abolished protease resistance of prp bc within min while denaturation in m urea showed a much slower time course. denaturation cuwes were used to calculate the gibbs free energy (as d ) in dependence of gdnscn or urea at different concentrations. the linear relationship between agd and the denaturant concentration is suggestive of a two state model involving the conformational change of a single protein domain. this is also reflected in the small number of side chains ( , ) additionally exposed to the solvent upon conversion of prp sc to its proteasesensitive isoform. our results suggest that minor rearrangements of the structure of prp sc are sufficient to abolish its protease resistance. of physiology, university of zurich, winterthursrstr. , ch- zt~rich in contrast to mammalian kidneys where inorganic phosphate (pi) is reabsorbed unidirectionally, flounder renal epithelial cells both re.absorb and secrete pi. in order to investigate on a molecular level the cellular pi handling we have cloned a re,absorptive nalp i transport system from flounder renal tubules. based on homology with the cloned na/p i cotransporter from rat we have isolated a edna clone (flounder napi-ii, bp) encoding for a protein of amino acids. in the eight transmembrane spanning domains the protein is % identical with the corresponding system from rat kidney, in the hydrophilic regions only %. expression of in vitro transcribed rna in xenopus oocytes specifically stimulated na-dependent pi transport. binding properties of na and pi as well as the ph-dependency were similar to the ones found in mammalian systems (rat, human, ok cells). by northern analysis three transcripts, . , . , and . kb in length, could be detected. rna from flounder renal tubules contained all of the transcripts, intestinal rna only the two lower bands, and non-epithelial tissue from kidney expressed only the . kb transcript. mammalian systems share only the . kb transcript but not the related species. the close functional relationship of flounder napi-ii with the known najp i transport systems and the pronounced differences in primary structure provide the basis for structure function analysis of na/p i cotransport. the amyloid /a protein precursor (app) is a transmembrane protein expressed in different isoforms throughout the organism. our work has consisted in a detailed study of the structure and biological function of app. specifically, we have shown that the secreted form (sapp) of app- is involved in the growth regulation offibroblast% and also stimulates neurite extension in b neurnblastoma cell line. functional mapping studies have shown that the domain responsible for both the growth-regulating and the neurotrophic activities of sapp- is the rerms site, arg- to ser- . the presence ofsapp binding sites on the surface of b ceils (through the active rerms site) indicate that the neurotrophic activity of sapp is mediated by a receptor, and the subsequent activation of a signal transduction mechanism. in addition, we have shown that the rerms site is also involved in the neuronal survival properties of sapp- . finally, we have initiated an in vivo study of app biological function, and shown that a peptide representing the neurotrophic domain ofsapp- can strengthen memory retention in rat through stabilization of synaptic structures in the frontal cortex. this work was supported by grants from the swiss national science foundation ( a- ), and nih (ag ). we previously showed that in smooth muscle cells, the (~i-ar is rapidly phosphorymted following exposure to agonist. to understand this biochemical event, we investigated the phosphorylation of the c( b adrenergic receptor stably expressed in rat fibroblasts ( . pmol/mg prot.). the =( b-ar was solubilized from p-labeled rat cells and immunoprecipitated with antibodies raised against the n-terminus part of the receptor. treatment of ceils with ~.m epinephrine showed a rapid and significant increase in receptor phosphorylation above basal. treatment of cells with a specific pkc inhibitor (ro- ) did not affect agonist.promoted phosphorylation while it completely abolished phorbol esterinduced receptor phosphorylation. this strongly suggests that pkc is not involved in agonist-induced c{ b-ar phosphorylation. the elb-ar contains several putative phosphorylation sites in both its third intracellular loop and c-terminus tail. to investigate the role of these domains, a trunoated receptor (lacking its last amino-acids) was constructed and stably expressed in rat cells ( . pmol/mg prot). this receptor mutant displayed similar ligand-binding properties and abirity to activate phospholipase c as compared to the wild-type receptor. phosphorylation experiments revealed that this mutant is not at all phosphorylated, neither by agonist nor by phorbol esters. agonistinduced decrease in cell surface receptors, measured by binding of the antagonist h-prazosin at ~ was however similar for both the wild-type and mutant receptor. this suggests that loss of phosphorylation sites does not affect receptor internalization. human intestinal lactase-phlorizin hydrolase (lph) is synthesized as a precursor molecule (pro-lph), which undergoes proteolytic processing during maturation. lph expressed in cos- cells was enzymatically active, the electrophoretic properties of lph-species synthesized by transfected cos- cells correspond to those in organ cultured human intestinal explants, in caco- ceils and in transfected mock ceils. they included the pro-lph and a proteolytically processed form, which had similar electrophoretic properties to the mature enzyme. the appearance of the putative mature form was inhibited by brefeldin a, ammonium chloride and chloroquine. in addition, only complex glycosylated pro-lph (pro-lphc) was inserted into the cell surface membrane. these data indicate that in cos- cells pro-lph is inserted into the cell membrane, is internalyzed and enters lysosomes where proteolytic events lead to the appearance of a mature-like enzyme. $ - assembly and transport of paba peptide hydrolase alpha and beta subunits in cos- cells eldedng, j.a., gr nberg, j. and sterchi, e., institut for biochemie und molekularbiologie, universit&t bern. paba peptide hydrolase (pph) (ec . . . ), a metalloendopeptidase from epithelial cells of human small intestinal mucosa, consists of two subunits, ~ and , which form homodimers and oligomers, cdna cloning has revealed homologies with developmentally important proteins from different species and has led to the definition of a new family of metalloendopeptidases, the astacin family (j. biol. chem. , , ) . here, we report on the expression of the cdnas of pphcz and pphj in cos- cells. when expressed on its own, pph(z is synthesized in a core glycosylated form only, suggesting that it is transport-incompetent and not able to exit the er. immune-fluorescence studies have confirmed that pph~ is not expressed on the surface of cos- cells. pphl , on the other hand, matures and is transported to the cell surface. when the two subunits are coexpressed, ppho~ can also be detected on the cell surface, suggesting that a heterooligomeric assembly is necessary for the surface expression of pphtz. truncated forms of both pphc~ and pphi~ could be detected in the medium of transfected cos- cells. in our previous work, we demonstrated by immunocytochemical reaction that sensory neurons expressed nuclear triindothyronin receptors (nt r) in embryonic as well as adult rats. in contrast, in sciatic nerve, schwann cells exhibited only transient nt r immunoreactivity from e to postnatal day . in the present work, we attempt to demonstrate by radioautography that the detected nt r are effectively the binding sites of [ i] labeled triiodothyronin. cryostat sections of dorsal root ganglia (drg) or sciatic nerve were incubated with . nm of l, , , '-[t i]triiodothyronin ci/m mol (nen), while the controls were incubated with a large excess (llxm) of unlabeled trfiodothyronin (t ). in aduit rat drg, radioautographs revealed that silver grains were exclusively restricted to neuronal cell bodies, while the schwann cells ensheathing the axons were free of significant label. in newborn rat sciatic nerve, silver grains accumulated over schwann cells; in contrast in adult rat sciatic nerve, schwann ceils were free of label. in control drg or sciatic nerve sections incubated in large exceas of unlabeled " " , all the radioautographs exhibited only scattered silver grains. in conclusion, our results show that sensory neurons and schwann cells are able to express functional nt r which bind thyroid hormones (snf - - ). characterization of the maturepreeursor glycophospholipid for glycosylphophatidylinositoi anchors of saccharomyces cerevisiae. many attempts to isolate the mature yeast precursor gpi glycophospholipid ready to be attached to newly translated proteins for gpi-anehoring have failed although such precursors were previously identified in mammalian cells and protozoa. here we show that metabolically labeled glycolipids of the expected structure can be observed if the incorporation, their accumulation and their extraction are optimized. two very polar, ( h)-mannose-labeled glyeolipids named cpi and cp were identified and purified. they were structurally characterized using phnspholipase treatments, partial deacylation with methanolie ammonia, hydrofluoric acid dephosphorylation, nitrous acid deamination, acetolysis, exoglycosidase treatments and combinations thereof to produce labeled fragments which could be analyzed by paper chromatography or thin layer chromatography. cp and cp both contain the identical core oligosaccharide mangl, (x->po - )manal, mamxl, (y->)mana-glcn-lansitol, x and y being hydrofluoric acid-sensitive substituents (most likely ethanolamine and phospheethanolamine, respectively). the inositol is acylated although no acyllaositol is present on protein-bound yeast gpi-anchors. cp and cp can also be observed after lableling with ( h)myo-inositol, the lipid moieties of cp and cp can be completely removed by mild alcaline hydrolysis altough the protein-bound gpi-anchors made by the pmi cells under identical labeling conditions are completely mild base resistant. this finding reinforces the notion that the ceramide moiety typically found on the majority of yeast gpi-proteins are added only after addition of the gpi precursor glycolipid to proteins. the distributions of the mannose- -phosphat e/igfll-recept or and a , slalyltransferase in hepg cells: no evidence for co-localization by confocal laser scanning microscopy. the organization of the trans golgi apparatus (ga) with respect to (recycling) man- -p/igfii receptor (mpr) and (resident) c~ , sialyltransferase (st) has been investigated by double immunofluorescence laser scanning confocal microscopy in hepg cells. they were chosen because biochemical evidence suggested sialylation of mpr upon recycling (duncan & kornfeld, j. cell biol. , , - ) implicating colocalization of both proteins. in the steady state st was confined to the ga whereas total (endogenous) mpr was localized to coarse vesicles without evidence for colocalization by confoeal microscopy. endocytosis of surface-labeled mpr was monitored in presence and absence of brefeldin a (bfa): without bfa, early endosomes ( ' of warming up) showed no, late endosomes ( ') little if any colocalization with st. in presence of bfa, the st-compartment disappeared whereas late endosomes persisted with some tubular emanations. under similar conditions, in "absence of bfa, endogenous mpr concentrated in the ga region suggesting colocalization with st. in presence of bfa the mpr compartment formed a redculum whereas the st compamnem disappeared. in conclusion, within the limits of the techniques used, exogenous mpr was not detectable in the st compartment whereas endogenons mpr concentrated in the ga region but obvious co-localization was exceptional. supported by grant - . of the snsf to egb. cultures of dissociated striatal neurons prepared from fetal rats were grown in the presence of neurotrophin- / (nt- / ) as well as the other known neurotrophins, ngf, bdnf and nt- . we found that acute administration of nt- / to seven days old cultures stimulates the hydrolysis of phosphatidyllnositol, an event involved in neurotrophin signal transduction. growth of stdatal cultures in presence of nt- / resulted in increased cell survival as indicated by elevations in total cell number, protein content and number of viable cells. nt- / exposure increased gaba uptake and staining intensity in gaba immunocytochemistry in these cultures indicating atrophic action on gabaergic neurons. to further identify responsive cell populations we analyzed for calretinin, a calcium binding protein known to colocalize with gaba in a number of neuronal cells. nt- / strongly increased the number of calretinin positive cells in cultures prepared from rats of embryonic day , as we]l as calretinin levels determined by western blot analysis. when cultures were prepared from embryonic day rats, nt- / very strongly increased the morphological differentiation of calretinin positive cells. while all effects produced by nt- / were mimicked by bdnf with similar potency, nt- was found to be only marginauy effective, our findings identify nt- / as a potent neurotrophie factor for striatal gabaergic neurons. fusion of cells and organelles is a general patho-biological phenomenon: muscle cells, transport vesicles, langhans cells and virus induced fusion from within (ffwi, during replication) and without (ffwo, by the particles). in vivo and after virus isolation only little fusion can be seen, only after cultivation fusing hsv can be detected; a selective pressure must be released. syn loci are known on the genome; the syn locus is located inside of the cell in the carboxy terminal part of glycoprotein b, all other fusion domains are outside the ceil. fusion inducing mutations are in aa , , , , and and were correlated to fusion from within and without as well as to selective cyclosporin a effects. a model is presented for the syn locus. -by recombination the fusion capacity could be transfected to fusion negative viruses. -hsv penetrates the cellmembrane by fusion by ligand-recepter interactions. ffwo+-strains penetrate at ~ cell/cell fusion was analysed and found to need also receptor-ligand interactions if induced by syn mutations. timing analysis of fusion events and by certain inhibitors allow further conclusions. quenching of lhcii isolated from dark-adapted (lhcii-d) or preilluminated (lhcii-l) rye leaves was similar in both preparations, but reversibility of this process within two hours of darkness was slower in lhcii-d. no differences in fluorescence quenching and full reversibility was observed for both lhcii preparations in reverse micellar solution. xanthophyll/chl concentrations in lhcii were consi-derably decreased under this conditions. excitation difference spectra (before and after illumination) of lhcii in asolectin liposomes showed typical changes of energy transfer between carotenoids and chl. a model is proposed according the xanthophyll cycle controls reversibility of light-driven excitation quenching as well as the xa~a~q~_x~tentof ~,,~nchi~g i/~ lhc~z. myelinogenesis is a complex, developmentally regulated process involving coordinate expression of myellnafion-related proteins. the myelin forming cells are the oligodendrocytes in the central nervous system (cns) and the schwann cells in the peripheral nervous system (pns). we differentially screened a rat postnatal day (p ) spinal cord edna library with probes derived from normal (plus probe) and from myelin-free (minus probe) p spinal cord mrnas. we succeded in the isolation of several novel edna clones whose corresponding mrnas were expressed either selectively by oligodendroeytes or by oligodendroeytes and sehwann cells. here we describe one of the edna clones (tentatively denominated ns ) whose mrna is specifically expressed by oligodandroeytes and schwann cells. the . kd transcript is expressed at highest level during myellnation and at much lower levels in the adult as it is known for other myelin-specific mrnas. sequence analysis of the full length edna sequence showed a % identity to the rat liver udpglucuronosyltransferase family, involved in the detoxificafion system. the amino acid open reading frame encodes a kd protein with a putative signal peptide and two transmembrane domains, whether this ns protein is involved in a detoxification reaction or in glycosilation of proteins and lipids with glueuronie acid is under investigation. neuroblastoma (n ) are sympathetic tumors of childhood characterized by mycn amplification in advanced stages. cd standard (cd s) and splice variants (cd v) represent a group of surface glycoproteins whose expression has been recently linked to metastasis. we analysed the expression of cd s and cd v on n tumors and cell lines by immunohistology and northern blot. results showed high levels of cd s on / nb, / n cell lines and / ganglioneuromas (mature, benign sympathetic tumors). lack of cd s staining was observed on stage iv, mycn amplified tumors only. in cell lines, expression was not detected on cells with a neuronal phenotype while high levels of cd s were expressed by cells with a glial differentiation, independently of mycn amplification. up-regulation of cd s was obtained with agents that induce a glial differentiation, while neuronal differentiation by retinoic acid was not accompanied by a change in cd s expression. no gd v were detected on tumors or cell lines. we conclude that cd s expression is down-regulated in a subset of nb ceils and tumors and that mycn product and additional mechanisms responsible for control of differentiation are involved in this regulation. the glycoprotein gc of herpesviruses is known to be non-essential for virus replication. non-essential herpesvirus proteins are suggested to perform "luxury functions" which may influence the outcome of an infection. we are aiming to analyse the function(s) of ibc specified by bovine herpesvirus (bhv ), bovine herpesvirus hv ) and caprine herpesvirus (caphv ). these viruses are closely related but differ in their pathogenic potential. in a first step we have cloned and sequenced the individual genes. the nucleotide and the deduced amino acid sequence homologies of two hv reference strains was found to be > %. the sequence homologies between bhv and bhv were > % and those between bhv and caphvl were > %. comparisons on the amino acid sequence level revealed that the differences were most prominent in the n-terminal parts, whereby the potential signal sequence region might be concerned. the putative glycosylation sites, however, were not affected, and the transmembrane sequences were similar in size and location. in order to characterize the significance of these differences, we are constructing gc-deletion mutants and recombinant viruses carrying a foreign gc gene. the in vitro and in vivo behaviour of these viruses will be compared to the wildtype virus. $ - o. moullet and j.-l. dreyer, instimt de biochimie, unlversit de fribeurg, ch- fribourg camp has been recently shown to promote a cell survival response by retarding apoptosis (berridge, m.v& el. ( ) exp. hematol. , - ) . on the other hand quinones are widely distributed substances of often potential toxicological significance. we have tested the effects of quinones on adenylate cyclase. the enzyme is rapidly inhibited by pbenzoquinone (ic = - ktm) or dicnlorophenol-indopbennl (/ ' = ilm), but -substituted quinones are inactive. the lc 's are decreased - times after membrane soinbilization but are not affected by gtp, gdp or analogues nor by cholera and pertussis toxins. the inhibition is not mediated by a g-protein or by the activation of a defined receptor. quinone inhibition stoichiometrically competes with forskolin activation of adenylate cyclase, equimolar concentrations of beth quinone and forskolin restoring the enzyme activity to its basal value. the cytotoxicity of benzoquinone, tested in vivo on hep-g , a human bepatocellular carcinoma cell line, could be prevented with forskolin. in plasma membranes quinones tightly bind to only one major and two minor proteins that were purified by page electrophoresis under native conditions. together these observations indicate that quinone action can be attributed to targetting to a limited number of proteins at tile plasma membrane in a highly selective way. by affecting adenylate eyclase, a modulation of the camp pool induces apoptosis as a result of the cytotoxicity via. t.congolense is an african,unicellular hemoflagellate, pathogenic for domestic animals such as cattle.within the host bloodstream,parasites adhere to the wall of the microvasculature,eausing severe organ damage.we have set up and characterized an in vitro assay in order to study the metabolic requirements,reeeptor-ligand interactions and the ultrastructure of this adhesion phenomenon.our findings suggest that adhesion is an active process requiring metabolic energy on part of the parasite, but is independent of the target cells.we also show that t.congolense possess a leetin-like domain localized at specific sites along the surface of the anterior end of the flagellum, which interacts with specific carbohydrate receptors, probably sialic acid and/or n-aeetylglueosamine residues,on the endothelial cell surface. this suggests that adhesion is likely to be mediated by a trypanosomal surface component distinct from the variable surface glycoproteins(vsgs). we also suggest that the cytoskeletal protein aetin is involved in this process. local immune response based upon intestinal parasitespecific t and b cells to giardia lamblia may be impomrant in parasite clearance. experimental infections qf mice (cr:nih:s) with g. lamblia (clone gem-h which e~presses a major kd antigen on its surface recognized b~ mab g / ) have demonstrated that the parasite undergoes surface antigenic variation in rive. experimental i~fection of immu/%odeficient mice (nu/nu mice and sci~d mice) and oontrol nu/+ littermates provided insights into associated immunological mechanisms: dominant surfade epitope-specific slga plays a major role in modulatin ] surface antigenic variation, and thymus-dependent t-lyn~phecytes in the induction of selfcure. athymio nu/nu mioe (zu. icr-strain) were reconstituted with immune peyer:~ patch lymphocytes obtained from self-healed littermat~ nu/+ mice. intestinal b-cells from these nu/+ mice @s well as from reconstituted athymic nude mice synthesized in vitro parasite-specific iga. this iga showed a predominant immunoblet reaction with the major surface antigen (a mr , polypep-tide) characterizing the giardia clone in question. nu/nu mice reconstituted wit~ immune cells acquired the potential to decrease significantly their intestinal parasite mass. thus the hypothesis on the causative role of intestinal iga in tl~ control of intestinal g. lamblia infection has found further experimental support. stephan jentsch, heinz tobler and fritz m er, institute of zoology, university of fribourg, p rolles, " fribourg the process of chromatin diminution in the parasitic nematode ascaris lumbricoides leads to the formation of somatic cells that contain less dna than the germ-line cells. during this process, which occurs between the third and the fifth embryonic cleavage divisions in five different blastomeres, the termini of the chromosomes are cut off and eventually degrade in the cytoplasm. to analyze this process at the molecular level we constructed a xzap somatic telomere library. the analysis of three cloned telomeres and their corresponding germ-line genomic regions revealed that chromosomal breakage takes always place within short specific chromosomal regions (cbrs) and is followed by the addition of the telomeric sequences (ttaggc)n to the breakage sites. the different cbrs do not crosshybridize to each other. a comparative nucleotide sequence analysis is now being performed to screen for the existence of conserved sequence motifs. in a parallel approach we analyze the chromatin structure of the cbrs and their flanking regions in developmental stages before and during the elimination process, putative recognition or regulation sequences important for the elimination process might be recognized as dnasel hypersensitive sites. once such sequences will be identified, it should be interesting to establish their role in the elimination process and to look for specific binding factors. chromatin diminution takes place in all presomatie ceils of the early embryo of ascaris lumbricoides and is followed by the addition of many repeats of the telomeric sequence ttaggc. chromosomal fragmentation is developmentally regulated and occurs within specific chromosomal regions (cbrs). one of these cbrs (cbr ) was analyzed in detail. agene located close to the cbr encodes a putative gtp-binding protein, whose promoter region is located within a distance of only kb from the telomeric ttaggc repeats of the corresponding somatic chromosome. a homologous gene was isolated from c. elegans. most interestingly, the c. elegans gtp-binding protein gene is also located at a telomeric position, namely at the end of chromosome v. in ascaris, transcripts of this gene are present in all developmental stages, though they seem to be stronger expressed in oocytes and early embryos than in later developmental stages and somatic tissues. a high degree of sequence conservation of these genes between the two nematode species and man (a corresponding partial cdna clone was identified in a human heart cdna library) suggests an important biological function. currently, we are using genetic methods to determine the possible function of this gene in c. elegans and to test whether its telomeric localization in both nematode species has any functional importance. it encodes a nuclear protein which is associated with she chromatin together with other probeins of the poiyoomb group. the mechanism of gene regulation caused by pelycomb seems to be similar to that of the modifier genes of the position variegation effect which also encode structural proteins of the heterochromatin. one of these proteins is hpi which shares with polycomb a region of similarity at the amino terminus called the ~omatin ~rganisation m~difier domain (chromo domain). the particular function of the chromo domain is not yet understood, but it seems to be important for the protein-protein interactions in chromatin associated complexes. furthermore, chromobox cdntai~ing genes were found in several species, suggesting that they are widespread in the animal and plant kingdoms. here we show chat ~. clemens contains at least one chromobox containing gene (cdna: cm h ). length and szrusture of the putative ~. elegs~ chromo domain protein are similar to those of the chromo domain containing prozeins of other species. the expression pattern of cm h is s:udied with antibodies raised against the putative chromo domain protein and by injecting a -ga! fusion construct inns the germ line of ~. eleman$. chromatin diminution in the parasitic nematode ascaris lumbricoides is a complex molecular mechanism, involving cleavage of the chromosomes at specific breakage regions, degradation of the eliminated chromatin and new telomere formation in all presomatic cells during the early embryonic development. the aim of the present project is to reproduce this dna elimination process in vitro, step by step or all in one, by using cell-free extracts of a. lumbricoides eggs. therefore, - cell extracts were established and their quality was assessed by the ability to transcribe s rrna genes (pol iii) and sl rna genes (pol li). faithful extracts were then tested for cleavage activity on dna substrates derived from different chromosomal breakage regions. finally, preliminary results revealed a possible non processiv rnase sensitive telomerase activity in the in vitro extracts, capable of adding specific nucleotide sequences to an oligonucleotide primer containing four repeats of the telomeric sequence traggc. as de novo synthesis of several kilobases of somatic telomere is likely to require a strong telomerase activity, we assume that this a. lumbricoides in vitro system is a good tool to isolate the telomerase protein and its corresponding gene or other implicated factors, s - university of berne, ch berne a common feature of all mycobacteria -whether pathogenic or not -is their richness and variety of lipids. among numerous lipids widely distributed, pathogenic mycobacteria exhibit a group of sulfated surface lipids thought to be determinants of virulence. therefore, we studied sulfolipid-bioslrnthesis in pathogenic and apathogenic (opportunistic) mycobacteria species by monitoring the kinetics of [ s]sulfate incorporation into cellular lipids, pathogenic mycobaeterla (two virulent m. tuberculosis strains) showed a marked sulfolipid-synthesis with highest rates during exponential growth, while apathogenic ones (an avirulent m. tuberculosis strain and an opportunistic species) manifested negligible incorporation of the label. the partial characterization of the [ s]-sulfated lipids of pathogenic mycobacteria by thin layer chromatographic separation, combined with autoradiographic detection, revealed the presence of several radiolabeled lipid components of different polarities and with altering expressionpatterns during growth. these results strengthen the hypothesis, that sulfolipids are involved in the pathogenesis of mycobacterial disease. maiada ( , ) . some features of human pf maiada are observed in mudne malaria, although the pattern of sequestration changes due to the different plasmodial species involved, in mice, we showed by immunohistocbemistry that the expression of vcam- and icam- is upregulated in brain, lung and heart of p/asmodium bergheiinfected baib/c and also in lps-pdmed cid mice. in $gid mice injected with labeled human erythrocytes (e), a higher rate of sequestration to brain was observed with pfe than with uninfected e. lps-pdming increased the retention of pfe in the brain and lungs significantly (t test), but decreased it in the spleen. we conclude that pfe express surface structures which allow them to adhere to the vasculature of the brain more efficiently than uninfected e. lps increases the number of adhesive molecules on the host endothelium. interestingly, sequestration in the cid mouse resembles that of pfe in man. f. roth, f. riniker, k. schopfer and t. burkart inst. med. microbiology, univ. of berne, ch berne it has recently been shown that bacterial lipids cancarry antigenic determinants. the study of antibody specificity to such lipid epitopes in vitro turns out to be difficult since hydrophobic antigens have to react with watersoluble molecules. we have developed a solide phase lipid antigen immunoassay (laia) that allows to quantify the antibody reactivities with an array of lipid antigens derived from mycobacterial cells. defined amounts of extracted lipids were immobilized as equidistant lines on a nitrocellulose sheet. small strips of nitrocellulose, each carrying the same sets of lipids were incubated with different human sera. bound antibodies were then reacted with [ -i]-iodine labelled antihuman antibodies and visualized by autoradiography. the reaction was quantified by densitometry. assay conditions were optimized for antigen and antibody concentrations and appropriate incubation times. in addition, the effects of different ingredients (salts, proteins, detergent) on antigen immobilization and epitope accessability were studied. the presented laia-system is rapid, sensitive and reproducible. it allows to compare the specificities of different bacterial lipid antigens and to quantify their reactivity with serumantibodies. the atomic force microscope is a very convenient tool for studying hard and flat samples with atomic resolution. biological objects, usually soft and rough, are sometimes difficult to image using this technique. in contrast bacteria, which are too small to be observed with high resolution using the optical microscope, are hard enough to be observed with an afm at a relative good level of magnification. this kind of microorganisms can be prepared for afm imaging using very rapid and simple techniques. this method could be a convenient tool to observe the effect of bioactive substances such antibiotics. we show in this poster an exemple with the action of penicillin on b. subtilis. within the family trypanosomatidae, leishmania is a protozoan pathogen producing a broad spectrum of human disease. its life cycle is divided in two stages. promastigote is the flagellated form which colonizes the gut of the sandfly vector, and amastigote is the non-flagellated form that is specialized for growth and survival in the vertebrate host macrophage. to understand gene regulation during the transition between the promasttgote and the amastigote stage, we were interested in the characterization of stageregulated genes. we isolated a gene, sw , from leishmania major whose mrna is more abundant in amastigote compared to promastigote. structural analysis of this gene showed that an additional polypeptide could be encoded by the opposite strand of sw . we confn'med the presence of an open reading frame on the opposite strand by in vitro translation of the sw antisense rna. in vivo, an amastigote sw antisense transcript and the corresponding polypeptide were also detected suggesting that the sw is transcribed in both directions and that stage specific transcripts and polypeptides could be produced. analysis of other gene segments tend to indicate that an intensive usage of the leishmania genome to produce various polypeptides could be a general phenomenon in trypanosomatidae. the four variants and / or posttranslational modifications of histone h of procyclic trypanosoma brucei brucei (protozoa, kinetoplastida) were purified by hplc reversed phase chromatograpy and characterized by their amino acid composition and partial amino acid sequence. differences in sequence of up to % as compared to histone h of higher eukaryotes exist. alkaline phospatase digestion and analysis of h by means of hpce was used to demonstrate the presence of phosphorylated modifications. the unique biochemical properties of h of t. b. brucei contribute to the structural differences seen in the chromatin of the parasite as compared to that of the higher eukaryote host. larvae of the parasitic wasp chelonus inanitus (braconidae, hymenoptera) develop inside embryonic and larval stages of the host spodoptera littoralis (noctuidae, lepidoptera). parasitism induces in the host a precocious onset of metamorphosis and developmental arrest in the precocious prepupa. the wasp injects, along with the egg, pdv and venom into the host egg. injection experiments with pdv and venom revealed that pdv play a crucial role in altering host development and in suppression of the host's immune system. the genome of the pdv of c. inanitus consists of at least i segments of double-stranded circular dna ranging in size from - kb. we analyzed the expression of viral genes in the course of parasitization with cdna made from mrna at various developmental stages of s.littoralis. we also investigated the localization of the expressed viral genes on the various segments. in addition, these bacteria were isolated and cultured from brain tissue. to identify the infectious agent we used, among other techniques, an atomic force microscope (afm). when the. isolation fluids derived from the cortex of three alzheimer's cases were examined with afm and scanning electron microscopes, we observed bacteria whose size and morphology fit to the order of spirochaetales. these images are presented and discussed on our poster. ~ottstein bruno, institute of parasitology, ch-berne. %lveolar echinococcosis ae is due to infection with the metacestode (larval stage) of echinococcus multilocularls. an immunodominant antigen em of e. multilocularis was characterized by mab-gii, respective studies revealed, that (a) antigen expression metacestode stage-sdeci-fic and (b} that expression was restricted to the laminated layer. the em -antigen i s a lectin-binding carbohydrate linked to a lipid residue. the "em positive" laminated layer proved to be a crucial factor in protecting the parasite from host immune-effector mechanisms: only e. multilocularis larval structures exp ressing em were able to induce secondary alveolar chinococcosis in rodents. subsequent experimental studies in resistant (c bl/ ) versus susceptible (c bl/ j and akr) mouse strains showed that resistance was as-sociated with synthesis of anti-em igg and igg i. sus-ceptibility of c bl/ j-mice was associated anti-em s and no igg . the parasite-specific in vitro lym-~hoproliferative i~une response remained stationary ~ith time after infection in c bl/ and decreased in r and aki< mice. day p.i. cd § -lymphoblast cells dominated in all meuse strains; day p.i. an in-creased nu/ ber of cd -/cd + and cd +/cd + cells were observed. only susceptible mice exhibited a significantly ~eereased response of lymph node cells to cona-stimula-~ien with time p.i. in conclusion, subtypes of parasitespecific cellular and humoral host immune responses seem leishmania species are protozoan parasites causing a spectrum of clinical manifestations in man ranging from cutaneous lesions to morbidity and death. the insect stage (promastigote) and intracellular stage (amastigote) differ in terms of morphology, physiology, antigenicity and gone expression. it is clear that the amastigote stage is uniquely adapted for survival within the inimical environment of the macrophage phagolysosome and that mechanisms regulating these adaptations must be called into play during the early stages of infection when the parasite undergoes transformation. in an effort to elucidate these regulatory mechanisms, we have constructed a "subtracted" cdna library which is specifically enriched for parasite transcripts expressed during the first hours of infection ("switch" genes). several clones have been characterized by sequence, northern, and southern analysis. three clones, sw , sw , and sw , are expressed at a several-fold greater level in the amastigote stage and potentially encode proteins which interact with nucleic acids. sw predicted protein is homologous with histone h proteins and sw and sw potentially encode ribosomal proteins. analyses of transcripts from this library are yielding clues not only to mechanisms underlying the regulation of parasite transformation, but also insights into post-transcriptional and translational control of gene expression in leishmania. $ - microbiology, university of geneva, medical school, c.m.u., avenue de champel, geneva . a viral rna representing the sequence of a defective interfering rna, naturally selected for efficient replication, i.e. only containing the replication promoter, was cloned under the control of the t rna polymerase promoter. the expressed transcript was then replicated in rive by supplying in trans the replication functions, equally expressed from plasmids. this system, completely accessible to genetic manipulations, allowed us to define a basic rule directing sendal virus replication, the "rule of six" (calain and roux, j.virol. : - , ig ). a second defective rna, representing a shorter version of the viral rna, in that it contains not only the replication but also the transcription promoters, was then cloned and replicated in the same system, although with lower efficiency. the replication of this "transcribing" viral rna was found to obey the "rule of six" as well. replicationitranscdpfion promoters were then achieved to define the cissequence requirements needed for efficient replication or for replication /transcdption. results obtained with these chimerical viral rnas will be presented and their contdbufion to the comprehension of the paramyxovirus replication and transcription processes bovine t-cells that become infected with the intracellular parasite theileria parva undergo lymphoblastoid transformation and acquire the ability to proliferate continuously. they cease to require specific antigenic stimulation, become independent of exogenous growth factors and cause tumors in nude mice. the il and il r genes are constitutively expressed and the transcriptional activator nfwd which regulates these two genes is continuously activated. these processes are all strictly dependent on the presence of the parasite in the host cell cytoplasm and cease upon the specific killing of the parasite by the theilericidal drug bw c. we have shown that the presence of the parasite results in the activation of the protein tyrosine kinases fyn and ick, which can participate in early t cell receptor signalling, suggesting that the parasite may use this signal transducti n pathway to induce continuous proliferation. cysteine proteinases of leishmania as targets for chemotherapy. jacques bouvier*t and judy sakanari*. *department of pathology, ucsf school of medicine, san francisco, ca, and tanimal health, ciba-geigy, ch st. aubin, switzerland. the overall goal of the study is to apply the structure-based approach to develop lead compounds and design novel enzyme inhibitors as potential therapeutic agents against leishmaniases. in this regard, we have identified and characterized the cysteine proteinases of leishmania major, and showed that they consist of excellent targets for drug design. we have purified a membrane-boand cysteine pmteinase not previously described in other trypanosomatids or protozoans and obtained amino acid sequence information from the purified enzyme. in addition, we have also characterized and purified the soluble cysteine proteinases of the parasite. consequently, we have isolated two genes encoding the soluble and membrane proteinases. the soluble cysteine proteinase gene occurs in multiple copies of . kb with flanking regions of . and . kb and is localized on one chromosome of approximately kb. the membranebound enzyme gene is . kb and occurs as a single copy on a chromosome of approximately kb. a three dimensional computer model of both genes wiu be generated and enable us to scan the vast chemical database for new lead componds. we akeady have identified several cysteine proteinase inhibitors that are effective compounds in killing both promastigote and amstigote stages of the parasite in the micromolar range in vitro without affecting the host cells. dollenn~ier, g., cassinotti, p. and weitz, m., institute for clinical microbiology and irananology, st .gallen/swit zerland hav is a ~r of the picornaviridae. its rna gencrne rf~y be divided into coding regions (pi, p and p ), qhe p region contains a protease cp ro that generates mature proteins by cis-cleavage of the initial polyprotein precursor. catalytic activity of cp r~ has been d ~aonstrated in a vacciniavirus/t ~qa-polymerase hybrid expression system. (balouter aided ali~ts of hav cp r~ sequence with that of other picornaviruses imply that amino acid residues his- and/or h) the complete hav open reading frame, hav specific expression products were identified by radioj_rmmnoprecipitation and lyy sds-page. the analysis with an antibody specific for putative nonstructural protein b revealed a , kda peptide. this is in contrast to its predicted size ( kda). however, analysis with anti-(putative) a antiserum confirmed an upstream location of the b n-terminus. the new bpeptide was also identified in lyeates of hav infected mrc- cells. hence, recombinantly expressed hav polyprotein underwent authentic processing and the predicted p organization has to be modified. we are interested in the development of animal models for one of the two major pathological changes found in the brains of patients with aizheirnar~s disease, the formation of fleuroitbrillary tangles, a main component of which is abnormally phosphorylaled tau-protaln. (tau itself is a protoin associated with mlcrotubuli.) two fundamental questions may be answered with our models: first of nit we woald like to reproduce the formation of tangles, and secondly we hope to answer the question, whether the formation of these pathological structures is sufficient for the bevalopment of senile dementia ot the alzheimer type (sdat). at present there are only a tow putative candidate genes known which may be in-voned in the hyperphosphor~lation ot tau in v/vo, one of which is the map-kinase erk . we have cloned this candidate kinase from rat txaln rna via an rt-pcr approach and expressed the cona under,the co.rat of tour different promoters in transgenis mice. these promoters were tested for maximal expression. a similar approach was chosen to express the human taut isofonn in the brain ot transganic mice. we have stated to analyze different tau an~ erk expressing lines, northern blot analysis has revealed high levels of expression for all transgenes (up to fnetoid {n comparison to endocjenous levels). the neuronal spectficry of the transgenio erk expression was c~nfirmsd by in situ hybridization analysis. ir~ addition several tauexpi'esalog tines were analyzed in wealernblots to determine the relative amount of htau= protein in the brain. sections of the brain were stained with tau-sp~c~c ardib~iies to identify the neurons expressing tau. from intemrosses of tau-with erk -expressing lines, we have already identified double-positive mice which we currer~y analyze by immunohistocbemistry and in western nots to examine the phosphorylaiton status of tau. we are well aware of the possibility that only animals trans~enic for both tau and erk might beveiop tang{es, whereas single transgenics mlsht not. pairs of individual neurons were recorded with sharp electrodes or in whole-cell configuratio~ in hippocampal slice cultures of rat. synaptic connections were studied between pre-and postsynaptie cells in ca and ca hippneampal fields by eliciting single aetinn potential in the presynaptic cell. monosynapfic excitatory connections were highly probable between ca and ca pyramidal ceils (p= . , n= ) with almost no feed-forward inhibition (p= . ). by contrast, disynaptic ipsps, blocked by cnqx or bieuculline were observed with a very high probability ( -- . ) between ca pyramidal cells. monosynaptic excitation was found ha % of cases between ca neurons (n= ) but only in % of cases (n=ll) between two ca cells. monosynaptie ipsps with very short lateneies (<lms) and blocked by bicuculline, but not by cnqx, were recorded within area ca ( / ) and within cai ( / ), but not between ca and cat fields ( / ). probability of transmitlzr release, studied in recordings where failures could be unambiguously distinguished from spontaneous or small psps, was found to be close to % at both excitatory and inhibitory synapses. specific inhibitors of presynaptie release at excitatory and hahibitory terminals (i.e. adenosine and opioid peptides, respectively) gradually reduced the anaplitude of the psps indicating that the release at excitatory and inhibitory unitary synapses is multiquantal. the cns of a neonatal opossum, monodelphis domestic& shows profuse growth of fibers across a lesion. as in other mammals, such repair is not seen in adults. experiments have been made to define the age of the animal and stage of development at which repair ceases to occur. the entire cns was removed from animals aged - days or - days after birth. the spinal cord was crushed and growth of fibers assessed days later by electrical recording of impulses conducted through the crush. repair after lesions to spinal cords of younger animals occurred in % of the trials ( animals, aged - days). in nervous systems from older animals ( - days after birth) the success rate was low (only out of preparations). similarly, the carbocyanine dye dil labeled numerous fibers in spinal cords of younger animals ( out of ), whereas only one preparation out of showed scant outgrowth into lesions in the older animals. our results suggest that there is a critical period for neuronal repair; at about days nerve fibers lose the ability to grow across lesions. it is an advantage that at this later stage the nervous system is still small enough to survive in vitro. features correlated with these changes are oligodendrocyte differentiation and myelin formation. with immunofluorescent staining as well as electron microscopy myelination becomes apparent at days after birth. we are now using time lapse-videomicroscopy to analyze molecular mechanisms that could play a part in promoting or preventing repair. supported by grants to j.g.n from the swiss nationalfonds ( - ) and from the internat. res. inst. for paraplegia. i. impulse conduction in axons has integrative functions. the dorsal root ganglion (drq) of the embryonic rat put into culture forms a monolayer and is thus accessible to eonventionalmieroelectrode technique, which allows testing the above hypothesis. we have chosen a dual approach by correlating ext~rimental el~ctrophysiological data from the chlture with results obtained from a compartrhental computer model. ii. the safety factor for conduction was found to be low. thus failures of ap invasion of the drg cell soma could occur at sites of impedance mismatch when a second stimulus felt into the relative refractory period of the first or when the axon was repetitively stimulated. the electrotonic resiuues (e.rs) of the failed aps had discrete amplitude levels, suggesting that the failures were always caused at the same site along the axon. these sites of low safety factor were found to be the branch point in the unipglar drg cell and the entrance of the stem piece into the soma in both cell types, the bipolar as well as the unipolar. a systematic comparison of oth cell types showed that the ap conduction through the latter is more reliable.'the length of this stem piece is inversely' correlated to the delay caused at the branch point, as tile electrical load of the soma is more efficiently masked by a long stem piece. the dendrites of motoneurons (and subsequently many other neurons) have been assumed to be electrically passive since the work of coombs, ecc]es and fatt in the 's. however, direct evidence has been impossible to come by until recently with the advent of appropriate dyes and equipment for measuring very small and very fast optical signals. we examined the propagation of action potentials, both spontaneous and evoked, in the dendritic trees of spinal cord neurons using a • array of photodiodes and a ccd camera as weft as conventional microelectrode techniques. organotypic slice cultures of embryonic rat spinal cord were stained with a voltage-sensitive dye (di- -anepps) or a calcium dye (fluo- ) and recordings were made from motoneurons identified by their location and morphology. the results indicate that there is in fact an increase in calcium in the dendrites that accompanies action potentials whether synaptically evoked or evoked by current injection at the soma. spikes in the dendrites up to i~m from the soma are much larger than could be explained by models assuming passive dendritic trees. work is currently underway to test the possibility that sodium conductances might contribute to the propagation of action potentials in the dendrites. cat auditory cortex contains multiple cochlear representations in both hemispheres that are interconnected through the corpus callosum (cc). the distribution of auditory callosal axons was studied on the mediosagittal level after injections of biocytin at electrophysiologically identified locations. single injections were done in ai ( cats), aaf, aii and paf; multiple injections in ai & aaf in one cat. an additional cat received injection in sii. the mediosagittal image of cc was subdivided into segments along a rostro-caudal axis. labelled axons crossing the midline were counted in coronal or horizontal sections. axons from auditory fields were found in the posterior / and those from the somatosensory cortex in the anterior third of cc. axons from a discrete injection (ca imm in diameter) spread over as much as i/ of cc. the rostrocaudal distribution of axons at the midline reflected roughly the rostrocaudal position of the field of origin, but apparently not the tonotopic order of a given field. the major efferent projections of substantia nigra pars reticulata (snr) convey information to the thalamus, to the tegmentum, to the superior colliculus, and to the spinal cord. such a range of targets suggests a highly organized activity within snr and this study investigates the temporal organization of spike trains. in the portion of snr between + . and + . mm interaural levels, we recorded single units along electrode tracks in young female wistar rats. most units (n= ) fired in a non-regular poisson process, although only a small number ( / ) was characterized by an average burst size with more than spikes. crosscorrelograrns were computed for pairs of units simultaneously recorded from the same electrode and for pairs simultaneously recorded from distinct electrodes. most crosscorrelograms ( / ) showed a symmetrical peak centered on time zero, which indicates a tendency to synchronous firing. when units were recorded from the same electrode tip we observed that / to / of their spikes were synchronous. about half of the significant interactions were characterized by a relatively long duration (> ms) and are likely to correspond to a different mechanism of synchronization. these results support the hypothesis that snr cells receive common afferences of two different types and their fine temporally organized activity yields coordinated activity between the target structures. the neuronal microtubule-associated protein map binds to microtubules via a domain containing or repeats of an amino acid sequence. we have previously shown that when cultured nonneuronal cells are transfected with map their microtubules become stiffer and are then capable of supporting process outgrowth when their cortical actin cytoskeleton is depolymerised. we hypothesise that this stiffening effect of map is caused by the amino acid sequences binding to neighbouring tubulin subunits in the walls of the microtubules thus reducing their freedom of movement relative to one another. to investigate this further we have created map mutants in which or repeats of the tubulin-binding motif were deleted. these were tested by transfection and expression in cultured cells. the results confirm that the number of repeats affects the stability, stiffness and the cytoplasmic arrangement of cellular microtubules. we were previously able to demonstrate the presence ot kinin-like immunoreactive structures in the mouse brain. in order to characterize them, we have now performed a donble-immunofluorescence study using both polyclonai auti-srudykinin (bk) and mouse monocloual anti-neeron-specificenolase (nse) antibodies. the latter is considered to be a specific neuronal marker. after fixation with bonin-hollande solution, the brains were removed, embedded in paraffin and serially sectioned at am. the sections were incubated with the antibodies, which were visualized by an indirect immunofluorescence technique using fitc for nse and by an avidin-biotin technique using texas red for bk. double-immunofluorescent cells were found in the thalamus ventralis, in the nucleus cechleuris ventralis and in the nucleus mesencephaficns nervi trigemini (v). bk-positive cells in the nucleus principalis v were nse-negative ih spite of their typical neuronal aspect. the other bk-positive structures (pia, ependyma, hnic~es) also were alwa~ nse-negatlve. most of the nsepbsifive bk-negative cells were seen in the cortex. the neuronal nature of many kinin-like immunoreacfive structures in the mouse brain is thus ascertained. in contrast to the amphibian optic nerve (on), on of adult mammals is unable to regenerate after injury. astrocytes disappear from and macrophages accumulate at the lesioned site (blaugrund et al., j. neurocytol, , , ) , we have followed the distribution of astrocytes and microglial cells in xenopus tadpole ons over d after mechanical crush. astrocytes were stained for cytokeratin and vimentin, and microglial cells for griffonia isolectin b . soon after crush, immunoreactivity for intermediate filament proteins was strongly increased. astroeytic processes extended into the lesion from both proximal and distal stump. at , d, astrocytes had crossed the injured region. in the distal stump, some of them contained vacuoles indicating phagocytic activity. while in the normal on microglial cells were ramified and scarce, . d after crush they were roundish and vacuolized, and scarce within the lesion but numerous distal to it. at d, their number was decreased and most of them had reacquired ramifications. conclusively, in the tadpole on, astrocytes are virtually never absent from the lesion site but rather extend rapidly into it. astrocytes may thus provide a guiding support for outgrowing axons, microglial cells that are frequent in the distal stump, do not accumulate at the lesion. the behaviour of the two cell types differs profoundly from that in mammals and is likely to be one of the factors that may contribute to successful neuronal regeneration of the amphibian on. ci mence, j. f. , ranieri, j. p. , aebischer, p. , and sigrist, h. , institute of biochemistry, university of berne, ch- berne; division autonome de recherche chirurgicale, chuv, ch- lausanne. small peptidic sequences of laminin (yigsr, ikvav) are known to promote attachment and/or neurite outgrowth of various neuronal cell lines (pc , nb , ng - ). new and established heterobifunctional photo crosslinkers were used to immobilize these peptides in a topically defined fashion onto biocompatible surfaces such as hydroxylated fluorinated ethylene propylene (fep-oh) and pely(vinymlcohol) (pva) . n-[m[( -trifluoromethyl) diazirine- -yl]phenyl]- -maleimido-butyramide (mad) was thermochemically linked to the synthetic peptide cdpgyigsr via its n-terminal cysteine, leaving the bioactive part (yigsr) free for cell receptor interaction. mad-cdpgyigsr was radioactively labeled by reductive methylation and purified by reversed phase hplc. patterned ( and #m) peptide immobilization was attained by photocoupling onto pva and fep-oh and visualised by autoradiography. light-structured biomaterial surfaces with molecularly oriented nerve growth promoting peptides were investigated for spatially controlled neuronal cell attachment and differentiation. it is striking to observe that these inhibitory or stimulatory effects were corroborated by binduced decrease or increase in -deoxyglucose uptake, respectively. this gives b a putative role in the limbic regulation. the sheet-like processes of oligodendrocytes wrap themselves spirally around central axons, thus formtng the myelin sheath. a single oligodendrocyte may donate internodal segments of myelin to each of - or more adjacent axons. it is not known, if one oligodendrocyte picks out axons randomly or if it prefers axons with a certain diameter or with a certain chemical specificity. we have studied this last possibility by combining intracellular injection of a fiuorochrome, and immunohistological detection of calciumbinding proteins (cabp's), markers for the axons ef certain subpopulafions of nerve ceils. lucifer yellow was injected into oligodendrocytes of the optic nerve, of living rat brain slices. the oligodendrocytes were identified by their electrophysiologieal properties. slices containing the iniected cells were immunostained /or parvalbumin or calretinin using second antibodies labelled with texas red. using co.focal laser-scanning microscopy combined with a three*dimensional reconstruction programm, the relationship between oligodendrocyte processes and axons positive for one of the cabp's was determined. for ultrastructural confirmation, single, lucifer-yellow injected, oligodendrocytes were photoconverted, and the cabp's-positive axor~ were revealed by gold-labelled antibodies. the few oligodendrocytes injected show the classic morphology, that is of a small cell body and few processes running parallel to the course of the axons. the proximity between glial processes and positive axons can be easily appreciated in confocal laser-scanning images. a preferential assodation of oligodendrocyte processes with parvatbumin-positive axons has not as yet been found. thus, the confirmation of the hypothesis that oligodendrocytes choose their axonal targets according to chemical cues awaits further work including the injection of a lar~er number of these cells. to initiate a molecular genetic analysis of brain development in insects, we are characterizing the mechanisms of embryonic brain development in the fly drosophila. early in embryogenesis, a small number of molecularly diverse brain neuroblasts generates the neurons of the brain. subsequently, pioneering brain neurons initiate axonal outgrowth along glialbound brain compartments. these pioneering neurons establish the primary brain tracts. the pioneering brain axons express the cell-cell adhesion molecule fasciclin i dynamically during outgrowth and initial fasciculation; a subset of the pioneering axons expresses fasciclin ii. the axonal framework of the embryonic brain tracts is used for outgrowth and fasciculation by subsequently differentiating axons and, thus, prefigures the tracts of the mature brain. a comparison of early brain development in drosophila and in vertebrates reveals common mechanisms for brain development. supported by the swiss nsf. institute ot histology, university of fribourg, p~rolles, ch- fribourg calretinin (cr), an ef-hand ca +-binding protein, is expressed in cajai-retzlus cells during development of the rat cerebral cortex. these cells are located in the marginal zone and are the ear{iest cortical neurons to be generated. they have been consldered as unusual on account of their morphology and their fate during corticogenesis. the functional significance of cajai-retzius cells and their destiny in adulthood is still controversial. in order to approach these questions we have applied organotypic culturing methods. slices of neocortex from brains of - days-old rat pups were cultured for - days applying the interphase technique (stoppini etal, j. neurosci. methods , ) or the roller-tube technique (g&hwiler, j. neurosci. methods , i ) . the fixed cultures were immunolabe ed with an antibody against cr. the obtained results showed that cr positive structures develop in vitro like in vivo. however, in organotyp[c cultures their distribution corresponded to a more mature stage than the situation on the day of the explantations. many cr-positive cells were seen in layer l they were horizontally oriented or had a pyriform shape. based on their morphology these ceils were identified as cajai-retzius cells. orpositive cells were numerous in layer i -l[i and showed mostly a bipolar morphology. some muripo[ar cells could also be observed. a fine net of crpositive fibers and puncta was found in layers [ and iv. the demonstration that cajai-retzius celts are detectable in these cultures will allow us to follow the fate of these cells dynamically and [nte~ene in their function by applying exogenous factors. cervical primary afferent input to vestibule-spinal neurones projecting to the dorsal horn: a double labelling study in the rat. wheat germ agglutinin-horseradish peroxidase (wga-hrp) was injected by pressure into cervical spinal ganglia c or c . in the same experiments fluorogold (fg) was iontophorefically injected into the ipsilateral dorsal horn of different cervical spinal cord segments. anterogradely labelled fibers (wga-hrp) were present mainly in the external cuneate nucleus, but also to a lesser extent in the caudal part of the medial and descending vestibular nuclei (mvn, dvn) . the fg injections, restricted to the cervical dorsal horn, revealed retrogradely labelled cells in the central part of the caudal mvn. a double exposure (fluorescent and polarisadon optics) under the microscope showed primary afferent fibers surrounding like baskets retrogradely labelled fg cells. the projection from cervical ganglia cells to the mvn represents a pathway for direct afferent information from neck receptors (in particular muscles) to vestibular nuclei and supplies the mvn with afferent information which could enable the vestibulospinal nenrones, projecting to the dorsal horn, to influence the local information processing. in the sciatic nerve, all myelinated and non-myelinated axons were snap-ir. in peripheral tissues, all nerve endings were positive. the preterminal schwann cells of motor axons were also snap- -ir. after sciatic nerve section, many myelinating sehwann cells also expressed snap- -ir. in conclusion, snap- is expressed by neurons in the pns. it seems to be also expressed by preterminal schwann cells and by myelinating schwann cells during regeneration. $ - repair of completely transected spinal cord of neonatal opossum in culture h.a. vischer, dept. pharmacology, iocenter, uni. of basel, switzerland woodward et al. (j. exp. biol. , have shown that fibers grow rapidly and profusely across a lesion made by crushing the spinal cord of the neonatal opossum monodelphis domestica. in such experiments careful controls must be made to establish that fibers were in fact broken by the lesion. tests have now been made to determine whether similar repair can occur after a more drastic lesion involving complete transection of the cord. after dissection of the entire gns from animals aged - days, the cord was cut into two with scissors at the c -c level. the two-ends were glued together with matrigel on a sylgard mold, which encompassed and held the cns. the rostral end was labeled with the fluorescent carbocyanine dye dil in order to visualize growing fibers. in preparations fibers grew over the cut into the separated part of the spinal cord. such growth could extend to ixm. fibers grew straight, branched and multidirectionally, or in fascicles. in another set of experiments cords were combined from different animals of the same or different ages. again, fibers could grow across the cut but they did so less frequently. these results set the stage for investigating fiber growth after rotating the two ends at a defined angle and for analyzing factors that influence the direction of growth. map la is a microtubule associated protein of kd. in rat brain two monoclonal antibodies, a and bw , recognized map la in neurons and their axonal and dendritic processes. in mouse cerebellum, monoclonal a recognized purkinje ceils and granule cells uniformly, as already described for rat brain. in contrast, monoclonal bw stained selectively some dendrites of purkinje cells, forming bands in the molecular layer. we compared this striation with that obtained with a monoclonal antibody, anti-zebrin ii, which recognized aldolase c. in the mouse vermis, zebrin stained in a striated pattern, but complementary to bw . these results demonstrate that map la is present in forms which are differentially distributed in the mouse cerebellum. these observations may be explained either by differences in metabolic states of neurons, or by differences in their regional functions, or by differences in the regional stability of microtubules. this work was supported by grant no - . from the swiss national science foundation. serum free aggregating brain cell cultures prepared from day old fetal rat telencephalon are used as a model to study brain development. in these cultures it is possible to distinguish ontogenetic events such as cell proliferation, differentiation and myelination, in the present study we examined synaptogenesis by analyzing the expression of synaptic proteins such as synaptophysin, snap- , synapsin i, and brain spectrin. different developmental stages were analyzed by western blotting. immunoreactivity for synaptic proteins was detectable already at day in culture, suggesting that the first synapses are already present at this early stage. the expression of synaptic proteins strongly increased between day and day in culture, reflecting a period of intense synaptogenesis. treatment of the culture with an elevated concentration of kci ( mm) increased the expression of synaptic proteins, suggesting a stimulation of synaptogenesis. these findings demonstrate the utility of this approach to study brain synaptogenesis, and possibly synaptic plasticity. drenhaus, u.i gunten, a.v., rager, g. ; institute of anatomy, university of ch- freiburg the retina of tupaia comprises three types of ganglion cells (rgcs) analogous to x-, y-, and w-cells found in other mammals. these cells differ in size and in their distribution pattern: large rgcs are frequent in temporal, small rgcs in nasal retina. as the fiber diameter correlates with the size of its respective rgc, it can serve as a parameter for the topographic representation of rgcs in the nerve. to study this we analyzed the optic nerves of three tupaia. using the electron microscope we recorded the density, diameter, and the position of fibers in the nerves. we found, that fibers with the largest diameter are located dorso-temporally and close to the center of the nerve. they are surrounded by zones of smaller fibers. the diameter decreases gradually towards the periphery and is smallest in the ventro-nasal region of the nerve. since the fiber diameter distribution corresponds to that of the size of rgc-types in the retina, we assume that the topographic relationships in the nerve and the retina are similar. (supported by s - stoppini luc, s. duport, l. parisl, c. oropes~, departement of pharmacology, cmu, geneva the micro environment of the central nervous system is important for neuronal function. in this context, the blood-brain (bbb) provides and maintains the extracellular medium that is compatible with normal neuronal and synaptic activity. due to the difficulties inherent using whole animal as an experimental model to study permeability and metabolic processes at the cellular level, major efforts have been engaged in recent years, in order to bring up suitable /n vitro models. we are presently developping an in vitro bbb by interfacing a coculture of endothelial cells monolayem and nervous organotypic cultures. the main idea of this study is based on the premise that the complex intercellular interactions between the different cell types pertaining to the nervous tissue and the neighbeurlng endothelial cell is of fundamental importance to promote a realistic bbb system. we expect that erganotypic culture of nervous tissue, combined to endothelial cell monolayers, will initiate and maintain a bbb phenomena/n vilro. we will test more specifically the hypothesis that neuronal activities [spontaneous or elicited) will influence gila] cell response which will in turn modify endothelial properties. preliminary results clearely show a good nervous tissue and endothelial cell survival. tight junction llke structures could be identified using freezefracture or conventional transmission electron microscopic thechniques. {work supported by chemodyne gent~ve ). the process of myelination is a prerequisite for the proper function of the brain since it enables rapid saltatory conduction of axons. in the central nervous system (cns) myelination is performed by oligodendrocytes. a differential screening approach was used to isolate new rat cdna clones that are expressed in this glial cell type. here we report the further characterization of two of these novel cdnas which appear to be expressed specifically in oligodendrocytes. the two characterized cdna clones (tentatively called cns and cop- . ) share an approximately bp region of sequence identity which suggests that they are dedved from the same gene by alternative splicing, within this area a putative open reading frame is located. we demonstrated by northern blot analysis and in situ hybridization that the two mrnas of the novel gene are expressed exclusively in oligodendrocytes. however, the mrnas show a different specific spatial localization within the cell. we compared mrna expression of myelin basic protein (mbp) and proteolipid protein (plp), with that of cns and cop- . in brain and optic nerve during development. it appeared that the mrnas of the novel gene were delayed significantly as compared to mbp and plp mrnas. streit. phyaiologisches institut, btihlplatz , bern in organotypic slice cultures of the embryonic spinal cord, rhythmic spontaneous activity arises when the inhibitory synaptie transmission is blocked. the rhythmic activity consists of bursts of regular oscillations of activity at - hz. this study investigates the origin of the regular oscillations. when stimulated electrically at hz, the synaptic transmission in the cultures showed strong depression by about % on average. the synaptie depression was highly variable among individual connections and tended do be less pronounced in frequently used connections. when random depression ratios with an average of % at hz were implemented in a computer model consisting of randomly connected excitatory cells, regular oscillations of activity did not arise, even in the presence of synchronous pacemaker cells. however, when individual depression ratios were adapted according to the rate of activity of individual connections, regular oscillations at - hz arose in the presence of few unsynchronized pacemakers. this finding suggests that the regular oscillations in the cultures originate in a network formed by the plasticity of synaptic depression. maier a., schlumpf m., beer h.f., sehubiger p.a. and lichtensteiger w., inst. of pharmacology, univ. of zurich, the ontogeny of expression of dopamine d receptor mrna in the rat brain and binding to the receptor was studied at time points from gestational day (gd) to postnatal day (pn) by quantitative receptor autoradiography and in situ hybridization.long evans rat pups and fetuses of time pregnant rats were frozen and sectioned coronally and sagitally at i um. d recepto~o~inding , determined with the selective antagonist ~ji-sch was first noted on gd in the developing striatum, basal ganglia and the olfactory tubercle, reaching adult levels at around pn . the expression of d i receptor ~na was studied by using a mixture of -~js-labelled oligonu-specific cleotides. on gd messages were noted in the developing stiatum, olfactory tubercle and retina. this study shows a good regional correlation between the development of receptor binding and mrna, however, mrna precedes detectable binding to the receptor by several days. earlier work had shown that q~ replicase forms complexes with q~, plus strand rna via interactions at two internal binding sites, the s-and the m-site, while binding of the '-end is mediated by host factor. in contrast, the '-end of the minus strand appeared to be directly accessible by replicase. we have prepared a series of plus and minus strand variant rnas containing either internal deletions or terminal deletions extending from the '-end. the template activities of the variants were determined by single-round replication assays. for the plus strand we find that while deletion of the s-site remains without effect (as expected from previous results), deletion of the m-site reduces template activity to % or less compared to wild-type rna; the residual activity shows a decreased dependence on the presence of host factor. the results agree with an important role of the m-site interaction both with replicase and with host factor for the formation of productive initiation complexes. the template activity of the minus strand was unexpectedly found to be strongly dependent on the presence of a segment between nt and from the '-end. this shows that recognition of the minus strand by rep(icase does not only involve interactions near the '-end but requires a previously unknown structural feature near the '-end of this template. $ - karsten graning and angela kr&mer d~partement de biologie cellulaire, universite de gen~ve, , quai emest-ansermet, gen~ve splicing of nuclear pre-mrnas takes place within multicomponent complexes (spliceosomes) that are assembled by interactions between the pre-mrna, snrnps and protein splicing factors. we have purified two splicing factors that function in the formation of the pre-splicing complex. sf a consist of three subunits of , and kda and corresponds to three proteins present in the functional s but not in the s u snrnp. it binds to the s u snrnp only in the presence of sf b, suggesting a two step assembly pathway of s u snrnp: binding of sf b to the s u snrnp generates a s intermediate particle that is converted to the active s u snrnp by the subsequent association of sf a. comparison of proteins enriched in the sf b fraction with polypeptides found in the purified s u snrnp suggests that sf b comprises at least five of the other s u snrnp specific proteins and together with sf a promotes the u snrnp/pre-mrna interaction. by edna cloning, two of the subunits of sf a have been identified as homologs of well characterized yeast proteins that function at the same stage of spliceosome assembly in yeast. splicing factor sf , a heat-stable -kd protein, functions early during spliceosome assembly. tryptic peptides of sf were sequenced and cdna libraries were screened with degenerate oligonucleotides. the information obtained by comparing the sequences of three overlapping cdnas suggests the existence of at least three mrnas that could be derived from a common pre-mrna by alternative splicing. in agreement with this assumption mrnas of the expected sizes that are differentially expressed depending on cell line or tissue are detected by northern blotting. in theory, three polypeptides could be generated that differ by the presence or absence of internal amino acids and in their ctermini. the deduced amino acid sequence is rich in prolines and contains several possible phosphorylation sites and a putative leucine zipper. proline-rich domains, which are also present in splicing factors psf and sap , as well as leucine zippers have been found in transcription factors and could mediate protein/protein contacts, suggesting that sf could function during splicing by interaction with other spliceesomal proteins. pre-mrna splicing is catalyzed by a multicomponent complex (the spliceosome) that consists of small nuclear ribonucleoprotein particles (snrnps) and non-snrnp protein factors. the spliceosome is assembled in a stepwise fashion on the pre-mrna. chromatographic fractionation of hela cell nuclear extracts and subsequent reconstitution of splicing in vitro has allowed the separation and isolation of snrnps and several protein factors. sf triggers the transition between splicing complex b, which contains all spliceosomal snrnps but is not competent for catalysis, and complex c, the active spliceosome. this transition involves a conformational change that leads to altered base pairing interactions between snrnas and pre-mrna. the purification of sf has been impeded by its low abundance; however, a correlation between sf activity and a polypeptide of kd could be established in the most purified fractions. we are currently investigating whether this protein represents sf . interestingly an atp-dependent rna-helicase cofractionates with sf through several purification steps. whether or not this activity is relevant for the splicing process is under investigation. selection of iron-responsive element rnas with high affinity for iron regulatory factor henderson, b.r., menotti, e., bonnard, c. , and kith_n, l.c., isrec, ch-i epalinges iron regulatory factor (irf) post-transcriptionally regulates iron homeostasis via binding to mrna iron-responsive elements (ires). the ire loop sequence ( '-cagugn- ') and "bulge" cytosine are phylogenetically conserved. we prepared a pool of , ire molecules randomized at these nucleotide positions, and employed in vitro selection to identify optimal sequences which bind human irf. we define two major classes of high affinity rna ligand, the optimal loop sequences of each are '-cagugn- ' (wild-type) and '-uaguan- '. this novel finding predicts base-pairing within the loop between positions i and . all nucleotide substitutions in the "bulge" or at loop positions , and decreased binding by % to %. in addition, binding specificity of rat irf differed with that of the related rat ire-binding protein, irf b. in vitro analysis of ire-like stem-loops identified by computer search has not yet revealed any new ire-containing genes. a $ - ' processing of histone pre-mrnas: a phylogenetic comparison reto kohler, petra duda and daniel schiimperli. zoologisehes institut der universtit/it bern, abteihing fiir entwicldungsbiologie, baltzerstr. , ch- bern, switzerland. we are using a phylogenetic approach to study the biochemical reaction by which animal histone pm-mrnas are processed at their ' ends. in mammals, the efficiency and specificity of this reaction is known to depend absolutely on the u snrna which interacts with conserved spacer sequences downstream of the proc~sing site. a highly conserved hairpin element which precedes the cleavage site serves as a binding site for an additional processing factor, but its importanea for efficient processing varies greatly between different historic genes and between extracts of different mammalian cell lines. to determine whether the relative importance of the hairpin and spacer dements have been conserved during evolution, we analysed the processing of histone genes from three different animal phyla in vitro, i.e. vertebrates (mouse), arthropods (drosophila melanogaster) and nematodes (c. elegans). in the mouse system, processing was strongly dependent on the presence of a mouse spacer element. in nuclear extracts of drosophila ke cells, processing occurred exclusively when a drosophila spacer element was present in the rna. processing efficiency was not reduced by foreign (mouse, c. elegans) hairpins. in whole cell extracts of ascaris lumbricoides embryos, an inverted situation was observed. ' processing products were only produced by rnas that included an upstream segment derived from a c. elegans histone gene, irrespective of the spacer sequences present. these surprising results correlate with certain unique features in the c. elegans upstream element. we are currently in the process of cloning ascaris lumbricoides histone genes, which will allow us to verify our results in a homologous system. in xenopus laevls ooeytes, wild type mouse u rna gets assembled into functional u snrnps, both when transcr~ed from an injected gene or when injected as/n vitro synthesized rna. if the sm binding site of u rna is converted into the canonical sm sequence (u sm opt), the rna assembles into a particle which accumulates more efficiently in the nucleus but wbach is nonfunctional. this u sm off particle inhibits the function of preformed endogenous u snrnps, most likely by nonproductive binding to histone pre-mrnas, histone pre-mrna processing can be demonstrated in c/s if u rna is placed ' of hlstone pre-mrna and injected into oocytes. only u sm wt sequences are active in this c/s processing. three proteins can be photoaffinity cross]inked to u sm wt rna, the common snrnp proteins g and b/b' and a u -specific protein of kd ( kd in mouse). these crosstinks map to closely spaced positions within the sm binding site with the u -specific crosslink being located most '. u sm opt rna does not become crosslinked to the u specific protein and is also more tightly associated with sm proteins. we now investigate the in vitro assembly of u snrnps using these characterized u rnas and a preparation of highly enriched snrnp proteins. u sm wt and u sm opt rnas associate with common sm proteins in this system, but the interaction with the u -speciflc protein could not be demonstrated. the double-stranded (ds)rna modifying enzyme, which can convert adenine to inosine, was first identified in xenopus laevis, but has since then been detected in different species and in mammalian cells. the enzyme is a specific adenine deaminase which uses dsrna as substrate and recently, it has been postulated to be responsible for a specific mrna editing reaction. we have purified this enzyme to homogeneity, approximately , fold from calf thymus whole cell extracts. the protein was purified primarily by ion exchange chromatography over six columns, with the final step being chromatography on a dsrna affinity column. the purified protein has a molecular weight of kd and is the only protein present when enzymatically active fractions are visualized on an sds-polyacrylamide gel stained with silver. the dsrna modifying enzyme is a very low abundant protein. we are in the process of further characterizing the purified enzyme and of cloning cdnas coding for it. detailed mutational analysis of histone rna ' end formation carmen spycher, andr furger, adrian streit, daniel albrecht, d. schiimperli, zoologlsehes iustitut der universtit/it bern, abteilung thr entwicklungsbiologie. baltzerstr. , ch- bern, switzerland histone rna ' ends are formed by cndonucleolytic cleavage resulting in poly(a)" mrnas with a highly conserved '-terminal hairpin loop structure. for the mouse h - gene major and minor processing sites are located and nucleotides downstream of the hairpin, respectively. for the cleavage reaction, the u snrnp interacts with the spacer element of histone pre-mrna by base-pairing through the ' end of its rna moiety, u rna. we have observed that this base-pairing potential extends further than previously recognised in either direction. we have made site-directed rnutagenesis in fltis potential hybrid region and around the processing site. rna processing and complex formation with the u surnp were analysed by incubation in nuclear extract from mouse k cells. our results indicate that base-pairing with u rna both in the classical spacer element and downstream of it are very important for histone rna processing. in contrast, sequences upstream of the spacer element do not seem to be involved in base-pairing with u rna, but mutations in this region may affect processing by other mechanisms. systematic mutations of the highly conserved four nucleofides immediately following the hairpin show no qualitative or quantitative effects in the processing reaction. alteration of nucleotides to around the minor processing site, however, result in a dramatic decrease in processing efficiency and alter the specificity of the cleavage site. in further experiments we will introduce specific nuclcetidc modifications at the two processing sites, which should allow us to characterize potential cleavage factors by chemical crosslinking. evolution callaerts p., glardon s., j.,oosli f., quiring r. and gehring w. cell biology, biozentmm, basel. the pax genes encode a family of dna binding factors which share the paired-box and play an important role in the control of development. they can be subdivided into four different classes which differ in the presence or absence of other conserved sequence motifs coding for a paired type homeobox or an octapeptide. the pax- gene, which contains a paired-box and a homeobox, has been cloned from humans, mice, rats and zebra fish. recently, we have cloned the pax- homologue of drosophila melanogaster. the five genes share a very high degree of sequence identity both in the paired-domain and the homeodomain. similar to its mammalian counterparts, the drosophila gene is expressed in the eye primordia and the nervous system. in addition, mutations in pax- affect eye development: aniridia in humans, small eye in mouse and rat, and probably eyeless in drosophila. this would imply that the pax- homologues share a conserved function in eye morphogeuesis in both vertebrates and invertebrates. furthermore, this suggests that pax- may have been present in a common ancestor. therefore, we are now trying to isolate pax- homologues from other, more primitive organisms by means of the polymerase chain reaction, and to determine whether they are implicated in eye development. putative candidates have been identified from a number of species and are being characterized. their sequence similarity and some evolutionary implications will be discussed. keegan liam and gehrin~ walter j., biozentrum, university of basel, klingelbergstr. , ch- basel, switzerland using an enhancer map screen we have identified two genes that may be direct targets of antennapedia involved in forming the adult leg. spalt major is repressed in the leg imaginal disc and tea, shirt is activated by antennapedia. we wish to determine whether the control of these genes by antennapedia is direct, spalt major is expressed in a ring in the antennal disc and is repressed by antennapedia in the leg disc. the antennal ring is repressed by ectopie antennapedia expression that transforms the antenna to a leg. we are defining an enhancer at spalt major that is required for antennal ring expression and repression by antennapedia. we are using various approaches to show that antennapedia binds to the antennal ring enhancer at spalt major. these include polytene chromosome banding studies using antibodies to antennapedia as well as in vitro dna-binding and genetic experiments. in search for novel regulators potentially involved in myogenesis, we identified a homeobox of the paired type in human muscle. subsequent cdna cloning revealed that we had cloned the full length coding region of human pax . our human sequence extends the known mouse edna both on the ' and the ' end. as a first step in order to test for a functional role of pax in myogenesis, we analyzed its expression pattern during chicken development. transcripts are present in the neural tube and in the dermamyotome of the developing somites. therefore, the pattern of expression of pax is conserved between mouse and chicken. next, we assessed the expression of pax in different mouse and human cell lines. we found pax transcripts specifically in myogenic cells and not in any other cell types. interestingly, pax is already present at the myoblast stage. moreover, ti/ fibroblasts converted to myoblasts by either transfection of myod or treatment with -azacytidine expressed pax , whereas the parental cells did not. while myod was able to activate pax in t / cells, expression of pax was not sufficient to induce the myogenic phenotype. nevertheless, our expression data are consistent with a role of pax during the mesodermal commitment to the myogenic lineage. the segmental organization of the drosophila head is achieved by a flow of positional information from maternal gene products to the zygotic gap genes. recent genetic analysis has identified three new gap genes involved in head development. one of these genes is the empty spiracles (ems) gene. mutations in eras cause severe defects in the head and the filzk~rper at the posterior end are missing. the eros gene encodes a putative transcription factor with a homeodomain. the eros rna is expressed in two phases of embryonic development. first expression is seen at the blastoderm stage in a single anterior band, correlating with its function as an anterior gap gene. later during embryogenesis eros is expressed in the posterior spiracles as well as lateral regions of each segment where the tracheal pits form and lateral neuroblasts originate. using g-gal fusions we could identify at least five different regulatory elements in the eros promotor region responsible for tissue specific expression of the gene. a bp element responsible for the early expression which is dependent on the maternal gene bicoid, the key gene of the anterior system, and the terminal gene tailless was identified and studied in detail. this element contains two bicoid and two tailless binding sites. the effects of muations in these binding sites on eros expression will be discussed.this analysis should allow us to elucidate the molecular mechanisms by which the morphogen bicoid regulates subordinate target genes like ems in the drosophila embryo. the pax- gene of the mouse encodes a transcription factor with both a paired-and a homeodomain. pax- is affected by several allelic mutations in the small eye (sey) gene. sey mutations cause a reduction of the eyes in heterozygotes, and homozygotes lack both eyes and nasal openings and die as newborns. the corresponding mutation aniridia in humans affects eye and cranlofacial development in a similar manner. we have isolated a pax- homolog from drosophila (dpax- ), which shares more than % sequence identity in the paired-and the homeodomain with the mammalian genes. also outside of these domains we find considerable sequence conservation arguing for a true pax- orthologue. the drosophila gene spans ~ kb and is expressed in the brain and the ventral nervous system of the embryo, and in the eye imagjnal discs and the brain of the larva. the gene was mapped to the eyeless (ey) locus. mutations at the ey locus cause either the partial or complete absence of the compound eyes or embryonic lethality. in one ey allele we have identified a transposable element in the first intron of dpax- , which affects dpax- transcription in the eye discs, suggesting that dpax- is encoded by the ey gene. this implies that pax- (sey) in the mouse is homologous to ey in drosophila. thus, despite of the different modes of eye morphogenesis, the same gene seems to control eye development in insects and vertebrates. the drosophila homolog of the vertebrate serum response factor (srf) was isolated by low stringency-hybridization. nucleotide sequence analysis revealed that the drosophila srf homolog (dsrf) codes for a protein which displays % of sequence identity with human srf in the mads domain, the region required for dna binding, dimerization and interaction with accessory factors. the dsrf gene is expressed during several phases of embryonic development. both the rna and the protein are maternally provided to the egg and slowly decrease in their levels during gastrulation. after germ band retraction, high levels of zygotic expression were observed in a distinct subset of peripheral tracheal cells distributed throughout the embryo. many of these cells are at the tip of tracheal branches and are in direct contact with the target tissues these branches tracheate. the dsrf gene was mapped to position c on the second chromosome, and overlapping deficiencies which remove the gene were identified. analysis of tracheal development in embryos carrying these deletions revealed a degeneration of most of the major branches of the tracheal system. although the initial migration of tracheal cells was not affected in those deficiency embryos, many tracheal cells appeared not to maintain their formerly correct position and continued to migrate. thus, the dsrf gene might play a role in the proper formation and maintenance of the major branches of the trachea. s - our experiments would be expected to provide insight into such problems as the evolution and function of transcription factors with multiple dna binding domains. ideally, we would be able to mimic "gene splitting" which occurred during evolution. the poan gene plays an important role during drosophila neurogenesis. it is expressed in specific subsets of sensory mother ceils (smcs) and neuroblasts. the smcs that express poxn differentiate into polyinnervated external sense (p-es) organs. in the absence ofpoxn, smcs differentiate into mono-innervated rather than poly-innervated external sense organs. as the poxn gene contains a paired-box, it probably encodes a transcription factor. since the function ofpoxn in the central nervous system is not known and no po~n mutants are available, an ems mutagenesis screen was initiated to isolate such mutants. based on the assumption thatpoxn null alleles are lethal, we screened for lethals uncovered by the deficiency df( r)wmg, which includes poxn, but survive over the deficiencies df( r)xte- and df( r)kl- flankingpoxn. in a further test, such lethals are screened for the absence of embryonic p-es organs. ifpoxn mutants are not lethal, they will escape this screen. therefore, we are inducing local hopping of p-elements that have inserted in the vicinity of poxn. the offspring of flies that display an altered eye phenotype are screened for p-element insertion into poxn. we hope that these approaches will generate mutants that will be crucial for future studies ofpoxn function in neurogenesis. chromatin structures and transcription of rdna in yeast saccharomyces eerevisiae reinhard dammaun, renzo lucchini, thee keller and jest m. sogo; institute of cell biology, eth-honggerberg, ch- ziirich the chromatin structure of yeast ribosomal dna was analyzed in rive by cross-linking intact cells with psoralen. we found that in exponentially growing cultures the regions coding for the s rrna precursor fall into two distinct classes. one class was highly accessible to psoralen and associated with nascent rnas, characteristic for transcriptionally active rrna genes devoid of nucleosomes, whereas the other class showed a cross-linking pattern indistinguishable from that of bulk chromatin and was interpreted to represent the inactive rrna gene copies. by cross-linking the same strain growing in complex or minimal medium, we have shown that yeast ceils can modulate the proportion of active (non-nueleosomal) and inactive (nucleosomal) rrna gene copies in response to variations in environmental conditions which suggests that yeast can regulate rrna synthesis by varying the number of active gene copies, in contrast to the vertebrate ceils studied so far. whereas intergenie spacers flanking inactive rrna gene copies are packaged in a regular uucleosomal array, spacers flanking active genes show an unusual cross-linking pattern suggesting a complex interaction of regulatory factors and histories with dna. $ - r. felleisen & b. gottstein; institute of parasitology, university bern, bern, switzerland most patients suffering from alveolar echinococcosis (ae) respond to infection with a marked igg synthesis directed against e.multilocularis metacestode antigen ii/ , which represents a novel member of the family of cytovillin related proteins. although the respective gene basically is also present and expressed in e.granulosus, most of the cystic echinococcosis (ce) patients do not recognize the antigen. this phenomenon was tackled by generating cdna derived from full length ii/ genes from both echinococcus species, performed by rt-pcr. sequence analysis revealed a very high degree of conservation of the primary sequence ( . % homology), cdna fragments were expressed as recombinant proteins and were comparatively assessed in elisa respective to antibody-binding characteristics. sera from patients suffering from ce were showing no significant differences in reactivity with the antigens derived from both species. therefore, parameters others than minor differences in the primary sequence seem to be responsible for the lack of antigen recognition with respect to ce. $ - patrick rigoni, sandro rusconi, institut f molekularbiologie h der universitat zarich, winterthurerstr. , z~rich, switzerland. trinuclcotide repeats are often found in the coding sequence of transcriptional regulators in both mammals and yeast, however, their function is yet unclear and data collected in our laboratory on the gheocorticoid receptor even suggest that they may not have any, and that their presence is probably the result of a selfish replicative behavior. nonetheless, we believe that these motifs can be used as tags to identify flexible protein domains typical of modular proteins such as transcription factors. another kind or repeat (coding for a poly-glutamine/alanine) has been discovered in the yeast regulators gall and ssn . from northern blots, we know that such caggcn repeats are present also in higher eukaryotes and we want to isolate them by constructing an enriched edna library using a ' race protocol. so far, no mammalian protein bearing the motif glrdala has been characterized. among the positive clones we hope to find the homologous or paralogous of gall and ssn that have escaped so far conventional cloning techniques. meanwhile, we have been testing the effect of coexpressed yeast gall on different reporter-activator combinations in mammalian cells. preliminary results show that galll but not the mutant form galllp can inhibit the activation properties of some (not all) gal chimeras. we are also producing antibodies directed against oligo-ala and oligo-gln and oligo-gln/ala motifs in order to better characterize natural proteins containing these repeats. we are interested in the early development of c. elegans, particularly in the contribution of genes whose homologues in vertebrates and d. melanogaster mediate positional infolzzations during development. therefore, we are working on ceh- which belongs to a cluster of at least homeobox containing genes. this cluster is considered to be equivalent to the homeotie cluster of drosophila and to the hox clusters in vertebrates. by generating ~-galactosidase transgenie lines, we have observed that ceh- is expressed very early during embryogenesis, embryonic expression appears to be restricted to the posterior half of the embryo, which is rather surprising, since the ceh- hemologues in drosophila and vertebrates are anterior-specific. during larval stages, ceh- is expressed in neuronal cells. these results are now in the process of being confirmed by immunolocalization of the ceh- protein product with a polyclonal antiserum. in order to clarify the function of ceh- in the c. elegans development, we have isolated a ceh- mutant from a tcl insertion library, in collaboration with r. h. a. plasterk from amsterdam. from this worm, which looks wt, we are now trying to obtain a deletion derivative. previous studies have shown that bovine herpesvirus (shv- ) infected cell pratein (bicp) acts -depending on the promoter -as a strong transactivator or as a repressor in transient expression assays. the cp polypeptide contains a cysteine-rich zinc finger domain (c hc ) which is conserved in a number of viral and cellular regulatory proteins including the icp homologs icr of herpes simplex virus type and protein of varicella zoster virus. this type of zinc finger (the so-called ring tinge was shown to bind zinc ions but functional requirement for zinc has not yet been demonstrated, a gap which we aimed to fill with the present study. transient expression assays were performed in oocytes which had been microinjected with thionein to chelate and deplete the intracellular pool of zinc. cp -induced cat activity of a promoter-cat construct was -fold higher than the basal cat activity of the reporter plasmid, in the presence of thionein, bicp -induced transaotivatlon was reduced old. with a set of control experiments we excluded that thionein might affect transcription and protein synthesis in general, to our knowledge this is the first demonstration of zinc-dependence for a member of the ring finger family. we have identified by pcr and edna cloning five pou genes expressed during early embryogenesis of zebrafislx four of these genes show extended homology to the brn- class of pou genes. preliminary evidence suggests that the brn- like pou genes overlap with most of their expression domains.the gene studied in most detail so far (zp- ) is first expressed on the ventral side of the future fore-and midbrain. slightly later, expression is also found in rhombomeres and in the hindbraln. these rbombomeres were identified by the specific expression of the krox- marker using a novel in situ hybridization protocol which allows the simultaneous detection of two different transcripts using different color substrates. in the hours old embryo a complex expression pattern is found involving a variety of brain structures and the spinal cord. the distribution of the transcript suggests that zp- is mostly expressed in glia cells. another pou gene we have identified (gp- ) defines a novel class of pou proteins. as a maternal mp, na it is initially ubiquitously present. after gastrnlation its transcript is found most notably in the developing hindbrain in rhombomeres and for which so far no good molecular markers were known. we are investigating the roles the identified pou genes may have in zebrafish hindbrain segmentation. we are currently attempting to manipulate gene expression in developing embryos by injection of rna or by the production of transgenic fish. we are also screening the zebrafish genome for new developmental marker genes. progress about these experiments will be presented. $ - willimann, t. and trueb, b. to gain insight into the regulatory mechanisms of collagen vi synthesis we have characterized the cis-acting elements of the chicken ctl(vl) collagen promoter. footprinting experiments with nuclear extracts from chicken embryos revealed three distinct elements, designated a, b and c, that were protected from dnase i digestion. the nuclear proteins that interact with the three sites were identified by gel retardation assays in combination with the use of various oligonucleotide competitiors as well as specific antibodies raised against well-characterized transcription factors. site a was found to be a target for transcriptional activator apf, whereas sites b and c were shown to be recognized each by two distinct nuclear proteins which belong to the spl multigene family. to address the question whether the three sites alone are able to direct transcription, a minipromoter construct was created in which the sequences of sites a, b and c were placed in front of a reporter gene. after transfection into chicken fibroblasts, this construct exhibited a high relative promoter activity when compared to a large genomic fragment containing the basic ctl(vi) collagen promoter. thus, the three sites are sufficient to induce transcription of this gene. octamer transcription factors (oct or otf) are a subset of the pou family of transcription factors which regulate expression of cellular and viral genes by binding to the octamer sequence atgcaaat. neurons and astroglial cells harbour, in addition to the ubiquitous oct-i factor, brain specific factors termed n-oct , , and . in the present study we determined the chromosomal localization of the gene encoding the n-oct transcription factor and characterized the structure of isolated n-oct genomie clones. the chromosomal mapping was performed by analysis of somatic cell hybrid panels and radioactive and fluorescence in situ hybridization of human metaphase chromosomes. it is interesting that the ' end of the n-oct coding sequence contains repetitive cag and ggc residues. several disorders have been discovered to be related to expansion of trinucleotide repeats. we are presently investigating if the n-oct cag or ggc clusters are hot spots for such mutation mechanisms and if so, which diseases could be associated with it. two dna regions upstream of the distal glucocorticoid receptor binding site interact with nuclear proteins that are tissue-specific (region a) or ubiquitous (region c). the characteristics of the dna-protein interactions have been studied in vitro. these sequences, in different combinations with the natural mmtv promoter, were tested in transfection assays of fibrcblast or mammary cell lines. we show that they are able to modulate the level of glucocorticoid-stimulated transcription. the proximal region of the mmtv promoter has binding sites for the transcription factors ctf/nf-i and oct- . p~asmids with a deletion or mutations in the oct- site were tested in stable transfections, that are most representative of the state of proviral dna with respect to both number of integrated dna templates and chromatin organization. we show that the oct- site is important for the basal level of promoter activity. the data further indicate that the functional outcome may depend beth on the relative ratio of factors to dna templates and on the relative affinity of binding sites, as determined by oligonucleotide competition footprinting. besides the fact that telomeres represent specialised structures important for chromosome stability, the particular significance of cloned c. elegans telomeres is that they will contribute to the completion of the physical mapping of chromosomes and that they provide one set of elements for the construction of artificial c. elegans chromosomes. the cloning of c. elegans telomeres, however, is impeded by the fact that tandem repeats of the sequence ttaggc are not only located at the ends of the c. elegans chromosomes, but also at many internal chromosomal sites. therefore, we have developed a special protocol for the construction of a c. elegans endlibrary in ~ zap. the resulting telomeric library was screened for recombinants containing ttaggc repeats and yielded about positive clones. so far, were analysed by partial sequencing and twelve of them satisfy all criteria required for telomeric clones. their ttaggc tandem repeats are located at the expected position, namely just adjacent to the blunt end cloning site in the polylinker. furthermore, the g-rich strand of the repeats is oriented ' to ' towards the end of the fragment, thus corresponding exactly to the defined orientation of eukaryotic telomeric sequences. finally, hybridisation data with one of these putative telomeric clones show that a subtelomeric fragment hybridises to bal -sensitive fragments on a southern blot with c. elegans dna, indicating that this clone represents a c. elegans telomere. $ - to investigate whether chromatin structure or transcription can interfere with replication, derivatives of the yeast trp ars minichromosome were constructed that contain either the ded or pet promoter firing against the origin of replication ars . while the pet constructs transformed yeast at high frequencies and were maintained as high copy number minichromosomes, the ded constructs transformed at low frequency and the constructs were integrated in the genome suggesting that ars function was impaired. insertion of the h -ars, a second origin of replication, rescued a ded construct as a minichromosome (yrpdh ).the chromatin structure mapped at low and high resolution of the ars region in yrpdh was indistinguishable to that of the pet construct yrpft . ananlysis of rna showed that transcription was going through ars in yrpdh and in yrpft , but the levels of transcripts in yrpdh were much higher. we conclude that transcription through are interferes with replication and prevents extrachromosomal maintenance. dna sequence of a repeated dna segment on circular and linear plasmids of borrelia burgdorferi w.r. z%ckert and j. meyer, abt. pzmom, zahn&rztliches institut der universit~t basel a plasmid-associated repeated dna segment of b. burgdorferi strain b was cloned. at least two copies of this segment appear to be present on the kb circular plasmid, whereas one copy is carried on the kb linear plasntid of this strain. dna sequence analysis revealed a region containing a novel bp putative open reading frame (orfl) on the kb linear plasmid, orfl displayed extensive sequence homology ( %) to putative open reading frames present on the clones obtained from the kb circular plasmid. heterogeneity is mainly caused by rd-base-wobbllng. flanking sequences share - % homology, including the ' end of an additional putative open reading frame irmaediately downstream of orfl. whether orfl and/or the related sequences are being transcribed and yield, in the case of orfl, an approximately . kd, lysine-rich polypeptide, is yet unknown. the repeated sequence seems to be specific for b. burgdorferi sensu lato and therefore may be useful in nucleic acidbased diagnostics of lyme disease. $ - similar to type ib oculocutaneous albinism in humans, overall production of pigment is greatly reduced in dark eyed albino and only obvious in eyes. we have studied the molecular basis of the c h mutation and show that expression of the tyrosinese gene is not affected. after sequencing tyrosinase cdna isolated from c h/c h homozygotes we uncovered a single base alteration from wild type leading to a serine to isoleucine exchange. the importance of this mutation was demonstrated by generating transgenic mice containing a mutated tyrosinase minigene. this showed that the single base change is sufficient to severely depress pigment production in transgenic mice. we therefore conclude that the point mutation is responsible and sufficient to generate the dark eyed albino phenotype. members of the pou-family of transcription factors are involved in developmental and differentiation processes. using a pcr-based cloning strategy with degenerated oligonucleotides we identified several pougenes from the lactating and involuting mouse mammary gland. three of these cdna clones which contained as yet unknown sequences were chosen for further investigations: clone . which has a high homology to pit-l, clone . which is related to the brn- gene and clone . which belongs to the class iii of pou-proteins. the expression levels were analyzed in different mouse organs and in several developmental stages of the mammary gland, including the postlactational process of involution. because of the low abundance of the specific transcripts we used a semiquantitative, reverse transcriptase-mediated pcr assay to investigate the mrna levels of the three novel pou-family members. distinct and specific expression patterns for all three members were obtained in the different investigated organs. interestingly, the expression of the pit- related cdna clone , was elevated in all developmental stages of the lactogenic-hormone dependent mouse mammary gland and in sceletal muscle, whereas its expression was low in other organs. repetitive sequences in dna can allow ectopic (outof-register) homologous recombination, which is often undesirable and can even lead to disease in humans. to prevent this, long homologies should often be interrupted during evolution. accelerated mutation of repetitive sequences by so-called ripping may be one of the mechanisms used to accomplish this in fungi. we are investigating if introns in vertebrate genes have an undiscovered role as interrupters of homology within and/or between genes, in addition to their established role in exon shuffling. by inserting homology-poor introns in an otherwise homology-rich region the genome could prevent ectopic homologous recombination. such a mechanism could be especially useful where there is an advantage in encoding highly repetitive protein sequences. it appears that within the collagen gene family there is indeed a correlation between repetitiveness and intron density. the influence of prenatal diazepam exposume (i, mg/kg/d, s.c.) from gestational day to on the development of the ~-opioid binding sites in striaturn, nucleus accumbens and midbrain of pn , pn and week old male and female rats was studied. week old prenatally diazepam exposed male rats showed a significant decrease of k-opioid binding sites in the nucleus accumbens. a sex-dependent difference in k-opioid binding site densities could also be detected between pn prenatally diazepam exposed male and female rats. we now investigate by in situ hybridisation whether changes in the level of mrna encoding fo~ prodynorphin, the precttrsor for various endogenous ~-opioid agonists, could be responsible for the decrease of ~-opioid binding sites in the nucleus accumbens of week old prenatally diazepam exposed male ~ats. $ - distribution and morphology of nitric oxide-positive neurons in the marmoset cerebral cortex during pre-and postnatal development j.p. hornung* and j. schultz** *institute of anatomy, university of lausanne, lausanne, and **university of fdbourg, fribourg nitric oxide, synthesized by the enzyme nitdc oxide synthase (nos), is a neuroactive substance and a limited number of neurons were shown to express this enzyme either by histochemistry or by immunocytochemistry. both techniques were performed on brains of marmosets with ages ranging from weeks before birth to adult. the morphology and distribution of nos-positive neurons were analyzed in all lobes of the cerebral cortex. the same pattern was revealed by histochemistry or immunocytochemistry. three populations of nos-positive neurons were revealed. first, a transient population of neurons in the molecular layer of the embryonic cortex, many having the morphology of the retzjus-cajal cells. a second population was made of large multipolar neurons in the deep layers of the cortex and the underlying white matter, which persisted from embryonic to adult life. a third, permanent, population was made of small, weakly reactive neurons in the supragranular layers appearing postnatally, this study demonstrates that no can exert its actions in the cerebral cortex through several pathways at different developmental stages. trimethyltin (tmt), a well-known neurotoxicant, was used to study the sensitivity of the different brain cell types (i.e. neurons, astrocytes, oligodendrocytes and microglial cells) to a neurotoxic insult. aggregating brain cell cultures of fetal rat telencephalon were treated during days with tmt at different concentrations, ranging from - m to - m. microglia were found to be the most sensitive cell type, since already at - m of tmt an increased number and clustering of griffonia simplicifolia-positive ceils could be observed. at - m of tmt astrocytes showed increased staining for glial fibrillary acidic protein, characteristic of gliosis. neurons and oligodendrocytes appeared to be the least sensitive cells, since a decrease in the activity of cell type-specific enzymes was observed only at - m of tmt. these results show that microglial cells and astrocytes respond to a toxic insult before any neuronal changes could be detected, and that the microglial reaction may be the first target of tmt. this reaction could then trigger the astrocytic response. vif is widely distributed in brain with suggested functions related to development. vip expression was studied during rat brain development using hybridization histochemistry with a met probe. vip mrna was found in thalamic nuclei on el , later recognized as the ventrolateral and reticular nuclei after further maturation during prenatal period, vip mrna was found in hypothalamus, especially suprachiasmatic nucleus on el and its expression matured over the next days. few cortical neurons contained vip mrna on e , they continuously increased in number and signal intensity over the perinatal period. vip gradually matured in the first three postnatal weeks and adult-like patterns were found on p , when cerebral cortex, ventrolateral and reticular thalamic nuclei, hypothalamus, esp. suprachiasmatic nucleus, were the regions with most prominent vip expression. these results demonstrate the relatively late appearance of vip gene expression in the rat forebrain, as compared with peptides like srif and cck, not suggesting a major role in earlv brain maturation. in primary cultures of mouse cerebral cortical astrocytes, a rapid glycogenolysis followed by a massive glycogen resynthesis ( six-to ten-fold over basal levels after hr) are induced by vasoactive intestinal peptide (vip) or noradrenaline (na). both actions of these neurotransmitters are mediated by camp. since the induction of glycogen resynthesis triggered by vip or na is abolished by inhibition of transcription and translation, we applied the -d gel electrophoresis technique to search for astrocytic proteins induced by vip or na. the comparison of s-labeled proteins from primary astrocyte cultures treated or not with vip ~tm revealed two newly synthesized proteins: a kda protein (pi= ) and a / kda protein (pi= ). only the latter is visible on a silver stained -d gel, which is a prerequisite to consider its purification and microsequencing. induction of the / kda protein by vip was time-dependent with a maximum at - hr. preliminary results indicate a similar induction by na and isoproterenol. in humans, the central analgesic effect of tramadol (t) is only weakly reversed by the ~t opioid antagonist naloxone (nx) (br j clin pharmacol ; : p). t analgesia may thus result from an action on monoaminergic pathways as suggested by in vitro and animal data. we therefore investigated the effect of ct -adrenoeeptor antagonism by yohimbine (y) on t analgesia. according to a randomised double-blind crossover and placebo (p) controlled design, healthy volunteers (n= ) received t ( mg orally), followed (+ h) by y ( . mg/kg intravenously), and y + nx ( . mg intravenously). analgesia was assessed over h by subjective pain threshold (numerical scale -ns-) and objective pain threshold (rill nociceptive reflex -riii-) monitoring. peak analgesic effect was observed at ca. . h (riii + . +semi. ma and ns + . + . ) vs p (rih - . + . and ns + . + . , p< . ) and lasted ca. h. y reversed t analgesic effects during ca. h (r/ii - . + . , p< . and ns + . + . , p< . ), whereas y + nx abolished t effects thoughout the study period (rill - . - . and ns + . + . , p< . ), y alone tended to lower pain thresholds (rill - . + . and ns - . + . , p> . anova). yohimbine, an r antagonist, reverses tramadol effects, thus pointing to the major role of monoaminergic modulation in tramadol anfinociception. a monoclonal antibody (in-l)was raised against neurite growth inhibitory proteins present in rat cns myelin (caroniand schwab, neuron : , ) . in- neutralizes the inhibitory ettect of cns tissue in vitro and in vivo, thus permitting regeneration of certain lesioned cns fiber systems. we have used this antibody to immunostain cryostat sections of the nervous system of the rat. we found that in- stains white matter and myehnated fiber tracts in the cns. the staining pattern corresponds to that of the known myelin specific antigens mbp (myelin basic protein), mag (myelin associated glycoprotein) and mog (myelin oligodendrocyte glycoprotein). the staining of in- in the cns is found in regions of the adult nervous system which have been shown to be inhibitory for axonal growth. interestingly, the regions which show a high abundance of in- antigens are low in staining for gap- , a marker for growth and plasticity in the developing and adult brain. in contrast, gap- is strongly expressed in unmyelinated fiber tracts or nuclei. prevention of oligodendrocyte development in rat spinal cord resulted in suppression of in- expression and elevated gap- levels. this suggests that inhibitory myelin proteins may negatively influence plasticity in the rat cns. to date, the existence of anp and npy in the adrenal medulla has been shown only for a few mammalian and anuran species. thus the present immunohistochemical investigation attempts to localize anp-like and npy-like peptides n the adrena gland of representative mammals, birds, reptiles, amphibia and bony fish by the use of antisera specific for mammalian anp and npy. furthermore, the catecholamine-containing cells were characterized by antisera against enzymes of catecholamine synthesis. anp-and npy-immunoreactivies were detected in adrenal chromaffin cells of all vertebrates studied while no immunoreactivities were observed in the adrenal cortex or in its homolog, the interrenal. the representatives of the different vertebrate classes exhibited marked differences in the proportions of anp-immunoreactive and npy-immunoreactive cells, in the presence of anp-and npy-immunoreactivities in noradrenalineand/or adrenaline-containing cells and in the amount of of cells containing both anp-and npy-immunoreactivities. our results give evidence for a long phylogenetie history of adrenal anp-and npy-like peptides. thus, they stress the physiological impact of these peptides as adrenal paracrine and/or endocrine hormones. human neuronal growth inhibitory factor (gie) is involved in the regulation of neuronal growth and is deficient in the brains of alzheimer's disease victims. this brain-specific metalloprotein consists of amino acids out of which are cys and has been found to contain copper and zinc. proteins with similar primary structure were isolated from bovine and equine brain. the amino acid sequence of these proteins shows about % homology to those of mammalian metallothioneins (mt), with two conserved inserts of thr and a glutamic acid rich hexapeptide in the n-and c-terminal regions, respectively. in contrast to mts, which usually contain only zn(i ), the presence of cu(i) and zn(ii) ( - moles total) in all gif isolations suggests the functional importance of both metals ions. to spectroscopically probe the native zn-binding sites, the zn(ii) ions were replaced by cd(ii). both bovine and equine cd/cu-gif derivatives exhibit similar absorption and cd spectra with features characteristic of cd-thiolate clusters. the release of h-arachidonic acid evoked by glutamate was characterized in cerebral cortical neurons in primary culture grown under conditions that prevent glial cell proliferation. in these cultures, % of the total ceils are immunocytochemicauy characterized as being positive for specific neuronal markers such as neuron-specific enolase and neurofilament. specific markers for astrocytes, oligodendrocytes or microglia were not detected. glutamate induces a concentration-dependent release of h-arachidonic acid with a ec of pm. the pharmacological profile of this glutamate response shows the involvement of two receptor types: the nmda and the ampa ionotropic receptors. the glutamate-evoked release of h-arachidonic acid is phsensitive. when the incubation buffer is alkalinized from . to . , both basal and glutamate evoked release of h-arachidonic acid are enhanced. the glutamate response is also enhanced as intracellular ph is alkalinized by pharmacological treatments: such as inhibition of c -/hco -antiport by dids. these results further stress the role of intracellular ph in glutamate-mediated neurotransmission. $ - multiceli culture system for the study of drug kinetics wechsler d., *schittny j.c. and honegger u. e., depts. of pharmacology and *anatomy, university of bern, ch- bern a cell culture system was developed for the investigation of cellular drug kinetics in cultures with up to cell types. it was used for the study of uptake, competitive uptake, release and redistribution of drugs. individual cell types (human skin fibroblasts, rat astrocytoma cells (c ) and rat hybfidoma ceils (oligodendrocyte x astrocytoma, roc)) were separately grown to confluency on glass circle sectors. in the same petri dish sectors in various combinations were exposed to media containing radiolabelled chlorpromazine (cpz). single and repetitive uptake and release of cpz were measured in each cell type after individual exposure or exposure in any combination of cell types: in hour competitive uptake studies fibreblasts reached . and . times the concentrations of c -and roc-cells, :respectively. in redistribution experiments the exchange of cpz from preloaded to unloaded cells was tested. the exchange rate was dependent on cell type and loading period. it decreased with increasing time of exposure suggesting the formation of more stable cpz stores. we have previously demonstrated that certain neurotransmitters such as vip, noradrenaline (na) and adenosine promote glycogenolysis in mouse cerebral cortical astrocyte cultures (brain res. : - , ) . the question that we then addressed was the nature of the metabolic substrate released by astrocytes following glycogenolysis. we therefore determined the presence of various metabolic substrated released from astrocytes under static conditions and in the superfusate of a laminar flow system developed in our laboratory. neither glucose nor any intermediate of the tficarboxylic acid cycle could account for the decrease in glycogen stores; astrocytes however were shown to release great quantities of pyruvate and lactate at a rate of nmol/mg protein/minute. noradrenaline but not vip promotes a significant increase ( %) in lactate release. the effect of noradrenaline is mimicked by isoproterenol and inhibited by propranolol, suggesting a i~adrenergic mediation. when astrocytes are incubated in an isotonic solution containing no energy suhstrate, the glycogen stores are rapidly depleted and lactate, but not glucose, is released in the medium. in conclusion, lactate appears to play a major role in cerebral energy metabolism homeostasis, as substrate released by astrocytes to prey!de energy fuel for neurones and other neighboring cells. it is known so far that calcium-binding proteins (cabps: calbindin, pan, albumin and ca/retinin) are expressed in some cells of the pineal gland of laboratory mammals. the exact phenotype of these cells is unknown. nevertheless, according to recent studies, it seems that some of them are ganglion cells. therefore we studied the pineal gland of different species (gerbils, rats, goats, cows and humans) using calretiain (cr) antibody which is considered as marker for neurons (andressen & al, cell & tissue rss, t: , ) , the cr-expressing cells were found in all oar species: in gerbils, rats, cows and humans they were scattered throughout the parenchyrna, whereas in goats they lined mostly the pericapillary spaces. according to our findings, it seems that most of the reactive ceils are rather neuron-like elements since morphological criteria for true neurons (axosomatic synapses, nissl bodies, bundles of neurofilaments) are missing. however one cannot exclude that among these cells some are intrapineal ganglion cells, because of ranvier's nodes found along the processes of some crpositive cells. thus, it is possible that these cells are involved in the modulatory control mechanism of the pineal neurohumoral activity and/er by accumulation of intracellular calcium in the formation of pincal calcifications. besides the excitatory amino acid transmitter glutamate, its sulfur containing analogue homocystcic acid (hca) could play a role in excitatory processes in the central nervous system. hca is present in and released from nervous tissue and is a potent neuronal excitant, predominantly activating n-methyl-d-aspartate receptors (do et al., ) . these properties are suggestive of a classic synaptic neurotrausmitter role. on the other hand, hca seems to be localized not in neurones but in glial cells (grandes et al., ; tschopp et al. ) . the efflux of hca is temporally delayed following activation of specific pathways (klancnik et at., ) ; these latter observations are not in accordance with the classic concepts of synaptically-mediated neurotransmission. a laminar flow superfnsian system of monolayer cultures was developed and the released materials from mouse cortical astrocytes, previously preincubated with [ s]-methioulne, were investigated. during application of either noradrenaline or the b-adrenergie agonlst isoproterenol, the extracellalar level of [ s]-methianine was decreased and that of labelled hca was increased. this effect was not observed with the -adrenergic agonist methoxamine, and could be blocked by the ll-adrenergic antagonist propanolol these results, combined with the delayed hca release, the glial localisatian of hca and its neuroactivity, strongly suggest a potential role of hca as a gliotransmitter, modulated by noradrenaline inputs. vasoactive intestinal peptide (vip) induces long lasting metabolic effects in the mouse cns. we have investigated a shorter neuromodulatory action of vip on the synaptic responses in coronal slices of adult mouse cingulate cortex by means of intracelhilar current clamp recordings. electrical stimulation of the underlying white matter evoked a multiphasic synaptic potential consisting of early epsp~ early ipsp, late epsp and late ipsp mediated by non-nmda, nmda, gaba a and gaba b receptors, respectively. vip ( . ~d~l) superfused for rain. had no effect, whereas concentrations over . ixm selectively and reversibly depressed the nmda-medlated responses in / cells. in the presence of cnqx ( [tm) and biencuuine ( ~tm), the isolated nmda-mediated late epsp was still attenuated by . ~m vip in / cells (mean:- % al~er rain.). in other cells vip induced a spreading depression (sd) and a strong, reversals inhibition of the late epsp (mean:- % after min.).these results indicate that in the absence of gabaergic inhibition vip can induce two types of effects in the mouse cingulate cortex, a prolonged decrease of the nmdamediated syaaptic response and a striking sd. interaction between vip and glutamate on c-fos mrna expression. |.-l. martin, d. gasser and p.] . magistretti. institut de physiologie, universit de lausanne, lausanne, switzerland. the regulation of c-los mrna expression by vip and glutamate was examined in primary cultures of neurons originating from the mouse cerebral cortex. in primary cultures of cortical neurons, vip increases camp levels and stimulates c-los mrna expression. interestingly vip stimulation is completely blocked by mk- , an nmda receptor antagonist but not by cnqx or ap , which antagonize ampa/kainate and metabotropic receptors respectively, c-los expression stimulated by glutamate shares the same pharmacology. these results suggest an involvement of endogenously released glutamate in the stimulation of c-fos mrna expression evoked by vip. furthermore, when added together, vip and glutamate interact synergistically to increase cfos mrna levels. consistent with the pharmacology of vip and glutamate, the synergistic interaction between vii' and glutamate on c-fos mrna expression is also inhibited by mk- . interestingly, this synergism is completely inhibited by h- , a protein kinase a inhibitor, whereas staurosporin and kn- which block protein kinases c and ca++/calmodulin dependent respectively exhibit smaller inhibitory effects. presynaptic proteins involved in neurotransmitter release (i.e. synapsin, b- ) and some postsynaptie gaba a receptor subunits possess phosphorylation sites for camp-dependent protein kinase (pka) and/or protein kinase c (pkc). the functional consequences of phosphorylation by these kinases is not known. we have studied the action of specific activators of pkc and pka, phorbol ester (pdbu) and forskolin, respectively, on gabaergic synaptic transmission in area ca of hippocampal slice cultures. pdbu significantly enhanced the amplitude of evoked monosynaptic ipscs (in cnqx and ap ), and both pdbu and forskolin increased the frequency of spontaneous miniature ipscs (m[pscs) in the presence of cnqx, ap and ttx. this effect by pdbu was antagonized by staurosporin, a protein kinase inhibitor. pdbu and forskolin did not change the amplitude or decay of mipscs, suggesting that only presynaptic kinases are involved. furthermore, pdbu enhanced mlpsc frequency by a comparable amount in the presence of the ca + channel blocker cd +, indicating that pdbu did not enhance gaba release from presynaptic endings by facilitating ca + influx into axon terminals. valiunas, v., sc~ilinsky fluri, g. and weingart, r. arachidonic acid (aa) reversibly impairs the gap junction conductance (g~) in cardiac cells. now, we examined whether aaacts directly or via its catabolites. experiments were performed on pairs of neonatal rat myocytes using a dual voltage-clamp method. we used blockers which interfere with enzymes of various catabolic pathways. pretreatment with i ~mpoca (blocks carnitine acyltransferase i) did not prevent the decline in g by i ~m aa; i.e. intermediates of p-oxidation are not involved. similarly, preexposure to i ~m indomethacin (blocks the cyclooxygenase pathway) did not prevent aa from uncoupling; this rules out an involvement of prostaglandins and thromboxanes. exposure to ~mndga (blocks the - ipoxygenase pathway) per se produced a decrease in gj. likewise, exposure to i ~m etya (blocks the - ipoxygenase pathway) also impaired gj. these effects cannot result from accumulation of endogenous aa because preexposure to ~m bpb (blocks phospholipase a ) did not prevent ndga-or etya-induced uncoupling. exposure to ~m skf -a (blocks the epoxygenase pathway) also produced a decrease in gj. hence, ndga, etya and skf -a, compounds with different biochemical actions and of different chemical structure, act on g~ either directly or via an unknown mechanism. $ - potential changes associated with electrical activity in nonmyelinated nerve fibres a. robert and p. jirounek d~partement de pharmacologie, cmu, gen~ve simuheneous measurements of the extracellular concentration of k + ([k+]e), and of the concomitant changes in the membrane potential (era) were measured in the rabbit vagus nerve during and after a period of activity. during a hz stimulation there was a large increase in the [k+]e , accompagned by a change in era, which roughly followed the depolarization expected from the increase in [k+] e . at the end of the stimulation, although the [k+]e was still elevated, the nerve developed a posttetanic hyperpolarization (pth). the pth was generated by the electrogenicity of the na+-k + pump, but its amplitude and time-course were strongly affected by two depolarizing currents: (i) an inward ba +-sensitive current of k + and (ii) an outward current of ci-. we hypothesize that during activity and during the early phase of recovery there was a ba +-sensitive uptake of potassium by the schwann cells. the ci" current, by short-circuiting the na+-k § pump, contributes to the control of the e m, and by this way to the driving force for the inward k § current. overdose of ifosfamide has ooo/red in a canc~c patient ( g i.v. iy / hours with g as ~utectarfe). urir~ collece{on was obtaj/ ed in hour ~ fcr t~o days. c~ was measured ~ a gas liquid d~romatogr~ method of the u ~ ~ (s~@pl. v, pag. - g) ~sir~ a ~e~s ar~ ni~, sensitive ths~mi(~ ic d~. ~ u~s p~mt ~ ~ ~ ~ ~ ~ m~/ h and r~/ h at day one ard day res~ec~vely. ~ _h=~=mmtly ~ has also bsm foa~ in mti~s ~ mgh e~e ~ ~ (~/o~e). w~n ~ ~s am/nist~ to rats (i~ m~/~ ~c m~f~ intraperit~neally), no ~ omild be detected in urine within ht~r~ after drug administl- (cicn. from these ~ data we conclude that c~ is extensi~_ly metabolized in rats and ~ mscbard_sm of ~ el ~mlnaticn in pati~rfc t=cj_ne m~cjlr[c reflect a flmicticrsl der~,~t of c~ m~t~ olisn in man to ifo~=~de ~tion. there are numerous reports of improved memory functions in rats following a dietary supplementation with choline. in some cases, this improvement can be related to enhanced cholinergic transmission (mack et al., behav neurosci., , - , ). but it is not clear whether there is a general improvement in memory or whether specific aspects of learning and memory are affected. this work was aimed at analysing the effects of perinatal cholinergic supplementation on the development of spatial abilities and upon adult performance. choline supplementation ( . g./l. in . m saccharine solution in tap water) was maintained during two weeks before birth. additional supplementation was maintained from the th to the oth week postnatal. spatial learning capacities were studied at the ages of , or days in a cimular swimming pool (morris place navigation task) and at the age of of months on a homing board. adult and aged rats were tested on a radial maze. selective aspects of the performance were markedly improved. this treatment had no specific effect upon spatial memory per se, but, instead, affected learning abilities by promoting efficient behavioural strategies hypothetically based on active representation and anticipation processes. the effects of the treatment remained evident up to months following the supplementation. barcelona, e- bellaterra females of both lines (n=b/grp) were injected sc daily between days - of pregnancy with vehicle (v), i(di) or (d ) mg/kg diazepam, or not(c). in these studies(a,b) their - mo old offspring were subjected to a -run session in a shuttlebox(a: males, females of each line/condition) or a min session in a hexagonal tunnel maze with strategically-placed barriers and a lit central arena(b_: - males of each line/condition). a: the freezing behavior of ld rats was reduced ~s lc, this behavior reverting more to escapes in males & avoidances in females, although v had an effect in the latter also. no within-line differences in inter-trial responses were seen, but pre-session activity was increased in female lvs and ldis, returning to lc levels for ld s. no differences existed among the h groups. b: whereas h maze activity exceeded that of l, no wtthin-line differences appeared. hd s entered the lit arena less than hcs or hvs, indicating an increased timidity after pnd, and a trend in the same direction existed for ldi/ld vs lv. two groups of abstinent female smokers, performing either a fixed rate or a subject paced version of a rapid information processing task were compared. both groups participated in two test sessions (in which the task was performed twice) to compare two different types of cigarettes which were smoked before and during the second task trial: a) the subject's habitual cigarette with a nicotine yield of at least . mg/cigarette and b) a nearly nicotine free test cigarette with a tar yield comparable to that of the habitual cigarette. reaction times to correct detections (series of three odd or even digits in a pseudorandom sequence of single digits) were longer with the subject paced version and not affected by the type of cigarette but shorter with the fixed rate version and even more decreased with the smoking of the habitual cigarette. on the other hand, the pre-to posttreatment increase in the subject paced processing rate was greater with the habitual than the test cigarette. it was concluded that the two task versions assess different cognitive functions. whereas the fixed rate version primarily assesses vigilance performance and seems to be more suitable to measure changes in reaction time, the subject paced version is more sensitive to changes in speed capabilities in rapid information processing. heart rate, physical activity, and cigarette consumption were continuously recorded under field conditions, whereas subjective craving was assessed six times per day and saliva cotinine was measured once daily. abstinence, habitual cigarettes (with a nicotine yield of at least . mg/cigarette), and nearly nicotine free test cigarettes but with a tar yield comparable to that of the habitual cigarettes were compare~) in a sample of twelve female smokers for two days each. whereas physical activity was similar for the three conditions, heart rate was highest with the habitual cigarettes, lowest on abstinence days and in between with the test cigarettes. cigarette consumption was similar for both types of cigarettes and subjective craving was higher on abstinence than on smoking days but no differentiation between the two cigarette types was obtained. saliva cotinine values were highest on the days with the habitual cigarettes but lower and similar with the test cigarettes and on abstinence days. it was concluded that heart rate and saliva cotinine depended only on the amount of nicotine absorbed, whereas subjective craving was reduced by smoking independently of the actual nicotine yield of the cigarette. (ptps) is the rate limiting enzyme in the synthesis of bh, in man, a cofactor for several hydroxylases involved in catecholamine and serotonin biosynthesis. the human and rat liver cdnas encoding the kda subunit of ptps were expressed and the recombinant enzymes purified to homogeneity. the apparent k~ for the substrate, pi and heat stability of the re-combinant enzymes were similar to the native enzymes. the rat enzyme was crystallized and the x-ray structure showed a hexameric native form arranged as a sandwich of two trimers. three histidine, a glutamic acid, and -also shown by site-directed mutagenesis -a cysteine residue characterize the active center of the enzyme thought to be mg +-dependent. according to the proposed ligands we replaced mg * by fe § or mn ~+ and observed an enhanced activity up to %. m. neerman-arbez, v. cirnlli and p. halban. laboratoires/eantet. centre m dical universitaire, gen~ve . pci( ) and pc , members of the mammalian family of pro-protein convertases homologous to the yeast kex , are both expressed in pancreatic islets of langerhans and are thought to be responsible for the conversion of proinsulin to insulin and c-peptide in beta cells. however, the insulin secreting beta ceils are not the only ceils present in these complex microorgans, prompting us to evaluate the expression of pc and pc in islet beta and non-beta cells. rat islet cells were sorted by autofluorescence activated flow cytometry to separate beta cells from non-beta ceils and conversion cndoprotease levels were analysed by western blotting. in beta cells, pc levels were higher than pc (pc /pc = . + . ) whereas the opposite was found for non-beta cells (pc /fc = . • confirming that pc is important for proinsulin conversion while suggesting a role for pc in the conversion of proglucagon, prosomatostatin and propancreatic polypeptide. transformed murine cell lines (insulin-producing beta-tc and glucagonproducing alpha-tc) were found to faithfully reflect the primary rat cells in terms of their pc /pc ratios. finally, post-translational modification of the convertases themselves was found to differ between cell-types, in particular, a precursor kd form of pc accumulated in beta cells whereas only the fully processed kd form was detected in the non-beta cells. the kringle ( + ) domains (k + ) were successfully expressed in e. coil a n-terminal hexahistidine tag was fused to insure the isolation of k + by affinity chromatography on a ni- +-ntaagarose-column. to be able to remove the hexahistidine tag a factor xa cleavage site was introduced between the histidine residues and the n-terminus of the kringle structures. the correct arrangement of the disulfid bonds was determined by amino acid-and sequence analysis. the lysine binding site was proven by affinity chromatography on iysine-bio-gel, as previously shown for k . the dissociation constant of k + was measured by intrinsic fluorescence titration with -aminohexanoic acid. a replication protein a copurifying dna helicsse from calf thymus anthl georgakl, narendra tuteja, blrglt sturzenegger and ulrich hobscher department of veterinary biochemistry university of z rich-lrchel winterthurerstrasse f ch- z rich, switzerland a dna helicase from calf thymus copurified with replication protein a through several steps of purification including deae-sephacel, hydroxyapatite and single stranded dna cellulose. it is finally separated from replication protein a on fplc mono q where the dna helicase elutes after replication protein a. we named this new calf thymus enzyme dna helicase f. characterization of the helicase f by affinity labeling with [a p]atp indicated that the enzyme has a catalytic subunit of kda. gel filtration experiments suggested that dna helicase f can exist both in a monomeric and an oligomedc form. the enzyme unwinds in the ' -> ' direction in relation to the dna strand it binds. all eight deoxyribonucleoside-and ribonucleosidetriphosphates could serve as an energy source. testing a variety of dnndna substrates indicated that the dna heitcase f preferentially unwinds very short substrates and is slightly stimulated by a single stranded '-tail replication protein a allowed the dna helicase f to unwind longer dna substrates up to bases suggesting that copurification of replication protein a with the dna helicase might be of functional relevance. , , , ', ', '-hexachlorobiphenyl ( -cb) shows limited elimination and extensive storage in adipose tissue in vivo (lipophilie unmetabolizable compound) while chlorpromazine (cpz) is known for its lysosomal storage. uptake and release of -cb and cpz were studied in t -preadipocytes (p) and t -adipocytes (a). t -cells were cultivated in dulbecco's minimal essential medium supplement with % fetal calf serum and grown to eonfluency. differentiation was stimulated by dexamethason and bezafibrate exposure for h. radiolabelled -cb ( ~tm) or cpz ( ~tm) was added to the medium. following a single dose - % of cpz were taken up within an hour into p and a. in contrast, -cb reached an intracellular plateau of - % of the dose after lh in p whereas dependent on the triglyceride content a accumulated up to % of the dose. -cb uptake into a was temperature dependent and not saturable with repetitive daily doses up to week. -cb uptake into p was fully reversible while the release from a was limited to % of the accumulated drug. in contrast, the release of cpz was higher from p than from a as a consequence of the different lysosomal activities of the respective cells. the use of t cells allowed to show a remarkable difference in cellular handling of neutral and basic lipophilic drugs and may be helpful to further elucidate basic mechanisms involved. chronic granulomatous disease: an attempt to reconstitute the function of the x-linked form of the disease. chronic granulomatous disease (cgd) is a group of congenital immunodeficiencies that predispose patients to recurrent severe bacterial and fungal infections. the cause of the disease is lack or impairment of -production in phagocytic cells due to mutations in any of the four components of the superoxide producing system. approximately % of all cgd patients suffer from an x-linked form of the disease where the phagocyte oxidase gp phox is affected. this project concentrates on the reconstitution of the deficient gene in phagocytic patient cells. expression of recombinant gp was assayed in vitro and in vivo. attempts to "tag" gp with a foreign epitope were unfortunately so far unsuccessful. the target cells for gene reconstitution are non-dividing monocytes. thus, we have chosen an adenovirus over other viral systems because of: a) its ability to infect nondividing cells, b) its genetic stability and, c) lack of human adenovirus related malignancies or side-effects. we constructed two recombinant adenoviruses: one recombinant expresses a lacz gene (control), another the gp lphox gene. we are trying the expression of recombinants adeno/lacz and adeno/gp in normal and patient-derived cells (for example ebvtransformed normal and patient b-cell lines), as well as the reconstitution of nadph oxidase function in peripheral blood monocytes of cgd patients. kinetics of molecular chaperone action schmid, d., gehring, h., *baici, a. and christen, p., biochemisches institut der universit&t z~rich, ch- zorich; *rheumaklinik, universit&tsspital, ch- z%rich molecular chaperones of the hsp type transiently sequester unfolded segments of proteins and promote their correct folding. target peptides labelled with an environmentally sensitive fluorophore allowed to follow their binding to the molecular chaperone dnak of escherichia coli in real-time. the two-step process is characterized by relaxation times z~ = s and z = s at ~m dnak and .i ~m ligand at ~ in the presence of atp, the formation of the complex is greatly accelerated and follows a single-exponential process with z : . s. the rate of dissociation of the complex was even more increased than that of association resulting in a decreased net affinity for ligands in the presence of atp. the binding-release cycle of dnak thus occurs in the time range of polypeptide chain elongation and folding and is too fast to be stoichiometrically coupled to the atpase activity of the chaperone (ko~ ~ = . min i at "c). supported by the olga mayenfisch stiftung, z%rich, and the hartmann m~ller-stiftung, z~rich. comparison of the activities of native bovine seminal ribonuclease and that secreted from e. coil schein, ch* (siat, technopark, pfingstweidstr. , ch ziirich) and haugg, m (lab. org. chemistry, e.tm., . seminal ribonuclease (bs-rnase) differs from the pancreatic rnase a in that it forms a cysteine-linked dimer and has a relatively higher specific activity in the cleavage of double-stranded (ds-) rna substrates. we expressed bs-rn in a secretion system similar to that described previously (biochem. j. : - , ) using the signal sequence of routine pancreatic ribonuelease. about i mg bs-rn was isolated per liter culture supernatant. human and bovine recombinant interfcron-~ (ifn-r) inhibit the degradation of ss-and ds-rna by bovine pancreatic rnase while they activate the activity of bs-rn on the same substrates (febs letters, : - , ) . recombinant bovine or routine pancreatic rnase (produced in our secretion system) are inhibited by ifn-y, while the recombinant bs-rn or a cysteine-linked dimer of rnase a is also activated by ifn-y. ifn-y activates bs-rn even in the presence of inhibitory concentrations ( . - ram) of mononueleotides. thus the mode of activation cannot be attributed to alleviation of product inhibition. kinetic studies using bp ds-rna as substrate suggest that ifn-~ interacts directly with the bs-rn to increase the rate of highmolecular weight producffsubstrate release, as the apparent ks increases proportionately to the increase in kcat. significantly smaller movements were observed in the simulation with the liganded enzyme. the structural differences between the protein in the crystal and the ensuing calculated structures might arise from the absence of lattice forces in solution. evolutionary relationships among pyridoxal- '-p-dependent amino acid decarboxylases sandmeier, e., hale, t.i. and christen, p. biochemisches institut der universit~t zgrich, z~rich. the pyridoxal- '-p (plp)-dependent amino acid decarboxylases (de) can be subdivided into four apparently unrelated groups. group i is represented by glycine dc, group ii comprises glutamate, histidine, tyrosine and aromatic-l-amino acid dc, group iii procaryotic ornithine and lysine dc as well as the procaryotic biodegradative type of arginine dc, group iv eucaryotic ornithine and arginine dc as well as the procaryotic biosynthetic type of arginine dc and diaminopimelate dc. (n-l) profile analysis, a more stringent application of profile analysis [gribskov et al. ( ) methods enzymol. , - ] established the homology among the enzymes of each group. a search with the profile of group ii indicated a distant relationship with aminotransferases and thus with the ~ family of plp-dependent enzymes. no evidence was obtained that groups i, iii and iv are related with other plp-dependent enzymes or any other protein in the database. apparently, the amino acid dc are of multiple evolutionary origin, in some cases even if they have the same substrate specificity. a. lo russo, a.c. passaquin, u.t. r~eg~, ecole de pharmacie, %hiv. lausanne, c~- lallsaraqe drug-induced local vasoccmstricticn appears to be respmnsible for the hypertensive side effect of the imn/nosti~pressant cyclosporin a (csa). we have therefore ex~rnir~ the [ca +] i increase caused by the vasoccr~trictor hormcme [ar~]vascpressin (avp) in vascular ameoth ~uscle cells (vsmc) treated with csa. [ca +] i levels were m~nitored either with a fluoresc~qce analyser using fura or by ca + efflux. pretreatment of v~mc with csa increased the avp induced rise in [ca +]i. a leftward and upwsxd shift of the ccncentraticm-respmnse curve of the avp-induced rise in ca + efflux was also observed after csa treatxr~%t. ilzg/cticn of csa dote~tiaticn w-as detectable after rain, took to h to become fully established and was reversed after washout. csa potentiaticm was cc~centraticn-deperdent with a threshold at - m. %]]is potentiating effect of csa may be the underlyin~ cause for csa-ind/ced hypert~ sicn. ) barja, f. and turian, g. i, ilab. gen. microbiology, dept. biochemistry, station exp. zoology, university of geneva, quai ernest-ansermet, geneva the actin has been found from the simplest forms of cell to the highly evolved ones. in higher organisms it is transcribed by multigene families but in yeast and several filamentous fungi there appears to be only one actin gene. it was therefore of interest to study the expression of actin gene in n. crassa in which three actin isoforms have been characterized (barja et al., , fems left. : - ) . after isolating genemic dna from wild type n. crassa a pcr was performed with primers corresponding to actin consensus sequences and an amplified fragment of the gene (verified by sequencing) was obtained. this fragment was 'used as a probe in a southern to assess the number of aetin gene(s). our results suggest that n. crassa has alsc only one actin gene. the blocking effect of the n-terminal decapeptide of msmooth muscle actin (sma) (ac.eeedstalvc) on the monoclonal antibody anti-asm- was compared with that of synthetic peptides modified by changing the n-terminal acetyl group or by substituting a single amino acid in position to . using immunofluorescence or immunoblotting techniques, anti-c~sm- activity was abolished after incubation with the native peptide and peptides modified in position (only when e~a) or . on immunoblots running bsa crosslinked peptides on denatured gels, only the natural peptide and peptides with substitution in position or were detected by the antibody. our results indicate that ac.(e)eed is the epifope for anti-c~sm-l. binding of anti-~sm- to sma increased actin polymedzation by decreasing the critical concentration. as control this action was not exerted on skeletal actin. this is the first example of the role of a precise n-terminal sequence in the polymerization of a single actin isoform. double immunofluorescence for msma and total actin on cultured aortic sm cells microinjected with the native peptide showed a selective disappearance of msma staining suggesting that this peptide traps a protein involved in msma polymerization. work is in progress to search for the putative actin-binding protein(s) physiologically interacting with the n-terminal sequence of o~-sma. ( within mitochondria, the mechanism of selective protein degradation is poorly understood. while the bulk of mitochondrial proteins are long-lived, some are degraded rapidly. the selective degradation of mitochondrial proteins is likely to be mediated by proteases similar to those found in bacteria; this is based on the hypothesis that mitochondria have evolved from bacterial endosymbionts, in conjunction with the finding that mammalian mitochondria contain an atp-dependent proteolytic activity similar to that in bacteria. one atp-dependent protease from bacteria, lon, is of particular interest because it is involved in regulated protein turnover. degenerate oligonucleotide primers corresponding to two regions of the bacterial lon gene were used to pcr amplify a kb fragment from yeast dna that encoded an open reading frame with striking similarity to the bacterial lon protease. by screening a yeast cdna library, we isolated a . kb clone potentially encoding a protein of amino acids. haploids bearing a disruptedlon gene were unable to respire or to grow on ethanol/glycerol. fractionation of rnjtochondrial matrix from wild-type cells revealed a peak of aip-dependent proteolytic activity which was absent in the disruptant. furthermore, we have identified matrix proteins that are degraded with a half-time of ~ min in intact wild-type ceils but are stable in the ion disruptants. electron microscopy of lon disruptants revealed the presence of many electron dense bodies in the mitochondrial matrix which are not found in lon + cells. hydrolysis of high molekular weight kin nogen by plasma kallikrein benner, s. and duffer, h., laboratory for organic chemistry, eth h nggerberg, ch- z/inch high molecular weight kininogen (hmwk) is cleaved by the serineprotease plasma kailikrein to generate the hypotensive peptide bradykinin in human blood. we established a measuring system for the time course of the hydrolysis of hmwk in its native and deglycosylated form and in the presence or absence of c~-inhibitor. analysis of the bradykinin release and the yielded protein fragments lead to the three following results: . the hydrolysis did not follow plane michaelis-menten kinetics due to a hmwk-fragment operating as a competitive inhibitor. . addition of the ci-inhibitor stopped the hmwk-hydrolysis immediately, which is in contrast to the physiological role of plasma kallikrein in blood. . so far we have no evidence that the high content of o-glycosylated side-chains of the hmwk-light chain has an effect on the kinin-liberation as proposed in literature. ( tetrahydrobiopterin (bh ) is the essential cofactor for the hepatic phenylalaoine hydroxylase, but also for tyrosine and tryptophan hydroxylases that are responsible for the production of nettrofransmitter precttrsors. furthermore, bh is required for the deavage of giyceryl ethers aztd is i~vo[ved in the biosynthesis of nitric oxide by the nitric oxide synthase. a vazia~t type of hyperphenylalani~emia is caused by a deficiency of bh . the most frequent form of this cofactor deficiency is due to lack of -pyruvoyl-tetrahydropterin synthase (ptps) activity. the human cdna for ptps was isolated, and the recombinant protein was found to be active when expressed in e. coil, thus allowing us to perform hmctional t e, ting of mutant proteins. primary skin fibroblasts derived from ~everal ptps deficient patieilts were investigated for the molecular nature of this autosomal recessive disorder. all fibroblasts had f "ps mrna expressed, and subsequent cdna sequence analysis revealed different mutatkons in the coding region of ptps (codon replacemenls, deletions, or nonseme codons) responsible for a lowered or no enzyme activity. in order to potentially correct ptps activity (and restore bi-i biosynthesis) we are establishing a recombhtant rel~oviral-mediated gene transfer system to efficiently introduce the wild-type human ptps-cdna in these fibroblasts. the thylakoid membrane (tm) contains i molecular species of phosphatidylglycerol (pg). the relative amount of these species depends on the plant species and growth conditions. using phospholipase a (pla ) at ~ to digest preferentially pg in the outer monolayer of spinach tm, we have shown previously that this monolayer was enriched in pg (ca ~). the purpose of this investigation was to determine the transmembrane distribution of each pg molecular species. to this aim, we have studied the hydrolysis kinetics of each species in the presence of pla in spinach and squash tm containing different relative proportion of molecular species. the hydrolysis rate and extent of the molecular species depended on the unsaturation degree of the fatty acid at the sn-i position as well as on the presence or absence of : ( t) at the sn- position. for instance, the hydrolysis rate of pg ig: / :l( t) was greater than that of pg : / : . these results will be discussed in terms of the thylakoid transmembrane distribution of each pg molecular species. (supported by the snsf - . ). from previous electrophysiological evidence, we concluded that metal ions (hgz+,cd +,ag +) at low concentrations (i - m) increase rapidly ( - s) cation (na + , k + ) conductance at the apical membrane of epithelial cells. we investigated here the effects of these metals on [ca +]i in mdck-i cultured cells, for a potential correlation between functional membrane changes and [ca +]i . metals ( - - - m) were added at the apical side of the epithelium, and [ca +]i was measured with fura- . hg + induced a rapid (peak - s) and marked increase in [ca +]i, whereas cd + entailed a slower (peak - min) and marked response. the time-course of effects for both metals was similar to the electrophysiological changes. in contrast, the pattern of effects of ag + on [ca +]i differed markedly from that on apical membrane conductance. thus, the effects of metals on [ca +]i are conspicuous but not tightly associated with changes in membrane conductance. c. bulliard and l-l. dreyer, institut de biochimie, universit de fribourg, ch- fribourg trans-plasma membrane oxydoreductases (pmo) play a significant role in energy transduction and post-receptor signal transmission, but the enzymes have not been characterized so far. we have achieved the purification of pmo from rat brain synaptic vesicles and from synaptic plasma membranes. the membrane enzymes were extracted by % lubrol xl tm, precipitated with ammonium sulfate (between and % saturation) and purified by means of hydrophobic chromatography on butyl-agarose and gel f'fltration on superose- , followed by page under native conditions. specific enzymatic staining of native page gels yields two homogenous diaphoraselike activities, pmo-i and pmo-ii . sds-page of the enzymes showed that pmo-i is made of two subunits of kda and kda whereas pmo-ii consists of a single sub-unit of kda. the ki)a subunits from pmo-i and pmo-[i are probably identical from their respective properties. partial n-terminal sequencing ( aa) of the kda subunit from pmo-i showed % homology with rat brain fructose-l, -bisphophate aldolase. these data indicate that pmo is closely associated with the glycolytic enzyme that co-purifies in all steps under suitable conditions. the biochemical functions of pmo's remain to be established. ferredoxin:thioredoxin reductase (ftr) is the key enzyme in the ferredoxin/thioredoxin system, the light-dependent regulatory system in oxygenic photosynthesis. it is a nucleus encoded ironsulfur protein, composed of two dissimilar subunits, one of which is the catalytic subunit containing a redox-active disulfide bridge functional in the reduction of thioredoxins and a fe- s cluster. using pcr we have isolated and characterized a cdna clone of bp from spinach and a clone of bp from corn coding for the catalytic subunits including the transit peptides. the first (spinach) resp. (corn) amino acids after the start exhibit typical features of chloroplast transit peptides. following the transit peptides are (spinach) or (corn) ami[~o acids representing the catalytic subunits. the primary structures are highly conserved between these two species with % similarity and % homology. seven of the eight cys residues found in the spinach subunit and important for the catalytic activity are at conserved positions. (snf - . and - . ) $ - r. zurbriggen and j.-l. dreyer, institut de biochimie. univarsit de fribourg, ch- fribourg most mammalian cells display transplasma membrane oxidoreductase activity (pmo). the enzymes use intracellniar reduced pyridine nucleoticle to reduce an unspecific extracellular electron accepter as substrate. in order to set up a methodology for purifying, characterizing and quantifying pmo's, the plasma membrane from a neuroblastoma cell line, nb a , has been biotinylated. subsequently the biotinylated proteins were purified by immanoprecipitation with avidin, antiavidin-antibodies and insoluble protein a. the protein recovery of an immunopurified membrane preparation was < . % of the protein content in the cell extract. specific pmo activity was increased to fold compared to the activity in whole cells. this approach demonstrates the presence o ' pmo within the cell plasma membrane. this activity accounts for about one third of the total cellular diaphorase activity and cannot be attributed to an increased perraeabilization of the plasma membrane induced upon biotinylation nor to intracelluiar activity from lysed cells. pmo also promotes cell growth at low substrate concentrations ( . -ll.tm). native gel electrophoresis of iarinobiotinylated and affinity purified plasma membrane extracts displays two diaphorase-positive bands, indicating that a homogeneous cell population may express several pmo activities at the plasma membrane. fluvastatin (fs) is a new hmg-coa reductase inhibitor. its affinity for major human drug metabolizing p monooxygenases (cyp c , cyp d and cyp a ) was determined in liver microsomes. fs showed low affinity (ki > ~tm) for cyp d and cyp a whereas cyp c was selectively and competitively inhibited (ki = . ~tm). the potential for in vivo fs interactions with cyp c substrates was therefore investigated in volunteers using dicinfenac (df) as a probe ( mg orally) on days , and . df and '-ho-df kinetics were determined by hplc/uv. df cmax increased over time ( . [sd . ], . [ . ] and . [ . ] mg/l on day , and , resp.). oral clearance was reduced on days and ( % and %, resp.). a time dependent decrease in urinary metabolic ratio ( '-ho-df/df) was noted ( . [ . ], . [ , ] and . [ . ] on day , and , resp. (p < . )) only over the first hours. fluvastatin therefore exhibits a two-phase interaction in vivo: an early transient and a late cumulative inhibition. the transient phase matches inhibition profiles predicted by quantitative models integrating in rive pharmacokinetics and in vitro inhibition data (q-dips). simple competitive inhibition by fs cannot explain the late phase. other mechanisms (i.e. fs or metabolite cumulation, mi complexation) are hypothesized. in some situations fs is expected to cause interactions with cyp c substrates, which are partially predicted by quantitative modeling of in vitro data (snsf project n o - . ). gen~ve, switzerland. previously, a parvalbumin mutant, pvf~ w, was constructed with a unique reporter trp in the middle of the hydrophobie center. in the present study three new parvalbumin mutants, derived from pvft~w and containing alterations essential for ca +-binding in either one, pv.c• and pv_m, or both, pv-co~, of the two ca +-binding sites, were analyzed in order to study the metal binding properties of individual ca~+-binding domains and their contributions to the structure and stability of the protein. both, pv_ct, and pv.~, bind i ca ~ § with affinity constants, kc,, of . , and . - m "~ in the absence of mg +. mg + shifts the ca~+-binding isotherms of pv.co to higher free [ca z+] according to the competition equation with km~ = m t. pv.m~ is much less effected by mg z* (kmg = m "t) and can be considered as possessing a ca +-specific site. nevertheless, in the absence of ca § both, pv.co and pv~, bind i mole of mg + with much higher affinity than predicted from the competition studies. pv_cd;~ binds neither ca . nor mg +. trp-fluorimetry and uv-difference spectroscopy revealed that the ca~*-loaded conformations of pv_co, pv.ef and pvr~o~w are similar, with the trp- deeply buried in the hydrophobie core. however, striking differences between the mutants are found in the metal-free and mg~+-loaded forms. the conformation of pv~t~;~ is not affected by ca ~ or mg z*. our results indicate that destroying the ca +-binding of the ef loop, but not of the cd loop, strongly destabilizes the protein in the absence of ca z § . biochemlsches institut der unlversitfit zfirich, switzerland, ch- . the sea urchin, strongylocentrotus purpuratus, metallothionein gene, mta, was constructed and expressed under the control of a heat shock promoter in a protease-deficient strain of e, coll. the nascent recombinant protein was stabilised by the addition of exogenous cd. the cd-containing protein was precipitated with ethanol and subsequently purified to homogeneity by gel permeation and ionexchange chromatography. the purified protein was identified as the desired gene product by tryptic peptide map analysis, sequence analysis and mass spectroscopy. typical yields of protein were mg per litre of culture. the presence of cd ions per molecule was confirmed by the sh/cd ratio and by the existence of h cd nmr resonances. the apparent association constants of sea urchin mt with cd, which has a charge of - , were found to be approximately times weaker than for rabbit mt, at ph . (mt) are small metalloproteins displaying as their main feature clusters of d i~ metal ions bound to the thiolate ligands of the abundantly occurring cysteine residues (cys). from a set of over sequences a general multialignment of class i mt has been obtained on the basis of the needleman & wunsch algorithm. all cys are conserved in the man%mals and over % of them in other vertebrates and invertebrates. on the basis of similarity/identity scores we can isolate groups of sequences. evolutionary relationships between species and groups have been calculated with the maximum parsimony method of felsenstein and with the distance matrix method of fitch and margoliash. the divergences observed among the mammalian mts indicate a partition into four distinct subforms which all may have arisen before the separation of the species in evolution and which in part may differ in function. $ - bourque, a., singh, a., dykeman, a., macmahon*, a., and foster*, w. atlantic veterinary college, charlottetown, canada, and *reproductive toxicology section, environmental health centre, tunney's pasture, ottawa, canada. hexachlorobenzene (hcb) is a known environmental pollutant that is produced as an industrial by-product of certain chemicals. when administered in high dosage, hcb induces alterations in the rhesus monkey ovary. a purpose of the study was to determine a no observable adverse effect level (noael) for the compound. twenty, cynomolgus monkeys, - years old were placed in five groups. hcb was given orally in concentrations of . , . , . , and mg/kg b.w. in gelatin capsules, daily, for weeks. one group of animals fed glucose only, served as the control. ovary specimens fixed in % buffered glutaraldehyde were prepared by conventional methods for transmission electron microscopy. ultrastructural alterations were revealed in the developing ova and follicular cells in all the ovaries from hcb-treated animals in a dose-related manner. clinical chemistry was unaffected by hcb. a noael for this reproductive toxicant is yet to be determined. a.b. was a recipient of the nserc undergraduate student award. an acidic haem-and zinc-containing protein has been isolated from bovine brain. native and sds-polyacrylamide-gel-electrophoresis reveal a monomeric protein with an apparent molecular weight of , kda. the absence of co-staining with tetramethylbenzidine implies that the haem group is non-covalently bound. the electronic absorption spectrum, with a soret-band absorption maximum at nm and c~-and i~-bands at and nm, respectively, is similar to that of fe(ll)-myoglobin, consistent with the presence of haemiron in the ferrous oxidation state. in air the protein was easily oxidized to the fe(lll) form with a subsequent shift of the soretband maximum to nm. atomic absorption measurements indicate approximately mole of zinc and mole of iron per mole of protein. the amino acid composition is similar to that of indoleamine- , -dioxygenase, although no such enzymic activity has been detected. in this study, we looked for this substance and its metabolites in young and old bovine lenses, because of their possible role in cataract formation. the substances were detected by hplc analysis. the kynurenine aminotransferase (kat) activity was determinated by the method of tobes (methods enzymol ~ : ) . the fluorescent substance formed from -hk was characterized by thin layer chromatography followed by reaction with rtinhydrin, uv and fluorescence spectrometry, and atom bombardment for molecular mass determination. -hk at concentrations of . -+ . , . _+ . , and _+ . btg/g of tissue was detected in iris/ciliary body, retina, and calf lens respectively. this substance is deaminated by kat. the activity of this enzyme was . _+ . , . _+ . , and . +_ ~tmol/g of tissue/hour in retina, iris/ciliary body, and lens respectively of old bovine eyes, but it was not found in calf eyes. the deamination of -hk resulted in the formation of a fluorescent substance which was identified as oxo-xanthurenic acid (oxa) with a molecular mass of daltons. the accumulation of oxa acid possibly interacting with lens proteins could induce cataract formation. snf - . , emdo, and hartmann-mtiller. the goal of covalent immobilization of oligonucleotide capture probes to solid supports has been accomplished by light-dependent, photolinker polymer mediated immobilization strategies. synthetic oligonucleotides were immobilized by facile layer coating procedures. the procedure does not require functionalization of the oligonucleotide probe or the matedal surface. oligonucleotides were linked to polystyrene with a biopolymer which was multiply substituted by photoactivatable diazirines. capture probe photoimmobilization ( nm irradiation) was strictly light and diazirine dependent. using radiolabeled oligonucleotides as capture probes and polystyrene as material surface, the light dependent coupling efficiency was %. nonspecific oligonucleotide binding was below %. photojmmobilized oligonucleotides retained the ability to form stable complexes with complementary dna strands, and target oligonueleotides of varying length were captured as well as dna fragments produced by pcr techniques. indoleamine , -dioxygenase (ido), a superoxide radical scavenger, is present in transparent human lenses and produces -hydroxykynurenine, a uv-b filter. the synthesis of -hydroxykynurenine by ido and its degradation by kynurenine aminotransferase (kat) were measured in transparent lenses and cataracts. post-mortem eyes of elderly persons between and years of age with transparent lenses were obtained from the eye-bank of the department. fresh cataracts were obtained from cataract surgery patients. ido activity was measured by the method of yamazaki et al (biochem j ; : ) adapted for hplc. kat activity was measured by the method of tobes (methods enzymol ; : ) . ido activity found in post-mortem transparent lenses (cortex and nucleus) was . _+ . nmol/mg protein/hour. in cataracts a low do activity of . _+ . nmol/mg protein/hour was found in the cortex but not in the nucleus. kat activity, found in the eight cataracts examined, was . _+ . nmol/mg protein/hour. inhibition of ido activity and induction of kat activity seems to be involved in human cataract formation snf - . , emdo, and hartmarm-m er photolinker polymer mediated covalent immobilization of antibodies and f(ab') fragments has been achieved by newly established lightdependent coupling procedures. anti alpha-l-fetoprotein monoclonal antibodies and f(ab') fragments derived from anti prostate specific antigen monoclonal antibodies were covalently linked to microplates. diazirine derivatized bovine serum albumin served as multifunctional light activatable linking agent. both immunoreagents, monoclonal antibodies and f(ab') fragments remained immunologically active after nm irradiation (irradiance . mw cm - for minutes). covalency of antibody binding was inferred from i) the photoreagent dependence, ii) the light dependence of the immobilization process, and iii) the reversibility of immunocomplexation after acid treatment. $ - two ~ of extracelluiar elecirical resistance in v~kwricular myocardil~ fleischhauer j.c., kl~ber a.g., dept. of physiol. bern in myocardial tissue, the electrical resistive properties of the extracellular space are important for local current flow during e~citation and therefore influence impulse conduction and the magnitude of the extracellular electrical field. to assess the r~tlti~t nature of the resistance of the extracellular space, electrical cable analysis was applied on an arterially perf~-sed rabbit papillary mascle. ~he resistivity of the intravascular space was changed ( to ~i~n) by variation of hematocrit frcrn to % in the perfusate. in order to change the voltm~ and hence the electrical resistance of the interstitial space, colloidosmotic pressure in the perfusate was varied by changing dextran {m r ) concentration (i to g/l). altering the interstitial space volume had a marked influence on the extracellular resistance (ro) , conduction velocity and the magnitude of the extracellular field. variations of the resistive properties of the intravascular space had no significant influence on ro, conduction velocity and the magnitude of the extraeellular electrical field. our results suggest that the microvascular tree is insulated from the interstitial space and local electrical current flow during excitation in heart muscle is confined to the narrow interstitial clefts. s - , s - s - , s - s - , s - bchini hooft van huijsduijnen s - s - s - s - s - , s - s - s - , $ - s - nook, ek s - , s - s - , s - s - , s - , s - s - , s - , $ - s - , $ - s - s - s - s - , s - , s - s - lrmler s - , s - s - , s - , s - s - lipp, p. s - so - s - , s - , $ - , $ - , $ - , $ - $ - ,$ - s - s - , s - s - , s - molar s - s - , s - e s - , s - s - , s - , s - e. s - s - , s - , s - s - s - s - s - , s - , s - , s - , $ - s - , s - s - , s - s - , s - stalder h. s - s - s - , s - , s - s - teheesen s - , s - , s - s - van dillewyn s - , s - , s - s - , s - s - , s - huber d., ojha m. and turian g. laboratory of general microbiology, university of geneva, sciences iii, quai ernest-ansermet, geneva , switzerland. ca +-dependent protease in allomyces is predominantly localized in the apical region of the exponentially growing hyphae (huber and ojha, submitted) . many cytoskeletal proteins like actin and tubulin are also mainly localized in this growing region (heath, l~ ). it is known that the ca +-dependent protease proteolyzes a number of cytoskele~ ton proteins in order to regulate the plasticity of the cell (mellgren, ) . we have studied the proteolysis of some cytoskeletal proteins by this protease purified from the aquatic fungus allomyces erbuscula. actin, tubulin and desmin, were found to be rapidly proteolyzed in vitro at a high substrate to enzyme ratio. immunofluorescence studies of proteolyais made in situ in exponentially growing hyphae showed modification of apieally localized cytoskeletal proteins. this colocalization of the ca +-dependent protease and cytoskeletal proteins in the same apical region is inferred in relation to hyphal elongation. jbiological databases grow exponentially. in order to provide access, as much data las needed and as little volume as possible shall be returned upon a query. the icurrentiy available dna and protein sequence databases are updated in a sophisticated network of procedures and expose a considerable amount of redundancy. research is performed on optimizing the creation of non-redundant data sets. the resulting data are disttibuted in switzerland, or made accessible to researchers who cannot utilize local resources. transparent access to the swiss resources, as well as paneuropean services, is provided with a job scheduling system which was developed in basel, the hierarchical access system for sequence libraries in europe ("hassle"), and various other computer programs which allow comprehensive browsing such as "gopher" and "www". training courses to use the resource effectively are running throughout the year. key: cord- -yjvw ee authors: shikata, n.; maki, y.; noguchi, y.; mori, m.; hanai, t.; takahashi, m.; okamoto, m. title: multi-layered network structure of amino acid (aa) metabolism characterized by each essential aa-deficient condition date: - - journal: amino acids doi: . /s - - - sha: doc_id: cord_uid: yjvw ee the concentrations of free amino acids in plasma change coordinately and their profiles show distinctive features in various physiological conditions; however, their behavior can not always be explained by the conventional flow-based metabolic pathway network. in this study, we have revealed the interrelatedness of the plasma amino acids and inferred their network structure with threshold-test analysis and multilevel-digraph analysis methods using the plasma samples of rats which are fed diet deficient in single essential amino acid. in the inferred network, we could draw some interesting interrelations between plasma amino acids as follows: ) lysine is located at the top control level and has effects on almost all of the other plasma amino acids. ) threonine plays a role in a hub in the network, which has direct links to the most number of other amino acids. ) threonine and methionine are interrelated to each other and form a loop structure. the recent advances in experimental technology made it possible to analyze various kinds of metabolites in biological samples comprehensively. the amounts of the metabolites in biological fluids and tissues change temporally in coordination with physiological conditions in complex metabolic and signaling pathways. multivariate analysis and pattern recognition studies have revealed that these metabolite profiles contain the phenotypic information which can be used as a signature for a physiological condition (nicholson et al., ) . amino acids are a group of metabolites which are important as substrates for protein synthesis as well as signaling molecules (felig, ) . the plasma amino acid concentrations, like other metabolites, have distinctive features for various physiological conditions. some diseases such as liver failure (holm et al., ) , renal failure (hong et al., ) , cancer (watanabe et al., ) , diabetes (watanabe et al., ) , muscle dysfunction (jimenez jimenez et al., ) and aminoacidemia (tudor et al., ) have been reported to have specific abnormalities in plasma amino acid profiles. there are some studies which make use of plasma amino acid profiles to diagnosis and distinguish abnormal subjects from healthy subjects, or subtypes and stages of diseases (noguchi et al., ) . one of the traditional examples of using plasma amino acid profiles for diagnostic markers is the fisher's ratio, which is a ratio of branched-chain amino acids to aromatic amino acids and is used for the marker of liver fibrosis (ferenci and wewalka, ; soeters and fischer, ) . these studies clearly show that plasma amino acid profile itself can be a useful tool for monitoring the physiological state of an organism. although these previous studies discuss how to distinguish different physiological states using the plasma amino acid profile data, the control mechanism behind the change in the profile is not thoroughly discussed. further investigations on the control mechanism of plasma amino acids should uncover the trigger reasons for diseases or propose a new treatment to improve the physiological conditions, however, the control mechanism is so complicated that the investigations have not been succeeded. the reasons for this complexity should come from the interrelatedness of the amino acids and a number of internal and external factors affecting their concentrations. amino acids are directly and indirectly related to each other within a large metabolic pathway and their concentrations in the plasma are equivalent to the whole sum of the metabolic flow in each organs and tissues. the metabolic flow in organs and tissues are affected by various factors such as nutrition, diseases, exercise, and body composition, and so does the plasma amino acid profile. also, there are still unknown metabolic pathways, which are being discovered not only experimentally but also with using automated methods (chou et al., ) . from these reasons, the changes in the concentrations of plasma amino acids can not be simply explained by the topological network structure of metabolic pathway map. in the previous study, we have introduced the correlation based network analysis of plasma and tissue amino acids (noguchi et al., ) and demonstrated the similarity of the behavior of two amino acids in plasma and tissues. in this study, we carried out the network analysis one step further and introduced the directional relationship between plasma amino acids. for this purpose, we used plasma amino acid profile data of rats fed on a single amino acid-deficient (minus-one) diet. in this experimental model, the plasma concentration of deficient amino acid decreases drastically, which also affects the concentration of other amino acids. this resembles the changes observed in the gene expression profile of single gene knock-out models (yeang et al., ) . taking advantage of this resemblance, the methods used for inferring gene expression network structure are adopted in this study for inferring the interrelated and directional network structure of plasma amino acids without considering any prior topological information on metabolic pathways. this data driven analysis should lead us to the further understanding of the dynamics of the plasma amino acid profiles and gives us some clues for the factors affecting the interrelated network under various physiological conditions. adult male sprague-dawley rats were maintained on a : -h light-dark cycle, with water provided freely. starting from weeks of age, the rats were randomly assigned and switched to control or one of the experimental diets (n ¼ each). the composition of the diet is based on ain g standard diet, slightly modified by replacing dextrin and sucrose by corn starch, and casein by amino acid mixture shown in table . each experimental diet (minus-one diet) is completely depleted of single essential amino acids; histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine. the rats fed branched-chain amino acid (bcaa) minus-one diets were kept for weeks, and those fed other essential amino acid (eaa) minus-one diets were kept for weeks under ad libitum feeding condition. rats were sacrificed h after feeding and blood samples were collected from postcaval vein and mixed with edta immediately to prevent coagulation. the plasma separated from the blood samples are mixed with volumes of % (w=w) trichloroacetic acid, and centrifuged immediately at c,  g for minutes. the supernatant was filtrated with ultrafree-mc filter (cat. no. ufc lgc , millipore), and the amino acid concentrations were measured by an automatic amino acid analyzer (l- ; hitachi, tokyo, japan). in this study, we are to use threshold-test analysis and the multi-level digraph analysis methods to infer amino acid networks. using graphic approach or diagraph method to study biology-related can make the problem more intuitive, facilitating illustration and stimulating imagination so as to help reveal the essence of the problem concerned. graphic approach has been successfully used to study enzyme-catalyzed system (chou, (chou, , chou and liu, ; kuzmic et al., ; lin and neet, ) , protein folding kinetics (chou, ) , hiv reverse transcriptase inhibition mechanisms (althaus et al., a, b, c; chou et al., ) , and analysis of base frequencies in the anti-sense strands of human protein coding sequences (zhang and chou, ) . recently, the images of cellular automata were used to investigate hbv virus gene missense mutation (xiao et al., a) , hbv viral infections (xiao et al., b) , represent biological sequences (xiao et al., b) , predict protein subcellular location (xiao et al., a) , and analyze the fingerprint of sars coronavirus (wang et al., ) . our strategy for the inference of interrelated amino acid networks can be summarized as follow: ( ) given data of fold-change in concentration of deficiency or over-consumption in one essential amino acid under the stationary state, the threshold-test analysis method is applied to infer binary relationships between target amino acids. ( ) the multi-level digraph analysis method infers consistent minimal binary relationships starting from many binary relationships derived from the threshold-test analysis method. a threshold-test analysis method treats the data representing the binary relations of change in the concentration of amino acids. these relations describe the effects of one amino acid on the concentration of the other amino acids and are mainly provided by the changes in the state of amino acid concentrations (fig. ) . upon setting an arbitrary threshold value of concentration ratio, one can extract binary relations between two amino acids directly by estimation of the relative change in concentration ratios (y f ) or by the statistical probability of change in average concentrations (y p ) from deficiency or over-consumption in one essential amino acid experimental data. the following method is explained using the concen-tration ratios (y f ) as an example. a set of amino acids is defined as s ¼ fa; b; c; . . .g. we assume here experiments are those of deficiency or over-consumption of one essential amino acid, and that the measurements of concentrations of many amino acids are performed simultaneously. the change in concentration of the amino acids resulting from the deficiency or over-consumption of one target amino acid relative to its concentration under normal conditions (control of amino acids) is examined and recorded. this change may be recorded as an increase, decrease, or no change. an concentration ratio matrix, e, is created from a set of deficiency in one amino acid (or over-consumption) experiments, in which each matrix element represents the real-valued ratio of amino acid concentration. for instance, the value of matrix element eða; bÞ indicates the relative change (the concentration ratio) in concentration of amino acid 'b' in comparison to its concentration under control conditions caused by the deficiency (or over-consumption) in amino acid 'a'. thus the matrix e is defined as e ¼ fða; bÞ; . . .g. the inference procedures of this network model are as follows: ( ) obtain the concentration ratio matrix e using several sets of the amino acid concentrations resulting from deficiency or over-consumption of one essential amino acid. ( ) using the concentration ratio matrix e, we determine whether a given amino acid affects another given amino acid. for example, if, following the deficiency of amino acid 'a', the ratio of amino acid 'b' becomes higher than a given threshold value (specifically, more than y f -times higher), or becomes lower than a given threshold value (specifically, less than =y f -times lower) we say that amino acid 'a' affects amino acid 'b' directly or indirectly, and the value of element (a, b) in the binary matrix r is set to ; r(a, b) ¼ . ( ) it should be noted that the condition amino acid 'a' affects amino acid 'b', that is r(a, b) takes the value , means both ''a change in concentration-value of 'a' leads to a change in that of 'b''' and also ''no change in the concentration-value of 'b' leads to no change in that of 'a'''. thus the probability of rða; bÞ takes the value , or more generally the probability that the value rði; jÞ takes the value , pðrði; jÞ ¼ Þ (where i; j ¼ ; ; . . . ; n in which n is the total number of target amino acid) is examined through all the experiments of deficiency or over-consumption of one essential amino acid. an arbitrary second threshold value for probability is set, r, and experimental events with pðrði; jÞ ¼ Þ > r are extracted statistically. a multi-level digraph analysis method infers amino acid networks by using a set of binary relations between amino acid (e.g. amino acid 'a' affects (maki et al., (maki et al., , . a systematical analysis of the binary relations between pairs of amino acids enables us to reconstruct a possible minimum architecture of the amino acid network that is consistent with all of the data. in fig. , the accessibility matrix r à is derived directly from the binary relation r. in the accessibility matrix r à , if there exists the relation that amino acid 'a' and 'b' affect each other, that is that r à (a, b) ¼ r à (b, a) ¼ , we cannot decide which amino acid is located at the upper stream. we therefore introduce an ''equivalence set'', which makes a single set of the group of amino acids affect each other, and this group is deemed to be a single amino acid. in order to partition amino acids into equivalence sets, we use the accessibility matrix r à . this matrix is a relative transitive closure of the binary relation matrix, r, where the matrix entry r à ða; bÞ indicates whether amino acid 'a' finally affects amino acid 'b' or not. the multi-level digraph analysis model is implemented on the basis of this accessibility matrix r à . figure shows the procedure for drawing a multi-level digraph. for the accessibility matrix r à in the figure, since 'c' and 'd' can be regarded as an equivalence set, we can combine them as 'c à '. in this manner, we can draw up equivalence sets in a semi-ordered (topologically sorted) accessibility matrix. a semi-ordered accessibility matrix between equivalence sets includes indirect amino acid relations. in order to remove them and to make a skeleton matrix, we process the semi-ordered matrix as follows: the value of line i and column j in a semi-ordered matrix a and skeleton matrix s are represented as aði; jÞ and sði; jÞ, respectively. if aði; jÞ ¼ , sði; kÞðk ¼ ; . . . ; nÞ is set to maxfaði; jÞ À að j; kÞ; g. thus, all indirect effects are removed from the semi-ordered matrix. in fig. , the relation between amino acid 'a' and 'c à ' are removed, we thus can construct the skeleton matrix s. finally we draw lines between nodes based on the value fig. . process of the multi-level digraph analysis method fig. . plasma amino acid concentration ratio. the concentration of plasma amino acid by amino acid minus-one diet is shown as the ratio to that of control diet of each element in the skeleton matrix. in fig. , the amino acids with parentheses indicate an equivalence set of amino acids. the sample plasma was obtained from the amino acid minus-one diet fed rats, whose plasma concentration of the deficient amino acid is less than half of that of the control diet fed rats. deficiency in one essential amino acid triggers the change in concentrations of all the other amino acids in plasma as shown in fig. . this change is converted to the directional relation from the deficient amino acids to all the other amino acids by the described method. two different values were adopted to set the threshold. one is the fold-change value (y f -value). larger y f -value means the severer filtering condition. the other value is the p-value (level of significance) for the average difference (y p -value). each experimental and control groups consist of samples, and for all the amino acids measured, the p-value, level of significance, for the average difference between the experimental group and the control group was calculated using dunnet's multiple comparison method. in this case, smaller y p -value means the severer filtering condition. network structure estimated by the multi-level digraph method from the binary interaction matrix, multi-scale digraph was drawn (fig. ) . in the analysis using fold-change value as the threshold, the number of amino acids (nodes) composing the network decreases as the filtering threshold becomes severer. in the analysis using the p-value (level of significance), the number of nodes does not change drastically as the threshold changes. in either analysis, some amino acids formed an equivalence group, in which the interactions (links) form a loop and the direction of the effect on one another cannot be fig. . network structure estimated by threshold-test analysis and multi-level digraph analysis methods. network structure of plasma amino acids is estimated using threshold-test analysis and multi-level digraph analysis methods. the amino acids whose minus-one diet experiments were performed are indicated in circles. the change in plasma amino acid concentration was converted to binary relational values, in the manner described in the results. the threshold to determine the binary value is set using a fold-change (y f -value) and b p-value (y p -value). for both cases, the figures to the right uses severer threshold in determining the binary values. lines with black arrowheads indicate the positive effects and the lines with white arrowheads indicate the negative effects decided. when the filtering condition becomes severer, the link forming the loop is disappeared and the equivalence group disperses into the smaller groups or individual amino acids. in the case using fold-change value, the equivalence group disperses at the threshold of . , and -level digraph with amino acids is drawn. in the case using p-value, the equivalence group does not completely disappear and at the threshold p ¼ . , which is the severest condition inspected, -level digraph with amino acids is drawn. since we should like to infer network model with as many nodes and links as possible, we decided to use p-value for the threshold for the further analysis. the same analysis was carried out using the partial dataset. by excluding one amino acid minus-one dataset, we can estimate the network structure without considering the effect of the excluded amino acid. the results are shown in fig. . at the threshold p ¼ . , the removal of threonine minus-one dataset changed the network structure drastically. the equivalence group disappears and the interactions between the amino acids which belonged to the group are alternatively revealed. this indicates that the relation responsible for holding the equivalence group together was the effect of threonine on the other amino acids, leucine, methionine, tryptophan and valine, in the group. at the threshold p ¼ . , the only amino acids forming the equivalence group are threonine and methionine. by excluding threonine minus-one dataset, the effects of methionine on other amino acids are revealed, and by excluding methionine minus-one dataset, the effects of threonine are revealed. integrating the information obtained from the analysis of partial dataset, the estimated network structure is further refined to the model shown in fig. . threonine directly interacts with the most number of amino acids, and lysine is located at the top control level in all the other amino acids. threonine and methionine is interrelated each other, which forms a feedback loop. using the data obtained under the essential amino acid minus-one condition, we have first estimated the coarse network structure of the plasma amino acids. the minus-one condition lowers the plasma concentration of the deficient amino acid to less than half, and this condition was maintained for a fairly long period. this drop in the concentration and the consequent changes in other amino acid concentrations are theoretically equivalent to the single gene disruption experiments in which the expression of the disrupted gene drops to nearly zero and the expression of the other genes are altered. in these experiments the genes whose expression levels were altered are suggested to be directly or indirectly regulated by the disrupted genes. similarly, the amino acids whose plasma concentration changed under minus-one condition are suggested to be directly or indirectly regulated by the deficient amino acid. combining the essential amino acid minus-one datasets, we were able to draw a coarse network model which can explain the change in the concentration of the plasma amino acids in our experiments. in the estimated plasma amino acid network structure, lysine is located at the top control level, which affects most of the amino acids, but is not influenced by any amino acids. this indicates the biological essentiality of lysine in an organism. lysine deficiency is the strongest signal and the wide range of amino acid metabolism has to be modulated to mitigate the damage. threonine interacts directly with the most number of the amino acids, acting as a hub in the network. this suggests the possible role of plasma threonine as a messenger which spreads the information of any essential amino acid deficiency to the whole body. when rats are fed minus-one diet, they will respond by changing their metabolic flow, to save and recycle the limited amino acid (kimball, ) and compensate for the shortage by making use of internal amino acid pool, such as skeletal muscle (kadowaki and kanazawa, ) . in this regulation of metabolic rate, threonine might play an important role. the concentration of threonine rises in any amino acid minus-one condition, except for threonine minus-one. thus it is highly possible that threonine may be used to regulate the pathway which will be needed in any essential amino acid shortages. as shown in fig. , we can speculate that threonine gives positive influence to methionine, and methionine gives negative influence to threonine. these positive and negative interactions make up a loop which causes the temporal oscillatory behavior of the threonine-methionine concentration. this kind of oscillation can play a role in a trigger switch in a biological system. one of the mutual relationships that link threonine and methionine is the common catabolic pathway downstream of -oxobutanoate. threonine is deaminated to form -oxobutanoate (kapke and davis, ; scarselli et al., ) , and methionine forms -oxobutanoate via l-homocysteine and cystathionine . this can partly explain the positive effect of threonine on methionine. when the concentration of plasma threonine decreases, the concentrations of -oxobutanoate may also decrease and the methionine catabolism may be stimulated. cystathionine beta-synthase usually catalyzes the reaction of replacing beta-oh of serine by homocysteine, however, it is reported that threonine can be substituted for serine in this reaction forming -methylcystathionine (borcsok and abeles, ) . cystathionine beta-synthase is a key enzyme in methionine metabolism which directs the metabolite flux towards catabolic trans-sulfuration pathway rather than methionine recycling salvage pathway (banerjee and zou, ) . the fact that threonine can be a counter substrate for this enzyme indicates the possibility of threonine having direct regulatory effect on methionine metabolism. another possible link between threonine and methionine is vitamin b . there are two enzymes which require vitamin b , l-methylmalonyl-coa mutase and methionine synthase. l-methylmalonyl-coa mutase catalyzes the conversion of l-methylmalonyl-coa to succinyl-coa. l-methylmalonyl-coa is one of the metabolites of threonine. methionine synthase catalyzes the conversion of -ch -tetrahydrofolate and homocysteine to tetrahydrofolate and methionine, respectively watanabe and nakano, ) . this may be a clue which explains the negative effect of methionine to threonine. the model reported in this study is constructed using the static plasma amino acid data obtained in a condition where plasma amino acids concentration had been equilibrated by the minus-one condition. it is a snapshot or a cross section, taken under this particular minus-one condition, of the dynamic plasma amino acid network. it is constructed without any prior topological information of the amino acid metabolic pathway, however, it is notable that some relations in the model, such as the direct relation of phenylalanine to tyrosine, can be explained by the pathway map. by comparing and integrating snapshots taken under various conditions, we can refine and validate the estimated network structure of the plasma amino acid. metabolic abnormalities in cobalamin (vitamin b ) and folate deficiency steady-state kinetic studies with the non-nucleoside hiv- reverse transcriptase inhibitor u- e kinetic studies with the non-nucleoside hiv- reverse transcriptase inhibitor u- e the quinoline u- is a potent inhibitor of hiv- reverse transcriptase redox regulation and reaction mechanism of human cystathionine-beta-synthase: a plp-dependent hemesensor protein mechanism of action of cystathionine synthase two new schematic rules for rate laws of enzymecatalysed reactions graphic rules in steady and non-steady state enzyme kinetics applications of graph theory to enzyme kinetics and protein folding kinetics. steady and non-steady-state systems graphical rules for non-steady state enzyme kinetics kinetics of processive nucleic acid polymerases and nucleases predicting networking couples for metabolic pathways of amino acid metabolism in man plasma amino acids in hepatic encephalopathy amino acid metabolism in liver disease the relationship between plasma homocysteine and amino acid concentrations in patients with end-stage renal disease prospective study on the efficacy of branched-chain amino acids in septic patients amino acids as regulators of proteolysis stereochemistry of the reaction of sheep liver threonine dehydratase. a nuclear magnetic resonance and optical rotatory dispersion study of its reaction pathway and products regulation of global and specific mrna translation by amino acids kinetic analysis by a recursive rate equation demonstration of a slow conformational change in liver glucokinase by fluorescence spectroscopy an integrated comprehensive workbench for inferring genetic networks: voyagene development of a system for the inference of large scale genetic networks metabonomics': understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological nmr spectroscopic data network analysis of plasma and tissue amino acids and the generation of an amino index for potential diagnostic use structure and function correlations between the rat liver threonine deaminase and aminotransferases insulin, glucagon, aminoacid imbalance, and hepatic encephalopathy elevation of serum cystathionine levels in patients with cobalamin and folate deficiency a study of aminoacidemia and aminoaciduria in epileptic children a new nucleotide-composition based fingerprint of sars-cov with visualization analysis serum amino acid levels in patients with hepatocellular carcinoma serum neutral amino acid concentrations in cirrhotic patients with impaired carbohydrate metabolism an application of gene comparative image for predicting the effect on replication ratio by hbv virus gene missense mutation using cellular automata to generate image representation for biological sequences using cellular automata images and pseudo amino acid composition to predict protein subcellular location a probability cellular automaton model for hepatitis b viral infections validation and refinement of gene-regulatory pathways on a network of physical interactions an analysis of base frequencies in the antisense strands corresponding to the human protein coding sequences authors' address: nahoko shikata, graduate school of systems life sciences, kyushu university, - - hakozaki, higashi-ku, fukuoka - , japan, fax: þ - - - , e-mail: shikata@brs.kyushu-u.ac.jp key: cord- -un ysc v authors: nan title: poster presentations date: - - journal: j pept sci doi: . /psc. sha: doc_id: cord_uid: un ysc v nan the boron neutron capture therapy (bnct) based on the interaction between b isotope and thermal neutron has been highly noted in recent years as one of promising techniques for treatment of cancers. p-( b)borono-l-phenylalanine (l- bpa), in which boron atom is enriched with b isotope, is now using clinically as an effi cient b carrier for treatment of patients in particular with malignant brain tumor and melanoma ( ) . in order to develop a practical method for the synthesis of l- bpa, we have recently examined the enantioselective method utilizing the negishi reaction based on the coupling of -( b)boronoiodobenzene derivative with -iodo-l-alanine derivative ( ) . from the standpoint of diagnosis of cancers, the magnetic resonance imaging (mri) is noted as one of common techniques. in particular, mri based on the measurement of f atom is becoming a remarkable one. to create practical materials utilizing as not only the b carrier but also mri probe, we had already synthesized the compounds containing both b and f atoms in a single molecule such as - [ -( b)borono- , -difl uorophenyl]-dl-alanine [dl- bpa( , f )] ., - [ -( l-proline is conformationally unique among coded amino acids in that its torsion angle is blocked (- ± ) by its characteristic fi vemembered pyrrolidine ring structure and the preceding torsion angle (tertiary amide) can undergo cis ( ) <-> trans ( ) isomerization much easier than the secondary amides of the usual peptide bonds. in addition, its torsion angle is commonly found either in the right-handed -/helical region (- ÷ - , or cis' conformation) or in the left-handed, semiextended, region [- ± , or trans', or poly-(l-pro) n conformation]. methylation at the c -position of a pro residue was suggested to block the preceding tertiary amide ( ) torsion angle of the resulting ( me)pro to the trans disposition and to restrict the , surface to the single region where the / -helices are found. we have synthesized a large set of n -blocked, ( me)pro-containing, dipeptide n'alkylamides having the general formulas p-d-( me)pro-xxx-nhipr and p-xxx-d-( me)pro-nhipr, where p is ac or boc and xxx is d-ala, l-ala, aib, gly, d-( me)pro, or l-( me)pro. the results of the present x-ray diffraction analysis clearly show that the region of the conformational map overwhelmingly preferred by ( me)pro is indeed that typical of / -helices, but the semi-extended, type-ii poly(pro) n helical region can exceptionally be explored by this extremely sterically demanding c -tetrasubstituted -amino acid. in addition, the known high propensity for -turn formation of the pro residue is even enhanced in peptides based on its c -methylated derivative. to complete the picture of the preferred conformation of ( me)pro, the synthesis and crystal-state investigation of a series of terminallyprotected homo-peptides from d-( me)pro are currently in progress in our laboratory the interest in guanidine-containing compounds is due to their important role in biological recognition processes. five-member cyclic n-amidinoamino acids represent hybrid structures, combining both proline rigidity and high positive charge of arginine guanidine group. structurally similar n-amidino-proline, n-amidino-pyroglutamic acid and cyclocreatine ( imino- -imidazolidine acetic acid) were chosen as objects of our study. the problem of their incorporation into peptide structure requires use of substituted derivatives or effective guanidylation technique. recently we have presented the synthesis of mts-protected n-amidinoproline. nevertheless, its application in n-termini modifi cation of peptides shows slow condensation rate in classical as well as solid phase synthesis for short peptides ( - residues) or completely fails in case of longer ones ( residues). on the other hand, polymer supported guanidylation of proline by bis-boc-protected carboxamidine- hbenzotriazole seems to be preferred route to n-amidino-proline containing peptides. three methods of n-amidino-pyroglutamic acid synthesis have been investigated and model dipeptide has been acquired on wang polymer by the cyclization of guanidine-glutamic acid. for selective boc-deprotection on wang resin the literature technique has been utilized. however, in this case esi-ms and nmr analyses evidence for side-reaction of triethylamine alkylation by polymer linker fragment. hplc analysis shows n-amidino-pyroglutamyl-phenylalanine stability at acidic and physiological ph but fast ring opening in water solution at ph . for cyclocreatine application in peptide synthesis we suggest the use of its p-toluenesulfonate having good solubility in dmf and showing effective coupling in cl-hobt/dic condensation. the examples of namidino-amino acids practical application in peptide synthesis will be presented. tryptophan is often a key pharmacophore which determines the affi nity of peptide ligands for their receptors. cyclic analogues of tryptophan which introduce local constraints and reduce the fl exibility of the indol moiety are very valuable tools to probe the bioactive conformation of the peptide ligands. one of the possibilities to freeze indol moiety of tryptophan is the synthesis of the additional -member ring by the formation of , , , -tetrahydro--carbolines. we report the synthesis of , -disubstituted , , , -tetrahydro--carbolines via the pictet-spengler reaction. methyl ester of tryptophan or dipeptides with n-terminal trp (trp-ala-ome, trp-leu-ome) were used as arylethylamine substrates and -amino aldehydes derived from l and d-amino acids were used as carbonyl components. we determined that there were no differences of the stereoselectivity of the pictet-spengler reactions in the case of trp-ome or trp-dipeptides. we also investigated the conformation of the newly created -membered ring in cis and trans diastereomers. the roesy spectra were used to assign the more stable conformation for each isomer. the conformations of cis isomers derived from tryptophan and dipeptides were the same and substituents on c- and c- were in both cases equatorial. the conformation of trans isomers were depended on the carboxyl part of trp. for methyl ester of tryptophan the ester group was axial, whereas for dipeptides we observed the opposite conformation with equatorial substituent on c- . our results show that the pictet-spengler reaction can be successfully performed for amino acids and peptides and the conformation of the newly created -membered ring depends on the substrates and the size of c- and c- substituents. helical-screw handedness of peptides composed of diastereoisomeric cyclic amino acids nagano helical-screw handedness in proteins is believed to result from thecarbon chiral center of l--amino acids. recently we have reported that the helical-screw sense of oligopeptides can be controlled without a chiral center on the peptide-backbone but by chiral centers at the side chain. that is to say, we designed and synthesized an optically active cyclic , -disubstituted amino acid (s,s)-ac( )c(dom), in which the -carbon atom is not a chiral center but chiral centers exist at the side chain. conformational analysis of the (s,s)-ac( )c(dom) peptides revealed that the hexapetide formed left-handed -helices both in solution and in the solid state, and the octapeptide assumed a left-handed -helix. herein we designed new two diastereoisomeric cyclic , -disubstituted amino acids; ( s, s)-and ( r, s)- -amino- -(methoxy)cyclopentanecarboxylic acid (ac( )c(om)) having chiral centers both at the -carbon atom and at the side chain. the amino acids ( s, s)-and ( r, s)-ac( )c(om) were synthesized starting from l-(-)-malic acid. that is to say, at fi rst, the malic acid was converted to diiodide compound, and bisalkylation of dimethyl malonate with the diiodide gave a cyclic diester. monohydrolysis of the diester, followed by curtius rearrangement produced separable mixtures of ( s, s)-and ( r, s)-ac( )c(om). we prepared both diastereoisomeric ( s, s)-and ( r, s)-homooligomers by solution-phase methods, respectively, and studied their preferred secondary structures using h nmr, ft-ir, cd and x-ray crystallographic analysis. zobel, ansgar; gaus, katharina; wollschläger, katrin; nieß, anke; juodaityte, jovita; sewald, norbert organic and bioorganic chemistry, bielefeld university, germany novel highly active and specifi c dna binding molecules have a considerable potential particularly with regard to applications in medical science. the major and minor grooves of the dna serve as recognition areas. it is of high interest for chemical biology to control dna-binding abilities of synthetic molecules. a possible modulator is light in combination with photoswitchable molecules. triostin a is a natural bisintercalator which belongs to the quinoxaline antibiotics originally isolated from streptomyces s- - . it consists of a bicyclic depsipeptide with n-methylated amino acids and a cystine bridge. its heteroaromatic quinoxaline moieties are able to intercalate gc-specifi cally into the dna inducing a change in conformation and therefore inhibit the transcription by blocking spezifi c enzymes. tandem is the n-unmethylated derivative of triostin a which binds at selectively to the dna due to the change in the hydrogen bonding pattern. the exchange of the cystine bridge of tandem with substituted azobenzene units leads to photoswitchable triostin a analogs. the synthesis of the depsipeptidic basic structure which contains the quinoxaline moieties is introduced as well as the coupling with azobenzene amino acids. the double cyclization step is accomplished under pseudo high dilution conditions. in order to differ between cisand trans-confi gurations of the molecule, nmr-and ir-spectroscopy was carried out as well as the photoswitchability of different analogues was tested by irradiation and rp-hplc analysis. for synthetic peptide vaccine prototype development: (pc) , which has an heteroatom in its structure and their bioconjugates obtained by microwave and carbodiimide methods were explained. for pc obtaining, the synthesis of the monomer, azabicyclo[ . . ]octane, (c), was carried out by organic methods which then will be used in polymer synthesis [ ] [ ] . consequently, polymers having different molecular weights and their water soluble bioconjugates were synthesized and the characterization of these polymers and conjugates were done by different methods such as uv, atr ft-ir, sec with four detectors and zeta sizer. for the chemical modifi cation of pc, bromoacetic acid was used and a water soluble polyampholyte synthesis was achieved with the quaternization of polymer. atr ft-ir spectra of pc was recorded and molecular weights, polidispersity values of polymers, mark-houwink constants and molecular diameters of the polymers were measured with size-exclusion chromatography with four-detectors (light-scattering, refractive index, viscosity and uv). with zeta sizer, size-analysis of polymer chains were done and their zeta potentials were measured. it is determined that molecular weights of the polymers were affected by the change in the amount and the type of initiators used and also from the polymerization media. analysing of having biodegradable characteristics of the synthesized polymers has been being reviewed. after synthesis and modifi cation of pc, its conjugates with antigenic peptides like avian infl uenza hemagglutinin (ha - ) ypydvpdya and rgdsggc cell receptor peptide were obtained. their characterization was also performed. japan; in alzheimer fs disease research, sparing water-solubility and uncontrolled self-assembly of amyloid peptide (a ) - are signifi cant obstacles to establish an experimental system that clarifi es a pathological mechanism of a - . to solve these problems, we herein disclose water-soluble "click peptides" based on an o-acyl isopeptide method. these peptides contain an o-acyl instead of n-acyl residue at the gly -ser of a - , and are converted to a - via an o-n intramolecular acyl migration triggered by phchange (ph-click) or photo-irradiation (photo-click). these peptides had remarkably higher water-solubility than a - . in addition, these peptides clearly adopted monomer state with a random coil structure, which were verifi ed by various physicochemical assays. importantly, we could establish an in situ system predominantly comprised of monomer a - as a result that the monomer click peptide was converted to a - quickly and quantitatively in accordance with ph-change. both selfassembly and conformational change of the produced a - in situ were observed with time. similarly, the photo-triggered click peptide afforded a - by uv-irradiation. because the in situ production of intact a - from the click peptides could overcome the handling problems of a - , this strategy would provide a reliable experimental system for investigating a pathological function of a - in alzheimer fs disease. the antioxidant effect of introducing phenylpropenoyl moiety in either in n-terminal group of analgesic oligopeptides or in c-terminal end of opioide active amino acid, modifi cated with polyamines have been evaluated. the series of hydroxycinnamoyl peptide and amino acid amides were synthesized by standard method used in peptide chemistry. obesity is a major public health problem associated with morbidity and mortality and continues to increase worldwide. the gastrointestinal tract and the pancreas release hormones regulating appetite and body weight. obestatin is a recently described amino-acids peptide derived from preproghrelin. it was identifi ed by bioinformatic prediction ( ) . obestatin decreases food intake and body weight gain, decelerates gastric emptying, promotes sleep in rat, inhibits water drinking. it has been described that obestatin effects on feeding behavior and an u-shaped dose-response relationship was found: low doses ( . - nmol/kg) and high doses ( - nmol/kg) were ineffective. the amino-acid carboxy-amidated human obestatin nalapropheaspvalglyilelysleuserglyvalglntyrglnglnhissergln alaleunh and corresponding rat obestatin hpheasnalapropheasp valglyilelysleuserglyalaglntyrglnglnhisglyargalaleunh were synthesized by different schemes. synthesis was developed on the basis of solid phase synthesis of protected peptide fragments followed by their assembly into the fi nal product. fragments - , - , - , - , - were built on the acid-labile -chlorotrityl chloride resin using the orthogonal fmoc/tbu strategy. fragment - (hcl•hargalaleunh ) was obtained in solution. the assembly of the full-length peptide was carried out by fragments coupling in solution or using solid phase method. the free peptide was obtained by treating the protected peptide with mixture of tfa: h o:tis ( : . : . ). the peptide was purifi ed with hplc and isolated by lyophilization with %+ purity according to analytical reversed-phase hplc. the mass of molecular ion determined by maldi-tof spectrometry was in fi ne agreement with calculated value. the inverstigation of biological actions of obestatins is in progress. straightforward synthesis of enantiopure tfm-amino acids from chiral cf brigaud, thierry; chaume, grégory; huguenot, florent; caupène, caroline university of cergy-pontoise, france trifl uoromethylated amino acids (tfm aas) are current synthetic targets due to their unique properties and their synthesis in enantiopure form remains a challenge. we will report that chiral -trifl uoromethyl- , oxazolidines (fox) are highly versatile synthons for the stereoselective synthesis of various functionalized -trifl uoromethylamino compounds such as -amino nitriles , -and -amino acids, diamines and amino alcohols. , moreover, trifl uoropyruvate-based oxazolidines proved to be very valuable building blocks for the stereoselective synthesis of both enantiomers of -trifl uoromethyl proline and -tfm-pyroglutamic acid, in enantiopure form. moreover, the synthesis of the (s)--tfm--allylglycine and the novel (s)--tfm norvaline were achieved in a few steps from this starting material. we will also report a recent straightforward synthetic route to tfm-dihydroxyprolines in enantiopure form from trifl uoropyruvate-based chiral oxazolidines. chitinases catalyse the hydrolysis of chitin, the natural homopolymer of ( , )-linked n-acetyl-d-glucosamine. chitin is a key structural component of the cell walls, exoskeletons, and eggshells of pathogenic fungi, insects, and nematodes, respectively, which all rely on the ability to hydrolyse chitin at specifi c points in their life cycles. chitinase inhibitors are now attracting considerable interest as novel fungicides and insecticides, as well as chemical tools to study human diseases as diverse as asthma and malaria. in this context, the cyclic pentapeptide natural products, argifi n and argadin, are two exciting inhibitors which pose some interesting synthetic challenges. the argifi n structure includes two sensitive -linked asp residues, as well as an unusual carbamoylated arg side chain, while argadin contains a unique aspsemialdehyde residue, that is cyclised to the peptide backbone to generate a potentially labile hemiaminal. we will describe improved routes to both compounds that allow us to avoid signifi cant side reactions such as aspartimide formation and homoserine-mediated backbone cleavage that are observed in our previously reported syntheses. [ , ] this is achieved by carrying out the assembly and cyclisation of both peptides, including key side chain derivatisation steps, entirely on solid phase. the application of the strategies devised to the automated synthesis of argifi n and argadin and related natural products will be described. during the last two decades combinatorial chemistry has become an important tool for the quick synthesis of large numbers of small molecules used, for example, in the generation of the new lead structures for medicinal applications. additionally, it allows rapid access to diverse chemical libraries with novel structures and properties. small molecules libraries are generally prepared on solid support simplifying tedious purifi cation steps of the intermediates and facilitating the entire synthesis. piperazines and keto-piperazines are amongst the important backbones in today's drug discovery. the piperazine framework has been defi ned in medicinal chemistry as a "privileged scaffold". it is a molecular backbone with versatile affi nity properties representing a frequently-occurring binding motif, and providing potent and selective ligands for a wide range of biological targets. the high number of positive hits revealed in biological screens with the piperazine scaffold urged chemists to develop plenty of different synthetic methods that allow fast and effi cient building of these heterocyclic systems on solid support as well as homogenous chemistry. however, the majority of these methodologies is not stereospecifi c and the complex mixtures of the stereoisomers are generated. in order to preserve important chiral centers in potential hits we propose to use pipearzine backbone, initially prepared in optically pure form, bearing various tethers with orthogonally protected groups applicable via solid phase organic chemistry (spoc). we will describe a novel synthesis of keto and diketopiperazine building blocks. these chiral building blocks are applied in "around-the-scaffold" modifi cation strategy by spoc, introducing valuable physico-chemical properties in independent diversity points. solid-phase strategies for constraining peptide structures can serve for elucidating the relationships between conformation and activity. for example, the systematic substitution of natural amino acids by their aza-amino acid counterparts has been accomplished using a fmoc strategy featuring couplings with aza-amino acid chlorides to provide insight into the biologically active conformers of the melanocortin receptor agonist ac-his-d-phe-arg-trp-nh as well as the calcitonin gene-related peptide antagonist [d , p , f ]cgrp - ( ) ( ) ( ) . employing cyclic sulfamidates, we have now developed fmoc strategies for the effective solid-phase introduction of alpha-and beta-amino gamma-lactam constraints into peptides. since their pioneering use in a somatostatin analog with about fold greater activity ( ), lactam restraints have been used to study a variety of relevant targets ( , ) . our strategies now provide effective means for performing lactam scans of biologically active peptides as demonstrated using the growth hormone secretagogue ghrp- , and an allosteric modulator peptide antagonist of the il- receptor. our presentation will focus on recent developments in the solid-phase chemistry for synthesizing such constrained peptide analogs and the relationships between peptide conformation and biology, elucidated by these novel methods. references: barcelona science park, university of barcelona, -barcelona, spain; linaclotide is a -residue peptide currently undergoing phase ii clinical trials for the treatment of gastrointestinal diseases such as chronic constipation (cc). linaclotide, which can be administered orally, is an agonist of the guanylate cyclase type-c receptor found in the intestine. from a structural point of view, this small peptide presents a constrained structure with the presence of three disulfi de bridges between cys -cys , cys -cys , and cys -cys . h-cys( )-cys( )-glu-tyr-cys( )-cys( )-asn-pro-ala-cys( )-thr-gly-cys( )-tyr-oh in order to reach the large amounts required for a marketed peptide, an effi cient synthesis needs to be attained. to optimize the synthesis, its fundamental limitations need to be determined and addressed. in the case of linaclotide, the key points are related to the numerous cys (some of them consecutive) present in the peptide, for two reasons: the potential risk of racemization upon assembling the linear chain, and the misfolding of the three disulfi de bridges. for that, the concourse of different protecting groups and folding conditions as well as the analysis of the disulfi de bridges in the fi nal folded peptide has been studied and will be discussed in this presentation. balalaie, saeed ; arabanian, armin ; mohammadnejad, mahdieh ; gross, juergen h. university, iran (islamic rep.); university, germany gnrh analogues have been used for the treatment of steroid-dependent tumors, such as prostate and breast cancers. the development of more potent gnrh analogues depended largely on the important made in the science of peptide chemistry. ugi- mcr reaction is known for the synthesis of amide bond. we wish to report herein an effi cient method for the synthesis of some gnrh analogues based on ugi reaction using fourcomponent reaction of n and c-terminus peptides, aromatic aldehydes and isocyanides. this ugi- mcr could describe to build up novel gnrh analogues deriving from triptorelin and gonadorelin. all of the products were purifi ed using preparative hplc and the structures were assigned according to maldi mass spectrometry data. peptide synthesis is based in a proper combination of protecting groups and in the right choice of the right coupling method. nowadays, almost all peptide bond formed are carried out in the presence of hydroxybenzotriazole (hobt) or its derivatives (hoat, cl-hobt). thus, hobt derivatives are used in combination with a carbodiimide or another coupling agent or built into a standalone reagent such as immonium ( thus, a replacement of hobt should be found for preparation of peptides for research purposes and, more important, for the production of peptide based apis. herein, several alternatives to hobt will be discussed taking into account the explosivity properties. furthermore, a new family of immonium salts, which incorporates an proton acceptor in its carbocation skeleton. the novel proton acceptor coupling reagent has shown superiority to the described previously. an oxygen in the carbocation moiety confers to the reagent more solubility, enhances coupling yields and decreases racemization, allowing the use of just one equivalent of base. examples on the use of the non-explosive replacement for hobt together with carbodiimides or built into phosphonium and immonium salts, with the proton acceptor, will be discussed. thiocarbamate-linked peptides by chemoselective peptide ligation using phenylthiocarbamate chemistry peptide chemical ligation chemistries, which allow the chemoselective coupling of unprotected peptide fragments, are useful tools for synthesizing native polypeptides or unnatural peptide-based macromolecules. native chemical ligation (ncl) ( ), staudinger ligation ( ) lead to the formation of a native peptide bond at the ligation site. other methods result in the formation of unnatural covalent bonds such as oxime ( ), thioester ( ) linkages. these methods are of great interest when native peptide bonds are not absolutely required. in this context, we examined the potential use of the phenylthiocarbamate group in ligation chemistry. the phenylthiocarbonyl group can be easily introduced into peptides on alpha or epsilon amino group using phenylthiochloroformate and standard solid-phase method ( ) . it reacts chemoselectively with cysteinyl peptides to give an alkylthiocarbamate bond. s,n-shift of the alkylaminocarbonyl group from the cys side chain to the alpha-amino group did not occur. the method was used for linking two peptides chain through their n-termini, for the synthesis of a cyclic peptide or for the synthesis of di-or tetravalent multiple antigenic peptides. synthetic peptide derivatives which contain secondary amide moiety at c-terminus are inhibitors of serine and cysteine proteinases and thus could be effi ciently used as ligands in affi nity chromatography in order to isolate these enzymes. there is a number of chemical syntheses of amidous peptide derivatives in the literature, but the reaction yields are usually low and reaction conditions and purifi cation procedures are too complicated. it is also known that the application of enzymatic catalysis allows to obtain high reaction yields with simultaneous simplifi cation of synthetic and purifi cation procedures, and at the same time preserves the optical purity of target compound. subtilisin sorbed on silochrome could be effi ciently used as catalyst of acylation of secondary amides by the esters of acylpeptides. subtilisin's wide substrate specifi city allows to carry out the syntheses with peptides which contain hydrophilic, hydrophobic, dicarbonic-and diamino-acids at c-terminus. piperidine, morpholine, indole, diethylamine and other secondary amines are used in the syntheses as the amino-components. the reaction yield equals about - % in dependence of the nature of c-terminal amino acid of acylating peptide, pka of amine and the hydrophobicity of the fi nal compound. the distribution of the reaction product in liquid and solid reaction phases is investigated, the optimal reaction conditions for enzymatic acylation of secondary amines are established. all the compounds obtained are characterized by its nmr and mass spectra. the inhibition constants for some proteolytic enzymes with the compounds of research are defi ned. unnatural amino acids are becoming increasingly important substrates in modern drug design, synthesis and discovery research. in particular, arylhistidines naturally occur in the active site of the heme-copper oxidases as well as in cytotoxic and antifungal marine peptides ( ) . a method of choice for the preparation of unsymmetrical biaryl systems is the suzuki-miyaura cross-coupling of an aryl halide with an arylboronic acid. it has been shown that microwaves signifi cantly enhance this reaction leading to higher overall yields and purities as well as shorter reaction times. although a great variety of biarylic compounds have been prepared following this approach, so far, it has not been applied to the arylation of the histidine imidazole ring. initially, we studied a methodology for the synthesis of -arylhistidines via a microwave-assisted suzuki-miyaura cross-coupling reaction in solution ( ) . taking into account the advantages of the synthesis on solid support, such as the avoidance of tedious work-up which are particularly valuable for palladium-catalyzed reactions, we studied the application of the above methodology on solid-phase. here, we report the suzuki-miyaura reaction between a -bromohistidine and an arylboronic acid on solid support. the reaction conditions were optimized by varying several parameters such as the solvent, the reagent concentrations and the reaction time. the optimized conditions were subsequently applied to the synthesis of peptides containing a -arylhistidine residue. this work constitutes the fi rst suzuki-miyaura coupling involving the imidazole ring of a histidine on solid support. microwave-assisted attachment of fmoc-amino acids to resins via triazine "superactive esters". the substitution level of the new functionalized resin was calculated following a standard protocol based on the spectroscopic measurement of the soluble chromophore piperidine-dibenzofulvene. coupling reactions to the resin proceeds at room temperature in hours. moreover we carried out the time-consuming attachment of fmoc-amino acids to the resin by an automatic monomode microwave (mw) instrument (liberty, cem), in fact microwave is proposed as a valid alternative to enhance effi ciency of coupling reactions and it has been widely applied to spps. novel peptide-hetorocycle conjugates: derivatives of -(benzimidazol- -yl)alanine ( ) . considering the biological activity and complexing abilities of benzimidazoles we developed a direct solid-phase synthesis of benzimidazole-peptide conjugates, expecting these new compounds to express novel biological properties ( ) . the peptide-heterocycle conjugates are obtained by on-resin reaction between aldehydes and peptides containing a specially designed -( , -diaminophenyl)alanine residue ( ) . the stoichiometric amount of aldehyde leads to '-substituted -( h-benzimidazol- -yl)alaninecontaining peptides, the increase in aldehyde content results in `, `disubstituted derivatives. the reaction with dialdehydes gives novel amino acid residues containing tricyclic systems, in the case of ophthalic aldehyde -the pyrido-[ , -a] benzimidazole. the compatibility of our method with the fmoc solid phase peptide synthesis protocols was proven by the synthesis of analogues of immunosuppressory fragments of ubiquitin and hla-dq [ , ] . the biological activity of the modifi ed oligopeptides was compared to that of original fragments . as well as to similar quinoxaline-peptide hybrids. the collision-induced dissociation of substituted benzimidazole-peptide conjugates leads to characteristic fragmentation ions, which may be used as a diagnostic tool in the fragmentation of the benzimidazole-peptide hybrids. cardona, valérie; oswald, benoit genzyme pharmaceuticals, switzerland dehydroamino acids are an important class of compounds due to their presence in many biologically active natural products including the antrimycins, tentoxin, phomopsin a, or the phosphatase inhibitors like microcystin and nodularin. in the last decade an increasing interest in -branched dehydroamino acids has been developed based on their importance as commodity chemicals and value as tools in structure relationship studies. the incorporation into a peptide of such unsaturated amino acids introduces an element of conformational rigidity, as well as changes in reactivity, allowing for the development of high affi nity ligands for receptors. a variety of methods exist for the synthesis of dehydroamino acids. some of these approaches rely on thermodynamic control to dictate the alkene geometry. if there is no strong thermodynamic preference, or if the desired product is not the thermodynamically favoured isomer, the existing synthetic methods are often ineffective. we describe here an effi cient stereoselective method for the synthesis of , -branched dehydroamino acids (iii) from -aryldehydroamino acids (i). the -aryldehydroamino acids (i) were prepared from commercially available z/boc-phosphonoglycine trimethylester and aldehydes using the schmidt protocol. the transformation of (i) into their -bromodehydroamino acid derivatives (ii) is performed by a hoerrner reaction in a very good yield giving the trans derivatives. suzuki cross-coupling on (ii) with a boronic derivative generated the high value building blocks (iii). a variety of side chains can be incorporated using this type of reaction. the stereochemistry of these compounds has been determined using noe enhancement experiments. we report here a scalable and high yielding synthesis of stereospecifi c , -aryldehydroamino acid derivatives. versatile methods for synthesizing acyl-tetramic acids peptide analogs. davidov, gali; mozes, tamar; khandadash, raz; byk, gerardo bar ilan university, israel acyl-tetramic acid derivatives have been identifi ed as potential antiviral and antibacterial compounds. in this work we have improved their synthesis starting from tetramic acid lactams using peptide coupling reagents such as bop under microwave heating for generating the carbon-carbon bond of the acyl-tetramate. using this procedure we have synthesized a number of new tetramic acid derivatives in solution and generated a series of acyl-tetramic acid building blocks that were introduced into peptides using conventional spps methods. additionally, we present here a new solid phase microwave assisted synthesis of acyltetramates on pre-synthesized peptides and amino acids. antibacterial and antiviral activity of the products will be presented. peptide sequence and include all side-chain groups. in practice this is diffi cult to achieve. the task of making a turn mimetic can be regarded from the perspective of the backbone dihedral angles -to make a beta turn mimetic the phi and psi angles of two consecutive residues have to be controlled. limiting the dihedral angle space is most effectively accomplished with a cyclic constraint -one or more may be used. in this case a single medium ring cyclisation from the (i) carbonyl to the (i+ ) amine -in place of the classical hydrogen bond -forces the four backbone dihedral angles into a turn conformation. due to the polyfunctional nature of peptide systems the introduction of unnatural constraints can be synthetically challenging. in the present case a number of side reactions were encountered and had to be overcome. some were well known such as diketopiperazine formation and others involving transannular interactions were unexpected and gave rise to interesting byproducts. ultimately the side reactions were overcome by choice of cyclisation position and synthetic modifi cations. auto-assembling antimicrobial cyclic pseudopeptides including aza- -amino acids antibiotic resistance of pathogens against conventional antibiotics is increasing at a rate that far exceeds the pace of new development of drugs. so, antimicrobial peptides, both synthetic and from natural sources, have raised interest as potential useful drugs in the future. however, due to proteolytic degradation, peptides are not ideal candidates for pharmaceutical development. that is why numerous researches try to develop non natural peptidic analogues for enhancing metabolic stability, bioavailability, and biological absorption. in this class of peptidomimetics, pseudopeptides consisting exclusively or including aza -amino acids have emerged as a promising new class of compounds that favour hydrogen bond formation and can enhance biological activities when compared to natural parent peptides ( ) . we have designed "mixed" cylic pseudopeptides composed of -and aza- -amino acids that target bacterial cell wall and induce the death of the pathogens. particularly, some of these cyclic pseudopeptides have broad spectrum antibactericidal activities on gram positive and gram negative bacteria with low minimum inhibitory concentrations (mic). on the other hand, this type of molecules is not haemolytic and cytotoxic at antimicrobial activity levels( ) now, we try to explain the mechanism of action of our pseudopeptides that act on the microbial membranes. with nmr studies we have demonstrate that in solution cycles auto-associate at high concentrations and we investigate their behaviour in presence of small unilamellar lipidic vesicules (suv) ( ) . this phenomenon of auto association seems to be facilitated at the lipid interface. liu, hongqiang ; pattabiraman, vijaya r. ; vederas, john c. university of alberta, canada; lantibiotics are a class of antimicrobial peptides containing lanthionine ( ) and/or -methyllanthionine ( ) residues in cyclic moieties, and are currently used for food preservation. they also have potential as human therapeutics. lacticin is a two-peptide lantibiotic produced by l. lactis subsp. lactis dpc , and its components (a and a ) are active against a wide range of gram-positive bacteria by a synergistic mechanism in sub nm concentrations. however, the oxidation of the thioether of lanthionine ( ) and -methyllanthionine ( ) is a major source of lantibiotic instability and loss of activity. to investigate structure-activity relationships and determine whether sulfur can be replaced, an analogue of lacticin a in which oxygen atoms replace sulfur was synthesized by solid phase peptide synthesis. utilizing the stereochemicaly pure oxa-lanthionine ( ) and oxa--methyllanthionine ( ) with orthogonal protecting groups, the conformationally constrained tricyclic moiety in oxa-lacticin a was constructed. the results of biological evaluation of the oxidatively stable analogue will also be described. controlling -helical secondary structure of oligopeptides and its use as a chiral catalyst replacement of -hydrogen atom of -amino acids results in ,disubstituted amino acids. as an , -disubstituted amino acid, achiralaminoisobutyric acid (aib) is well-known, and widely used to construct helical secondary structures of oligopeptides. the helical secondary structures constructed by using aib usually show -helices, but nothelices in the case of short oligopeptides. recently we designed and synthesized a chiral cylic , -disubstituted amino acid (s,s)-ac c dom , in which the -carbon atom is not a chiral center but chiral centers existing at the side chain. homooctapeptide composed of (s,s)-ac c dom formed a left-handed -helix both in solution and in the solid state. furthermore, in the case that the (s,s)-ac c dom was incorporated into l-leuhexapeptide, the hexapeptide cbz-{l-leu-l-leu-(s,s)-ac c dom } -ome preferentially formed a right-handed -helix in the crystal state, whereas the hexapeptide cbz-{l-leu-l-leu-aib} -ome formed a right-handed -helix. the fi nding that the propensity of cyclic amino acid ac c dom is to form -helix over -helix, stimulated us to use the -helical oligomer containing the cyclic amino acid as an asymmetric catalyst. we prepared several l-leu-oligopeptides containing , -disubstituted amino acids, analyzed their preferred secondary structures, and studied a enantioselective reaction of prochiral substrate using -helical oligopeptides as a chiral catalyst. fbp domains: spps using pseudoproline and depsipeptide approaches, stability and structure of glutamine-rich analogs (nature biotech., ) . one peptide will soon be tested in clinical studies. the success depends on effi cient synthetic access to large amounts of the peptide. systematic modifi cations of the lead structure hbvpres/ - have to be performed resulting in a panel of peptide variants to be studied with respect to their applicability, pharmacokinetics and serum stability. we selected a series of peptides carrying deletions, point mutations, d-amino acid exchanges and sequence permutations. the syntheses of derivates were performed by fmoc-solid-phase synthesis on a rink amide am resin using hbtu/dipea activation on an applied biosystems a peptide synthesizer. after completion of the peptide sequence, stearic acid was attached to the n-terminus. the products were deprotected and detached from the resin by tfa treatment and subsequently purifi ed by hplc. the stability of the peptides was determined in human serum. we were able to synthesize all hbvpres lipopeptides in acceptable yields ( - %). preparative hplc was used to obtain intended compounds in high purity as determined by hplc and esi mass spectrometry. the serum stability studies revealed exceptionally long biological half-lives (t / > h for all lipopeptides) mainly due to the fatty acid residue. the peptides belong to a class of intrinsically unfolded peptides and are therefore not prone to aggregations within the synthesis. consequently solid phase peptide synthesis provides a suitable access to the peptides described. it can be speculated that the peptides are protected against degradation due to an association via their lipophilic end to serum proteins. sugar derivatives for self-assembling -cyclic peptides brea last years, numerous inorganic and organic nanotubes have been developed. self-assembling peptide nanotubes (spn) made from cyclic peptides have structural and functional properties that may be suitable for various applications in biology and material science. recently, our group have reported , -cyclic peptide ( , -cp) that forms highly stable homo-and/or heterodimers with partial hydrophobic cavities. in the present communication we will describe the design, synthesis and applications of a new class of self-assembling -cyclic peptides containing sugar derivatives that modify both the inner and the outer surfaces of the resulting supramolecular entities. peptido .rotaxanes: can a tetramide macrocycle travel to a station by wrapping up around a helical peptide thread? we are currently expanding this fi eld by synthesizing and studying the properties of new sets of peptido .rotaxanes. the initial set examined includes symmetrical, achiral compounds with a fmoc stopper at each terminus, threads built up with a central fumaric diamide station and two helical aib ( -aminoisobutyric acid) homo-peptides of different lengths (from to residues), and an aromatic tetramide macrocycle. these supramolecular systems have been characterized spectroscopically and two of them by x-ray diffraction as well. the fundamental interactions between macrocycle and thread (the same interactions offering the major contribution to the template-directed preparation of this family of molecules) are intercomponent h-bonds comprising the four amide groups in the ring and the two amide bonds in the fumaric derivative. the second, more complex, set of peptido .rotaxanes examined is represented by non-symmetrical compounds involving a thread based on a central helical -(aib) -peptide linker and two stations of opposite chirality (a -d-leu-gly-gly-tripeptide at the n-terminus and a fumaric diamide-l-leu moiety at the c-terminus). as stoppers and the macrocycle, we selected two diphenylacetyl groups and the usual aromatic tetramide, respectively. as spectroscopically assessed, the macrocycle initially positions on the fumaric diamide-l-leu station. subsequently, by using photons as stimuli to induce the fumaric<->maleic equilibrium, we were able to switch partially the relative macrocyclebinding affi nity in favor of the -d-leu-gly-gly-tripeptide station. this is the fi rst example of a rotaxane where the ring makes a journey to one of the stations by wrapping up around a helical peptide thread. synthetic novel n phenyl and bi-phenyl tetracarboxamides bis peptides. an approach to dna threading intercalators as expected potential pharmaceutical carriers for catatonic agents. naphthalene diimides were reported to bind to dna opposite grooves via the threading intercalation mode ( , ) . on the other hand, peptides constitute an excellent class of molecules for rapid drug discovery and lead optimization. the title bis dipeptides may, consequently, offer signifi cant dna biological probes. potential anticancer drugs or drug carriers could thus be presumed. herein, the title bis peptides a and b were suggested and synthesized as new prototype candidates of such class compounds. pre-assembled and purifi ed peptides via the conventional methods of peptide synthesis are, subsequently, coupled to either , , , -benzene tetra-carboxylic dianhydride or , , , -naphthalene tetra-carboxylic dianhydride. the expected structures of obtained a and b as preliminary candidates were confi rmed via the chemical, chromatographic and spectroscopic methodologies. the chemistry of both a and b will be discussed. synthesis of other candidates, the corresponding biological, pharmacological and physicochemical investigations are under realization. x = phenyl or bi-phenyl ring [compound a and b respectivel the fragment pth( - ) is suffi cient to bind and activate the pth type i receptor (pth r). the molecular mechanisms by which pth binds to and activates the pth r have been extensively investigated. recent investigations focusing on the interaction of n-terminal modifi ed fragments pth( - )nh with pth r showed that certain modifi cations can increase signalling potency and that enhancement of the -helicity in the pth( - ) sequence yielded potent analogues of pth( - )nh . the design of cyclic analogues represents a widely used strategy to increase peptide stability and potency. the structural constraint induced by cyclization reduces conformational fl exibility and may enhance potency, selectivity, stability and bioavailability as well as membrane barrier permeability. initially, the work was concentrated on conformational constrains in n-terminal such as the introduction of ctetra-substituted amino acids. global restrictions in the conformation of a peptide are possible by limiting the fl exibility of the peptide strand through cyclization. to this purpose, the amino acid side chains that are not involved in receptor recognition are connected together or with the peptide backbone. previous works on the role of side chains of aa determined through d-scan, analogues which maintained better -helical structure, contained d-gln in position and . recently gardella and co-workers reported that an analogues of pth( - ) cyclized between and residues exhibited almost the same activity of linear analogues. so, in this work two new potent cyclic analogues in position and were synthesized directly on spps, using side chains of lys and glu or ser. then they were biologically tested and analyzed by cd, according to our previous work. boudreau, marc; vederas, john university of alberta, canada the neopetrosiamides a and b are two diastereomeric tricyclic peptides that inhibit amoeboid invasion of human tumor cells. they were isolated by anderson and co-workers from the marine sponge neopetrosia sp. collected in papua new guinea, with their subsequent structure elucidation by the same group in . the peptides are residues in length and contain three disulfi de bonds, as well as the unusual amino acid methionine sulfoxide at position . the two peptides differ only by being epimeric at this sulfoxide functionality. we report the total synthesis of neopetrosiamides a and b as well as an analog wherein the methionine sulfoxide has been replaced by norleucine, the carbon analog of methionine. the strategy involved solid phase peptide synthesis to generate the linear species, and selective formation of the disulfi de bonds from orthogonally protected cysteine residues. synthesis of mimetics of the antibody d a : a new class of antitrombotics. in the lab for tromboses research of the kulak (belgium) an antitrombotic antibody (ab) called d a has been characterized ( ) . the ab inhibits the interaction between the plasma peptide von willebrand factor (vwf) and by injury exposed collagen, a prerequisite interaction in thrombusformation in arteries (high blood shear). it has been shown in vivo that the ab d a has an antitrombotic effect without showing the bleeding complications that known antitrombotics exhibit. this makes the ab an attractive lead for antithrombotic drug design. by x-ray and mutagenesis studies, the paratope (active site) of the antibody has been determined. amino acids, discontinuous in the primary structure of the antibody but a quasi linear unit in space, play an important role in the interaction. incorporation of the functional groups of the amino acids and fi xation of the secondary structure of the paratope will be the aim in the design of the paratope mimetics. with this rational design we will try to develop an orally available, synthetic paratope peptidomimetic with the same antitrombotic effect as the antibody. the development (solid phase and solution peptide synthesis) of the mimetics is a stepwise process. in each step conformational restrictions are introduced. in this we hope to divert away from the peptide and go to a drug like molecule. peptide ligands for sh -and ptp domains containing phosphotyrosine are of great interest to infl uence the activity of kinases, phosphatases and other functional proteins. backbone cyclization can help to stabilize these ligands against proteolytic degradation and to form their bioactive conformation. till now backbone cyclization was performed with bifunctional and in few cases with trifunctional amino acids but not with phosphotyrosine. the assembly of such peptides requires preformed building units. because the necessary reductive alkylation of phosphotyrosine derivatives leads only to very low yields we used n-functionalized pseudodipeptides with the unprotected phenolic hydroxyl group. for fragment condensation we synthesized building units of the common structures: fmoc-aaØ[co-n(x)-(ch )n-nh-alloc)tyr(oh)-oh and fmoc-aaØ[co-n(ch )m -cooall)tyr(oh)-oh. depending on the steric hindrancy of the n-terminal amino acid these pseudodipeptides were synthesized in solution or at sasrinresin. they were purifi ed by fl ash chromatography and analytically characterized by hplc, esi-ms and nmr. we tested our strategy on the synthesis of an octapeptide-ligand for the n-terminal sh -domain of the phosphatase shp- : glu-gly-leu-asn/abu-ptyr-nle-asp-leu-nh . couplings of the dipeptide units were performed with pybop, stepwise coupling to the peptide fragments with unprotected phenolic hydroxyl group with pentafl uoro phenylesters. after fi nishing the assembly the obtained polymer bound octapeptides were consecutively cyclized, phosphorylated, removed from the resin and purifi ed by hplc. based on elucidated side reactions the synthetic strategy was optimized. the obtained backbone cyclic and phosphorylated ligands were tested for their infl uence on the phosphatase activity of shp- . the found enzymatic activities are correlated to size, direction, hydrophobicity and conformational fl exibility of the lactam bridges. biology and medicine. in this context, we have devised a new family of compounds named « polyamide amino acids » (paas), constituted by a pna (peptide nucleic acids) backbone mimicking rna sugarphosphate backbone, onto which aminoacid residues are linked. therefore, these paas could constitute a new type of rna ligands, liable to specifi cally interact with an rna target through an original interaction mode. to assess the ability of paas to be potential rna binders, we fi rst prepared tetra-paas (t -t ) via solid-phase synthesis, starting from paa monomers deriving from alanine, phenylalanine, lysine and arginine residues. interactions of the tetramers with a hiv- tar rna fragment, taken as a target model, was investigated by fl uorescence spectroscopy and circular dichroism. to give insights about specifi city, binding affi nities to tar were also assessed using an excess of a trna mixture. results showed kd values varying from . to m, indicating the importance of the aminoacid side chains in the interaction. thermodynamic analyses revealed that even if electrostatic interactions play a part in the complex formation, the binding is enthalpy-driven, highligthing the importance of non-electrostatic interactions in the recognition process. these results are of special interest since rna/ ligand association specifi city is typically assumed to be due to shortrange non-electrostatic interactions. moreover, t -t were shown to be specifi c to tar in the presence of an excess of trna. all together, these results are encouraging and they highlight the potential of paas as rna ligands. indeed, the use of only four paa monomers as building blocks leads to tetra-paas displaying both affi nity and specifi city for their rna target. studies on the solid phase synthesis and selective detection of peptide derived amadori products by mass spectrometry stefanowicz mass spectrometric analysis of glycation products of proteins and peptides attracts increasing attention ( ) . peptide-based amadori products could be used as markers of diabetes mellitus, which makes them the subject of interest in clinical chemistry. recently, several procedures of siteselective synthesis of amadori-modifi ed peptides has been published [ , ] . in this communication we will present the synthesis of a new, fully protected derivative of glycated lysine which was successfully applied as a building block for incorporation of the glycated lysine moiety into peptide chain according to the standard fmoc-solid phase synthesis protocol. we will also present a new and straightforward method of selective detection of peptide-derived amadori products by esi-ms basing on characteristic neutral losses in the sugar moiety. the proposed approach in contrast to the neutral loss scanning, which requires triple quadrupol mass spectrometer, can be performed on the instruments with higher resolution and sensitivity, like q-tof . new apa inhibitors interacting with the s subsite bring new insights in the apa substrate specifi city mediated by the calcium ion. aminopeptidase (apa, ec . . . ) is a membrane-bound zinc metallopeptidase involved in the maturation of brain angiotensin iii, a peptide which exerts a tonic stimulatory action on blood pressure in hypertensive animals ( ) . therefore, developing inhibitors of this enzyme should result in new antihypertensive agents with possible new application in the treatment of certain forms of hypertension ( ) ( ) . we and others have previously reported the design of such inhibitors ( ) ( ) . nevertheless, the improvement of the affi nities of the inhibitors is relatively impaired since the three dimensional ( d) structure of the enzyme is not available. with the aim of getting insight into inhibitors optimization, a d model of apa was constructed in our group as an alternative ( ) . furthermore, it is well established that apa substrate specifi city and enzymatic activity are calcium dependent. in order to render this model more accurate and so on to identify the amino acids residues involved in calcium binding, we have designed new inhibitors able to explore the apa s subsite to better understand its specifi city. we will report the synthesis of these new inhibitors, as well as an inversion of the specifi city of apa s subsite in absence of calcium. these results and the identifi cation of the amino acids implicated in calcium binding will be discussed on the basis of site-directed mutagenesis studies. one of the designed inhibitors, ni (ki = nm) is to date the more potent inhibitor of apa activity measured without calcium. altogether, these data should allow the improvement of apa inhibitors by structure aided design. synthesis, resolution, and absolute confi guration of bpaib, a benzophenone-containing c -tetrasubstituted -amino acid photoreactive amino acids with benzophenone side chains, the prototype of which is bpa ( -benzoyl phenylalanine), have found numerous applications as photo-probes for covalent modifi cation of enzymes and receptors, as well as in intramolecular quenching by a nitroxide free radical in trichogin peptide analogs. however, the remarkable fl exibility of the bpa side chain may question the extrapolation of results of photo cross-linking experiments and photophysical data to protein mapping and intramolecular distances, respectively [m. saviano et al., chembiochem, , , - and references cited therein]. to overcome this problem, we designed a new "constrained bpa" amino acid, bpaib, belonging to the sub-class of the ci <->ci cyclized, c -tetrasubstituted -amino acids (strong -turn and helix inducers in peptides). racemic boc-bpaib-oh was prepared by bis(alkylation) of ethyl isocyanoacetate under phasetransfer conditions with -benzoyl- , -(bis)bromomethyl benzene as alkylating agent, followed by acidic hydrolysis, n -boc protection, and saponifi cation of the ester function. resolution was achieved through poster abstracts the terminally-blocked dipeptide bz-bpaib-l-phe-nhchx with chromatographic separation of the diastereomers and acidic hydrolysis. x-ray diffraction analysis of a crystal of a z-bpaib-l-phe-nhchx diastereomer allowed the assignment of the absolute confi guration of the bpaib enantiomers. photo-crosslinking studies with this residue are currently in progress. n-methylation of n -acetylated, c -ethylated, fullyextended homo-peptides: synthetic and conformational aspects moretto peptides characterized by single or multiple n-methylated, ctrisubstituted (protein) -amino acids are one of the subject of increasing interest in medicinal chemistry. several naturally occurring peptides, remarkably stable to proteolytic attacks, are based on n-methylated peptides. n methylation of the -conh-function is a useful tool for discriminating solvent exposed from intramolecularly h-bonded secondary amide groups in peptides. we are currently extending this reaction to linear peptides based on c -tetrasusbtituted -amino acids. after having investigated synthesis and conformation of the n-methylated homo-peptides from the c -methylated, helicogenicaminoisobutyric acid and c -methylnorvaline residues [a. moretto et al., biopolymers (pept. sci.) , , - ], in this work we examined the n-methylation reaction on homo-peptides from c , -diethylglycine (deg), known to overwhelmingly adopt the fully-extended, multiple c , conformation. we studied the following peptide series: ac-(deg) n -n(et) , with n = - . under the experimental conditions used, only monomethylation (on the n-terminal, acetylated residue) takes place. our ft-ir absorption, nmr, and x-ray diffraction analyses support the view that the fully-extended conformation preferred by the original peptides is dramatically perturbed in all of the derivatives mono-methylated at position . computational study on secondary structure of oligopeptides containing chiral , -disubstituted -amino acids we have shown mcmm conformational search method and amber* force fi eld using macromodel is useful to predict secondary helical structures ( -helix, -helix) of oligopeptides prepared from ,disubstituted -amino acids. moreover, we have studied conformational analysis of oligopeptides containing chiral , -disubstituted -amino acids to predict the helical screw sense of helical structures. we calculated , -disubstituted peptide using mcmm conformational search with various force fi elds (amber*, mmff, opls) . in the case of using amber* force fi eld the results were in agreement with those of x-ray and were most stable conformation evaluated by - g level molecular orbital calculation. these results indicated that computational simulation using conformational search calculations with amber* force fi eld is most useful for conformational analysis of oligopeptides containing , -disubstituted -amino acids. a new colorimetric test for solid-phase amines and thiols. simple colorimetric tests still remain the most convenient tests for providing information on the course of solid-phase reactions. a colour test, which indicates the presence or absence of a functional group by visual detection, is a simple, practical tool to monitor the completeness of the reaction. although the variety of colour tests for amines, there is still a need for a reliable and sensitive test in some synthetic approaches. here, we wish to report a new simple, fast and sensitive colorimetric assay for the visual detection of solid-phase bound primary, secondary amines and solid-phase bound thiol groups using -alkyl- -aryl-imidazo[ , -a]pyrimidinium salts. in the search for a new synthetic approach to polysubstituted -aminoimidazoles ( ), we have reported a new procedure, employing substituted -aminopyrimidines and -bromocarbonyl compounds. the intermediate imidazo [ , a] pyrimidinium salts in the synthesis of substituted -aminoimidazoles appeared to give a strong colour when reacted with primary and secondary amines. due to the strong colour change of amino functionalised resins after reaction with the "desc" reagent ( ), a protocol for the detection of resin bound primary and secondary amines has been developed. the "desc" test is a new, practically, simple, quick and reliable test for the visual detection of resin bound primary and secondary amines in a sensitive way. the "desc" reagent is accessible from the cheap starting materials and can be prepared in two steps. tetrafl uoroborates of chiral n-triazinylammonium salts were found stable and useful in enantioselective (ee up to %) peptide synthesis from racemic amino acids in solution. several chiral n-triazinylammonium tetrafl uoroborates were obtained as stable l and/or d selective coupling reagents ( ) . broad range of common amines (strychnine, brucine, sparteine etc.) were found useful as a chiral auxiliary, opening access to amino acids of both required confi gurations. we have attempted to expand the scope of application of this family of reagents expecting their effi ciency in enantiodifferentiating reactions in solid phase peptide synthesis (spps). tetrafl uoroborates of chiral n-triazinylammonium salts were obtained by treatment -chloro- , -dimethoxy- , , -triazine with tetrafl uoroborates of appropriate tertiary amines in the presence of sodium bicarbonate ( ) . enantiodifferentiating reagents were used in synthesis on wang resin under classic condition for triazine based coupling reagents ( ) . the most important advantage of application of chiral n-triazinylammonium tetrafl uoroborates is the predictability of confi guration and repeatability of enantiomeric purity of incorporated amino acid. even if only threefold excess of racemic carboxylic acid, enantiomeric purity of coupling products in spps reached ee - . the demand for diversity of non-proteinogenic amino acids, willingly used as new building blocks, is severely restricted by laborious procedures leading to enantiomerically homogeneous individuals. typical sources such as isolation from natural products, biotechnological methodology, even the asymmetric syntheses posses a limited value because complex or tedious synthetic procedures or limited access to the pool of chiral auxiliaries. herein we present the novel, general approach allowing enantioselective incorporation of any enantiomer of amino acids directly from racemic substrate. according to our procedure, the chiral auxiliary is used only at the stage of enantioselective activation. the departure of chiral auxiliary yields in the traceless way, the activated derivative of n-protected amino acid identical with those which is obtained with the well known, classic, achiral triazine coupling reagent ( ) . therefore, the traceless chiral coupling reagents can be successfully applied directly in the coupling stage without additional studies of their reactivity. several chiral n-triazinylammonium tetrafl uoroborates were obtained as stable l and/or d selective coupling reagents. broad range of common amines (strychnine, brucine, sparteine etc.) were found useful as a chiral auxiliary, opening access to amino acids of both required confi gurations. the most important advantage of application of chiral n-triazinylammonium tetrafl uoroborates is the repeatability and predictability of enantiomeric purity and confi guration of incorporated amino acid. freeman, noam s.; hurevich, mattan; gilon, chaim hebrew university jerusalem, israel azapeptides are peptide analogues in which one or more of thecarbons, bearing the side chain residues, has been replaced by a nitrogen atom. azaamino acid residues conserve the pharmacophores necessary for biological activity while inducing conformational changes and increased resistance to proteolitic degradation. these properties make azapeptides an attractive tool for structure-activity relationship studies and drug design. a general approach for solid phase synthesis of azapeptides has been developed based on the in-situ activation of n- -( , dimethoxyphenyl)propan- -yloxycarbonyl (ddz), n €™-substituted hydrazines, with phosgene, followed by introduction to n-terminus of resin-bound peptide. the ddz-aza-amino building units include aliphatic, aromatic and functionalized side chains, protected for synthesis by the fmoc strategy. solid-phase azapeptide synthesis is demonstrated including selective mild deprotection of ddz with mg(clo ) and coupling of the next amino acid with triphosgene. the mild ddz deprotection is also orthogonal with boc chemistry. we describe the synthesis of n €™-substituted ddz protected hydrazines which have wide applications in the synthesis of azapeptides as well as in general synthesis of substituted hydrazines and aza containing peptidomimetics. ddz protected hydrazines offer the possibility to construct novel and unique drug-candidate structures such as branched and cyclic azapeptides. fast improved synthesis of insulin-like peptides barlos the a-and b-chains of insulin-like peptides were synthesized by various methods. the best results were achieved by dividing the sequences of the a-chain into three protected fragments and that of the b-chain into two fragments. the fragments were prepared on -chlorotrityl resin and/ or -methoxybenzhydryl resin using fmoc-amino acids. condensation of the fragments was carried out either by solution phase or solid phase techniques. the joining of the a and b chains was also studied using either biomimetic or chemoselective oxidative folding methods. a new approach for amides and peptides chemical synthesis by means of phosphonic acid/alkylene oxide chemistry few different derivatives of l-phe were synthesized, using originally discovered method of phosphonic acid/alkylene oxide chemistry. this method provides the successful synthesis of variety amides and dipeptides without preliminary protection of alfa-nh group of lamino acids. the supposed mechanism displays the formation of nphospholane carboxyanhydride ( -oxy- -aza- -phospholane- -one), or an o-hydroxypropyl h-phosphonate salt of an amide of l-phe. the nphospholane carboxyanhydride is protected at the n-terminus, as well as is activated at the c-terminus. this pathway favors the desired reaction with a variety of nucleophiles from the n-to c-terminus. this method also allows the synthesis of different esters of amino acids employing alcohols as nucleophiles previously described by us ( ). formation of disulfi de bonds in synthetic peptides is one of the more challenging transformations to achieve in peptide chemistry, in view of possible formation of oligomeric by-products and other side reactions, as well as occasional solubility problems in aqueous oxidizing media. the recently introduced polymer-supported oxidant, clear-ox™, has proven to be a very valuable tool in the preparation of disulfi de-bridged peptides. [ , ] oxidations using clear-ox were carried out at ph ranging from to in water/acetonitrile solutions, with concentrations - times higher than comparable solution oxidations. the latest progress in the preparation of various monocyclic and bicyclic peptides will be presented. the amyloid -peptide (a ) is believed to play a causal role in alzheimer's disease (ad). one of the hypotheses of a neurotoxicity is that it induces the generation of reactive oxygen species (ros) and hydrogen peroxide (h o ) formation by binding to metals such as copper and iron. it is hypothesized that the sole tyrosine residue plays an important role in a -mediated toxicity, due to its ability to form a dityrosine cross-link from tyrosyl radicals generated in the highly oxidative environment in the brain. hence, studies of the dityrosine cross-linked a peptide dimers would increase our understanding of the oxidative alteration and dimerisation of a in amyloid formation and ad related neurodegeneration. we are investigating the synthesis of the a peptide dimers containing the dityrosine cross-link. dityrosine, suitably protected for solid-phase peptide synthesis (spps), has been prepared from iodotyrosine using a miyaura borylation-suzuki coupling method. studies on the synthesis of dityrosine-linked peptide dimers through incorporation of dityrosine in spps are underway. initial model studies employing , -diaminopimelic acid (dap) have validated this approach. preparation of the model daplinked a peptide dimers will be discussed, as well as progress towards the dityrosine-linked a peptide dimers. nepomniaschiy, natalia; brik, ashraf ben-gurion university of the negev, israel the staudinger reaction, discovered nearly a century ago, occurs between a phosphine and an azide to form an aza-ylide. this transformation was further explored and developed, leading to several reactions of highly synthetic importance. traceless staudinger ligation is one example of such modifi cations, in which an ester moiety is placed within the phosphine structure to capture the nucleophilic aza-ylide, by intramolecular cyclization, leading to a stable amide bond. while the aza-ylide intermediate is known to be stable in organic solvents, it tends to hydrolyze rapidly under aqueous media to furnish the primary amine product. in the traceless staudinger ligation, however, the reduction of the azide to amine is a competing side reaction, as it would reverse the capture step leading to two peptide fragments. we have found that such reduction step would be benefi cial if an electrophile and an azide (rather than the phosphine) are placed within the switch peptide system. investigating various reducing reagents led us to the discovery that tris( -carboxyethyl) phosphine hydrochloride (tcep) is excellent azide reducing reagent. both the reduction and the acyl transfer steps are rapid and occur in a few minutes. applying the tcep-mediated triggering in switch peptides, derived from the a to understand the currently unclear processes of pathological folding, self-assembly, and aggregation of amyloid peptide will also be reported. fast fmoc-deprotection reagent for peptide synthesis diffi cult sequences have long been known in solid phase peptide synthesis, theses sequences can experience sever aggregation both during synthesis and under purifi cation. the aggregation is believed to depend mainly on -sheet formation or/ and hydrophobic properties of the peptide. now a days there are several strategies to circumvent theses problems, one of the most successful is to incorporate a backbone protective group as the hmb group, develop by johnson and co-worker( ), and thereby prevent aggregation due to -sheet formation. an additional charge will increase the solubility of peptides towards aqueous solutions and thereby facilitate the purifi cation by hplc (using acetonitrile and water system) and analysis (by maldi-tof mass spectrometer) of the peptide. here we present the nmec (n-methyl-n-[ -(methylamino)ethyl] carbamoyl) group( ) that is a orthogonal protective group for tyrosine side chain and the hydroxyl moiety of the hmb group. the nmec group is fmoc compatible and is protected during the synthesis with a boc group. the fi nal cleavage from the resin with tfa renders the amine of the nmec group protonated and will increase the solubility of the peptide during purifi cation and analysis. the nmec group can be easily removed with a mild alkaline treatment by a cyclization elimination reaction. the nmec group has been employed in the synthesis of diffi cult and hydrophobic peptides as the membrane spanning sequence of the calciton receptor, amyloid ( - ) and amyloid ( - ) with success. [ , ] . for example, infl uenza a virus-related peptide (h-gilgfvftl-h) with a diffi cult sequence was synthesized using o-acyl isodipeptide unit. analysis of the crude peptide revealed high purity of the product with no by-product derived from the diffi cult sequence or epimerization. using ch cl as solvent in coupling the isodipeptide unit, a - was also synthesized with almost no major side reaction .. racemization-free segment condensation method was developed by employing n-segments possessing a c-terminal urethane-protected o-acyl ser/thr residues .. the synthesis of long peptides/proteins by racemization-free segment condensation has thus become possible at ser/thr residues and not only c-terminal gly/pro residues. growing interest to peptide substrates, inhibitors and other peptidomimetics required new highly effective synthetic techniques. the important goal of enzymatic peptide bond formation is the optical purity of the peptide, which facilitates the product isolation. thus using enzymatic condensation as a last step in peptidomimetics production is preferable. fixing of enzymes on/in suitable insoluble supports has many advantages: high operational stability, ease of separation, possibility of recycling and improved activity in low water media. chitosan, natural hydrophilic polysaccharide, was used as a matrix as it has distinct advantages over the other supports due to (a) its renewable nature -it is available in large quantities as a waste product from fi shing industry and (b) its excellent fi lm-forming ability, allowing attachment to reactor walls for advanced processing. serine proteases subtilisin and chymotrypsin, and cysteine protease papain were immobilized onto chitosan. the fi lms of biocatalysts were prepared by drying the mixed solution of chitosan and enzyme in acetate buffer ph . . treatment with glutaraldehyde was found to give material with high stability and good mechanical properties. the obtained biocomposites showed high amidase activity against specifi c chromogenic peptide substrates and protein substrate azacasein. immobilized subtilisin and chymotrypsin also possess esterase activity against p-nitrophenyl acetate. the longterm storage in aqueous buffer and acetonitrile had a little effect on the hydrolytic activity of subtilisin-based biocomposite. the dependence of subtilisin/chitosan hydrolytic activity on temperature and ph was studied. obtained samples possessed high synthetic activity and were capable to catalyze peptide bond formation in dmfa/mecn mixture in reaction zaalome+fpna zaalfpna for subtilisin, zaaome+lpna zaalpna for papain. the product yield reached - % in h. acknowledgement: this work was supported by rfbr - - synthetic chemistry or on solid phase and b) the chemical ligation of the [cys (acm)]-( - )-thioester with the [cys (acm)]-( - ) segment, both deprotected and hplc purifi ed. the required intermediate peptides were prepared by optimized fmoc-based methods either stepwise or by convergent synthesis. for the formation of the two disulfi de bridges a two step procedure was investigated, involving a dmso oxidation step to form the cys -cys linkage, followed by iodine oxidation to form the cys -cys bond. native chemical ligation at valine haase, christian; rohde, heike; seitz, oliver humboldt-universität zu berlin, germany native chemical ligation is perhaps the most useful technique for peptide segment coupling in water. ( ) a c-terminal peptide thioester reacts with an n-terminal cysteine. the requirement for this rare amino acid represents the major bottle neck to the synthesis of native proteins. several strategies have been developed to overcome this limitation. in the extended ligation, n-terminally attached auxiliaries allow access to other ligation junctions by mimicking the cysteine-thiol moiety. the ligation-desulfurization approach employs -thiol amino acids for fragment coupling followed by sulphur removal. herein, cysteine acts as precursor of the abundant alanine( ) and phenylalanine can be obtained by using its -mercapto derivative. ( ) . we demonstrate the application of penicillamine in the generation of valine junctions employing the ligation-desulfurization approach. the , -dimethylcysteine building block is commercially available with various protecting group patterns suitable for routine solid phase synthesis of peptides. the ligation at penicillamine proceeded surprisingly fast despite the steric demand at the thiol group. even leu-val ligation sites, which appear in hydrophobic peptide segments, are accessible. we also present an improved method for achieving metal-free desulfurization and show applications in the synthesis of valine-containing peptides.( ) references: . p. e. dawson the application of n-alkyl cysteine (nac)-assisted thioesterifi cation reaction to the synthesis of polypeptides by the thioester method azide as a protecting group for lysine side chains on the solid phase peptide synthesis oriented toward the peptide condensation by the thioester method peptide ligation chemistry has been developed based on the use of peptide thioester as a building block in the thioester method ( ) and native chemical ligation ( ) . we found that a cysteine-containing peptide is transformed into the corresponding s-peptide (peptide thioester) by the n to s acyl shift reaction ( ). on the other hand, in , zanotti et al. reported that a diketopiperazine thioester, cyclo(-cys(coch ph)-pro-) ( ) was formed when a dipeptide p-nitrophenyl ester, phch co-cys(st-bu)-pro-onp ( ), was treated under reductive aqueous conditions ( ). thioester would be formed via intramolecular n-s acyl shift reaction followed by diketopiperazine formation. based on these observations, we designed a cysteinyl prolyl ester (cpe) autoactivating unit for preparation of the peptide thioester and for the peptide ligation [ , ] . a peptide containing the cpe unit, peptide-cpe , was transformed into a peptide thioester of diketopiperazine, cyclo(-cys(peptide)-pro-) , then thiol-thioester exchange reaction with an external thiol produced the peptide thioester and a diketopiperazine, cyclo(-cys-pro-) ( ) . the poster abstracts peptide-cpe was also able to ligate with a cysteinyl peptide , via the thioester in one-pot, to give a polypeptide . ( ) has enabled the synthesis of entire proteins including those carrying posttranslational modifi cations. one of the most frequently encountered modifi cation of eukaryotic secretory and cell surface proteins is the attachment of oligosaccharides to asparagine residues (n-glycosylation). despite many efforts in this fi eld the function of nglycosyation is poorly understood, which is mainly caused by the lack of pure glycoproteins. since purifi cation of natural glycoproteins is quite tedious due to heterogeneity in the sugar part, the total synthesis of homogeneous glycoproteins has become an attractive target ( ) . we have addressed this topic by choosing rnase c as a model glycoprotein. instead of the oligomannosidic type rnase c is containing a complex type n-glycan. for synthetic reasons ligations had to be conducted in a sequential manner ( ) by fi rst reacting the recombinant - cys-segment ( ) with an n-terminally protected and glycosylated thioester - .. selective deprotection of the ligation product - and ligation with synthetic thioester - in a one pot manner was accompanied by unexpected side reactions. these drawbacks were fi nally overcome leading to full-length glycosylated rnase c displaying enzymatic activity. a new pseudo-native ligation: multiple, successive azidealkyne cycloadditions. ( ) the reaction has become a classics. as triazoles are stable to acid and basic hydrolysis, and reductive and oxidative conditions, the reaction has been widely used in chemistry and biochemistry, proving to be an useful tool in combinatorial, bioconjugate or medicinal chemistry. the reaction has been applied in the peptide fi eld as an easy way to synthesize cyclic, labelled and side chain modifi ed peptides including effi cient synthesis of pseudo-glycopeptides. conformational and structural studies prove that the triazole ring is an excellent trans-amide surrogate and thus, can be considered as a new pseudo-native linkage. ( ) in order to explore the potential of the reaction as a way to successively assemble several peptide chains to generate mimics of proteins, we have elaborated a new strategy for consecutive triazole formation based on a semi-orthogonal alkyne protection scheme. ( ) so far, we have improved our fi rst one-pot successive cycloaddition approach performing the fi rst three successive cuaac in mild conditions thanks to a highly selective deprotection of two different alkyne groups. the application of this strategy represents a new promising method for the chemoselective pseudo-native ligation dedicated to the synthesis of protein chimera or for the decoration of molecular templates. solid phase synthesis using lightly crosslinked polystyrene supports has proved to be a successful route to the manufacture of peptides. the methodology introduced by merrifi eld nearly fi fty years ago has remained largely unchanged. during the last twenty years or so, it has been argued that incorporating a hydrophilic polymer within the conventional hydrophobic polystyrene matrix is benefi cial. others have suggested that polyamide or polyether-based resins may prove to be superior to polystyrene. despite this, large-scale peptide manufacture has generally relied on traditional polystyrene supports. this is due in part to the diffi culty in producing composite supports economically in large volumes, but also due to the handling diffi culties that are often associated with very high swelling polar polymers. other problems arise from the fact that many of these types of resin are relatively low loading and so yields are greatly diminished. in this poster we offer a solution to these problems: a polystyrene derived support which is modifi ed to create economical, amphipathic resins suitable for large scale peptide synthesis. attachment of appropriate handles or linkers enables both peptide acids and peptide amides to be produced. the synthesis of a variety of peptides is demonstrated. peptide synthesis in water using boc-amino acids nanoparticles reactants. here, we studied in-water solution-phase method using water-dispersible nanoparticulate boc-amino acids, which are the most common building blocks but are diffi cult to use for peptide synthesis in water. water-dispersible boc-amino acid nanoparticles were prepared by pulverization using a planetary ball mill in the presence of peg, and the size of the resulting water-dispersible nanoparticles was determined by dynamic light scattering analysis. the scanning electron microscopy image of water-dispersible boc-phe-oh nanoparticles also revealed nanosize particles. we studied in-water coupling reaction using waterdispersible boc-amino acid nanoparticles, and leu-enkephalinamide was successfully synthesized in water according to the boc chemistry. an alternative way for conopeptide formation kádár, kinga ; panyi, györgy ; tóth, gábor k. university of szeged, hungary; university of debrecen, hungary the chemistry used to oxidize the free thiol bonds to the corresponding disulfi de bond in a controlled fashion remains a signifi cant challenge in spite of many advances in peptide chemistry. the primary reason of this lies in the diffi culties involved in the formation of multiple regioselective disulfi de bonds. in this work we focused on the elucidation of synthetic strategies for the preparation of multiple disulfi de containing peptide venoms regulating the ion channels of immune cells-anuroctoxin and tc -, and a neuropeptide with regulatory functions-orexin a. for the synthesis of these naturally occuring cys-rich peptides we had chosen the oxidative folding being the most simple of the methods available. in the case of anuroctoxin we isolated the native form of the peptide toxin with high selectivity and we optimized the folding conditions, so within an hour the majority of the linear peptide folds in the cyclic form with all four disulfi de bonds in the correct form. the regioselectivity of the isolated isomer was verifi ed with biological measurements. for the synthesis of orexin a we optimized the folding conditions and, using a polymer-supported oxidant-the clear-ox resin-, within two hours we could isolate the correctly folded isomer as the major reaction product. the correct structure was proven by coelution of the isolated isomer and the commercially available orexin a. but oxidative folding does not always give the correctly folded isomer. the best proof for this is the tc scorpion toxin which albeit our tryings always gave the misfolded isomers if we used the linear, unprotected peptide. therefore a new chemical synthesis using different side-chain protection needs to be taken into consideration. the synthesis of orthogonally protected tc and its use for the preparation of the correctly folded peptide to be underway. poster abstracts have fundamental importance in biological recognition processes. one of the most challenging task among of them the rational preparation of the glycosylated peptides especially having oligosaccharide moieties. there are two main strategies for the synthesis of glycopeptides: the synthon and global (convergent) method. both of them can be implemented in liquid or solid-phase. since the glycosylation could appear on o and n atoms of the amino acid side-chain, due to the different reactivity of the glycosidic linkage different chemical strategies will necessitate. in this presentation we compare several chemical strategies for the preparation of two model peptides (leu-lys-asn*-gly-gly-pro, gly-val-glu-asp-ile-ser*-gly-leu-pro-ser-gly,*site of glycosylation). as glyco-part several mono, di and trisaccharide including chitobiose, galactosilxilose, mannosil-n-acetyl-glycosil-n-acetyl-glucosamine were used and several of the used strategies led to successful preparation of these glycoconjugates. site-specifi c pegylation of human igg -fab using a rationally designed trypsin variant in the present contribution we report on a novel, highly selective biocatalytic method enabling c-terminal modifi cations of proteins with artifi cial functionalities under native state conditions. the approach is based on the fourfold trypsin variant k e/n h/e h/d k which was generated by site directed mutagenesis and initially specifi ed in terms of selectivity and activity using a peptide library of the general structure bz aax aa x bb x cc aag-oh. the trypsin variant was found to bear a restricted proteolytic activity towards the rare recognition sequence yrh with an occurance of less than . % in native proteins. the specifi city is zinc ion dependent due to an artifi cial metal binding site in the s ´-subsite ( ). placing of the recognition sequence at the cterminal region of respective proteins via standard mutagenesis protocols provides suitable precursor targets for site-specifi c modifi cation via a transamidation reaction. due to the great demand for polymer-modifi ed antibody fragments for pharmaceutical purposes we evaluated the function of the approach on example of the c-terminal pegylation (peg...polyethylene glycol) of the human igg fab fragment. the fragment itself was expressed with the recognition sequence and an additional strep-fusion enabling effi cient purifi cation. the derivatization of the antibody fragment with kda peg via the trypsin variant (mw ~ kda) proceeds effi ciently with a total yield of isolated modifi ed protein of %. interestingly, no undesired cleavage reactions could be detected leading to fully active modifi ed antibody fragment. references: . willett, w.s., brinen, l.s. et al. ( ) . "delocalizing trypsin specifi city with metal activation." biochemistry ( ): - . -chloro- , -bis-( , , -trifl uoroethoxy)- , , -triazine and n- , -( , , -trifl uoroethoxy)- , , -triazin- yl)ammonium tetrafl uoroborates as highly effi cient coupling reagents jastrzabek, konrad; kolesinska, beata; kaminski, zbigniew, j. we expected that modifi cation of substituents in the triazine ring improve activity, stability and solubility of new triazine based coupling reagents. in order to increase activity we prepared -chloro- , -( , , trifl uoroethoxy)- , , -triazine by treatment of cyanuric chloride with , , -trifl uoroethanol. taking advantage of modular structure of triazine coupling reagents the entire family of n-( , -( , , -trifl uoroethoxy- , , -triazinyl- )ammonium tetrafl uoroborates have been obtained. effi cient coupling reagents should be useful for amide bond formation between broad range of substrates, work in stoichiometric quantities, be soluble and stable in most of the solvents. it should function effi ciently in solution as well as in spps, to get high purity crude products and minimize racemization of the products. we found n-( , -( , , trifl uoroethoxy- , , -triazinyl- )ammonium tetrafl uoroborates useful for activation of carboxylic components. the participation of triazine "superactive ester" as intermediate in the condensation has been proved in the model experiments. utility of reagents n-( , -( , , trifl uoroethoxy- , , -triazinyl- )ammonium tetrafl uoroborates were confi rmed by peptide synthesis in solution in high yield. in our study we focused our attention on the performance (in terms of purity of the crude and extent of racemization) testing the solid phase synthesis of acp( - ) and model peptides with aibaib fragment, which are a good example of diffi cult peptide sequence. purifi cation of chemically synthesised proteins by use of cleavable imac-based n -terminal protein protecting groups (tags) ( ), there remains a challenge in terms of time and cost, for the separation of impurities from the chemically synthesised product in spps. this is particularly acute in the case of protein synthesis where the crude product mixture contains capped (acetylated) truncates of comparable size and structure to the protein product. such a common situation results in extensive, and expensive, purifi cation techniques such as large scale hplc. to this end we designed a hydrophobic tag, tbfmoc ( ) , which incorporated the base-labile characteristics of the fmoc ( ) group. the tbfmoc group causes retention on hydrophobic columns and hence separation from untagged impurities. although this was effective, we were of the opinion that a complementary tag was required which would have the characteristics of metal-complexation and cleavage by -elimination for subsequent removal of the tag from the protein product after imac. considerations implicit in the design of such an n -terminal protecting group, or tag, will be discussed , and applications to the chemical synthesis of chemokines will be dealt with in the presentation. in last few decades, acridines which are known as anticancer, antimicrobal and antiviral agents have been in the center of interest of scientists. the heterocyclic molecules demonstrate a noteworthy group of compounds which interact with biological targets e.g. topoisomerase i, ii [ , ] . we wish to pay our attention to a new group of tuftsin-acridine conjugates. tuftsin offers a wide range of biological activities such as supporting of immune system, bactericidal, tumoricidal activity. however tuftsin is unstable in plasma which reduces its effi cacy. that is the reason for search of new analogues that were more resistance to proteolysis degradation ( ). tuftsin-acridine conjugates were synthesized using a fmoc solid phase strategy. the elongation of peptide chain was based on twostep procedure: deprotection and coupling reaction with dic and the additive of hobt in dmf/dcm/nmp mixture. tuftsin analogues were modifi ed at -amino group of lysine via introduction of the simple amino acids to obtain isopeptide bond. tuftsin analogues were conjugated to the acridine molecule via fl exible linkers. the carboxylic group of linker was connected to n-terminal group of peptide-resin using standard method for amide bond formation. the coupling reaction was performed with peptide-resin and the -fold excess of -nitro-acridine derivative using tbtu, hobt in the presence of diea in dmf for h. simultaneous deprotection of peptide side chain and cleavage from resin was done with the tfa cocktail. final products were purifi ed by spe and characterized by elemental analysis, ms and h-nmr spectroscopy. the effect of microwave irradiation under controlled temperature conditions on the solid-phase synthesis of peptides was investigated. for optimization studies a model peptide (h-gly-ile-leu-thr-val-ser-val-ala-val-oh) was selected which suffers from poor synthetic effi ciency under standard spps conditions. synthesis of the nonapeptide was performed using various combinations of solid supports (polysterene, tentagel, chemmatrix) and solvents employing fmoc/but orthogonal protection strategy. applying controlled microwave heating, the reaction times were signifi cantly reduced while maintaining a high purity of crude product with no racemization being observed. the optimized microwave synthesis method was succesfully applied for longer, aggregated peptides. microwave coupling and cleveage were accomplished in a dedicated reactor setup that allowed accurate internal reaction temperature measurment using a fi ber-optic probe system. comparison studies between microwave-and conventionally heated reactions will be presented. during the last few years microwave assisted peptide synthesis has became popular and it was reported to be useful in some cases. there is also an ongoing discussion about an additional "microwave effect". based on this background we have synthesized several model peptides on a microwave synthesizer and compared with the synthesis on conventional batch and continuous fl ow synthesizers with and without microwave irradiation. during this investigation we have also compared reaction rates and purity of the peptides synthesized under the infl uence of microwave irradiation or conventional heating on this different synthesizer systems by various conditions. in no case we can fi nd any additional postulated "microwave effect". the results of this investigation will be presented and discussed. conotoxins form a large family of peptide toxins from cone snail venoms that act on a broad spectrum of ion channels and receptors. the subgroup -conotoxins specifi cally and selectively bind to subtypes of nicotinic acetylcholine receptors (nachrs), which are targets for treatment of several neurological disorders. the aim of this work is to develop an improved method able to generate conotoxins in high yield and purity. this will overcome a key barrier currently preventing the effi cient synthesis of small focused libraries in order to investigate the structureactivity relationship (sars) of those peptides. the development of general synthetic strategies for the preparation of conotoxins and analogues are essential to effi ciently approach important questions within the area of neurobiology and for the development of novel drugs for treatment of various neurological diseases. a new and highly effi cient synthetic strategy for the synthesis of alpha-conotoxin-mii has been developed. this strategy combine solid phase synthesis with microwave assisted heating to produce the two disulfi de bonds peptide in high yield and purity. we fi rst report here the use of microwave assistance in order to form a disulfi de loop. this technique demonstrates the advantage of preparing the fi rst disulfi de bridge while the peptides are resin bound. this step is critical to provide the dicyclic native peptide, that was performed by a followed classical in solution oxidation by iodine strategy. an enhanced total procedure for the synthesis of ctxmii is recommended, which can be effi cient applied at several small disulfi de rich peptides of biological importance. solid phase peptide synthesis in aqueous environment using microwave assistance heating galanis, athanassios s. since the fi rst reports on the use of microwave heating more than years ago, microwave-assisted organic synthesis (maos) has become an important tool for rapid and effi cient synthesis of organic molecules. microwave assisted heating has been further applied to peptide synthesis in order to accelerate the rate of synthesis and to improve the yields for the synthesis of diffi cult sequences. as far as water as solvent is concerned, numerous recent publications report the combination of water as an environmentally benign solvent for chemical transformations with the use of microwave irradiation as an effi cient heating method. we report herein, the combination of the microwave-assisted heating and the use of water as solvent for solid phase peptide synthesis. a variety of common amino acids derivatives and coupling reagents have been studied in order to optimize coupling reactions in water by microwave-assisted heating. we also describe the total synthesis of a small peptide using water as solvent. the solid-phase synthesis using the environmental friendly aqueous medium dramatically reduces the cost of the synthesis and could be broadly applied in research or in industrial production of peptides. vanier, grace; collins, jr., michael; singh, sandeep; collins, jonathan cem corporation, united states the application of microwave energy for solid phase peptide synthesis (spps) represents a major breakthrough for overcoming incomplete and slow reactions typical of conventional spps. microwave energy has been applied successfully in a manual and automated approach for enhancing synthesis of peptides and peptidomimetics. common side reactions such as racemization and aspartamide formation have been studied and shown to be easily controllable with optimized methods that can be applied routinely. we will present the latest research on improving the synthesis of diffi cult peptides with microwave energy. synthetic antifreeze glycopeptides and analogues: synthesis, structural analysis, and functional studies norgren, anna s. glycosylated peptides are involved in various biological processes such cell adhesion and differentiation. in contrast to mucin-type oglycan peptides which are present on the membrane of mammalian cells antifreeze glycopeptides (afgps) are a little investigated example of glycosylated peptides containing similar components. afgps usually consist of a varying number of repeating units of (ala-ala-thr) with minor sequence variations and the threonine hydroxyl oxygen glycosylated with the disaccharide -d-galactosyl-( - )--n-acetyl-d-galactosamine. antifreeze activity has been proven by different experimental observations like suppression of recrystallisation and ice nucleation, thermal hysteresis and change of the crystal habitus. although it is known that the n-acetyl group at the c position of the galactosamine, the -confi gured glycosidic bond to the threonine hydroxyl group and the -methyl group are essential, the adsorption mechanism is not yet understood. ( ) . in contrast to fragment condensation, solid phase peptide synthesis gives the possibility to prepare one defi ned product and to introduce structure inducing amino acids such as proline. the disadvantage of spps with the bulky glycosylated amino acids is the low coupling effi ciency. this problem was overcome by using more active coupling reagents and microwave-enhanced methods during coupling leading to suffi cient coupling effi ciency without having to apply great excess of the glycosylated amino acid. after purifi cation the peptide structure was examined by cd and nmr in water and dmso at different temperatures. additionally, the substances were microphysically analysed according to their recrystalisation inhibition activity and their infl uence on the crystal habitus. microwave technology applied to spps has been recently proposed as valid support to the enhancement of coupling rates. we also used microwave energy in conjugation process between polyelectrolytepeptide bioconjugation with edc, hbtu. the use of peptide epitops of viruses particles in the composition of polymeric conjugates as vaccine has several potential advantages over whole viral or bacterial preparation. recently, our group report a novel approach to a totally synthetic vaccine which consists of fmdv (foot and mouth disease virus) vp peptides, prepared by covalent conjugation of peptide biomolecules with membrane active carbochain polyelectrolytes in the present study, peptide epitops of vp protein both - (p ) amino acid residues (ser-lys-tyr-ser-thr-thr-gly-glu-arg-thr-arg-thr-arg-gly-asp-leu-gly-ala-leu-ala-ala-arg-val-ala-thr-gln-leu-pro-ala) and triptophan (trp) containing on the n terminus - amino acid residues (trp- - ) (p ) were synthesized by using the microwave assisted solid-phase methods. synthesis of peptides were performed by microwave assisted spps. peptides characterized by lc-ms and purifi ed by rp-hplc. bioconjugation between polyelectrolytespeptide were synthesized by two different microwave assisted method. the fi rst one is classical carbodiimid activating method. the second one is hbtu activating method. the second method is a novel and effective method for bioconjugation process. peptides and polyelectrolytespeptide bioconjugates analysed and comparised by gpc system with four dedector (uv. refractive index, light scattering, viscosity). microwave-assisted solid-phase synthesis of peptide probes to detect specifi c biomarkers: shifting off limitations affecting conventional synthetic strategies biomarkers play a key role in development of diagnostic/prognostic tools. in fact, since their involvement in several diseases such as autoimmune diseases (i.e., multiple sclerosis, rheumatoid arthritis) and neurodegenerative diseases as alzheimer's disease, biomarkers represent a great promise for the development and set up of tuned therapies. for autoimmune diseases, we proposed the development of synthetic posttranslational modifi ed peptides as antigenic probes for characterization of specifi c and high affi nity autoantibodies as biomarkers. by an innovative "chemical reverse approach" we propose optimisation of antigenic probes by a statistically signifi cant screening of sera guided by autoantibodies circulating in patients' biological fl uids ( ) . spps via the building block approach is the principal strategy leading to modifi ed peptides. high purity is a condicio sine qua non for effi cient biomarker detection. these syntheses can present several diffi culties as result of internal aggregation of resin-bound peptides during elongation steps, reducing reagents penetration, and signifi cantly decreasing reaction rates in both acylation and deprotection steps. such events strongly affect purity of the crude peptides and therefore, fi nal yield. we demonstrated that application of microwave (mw) energy in spps of modifi ed complex peptide probes exhibits several advantages improving coupling rates, possibly because of the decrease of chain aggregation during the synthesis ( ) . we report the mw-assisted synthetic conditions of diffi cult peptides (i.e. glycosylated, citrullinated, multiple antigenic, amyloidogenic peptides, etc.) by which we strongly improved preparation of diagnostic/prognostic probes in terms of crude purity, fi nal yield, and time-consuming compared to conventional protocols. fidan, zerrin; volkmer, rudolf charité berlin, institute for med. immunology, germany the alpha-helical coiled-coil structure is a versatile protein-interactiondomain, in which the coiled-coil is composed of at least two righthanded amphipathic alpha-helices, which are coiled up around each other into a left handed supercoil. this widespread structural motif is involved in many biological procedures like transcription, scaffolding or signalling. in addition, the most characteristic and important identifying feature of a coiled-coil is the recurrence of a periodic heptad repeat sequence. coiled-coils are able to form complexes up to heptamers of different orientation. based on this signifi cant occurrence and also their structure coiled-coil peptides have the ability to function as molecular recognition molecules. our goal is to use self-associating coiled-coil sequences as molecular building blocks (lego brick). the ligation of desired functionalities on coiled-coil sequences opens up the opportunity to add different functionalities on artifi cial complex peptide molecules that stick together via coiled-coil moieties. this makes coiled-coil sequences to useful tools during complex oligopeptide assembly. we want to present novel chimeric oligopeptides which are build up by two segments, one responsible for association the other for functionality. as a model for the association segment several gcn -leucine zipper mutants ( ) and as functionality segments amongst others ww domains were used. both segments were linked by the native chemical ligation approach. we will present in detail the synthesis of a gcn -leucine zipper mutant and also the following c-terminal thioester formation for the subsequent ligation step. furthermore, we will present the synthesis of the functional device, a ww-domain with a n-terminal cystein residue and also the native chemical ligation of both synthesized peptide fragments supported by hplc and mass-chromatography. dialysis-related amyloidosis, a disease arising from long-term dialysis is characterised by gradual accumulation of -microglobulin ( m) amyloid fi brils in bones and ligaments. although -m is known to form amyloid fi brils in vitro under acidic ph conditions, seeding of preformed amyloid fi brils or as an effect of ionic strength, the mechanism underlying aggregation of soluble -m into insoluble fi brils under physiological conditions is largely unknown. interestingly, eakin and co. recently showed that the chemical basis of the amyloidogenesis process is a backbone isomerisation of the conserved pro . our aim is to understand the molecular mechanism associated with -m amyloidogenesis using fourier transformed infrared (ftir) spectroscopy and site-directed-isotope labelling, a method in which the vibration of the labelled residue is shifted from its original position in the spectrum. we already demonstrated that ftir spectroscopy along with site directed-isotope-labelling is a promising approach to obtain information on the microenvironment of tyrosine side chain residues. to use this approach in the case of -m amyloidogenesis, we decided to synthesise an isotopically labelled -m at crucial residues such as pro that should undergo drastic variations in their microenvironments during the misfolding. with its residues, -m is over the limits of a reasonable synthesis using spps but the two suitably positioned cysteines in its sequence offer the possibility of a chemical synthesis using native chemical ligation. thus, we were able to realise the chemical synthesis of an isotopically labelled -m in a good yield using a three segments strategy with disconnections at the two cysteines and thiazolidine as a masked cysteine for the middle segment. the synthetic -m obtained was in full agreement with the characteristic -m ftir spectra. a novel cyanophycin synthetase from thermosynechococcus elongatus bp- catalyzes non-primer-dependent cyanophycin synthesis. cyanophycin (multi-l-arginyl-poly[l-aspartic acid]) is synthesized by cyanophycin synthetase (cpha). it was believed that cyanophycin synthetase requires asp, arg, atp, mg + and primer (low-molecular mass cyanophycin) for cyanophycin synthesis and catalyzes the elongation of low-molecular mass cyanophycin. despite extensive studies of cyanophycin, the mechanism of primer supply is still unclear. in the present study, we searched for a cyanophycin synthetase that synthesizes cyanophycin from asp and arg without added primer in vitro. cyanophycin synthetase from thermosynechococcus elongatus bp- (tlr protein) was produced by an escherichia coli geneexpression system as a c-terminal his-tagged protein. we found that tlr protein synthesized cyanophycin without added primer. the tlr protein had strict substrate specifi city and used only asp and arg as substrates. the optimal ph was . , and mg + or mn + was essential for cyanophycin synthesis. atp could not be substituted by gtp, ctp, or ttp. the molecular mass of the tlr protein as estimated by gelfi ltration chromatography was } kda. thus, the tlr protein appeared to be a homo-tetramer of -kda subunits including the his-tag sequence. the tlr protein had thermal stability and fully retained its activity after a -min incubation at ‹c. additionally, we examined cyanophycin synthesis at ‹c, ‹c, ‹c, and ‹c. sdspolyacrylamide gel electrophoresis showed that the molecular mass of cyanophycin increased with increased reaction temperatures. synthesis of peptide thioester by fmoc chemistry through hydroxyl side chain anchoring barta , ) . azapeptides are usually synthesized on solid phase. a method, permitting the convergent synthesis of azapeptides starting from unprotected fragments, would offer the possibility to study aza amino acids effect in complex polypeptides or proteins. we have focussed our study on ligation reactions leading to the formation of an azagly residue at the ligation point, as gly is a frequent amino acid in peptides or proteins. the fi rst synthetic strategy relies on the reaction between a peptide thioester and a n-terminal azaglycine peptide. experimental conditions were found which permitted the chemoselective formation of azapeptide but with racemisation of the c-terminal amino acid of the thioester fragment. alternately, a synthetic strategy based on the chemistry of the phenylthiocarbonyl group, permitted the successful synthesis of azapeptide without racemisation. ( ), native glycoforms are of therapeutic interest. our retrosynthetic strategy implies sequential native chemical ligation ( ) and the split of il- into three fragments. an n-terminal fragment il- ( - ) thioester and the cysteine fragment il- ( - ) were expressed in e. coli. the carbohydrate carrying fragment il- ( - ) was synthesized via spps. to obtain the recombinant fragments specifi c termini were required. the n-terminal il- ( - ) was fused between two inteins and after expression and refolding from inclusion bodies the target peptide was released as a c-terminal thioester after dual intein cleavage. the cysteine fragment il- ( - ) was expressed as a single intein fusion also leading to inclusion bodies. this enabled the release of il- ( - ) with an n-terminal cysteine after intein cleavage of the refolded fusion protein. the central segment il- ( - ) contains a sugar residue attached to the n-glycosylation-site at asn . this fragment features both a c-terminal thioester and an n-terminal cysteine protected as a thiazolidine. sequential ligations leading to the full length il- glycoprotein will be presented. synthetic chemistry syntheses of shorter fragments which are fi nally linked together ( ) . this "small building block approach" should also simplify synthesis of modifi ed prion protein. in our plan of mouse prion (moprp) synthesis, we have employed consecutive chemical ligations. we have started with moprp( - ) in which it is possible to study native chemical ligation between cysteine in the position of and methionine in the position of ( ) several moprp( - ) peptide thioesters with aryl and alkyl thiols were prepared for further study of the native chemical ligation of moprp( - ) and moprp( - ). the optimal ligation conditions were found by a kinetic study which was carried out in a range of ph and with various thiols. infl uence of electron withdrawing and electron donating groups was studied in both aromatic and aliphatic thiols and it was found that it is possible to affect the ligation by choosing an appropriate thiol. ligation optimal conditions, kinetic study results, and suitability of various thiols for the ligation are discussed. low-cost industrial chemo-enzymatic synthesis of pharmaceutical, nutraceutical and diagnostic oligopeptides the production costs for oligopeptides and derivatives thereof by chemical synthesis are extremely high. therefore dsm pharmaceutical products embarked on a research programme on chemo-enzymatic peptide synthesis. important advantages of this technology are that no expensive stoichiometric coupling reagents nor side-chain protection are required and that no racemisation occurs. focus is on elongation in the n ¨c terminal direction using novel enzymatic c-protecting group interconversion methods. one of the preferred building blocks are amino acid amides, but selective deprotection of peptidic c-terminal amides is a challenge. we discovered that using a peptide amidase c-terminal peptide amides can be directly converted to methyl esters in almost pure methanol. thus, separate enzymatic hydrolysis and reactivation steps are no longer required. other versatile building blocks are amino acid t-butyl esters. we discovered that peptide c-terminal t-butyl esters can be transformed, using commercially available proteases, to activated alkyl esters such as methyl-and benzylesters which can be directly used in another protease-mediated coupling reaction. furthermore, we found that using commercial enzymes side chain unprotected peptides with a free c-carboxyl terminus can be directly transformed to cterminal thioesters and c-terminal aryl amides in the presence of the corresponding thiol and aniline, respectively. finally, a real-life example of large-scale industrial chemo-enzymatic peptide production will be shown. it is known that chiral amino acids, as well as their dipeptides, may catalyze the asymmetric condensation of glycolaldehyde in water. on the basis of the particularly large erythrose enantiomeric excesses (ee) obtained when utilizing the chiral l-val-l-val catalyst and given the possibility of an abundant delivery of other types of amino acids to the early earth, we have studied the catalytic effect on this synthesis of the peptides based on c -methylated -amino acids, such as iva (isovaline or c -methyl, c -aminobutyric acid) and c -methylvaline, ( me)val, that are abundant in meteorites. results of the catalysis experiments showed the all c -methylated peptides to the tetramer level exhibit signifi cant chiral infl uence on the synthesis of tetroses and mimic the effect of the l-val-l-val catalyst in having a larger erythrose ee than threose ee, as well as in their confi guration relationship with the sugars (the product erythrose acquires ee of confi guration opposite to that of the catalyst in case of peptides, while it is the same for amino acids). interestingly, the largest ee ( % for erythrose) was obtained with the iva homo-tetrapeptide under mild conditions. the homo-dipeptides of both iva and ( me)val also produced a signifi cant ee ( % for erythrose) that appears to increase with time. because c -methylated amino acids are non-racemic in meteorites, do poster abstracts not racemize in aqueous environments, and are known to be ( )-helix formers in peptides with as few as four residues, these results suggest that meteoritic, c -methylated, -amino acids may have contributed to molecular evolution upon delivery to the early earth by catalytically transferring their asymmetry to other prebiotic molecules. synthesis' and application of peptide adhesives for wound healing natural polymers with repeating peptide sequences, like spider silk and mussel glue have interesting properties that can be used for biomedical applications. however, the isolation from their natural sources is rather diffi cult and the desire to introduce modifi cations necessitates the development of novel methods to synthesize such polymers with repeating peptide sequences. recently, we have shown that the microwave-assisted copper(i)-catalyzed , -dipolar cycloaddition can be used in the polymerization of the model dipeptide azido-phe-alapropargyl amide. by varying the reaction conditions we could either obtain small cyclic oligomers ( - aas) or linear polymers which consisted up to aas residues ( ). to broaden the scope of this polymerization reaction, two novel biodegradable monomers, azido-phe-ala-lys-propargyl amide and azido-phe-ala-glyc-lys-propargyl amide were polymerized. both monomers are water-soluble and contain recognition sites for the proteases trypsin and chymotrypsin; moreover, the tetrapeptide can be chemically hydrolyzed depending on the ph of the solution. the molecular weight of the polymers could be tailored between - kda ( to aas residues). the enzymatic degradability of the polypeptides was monitored by a ninhydrin-based colorimetric assay and by maldi-tof. the results showed that the peptidic polytriazoles were smoothly degraded by trypsin and chymotrypsin. from these data we can conclude that the microwave-assisted copper(i)-catalyzed , -dipolar cycloaddition reaction is an effective tool to synthesize biodegradable functional polymers which provides new opportunities to design (novel) biomedical materials. details of monomer synthesis, polymerization reactions and degradation studies will be presented. peptide synthesis on cellulose membranes, known as spot synthesis, is an ideal tool to determine biological interactions and/or key positions in proteins. in general we can differ in two kinds of synthetic peptides with different possibilities: over the past years synthetic peptide library techniques have emerged as powerful approaches to determine t-cell epitopes and to specify peptide binding to mhc-i and/or mhc-ii molecules. the sequence-based approach is a straightforward method to directly identify t-cell antigens belonging to a specifi c target (e.g. a virus of interest) without the need of any further deconvolution steps. the entire protein sequence is presented as a set of overlapping peptides (peptide scan), which are subsequently screened for t-cell stimulation. several cd- t-cell epitopes in selected cmv proteins have been identifi ed using standard spps techniques. however, the peptide sequences synthesized by standard spot synthesis can only be cleaved without authentic c-termini (ß-alanine or glycine as c-terminal amino acid). here, we summarized three different established methods to generate cleavable peptides with authentic c-termini in adequate amounts, with suffi cient purity and within a justifi able time-scale. the standard spot synthesis to generate membrane bound peptides use glycine or ß-alanine membranes to map epitopes of b-cells, allergenic proteins, antibodies etc. furthermore, we have also established the "method of inverted peptides" to screen protein domains requiring peptidic ligands with free c-termini such as pdz-or . . domains. another highlight of peptide libraries is the screen of posttranslational modifi cation in proteins by phosphorylation at serine, threonine, and tyrosine residues which plays a role in eukaryotic cell cycle regulation, cell differentiation, apoptosis, or cytoskeletal regulation. labeled peptide libraries, consisting of -helices, -loops and -sheets peptides, have been successfully prepared for use in peptide arrays that act as a protein-detection system and have been applied for proteinrecognition studies [ ] [ ] [ ] . it is known that more than half the proteins in a mammalian cell are post-translationally modifi ed by such processes as phosphorylation, glycosylation, and acetylation. such modifi cations play an important role in various bio-recognition, for instance glyco- proteins are involved in cell-adhesion, infection, and biological protection. hence the designed peptide library used for arrays has been expanded to include fatty acid attached peptides and glycopeptides. fatty acids were easily introduced by conventional fmoc-spps, while the synthesis of glyco-peptides was not easy. in the present paper we describe the effi cient construction of o-glycoside structured and labeled peptides by fmoc-spps using a building block strategy. glycosylated threonine has been used as a building block and was prepared in large amounts. glucose, mannose, galactose and lactose were acetylated and coupled to the hydroxyl group of fmoc-thr in the presence of bf oet . the resulting protected sugar-amino acids were manually assembled on to the peptidyl resin containing a fl uorescent dye using a peti-syzer® (hipep laboratories). after construction of the glycol-peptides the acetyl groups on the sugar were removed by hydrazine hydrate, and the peptides were then cleaved and purifi ed. the resulting glycopeptides were characterized by lcms. the present protocol has been used to prepare ca. one hundred glycopeptides that have been tested against various carbohydrate-binding proteins. parallel small scale peptide synthesis meets a fast, lowcost purifi cation method for the production of high quality peptide microarrays to analyze dna/protein interactions herein, a parallel synthesis in a small scale ( , mol) which takes place in -micro well plates is demonstrated. with regard to a fast but yet effi cient, low-cost purifi cation in a parallel manner a ''fmoc-on'' purifi cation method, which is integrated into the synthesis process, was developed. peptides were cleaved from the resin with their fmoc-group still on. the crude products were transferred by extraction into another -micro well plate fi lled with purifi cation material. purifi cation takes place due to the high affi nity of the terminal fmoc-group to this material. hplc measurement shows a high recovery ( %) and purity ( %). cleavage of the fmoc-group during the purifi cation process is also possible. here, hplc measurement demonstrated a recovery of % and a purity of %. a fully automated synthesis of more than peptides in a -micro well plate combined with a parallel ''fmocon'' purifi cation is shown. the peptides were used for the production of microarrays to analyze dna/protein interactions. the "fmoc-on" purifi cation allows easy and cost-effi cient purifi cation of peptides for all kind of biological applications. is an antibiotic-macrolide related to a group of tylosin (tyl) which structure is based on -member lactone with carbohydrate substitutes attached. macrolides are well known translation inhibitors, they bind to ribosomal tunnel (rt) in a way that their lactone ring is located orthogonally to the long axis of the rt, covering most of its cleft; hence, the mechanism of protein synthesis inhibition by macrolides relies on the mechanical obstruction they provide to the passage of nascent polypeptide chain through the rt. recently we have designed and synthesized a number of peptide derivatives of macrolides where the peptide part modeled the growing chain, while the antibiotic served as an "anchor" for positioning the peptide at the specifi c site of rt. these derivatives are of interest both as antibacterial agents and as potential probes for investigation of the interactions of nascent peptide chain with the specifi c sites of the rt and their infl uence on the translation. now we report a new type of peptide -macrolide conjugates in which peptide binds by its -amino function through a spacer to the '-hydroxyl group of the mycaminose residue of des. two proline-containing peptides are chosen: gp and ggp. proline residues of these peptides are supposed to interact with the peptidyl transferase center region when the complexes of these '-peptidyldesmycosins with bacterial ribosomes are formed. the fi rst step of the synthesis was acetylation of '-, "-, "and "'-oh-groups of tyl by ac o in pyridine following by hydrolysis in n sulphuric acid resulted in des with all protected oh-groups except '-oh-group of mycaminose. this free hydroxyl group was used for reaction with succinic anhydride leading to formation of reactive carboxyl group. the next step was a reaction between this carboxyl function with -amino groups of peptide methyl esters. the structures of new '-peptide derivatives of des were proved by ms maldi and nmr-spectroscopy. synthesis . ]octane, (c), was carried out by organic methods which then will be used in polymer synthesis [ ] [ ] . consequently, polymers having different molecular weights and their water soluble bioconjugates were synthesized and the characterization of these polymers and conjugates were done by different methods such as esi lc-ms, uv and atr ft-ir spectroscopy. for the chemical modifi cation of pc, bromoacetic acid was used and a water soluble polyampholyte synthesis was achieved with the quaternization of polymer. it is determined that molecular weights of the polymers were affected by the change in the amount and the type of initiators used and also from the polymerization media. analysing of having biodegradable characteristics of the synthesized polymers has been being reviewed. atr ft-ir spectra of pc and pc-hemagglutinine peptide was recorded. for peptide synthesis we have used microwave assisted solid phase peptide synthesis method. lc-ms was used to determinate of molecular weight of hemagglutinin. after we have obtained m/z values of peptide we used preparative hplc for purifi cation of hemagglutinin. after synthesis and modifi cation of pc, its covalent conjugates with hemagglutinin (ha - ) were obtained by carbodiimide activation. its characterization was also performed. the peptide coupling reagent fi eld has clearly evolved in the last decade from carbodiimides to onium (phosphonium and uronium) salts. the era of industrial coupling reagents began in with the introduction of dicyclohexylcarbodiimide (dcc), which at that time was already known and well studied, as a reagent for the formation of amide bond. unfortunately, carbodiimides did not comply with the concept of ultimate coupling reagents because its high reactivity provokes racemization and side reactions during the coupling reaction. at the beginning of the 's, -hydroxybenzotriazole (hobt) was proposed as an additive to dcc to reduce racemization and from then on other benzotriazole derivatives such as -hydroxy- -chlorobenzotriazole (cl-hobt) or -hydroxy- azabenzotriazole (hoat) have also been used. the obt active esters are less reactive than the o-acylisourea, but are more stable and less prone to racemize. the use of the most reactive aminium salt, hatu , is inconvenient because of the price, which makes its use detrimental for industry. hctu/tctu, based on cl-hobt, are a good alternative to hbtu/tbtu, because of the presence of the chlorine atom that stabilizes the structure, hence, making these reagents less hazardous. herein, pyclock, the phosphonium salt of the cl-hobt is introduced p - n-methylation of biologically active peptides is of interest because it results in increased conformational integrity and stability against enzymatic degradation. furthermore, n-methylated peptides have a decreased ability to form h-bonds to water molecules and, consequently, a better ability to cross biological barriers. this is exemplifi ed by the naturally occurring peptide cyclosporine which is orally active. in an effort to improve the blood-brain barrier permeability of the cyclic enkephalin analogues h-dmt-c[d-cys-gly-phe-d(or l)-cys]nh (dmt = ', '-dimethyltyrosine), we prepared analogues that were n-methylated at phe and/or cys . n-methylated cys derivatives were prepared either by direct methylation (ch i)/nah) or by using oxazolidinone chemistry. single n-methylation of the two cyclic peptides at phe or d(or l)-cys produced four analogues that all showed very high mu and delta opioid agonist potencies in the guinea pig ileum and mouse vas deferens assays. the two analogues n-methylated at both phe and d(or l)-cys also retained high agonist activity at both receptors. results from molecular mechanics and molecular dynamics studies on the conformational behavior of these peptides will be presented. during the last years the number of deaths due to hemostatic impairments such as coronary angioplasts, coronary thromboembolisms, myocard heart attack, pulmonary embolism etc. has become equal to those caused by cancer formations. haemostasis is a key process whose correct functioning is an important defence mechanism of the human organism. it is a blood coagulation process activated in case of injury of the blood system. if it is functioning correctly vascular-motor and cell reactions are triggered and the blood coagulation cascade is activated. one of the most important enzymes in the blood coagulation cascade is factor xa. that's why its inhibitors are promising alternative against thrombotic disorders. in our previous work, we reported the synthesis of hybrid structure between isoform and of antistasin and the active sequences d-phe-pro-arg; d-arg-gly-arg; phe-ile-arg and tyr-ile-arg ( ). beside the analogues with c-terminal cooh group, the peptide d-phe-pro-arg-pro-lys-arg-nh was synthesized. the biological activity of the last one was times bigger than natural isoform of ats and some times more active than all other synthesized analogues. in the current work we described synthesis and biological activity of c-terminal amide analogues of all early synthesized peptides in order to deduce the structure-activity relationship. the anticoagulant activity according to the aptt and ic was determined. the neo-angiogenesis is the formation of new blood vessels from a pre-existing vasculature which enables the supply of the nutrients and oxygen necessary for tumor growth. consequently, inhibiting the blood vessels sprouting in order to starve the tumour constitutes an attractive anti-tumoral therapy. the vascular endothelial growth factor or their receptors (vegfr- and ) are key mediators of neo-angiogenesis and constitute nowadays validated targets for anti-angiogenic therapies ( ) . despite this fact, strategies aiming to develop pure receptors antagonists and not inhibitors of the tyrosine kinase activity remain scarce. we have previously designed antagonists that interact with the extra-cellular domain of vegf receptor and thus prevent vegf binding ( ) . these antagonists were proved to be potent and able to interfere with the vegf signalling pathway. because these compounds were rationally designed by using the co-crystallized structure of vegf with the immunoglobulin like fragment of vegfr- , we raise the following question: do these antagonists really target the immunoglobulin like domain of vegfr- ? in order to respond to this question but also with the future aim of optimizing our antagonist in a rational way we need to co-crystallize at least one of our antagonists with the d fragment of the vegfr- . furthermore, since human vegfr- d is a potential target in the development of angiogenesis modulators, the availability of this protein domain, and mutants, should be useful in the screening and development of novel specifi c ligands. up to now, the -amino acid polypeptide chain of vegfr- d has been only produced by gene expression ( ) . here, we report the fi rst solid phase peptide synthesis of the vegfbinding domain of the vegf receptor and the in vitro experiments which permitted to verify its biological activity. chung, nga n. different types of turns are important elements of secondary structure in peptides and proteins. the most common ones are different kinds of -turns involving four consecutive amino acid residues. the dipeptide unit containing the second and the third residues of such turns exists in the cis-conformation. our cispeptide bond motif -aminopyroglutamic acid promotes the -turn type vi/vi', and can be treated as a hybrid of glycine and alanine. this feature prohibits its utilization as a replacement for any possible dipeptide unit. in order to convert our compound into a more valuable tool for probing the existence of a particular peptide bond in the cis-conformation, we have elaborated the synthesis of nmonoalkylated derivatives of -aminopyroglutamic residue through a reductive alkylation reaction performed on solid support. following this idea, we have obtained analogues with n-benzylated and n-(phydroxy)benzylated -aminopyroglutamic residues as scaffolds for the phe-ala and tyr-ala dipeptides units, respectively. only one of enkephalin analogues, [n(bzl)-(r,r)-apy - ]-enkephalin amide, possesses similar agonist potency as leu-enkephalin in the gpi assay, indicating that the c-terminal part of this peptide may assume the cisconformation upon binding to the receptor. this is the fi rst active opiod analogue containing a -aminopyroglutamic acid residue. supported by kbn (poland) grant p f and nih (u.s.) grant da- mif- (l-prolyl-l-leucyl-glycine amide, plg) is a brain peptide, presented in hypothalamic tissue, as well as the c-terminal tripeptide of the neurohypophiseal hormone oxytocin and inhibits the release of melanocyte-stimulating hormone (msh) in some systems. recently, some chemical modifi cations of mif- to enhance opiate agonist/ antagonist actions as well as binding activity of analogues have been reported. on the other hand, the concept of structural modifi cation in peptide fragments to confer them specifi c properties is of current interest in the study and design of new bioactive targets. a well known example is the incorporation of unnatural amino acids into the molecule of natural biologically active peptides leading to analogues with signifi cant theoretical and practical importance. with this idea in mind we studied possibilities of introducing unnatural amino acids canaline (can), norcanaline (ncan), canavanine (cav), nor-canavanine (ncav) and slys into mif-moiety in order to achieve a better analgesic effect. to obtain the peptide mimetics, both fmoc-and boc-based spps approach were used. analgesic activity was determined by the paw-pressure (pp) and hp tests. the experiments were carried on male wistar rats. the changes in the mechanical nociceptive threshold of the rats were measured by the randall-selito paw pressure test using analgesimeter (ugo basile). it was found that substitution of leu in position of mif-molecule by unnatural amino acids increased the pain threshold. the analgesic effect with cav-substitution was highest, whereas parent mif- showed only a minute increase of pain-threshold. the compound m had the strongest effect on learning and memory and the compound p showed the strongest and fastest analgesic effect. the compound m and p were also able to modify the effect of the model cns-drugs (hexobarbital and pentylenetetrazole). theoretically and experimentally determined octanol/water logp values of the compounds correlate with their cns-effects. m and p had higher logp than m and p and showed better antinociceptive and anticonvulsant activity in vivo. compounds possessed also chelating activity towards fe ions. the stronger chelating activity of the compounds with the -spacer clearly correlated with their better neuropharmacological activity in vivo (in comparison to the compounds with the -spacer). the position isomery may also contribute for the variations in their pharmacological activity. the limited number of the compounds does not allow derivation of well-defi ned structure-activity relationships, however, their d models show possibility for many low energy conformers with different atoms involved in formation of fe chelating complexes. a major challenge in opioid peptide chemistry is the synthesis of novel compounds mimicking the endogenous peptide ligands. these new peptidomimetics should be biologically active and more stable against enzymatic degradation than their parent ligands. one of the possibilities is the introduction of -amino acids into the peptides sequence. monosubstituted -amino acids ( -or -) due to their similarity in structure to -amino acids, moreover their tendency to give folded structures even in short peptides and to the stability towards mammalian peptidases may be very useful in the creation of the new potentially active compounds. we have developed simple and effi cient two step conversion of the cyanoacetate into fully protected -amino acids. the procedure involves knoevenagel condensation of the methyl cyanoacetate and aromatic aldehydes (for aromatic path) or alkylation of the methyl cyanoacetate with various alkyl halides (for aliphatic path) at fi rst then reduction and boc-protection (performed in one pot) of the resulting fi rst stepproducts. we focused our attention on applying above method to the synthesis of different -amino acids (as homologues of -amino acids) as elements for the synthesis and structure -activity relationship study of endomorphin analogues. small library of analogues has been created in which -amino acids in every position with exception of pro were substituted by their respective -analogues. as templates, endomorphin- (tyr-pro-trp-phe-nh ), endomorphin- (tyr-pro-phe-phe-nh ) and its d-ala -analogue (tapp) have been used. in this communication the pharmacological consequences of such modifi cation in endomorphins will be discussed. solid-phase synthesis and effects of amino acid and peptide analogues of non-protein amino acid canavanine on nociception non-protein amino acids have been widely used as components of peptides to enhance biological activity, proteolytic stability, and bioavailability. it is well known that unnatural amino acids with guanidine functionality exhibit diverse pharmacological effects when introduced in biologically active systems. our previous efforts were focused on the preparation and the characterization of unnatural amino acids, particularly those containing a basic functionality in the side chain. we have synthesized numerous unnatural amino acids, structural analogues of arginine and lysine, which demonstrated certain biological effects. recently, as part of our ongoing research focused on the search of novel arginine mimetics, we developed an effi cient approach for solid-phase synthesis of unnatural amino acids nor-canaline (ncan) and nor-canavanine (ncav). next we studied the possibilities of introducing ncan and ncav into the molecule of biologically active peptides. we also studied their antinociceptive effects using the paw pressure (pp) and hp tests. in the last few decades, considerable attention has been devoted on the potential role and activity of certain environmental pollutants (edcs) in increasing anomalies that involve the endocrine system of wild species and man. ( )the development of a fast high-throughput detection system for the quantitative analysis of edcs requires the development of a novel sensitive solid phase edcs extraction method. because of the high affi nity of edcs with the estrogen receptor, this has been greatly investigated. this study has leaded the recognition of the amino acids located in the ligand binding domain of the receptor which interact with edcs. use of a dipodal scaffold molecule and different combination of the crucial amino acids, will allow the generation of a library of small to medium size biomimetic receptors. in this communication, we will disclose our fi rst results in the preparation of a small library of biomimetic receptors, with and without the dipodal scaffold, to evaluate their affi nity towards edcs and the role played by the scaffold structure. romeralo tapia, rosa ; van der eycken, johan ghnet university, belgium; ghent university, belgium endocrine disrupting chemicals (edc's) are an important class of pollutants, which have in common that they show affi nity for the hormone binding domain of the estrogen receptor, thus disturbing the endocrinal system. detection in waste water has not been succesful due to their low concentration. therefore, new sensitive screening methods are highly demanded. a possible solution is the use of estrogen receptor mimics for affi nity chromatography. to this end, scaffold will be synthesized and used for building a tetrapodal peptidomimetic library. amino acids known to be important for the estrogen-receptor interaction will be preferably incorporated. first of all, we set out to prepare one dipodal peptide library member . the orthogonally protected scaffold was synthesized in one single step starting from the commercially available amino- -nitrobenzoic acid. using fmoc solid phase chemistry on wang resin as the solid support, this dipodal scaffold allowed the attachment of two different oligopeptide chains.employing this methodology the synthesis of a small library will be performed, and the affi nity of the different peptidomimetics for edc's will be investigated. on-resin microwaves-assisted ring closing metathesis for the synthesis of octreotide dicarba-analogues di . octreotide is an antitumoral agent used mainly as a carrier of radionuclides for cancer diagnosis and therapy. it shows the same disulphide bridge of the parent srif that is prone to be opened by oxidizing and reducing agents. this prompted us to search a more stable tether bridging the active motif of the cognate molecule. the premier reaction of rcm was performed in an oil bath under severe experimental conditions i. e. anhydrous argon atmosphere and long reaction times [ ; ] . the microwaves assisted version was, instead, effi cient for the cyclopeptides yield and required very short reaction times. we also evaluated the effi cacy of different grubbs catalysts in the microwaves assisted rcm, operating in different conditions of temperature and time. in the search for potential antimicrobial drugs, we have been dealing with two microbial enzymatic systems: n á -succinyl-l-diaminopimelic acid desuccinylase (dape , ) and n á -acetyl-l-ornithine deacetylase (arge , ), which might be promising targets for potent and selective enzyme inhibitors based on the modifi cation of n á -succinyl-diaminopimelic acid (dap) and n á -acetyl-ornithine (orn). the inhibition of both the enzymes would possibly interrupt the pathways leading to development of bacteria due to a role of meso-dap as essential component of peptidoglycan based bacterial cell walls and orn, as well, being a component of biosynthetic pathway for arginine that can serve as a source of both the carbon and nitrogen in microorganisms. our effort was focused on the synthesis and characterization of two series of n á -substituted derivatives of dap and orn, potentially interfering with the hydrolytic action of dape and arge in the processes of bacterial growth. in this introductory study, the compounds prepared were also assayed against bacterial strains escherichia coli and bacillus subtilis and inhibitory activity of orn derivatives was found with regard to differences in the structure of n á -substituent. the fi rst report on practically effective bradykinin (bk) antagonists for b receptors was published in . the key to conversion of bradykinin into an antagonist was replacement of pro with an aromatic d-amino acid; d-phe was fi rst used. however, our studies demonstrated that the d-amino acid residue at position of the bradykinin antagonist, until recently considered to be necessary for b antagonism, can be replaced by suitable l-amino acid or achiral residue or, together with the amino acid occupying position , by a sterically restricted dipeptide unit. having all this in mind we synthesized and bioassayed two new analogues of bradykinin. the peptides were designed by substitution of position of bradykinin b receptor antagonist ([d-arg ,hyp ,thi , ,d-phe ]bk), previously described by stewart's group, with structural isomer of proline: -iso-pro or its homologue: -homo-pro. it's worth emphasizing that position in bradykinin molecule is occupied by proline residue. our previous results demonstrated the importance of the position in the peptide chain into which the sterically restricted -aminocyclohexane- -carboxylic acid residue (acc) was inserted. these fi ndings prompted us to investigate how introduction of l-pipecolic acid residue (l-pip) in position or of stewart's antagonist will affect pharmacological properties of resulting compounds. in comparison to the acc residue, the ring of l-pipecolic acid also consists of six atoms, but includes the nitrogen atom. bearing in mind that acylation of the n-terminus of several known b blockers with a variety of bulky groups has consistently improved their antagonistic potency in the rat blood pressure assay, the aforementioned four analogues were also synthesized in the n-acylated form with -adamantaneacetic acid (aaa). the activity of eight new analogues was assessed in isolates rat uterus and in rat blood pressure test modifi cations of the myelin basic protein epitope mbp - divert th to th : immune responses in peripheral blood mononuclear cells (pbmc) from multiple sclerosis patients. postranslational modifi cations (citrullination, phosphorylation, deamidation, methylation, glycosylation) are common biological processes that alter specifi c parts of a protein after synthesis. nearly all known proteins undergo some form of postranslational modifi cation and almost all amino acids can be altered by one or more of these processes. the modifi ed protein thus, contains new or rare amino acids or new specifi c side groups that can have critical infl uence on the structure and function of the protein molecule. conversion of arginine to citrulline, an important postranslational modifi cation, was fi rst described by fearon. arginine residues in proteins can undergo this modifi cation and the resulting citrulline remains part of the protein in the position of arginine. citrulline is not a natural amino acid in proteins, and may induce immune responses. such responses have been recently implicated in the pathogenesis of autoimmune/infl ammatory diseases such as ms and rheumatoid arthritis . herein we investigated cytokine secretion in peripheral blood mononuclear cells (pbmc) of ms patients and controls, and attempted to correlate cytokine polarization with the nature of the antigenic stimulus. we synthesized peptide analogs that map to the myelin basic protein ( . analogs p and p resulted from the citrullination of the and arginine residues in epitopes p and p . we then tested ms and control pbmc with various concentrations of the peptides and investigated cell proliferation by the brdu proliferation assay and cytokine secretion by elisa. we suggest that citrullination of self-antigens maybe an important step in triggering disease in susceptible individuals. incorporation of aza- -amino acid into rfa( - ), the endogenous ligand of gpr : structural analysis. aza- -peptides, mixing -and aza- -amino acids (the aza analogs of -amino acids), represent a novel and exciting type of peptidomimetics. in particular, we have shown that aza- -amino acid induces a n-n or hydrazino turn, stabilized by an eight-membered-ring intramolecular hydrogen bond between the carbonyl acceptor group of the residue i- and the amide proton of the residue i+ . interestingly, this n-n turn promotes a well-defi ned -turn formation (hydrogen bond between the co of the residue i- and the hydrazidic proton of the aza- -moeity) when an -amino acid is foregoing. rfa, a novel neuropeptide of the rfamide superfamily, exhibits high affi nity for gpr and induces a potent orexigenic effect in mice. in biomimetic environment, rfa encompasses an -helix between pro and arg residues and a canonical -turn centered on ser . rfa( - ), whose sequence is strictly conserved across species, is about times less potent than rfa. this heptapeptide shows important distortions of the -turn that may be responsible for its weak potency. the aim of this study was to restore the -turn formation in rfa( - ) (ggfsfrf-nh ) by the presence of an aza- -amino acid. for this purpose, we have (i) synthesized the aza- -counterpart of each residue of rfa( - ), (ii) individually incorporate the surrogate into the heptapeptide and (iii) investigated the d structure under nmr restraints of the hybrid peptides. the results will be presented with a particular attention on the serine position. the analysis of the structure-antithrombotic activity correlation for peptide receptors of adhesive glycoproteins with the general formula arg-xaa-asp gribovskaya, olga; martinovich, vera; golubovich, vladimir institute of bioorganic chemistry, belarus structural analogues of the arg-gly-asp sequence with the general formula arg-xaa-asp, where xaa -ala, d-ala, -ala, -abu, -ak, pro, d-pro, asn-trp, were synthesized using classical methods of peptide chemistry in solution, the levels of their antithrombotic activity were determined and stable conformations were calculated using a pairwiseadditive approximation method. interatomic distances in the obtained conformers were calculated between various atoms in the functional groups, including carbons in the carboxylic groups, nitrogens in the -aminogroups, amino and imino nitrogens in the guanidine group, distances in total. the analysis of correlation of the calculated interatomic distances and measured values of antithrombotic activity was carried out using statistical methods. we show that the distance between the amino nitrogen of the guanidine group of arginine and the -carboxyl carbon of aspartic acid determines the antithrombotic activity. it has the optimum value in the arg--ala-asp peptide, which was the most active among the synthesized analogues of the arg-gly-asp sequence (ic - . mkm, adp, . mkm). microbial proteases with narrow specifi city as an instrument of peptide chemistry kotlova the problem of selective removal of short n-terminal peptides from the gene-engineered proteins is actual by many reasons. at fi rst it is connected with gene-engineering method of protein preparation in form of its precursors. the problem of n-terminal formyl-met removal from gene-engineered proteins, which are produced by bacillacae may be solved by introduction of specifi cally-cleaved insert after formyl-met. subsequent specifi c removal of such short n-terminal peptide results in mature protein. moreover, the analysis of cleaved short peptides could characterize the process of mature protein formation. we suggest microbial enzymes with narrow specifi city to solve the mentioned problem. for this aim we have isolated the trypsin-like enzyme and postproline-specifi c endopeptidase from aspergillus sp. and glutamyl-specifi c protease from b. intermedius. these enzymes have high specifi city approved by hydrolysis of short synthetic peptides and long polypeptides. for example, specifi city of postproline protease was established by hydrolysis of mellitin. the fi nal peptide mixture was analyzed by rp-hplc and mass-spectra. the use of enzymes with narrow specifi city is demonstrated in the analytic method of detection of formyl-met presence at the n-terminus of gene-engineered -interferon. trypsine hydrolysis of -interferon leads to more than peptides are formed including the -membered n-terminal peptide. being hydrolyzed with glutamyl-specifi c enzyme -interferon is converted to peptides, including -membered n-terminal peptide. application of postprolinespecifi c enzyme for -interferon hydrolysis leads to formation of short -membered peptide and long fragments. experimental data show that in case of analytic control of -interferon maturity the utilization of postproline-specifi c protease is preferable. in other cases the enzyme selection is ruled by the features of protein primary structure. bimodal action of cystatin related epididymal spermatogenic (cres) protein and its reactive site loop derived s-s bridge bicyclic peptides on proprotein convertase- (pc ) activity several precursor proteins found on sperm surface and reproductive tissues were proposed or confi rmed as substrates of pc . these include adam proteins, growth factors proigf and and hormonal protein pacap. lack of pc leads to impaired fertility in mice, suggesting its important role in reproduction. pc is thus considered an important target for development of nonhormonal contraceptive agents. pc -inhibitors are expected to fi nd therapeutic, clinical and biochemical applications. a lot of interest has grown to develop specifi c pc inhibitors. recently natural inhibitor of serpin family have been described for pc , , and furin, but not for pc . however, a new serpin, cres, has been reported from epididymis fl uid, where pc may be found. so far, cres has only been shown to inhibit pc , which is not present in reproductive tissues. we propose that cres may represent a natural regulator of pc , based on localization and other studies. in this study we generated recombinant human cres protein ( aa), and its reactive-site loop (rsl) derived acyclic and cyclic -mer peptides with various s-s linkage combinations. cres inhibited pc with ic ~ um, while rsl-peptides were found to be more potent with ic - nm, depending on the s-s linkage locations. furthermore, we noted that at lower concentrations, cres and its peptides fi rst enhanced pc activity before any inhibition occurred. this bimodal behavior may be due to cleavage. molecular modeling showed strong interactions between cres and pc via some of their key residues. overall, we noted that "rsl" is the most crucial element required for pc inhibition by cres. funds were from from cihr (ab). the de novo design of peptides and proteins has assumed considerable interest didehydroresidues, in particular alpha, in the recent years. alpha, beta-beta-didehydrophenylalanine (deltaphe) are being considered important conformational constraints inducing tools in de novo peptide design. deltaphe is a noncoded, achiral residue, an analog of the naturally occurring phenylalanine amino acid with a double bond between calpha and cbeta atoms. introduction of deltaphe in peptide sequences is known to induce conformational constraint, both in the peptide backbone as well as the side chain, and to provide the peptide with increased resistance to enzymatic degradation. in small peptides containing eta turn structure, and in peptides containing a single deltaphe, a type ii b more than one deltaphe residues, helical structures are stabilized. a number of deltaphe peptides varying in length, content and position of deltaphe residues have been found to contain helices of both screw senses. following these design principles, we were able to design, synthesize and characterize super secondary structural motifs like helix-turn-helix, helical bundle and glycine zipper. we have extended this work to the de novo design of peptides with antibiotic and anti-fi brillization activity. a series of cationic peptides containing deltaphe residues have shown remarkable antibiotic activity and are being developed further. deltaphe containing peptides may have longer in vivo half life owing to their ability to resist enzymatic degradation. more recently, we have observed that small peptides containing deltaphe self-assemble in nanotubular and nanovesicular structures. these nanostructures have also been used to entrap small drug like molecules. we have fully characterized these systems and are exploring their potential as delivery agents in biological systems. the synthesis and design of peptidomimetic oligomers that adopt designed, compact conformations (foldamers) have become a challenging task in recent years. among them, -peptide foldamers exhibit rich diversity of secondary structures. [ , ] a relationship has been established between the backbone chirality pattern and the prevailing secondary structure, which underlines the role of stereochemical control in the -peptide foldamer design.( ) an important challenge is to introduce proteinogenic side-chains to generate diverse anchor points on the molecular surface promoting their application in drug discovery. it has been proved that stable helices can be obtained when sequences were coupled with -amino acid enantiomeric pair motifs in a lego approach.( ) in this work, -amino acids and open chain functionalized -amino acids were inserted between the enantiomeric pair design element for and , respectively. confi gurations in the backbone were designed to promote helix formation. nmr and ecd measurements augmented with ab initio calculations revealed that forms a h - helix and forms a h / helix. these helices are unprecedented in the literature. to prove that the stereochemical patterning is crucial in the folding, we perturbed the confi guration motifs of the backbones by swapping the sequence of the central -amino acids. the peptides with exchanged residue order could not fold into helices. these novel structures can be useful scaffolds for biomedical applications. introduction small length synthetic peptides, based on sp c-terminal fragment, increase the secretion of tnf-and prevent the proliferation of several cancer cell lines. we have already shown the antiproliferative activity of tri-and tetra-peptoids in breast and prostate cancer cells. the aim of this study was the synthesis of hexa-peptoids, analogs of sp c-terminal region, containing the residues d-trp and tic and the peptoid ones nhn(r)ch co, nphe and nala in their sequence and their evaluation against cancer cells proliferation. methods and results all the syntheses were carried out stepwise using the fmoc/ but methodology on the solid support -cltr resin and dic/hobt as coupling reagent. all analogs were purifi ed (hplc) and identifi ed ( cyclotheonamides constitute a family of structurally related cyclic pentapeptides of marine origin that inhibit trypsin-like serine proteases. their binding mode has been elucidated by the x-ray structure of cyclotheonamide a in complex with trypsin. an extended peptide conformation which is stabilized by macrolactamization allows to address in a substrate-like manner beside the s pocket also the s ' and s pocket. the s ligand, (s)- -amino- -guanidino- -oxo-hexanoic acid, interacts via its guanido function with asp at the bottom of the s pocket. in addition, the ketone covalently modifi es the gammaoxygen of ser by hemiketal formation ( ) . recently, two novel cyclotheonamides have been isolated from a marine sponge of the genus ircinia. one of them, cyclotheonamide e , is a potent inhibitor of human beta-tryptase ( ) . in this study, cyclotheonamide e was modifi ed at two positions: (i) the s ligand was replaced by beta-homolysine or as betahomoarginine to obtain reversible acting tryptase inhibitors, and (ii) the alpha amino function of (s)- , -diamino propionic acid, which is not part of the cyclic backbone, was used as anchoring point for basic p residues to exploit interactions with the negatively charged glu of tryptase. these analogs were synthesized by a combination of solid phase and solution phase chemistry .. synthetic details as well as the inhibitory profi le of these novel cyclotheonamide e analogs will be discussed. oostatic peptides containing d-amino acids: activity and degradation in the fl esh fl y neobellieria bullata bennettová deteriorating effect of c-terminus truncated p and p analogues [ , ] of decapeptide h-tyr-asp-pro-ala-pro -oh ( ) on ovarian development (i.e. oostatic effect) of insect species diptera, orthoptera and hemiptera has stimulated an analysis of metabolic degradation of corresponding peptides after application to the insect body [ ] [ ] [ ] [ ] . radiolabeling in different positions of the peptide chain allowed determination of the degradation decisive steps -the fast splitting off the c-terminal pro from the p followed by successive cleavage of tyr and asp from the nterminus. after introduction of corresponding d-amino acids into peptide chain of p, we could see the same or even stronger oostatic effect of the analogues in comparison with parent peptide after application on the fl esh fl y neobellieria bullata. in the degradation assay, an elimination of enzymatic cleavage of peptide bonds pointing from the central labeled pro residue to either d-ala or d-asp residues in the neighbourhood was observed, resulting in total increase of the analogs stability. structure-activity relationships( sar) studies of camk ii inhibitors. in fact, in vitro it can phosphorylate up to proteins, including enzymes, ion channels, transcription factors and a number of these proteins appear to be physiological substrates. for example, cam-kii is highly concentrated in the postsynaptic density of glutamatergic synapses where it phosphorylates and potentiates current through the a-amino- -hydroxy- -methyl- -isoxazolepropionic acid-type glutamate receptor ion channel (ampa-rs). in fact, this family is encoded by four genes ( , , and ), whereas the and isoforms are expressed in diverse tissues and , isoforms are most prominent in neural tissues. the identifi cation of camk ii inhibitors is important to better defi ne its physiological. in literature is reported a -aa brain-specifi c protein that and potently inhibited kinase and bound the catalytic domain of cam-kii activity with an ic of nm. the inhibitory protein (cam-kiin), and a -residue peptide derived from it (camkiintide, krppklgqigrakrvvieddriddvlk), was highly selective for inhibition of cam-kii with little effect on cam-ki, cam-kiv, cam-kk, protein kinase a, or protein kinase c. cam-kiin interacted only with activated cam-kii (i.e., in the presence of ca +/cam or after autophosphorylation). here we report a structure-activity relationships study using as template the camkiintide. we synthesised a peptide contains all amino acids present in camkiin-tide, however, in random sequence (camkiin-tide scramble). then we operated a progressive deletion of amino acids from c-terminal and dall'n-terminal to detect minimum active sequence. moreover camkiintide and cankiintide scrumble was made cell-permeable by n-terminal addiotion of an antennapedia (rqikiwfqnrrmkwk). here we report the biological results of our study. ptprj: a receptor-type protein tyrosine phosphatase as a target of new peptides with antitumoral activity to be signaling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. it has been demonstrated that ptprj (also known as hptpeta, dep- , cd ) is able to inhibit cell growth promoted by specifi c ptk some of which are over-expressed in certain types of cancer. moreover a role for ptprj was also assessed in vasculogenesis; infact its reduced activity in enhanced vegf-induced vegfr activity leads to increased cellular responses, supporting a ptprj-vegfr interaction. on these basis it is important the identifi cation of molecules with agonist activity which are able to stimulate the function of residual ptprj in malignant cells and stopping the proliferation and possibly trigger programmed cell death. using as a template a peptide, already identifi ed, with agonist activity against ptprj(h-[cys-his-his-asn-leu-thr-his-ala-cys]-oh), here we report a structure-activity study carried out through endocyclic modifi cations (ala-scan, d-substitutions, single residue deletions, substitutions of the disulfi de bridge) and the preliminary biological results of this set of compounds. faulty of chemistry, university of gdansk, poland; faculty of chemistry, university of gdansk, poland isolated in from the sunfl ower seeds trypsin inhibitor sfti- is up to date the smallest naturally occurring peptidic proteinase inhibitor and therefore is an excellent starting structure to design peptidomimetic serine proteinase inhibitors. peptomeric library consisting of monocyclic analogues of sfti- was designed and synthesized by the solid phase method with the intension of select chymotrypsin and cathepsin g inhibitors. all peptomers contained in positions and nbenzylglycine (nphe) that mimics proteinogenic phe. in the synthesized library different peptoid monomers were introduced in the segment - . this is a turn region that makes an important contribution to structural integrity and rigidity of sfti- . deconvolution of the library against both proteinases by the iterative method in solution revealed that the highest chymotrypsin inhibitory activity displayed analogue with n-[ -( -aminoethyl)morpholyl]-glycine (naem) in position . this analogue was even more active than the one with pro in this position which is absolutely conserved in bownan-birk inhibitors. while, deconvolution carried out against cathepsin g indicated that analogue with npiperonylglycine (npip) in position and and with n-butylglycine (norleu) in position presents the highest inhibitory activity. acknowledgements: this work was supported by ministry of science and higher education (grant no. /h / / ). a platform for the design and optimization of new antimicrobial peptides effective against specifi c pathogens has been set up. the main features expected for these peptides are low environmental impact, broad spectrum of activity, reasonable bacterial selectivity, and low eukaryotic cytotoxicity. our approach also includes the use of a design of experiments protocol in order to fi nd peptide sequences that fi t these features. the obtained peptides would represent an alternative to currently used antibiotics or pesticides. this work focused on fi nding new control agents against economically important plant pathogenic bacteria such as erwinia amylovora, pseudomonas syringae and xanthomonas vesicatoria for which the available methods are not suffi ciently effective. nowadays, their control is mainly based on copper compounds and antibiotics. although antibiotics are highly effi cient, they are not authorized in several countries and resistance has been developed on plant pathogens. we have synthesized combinatorial libraries of cyclic decapeptides and linear undecapeptides. these libraries have been screened for antibacterial activity and eukaryotic cytotoxicity, and have led to the identifi cation of peptides with mic values of . - . microm. notably, cyclic peptides active against e. amylovora have been found, constituting the fi rst report of this type of peptides with activity towards this bacteria. the best peptides are bactericidal, display a low eukaryotic cytotoxicity at concentrations - times higher than the mics, and show a low susceptibility towards protease degradation. best peptides have been tested in vivo by evaluating their preventive effect of inhibition of p. syringae, x. vesicatoria and e. amylovora infections. the most active peptide is slightly less effective than streptomycin, currently used in fi eld. therefore, the best analogues can be considered as good candidates for the development of antibacterial agents for use in plant protection. selection of chromogenic and fl uorogenic substrates of neutrophil serine proteases using combinatorial chemistry approach human serine neutrophil and mastocytes proteases such as cathepsin g, neutrophil elastase, proteinase and -tryptase are involved in several physiological processes. unwanted activity of those enzymes yields to severe pathological states like infl ammation, wegener granulomatosis or various types of cancer. therefore monitoring of the activity of these proteases is crucial for the proper therapeutical treatment. the simplest method for determination of protease activity is the use of synthetic substrates. they are also useful for characterization of the enzyme specifi city. in this work we report selection of chromogenic and fl uorogenic substrates of human serine neutrophil and mastocytes proteases applying combinatorial chemistry approach. peptide libraries were synthesized by the portioning-mixing method. deconvolution of synthesized libraries was performed using iterative approach in solution. -amino- -nitro benzoic acid attached to the c-termini of synthesized peptides served as a chromophore released upon the interaction of peptide with enzyme. additional introduction of -methoxy- -coumaryl acetic acid or -amino benzoic acid on á-amino groups of the chromogenic substrates converted them into fret displaying compounds. the most active substrates were subjected to further modifi cation applying non-proteinogenic amino acids. as a result, one of the most selective substrates of these proteases was obtained. we also attempted to construct a simple tools to detect activity of human serine neutrophil and mastocytes proteases by immobilization, through amide and peptoid based linkers, of selected substrates on solid phase. angiogenesis modulation: identifi cation of tetrameric tripeptide as inhibitor of vegfr- by the screening of peptide combinatiorial libraries vascular endothelial growth factor receptor- (vegfr- , flt- ) and their ligands are involved in complex biological processes associated to severe pathological conditions, like angiogenesis, infl ammation and metastasis formation ( ). thus, the search for antagonists of flt- has recently gained a growing therapeutic interest. in order to identify new molecules able to selectively bind flt- and neutralize its activity, a screening of a combinatorial tetrameric tripeptide library built with nonnatural amino acids has been carried out by a competitive elisa-based assay. the library has been designed on a branched tetrameric structure in order to obtain molecules with a high recognition surface and, using building blocks, a complexity of . different peptides has been achieved. peptide mixtures composing the library have been utilized as competitors of the flt- /plgf (placental growth factor) interaction and the most active components have been isolated following an iterative deconvolution procedure. the selected most active hit shows a selective binding to flt- over kdr and inhibits in vitro its interaction with both plgf and vegf-a. the peptide is fully stable in biological environments, prevents flt- phosphorylation and blocks huvec capillary-like tube formation stimulated by plgf or vegf-a. in vivo the peptide inhibits the vegf-induced neoangiogenesis in chicken embryo chorioallantoic membrane assays and also stimulates cornea neovascularization (cnv) by displacing vegf from a complex with sflt- . all data suggest that the compound has potential applications in diseases characterized by pathological angiogenesis, such as tumor growth and ischemic retinopathy. heinlein, christian; unverzagt, carlo bioorganische chemie, universität bayreuth, gebäude nw i, bayreuth, germany since the purifi cation of homogeneously glycosylated glycoproteins remains a diffi cult task the synthesis of entire glycoproteins is a fi eld of emerging interest ( ) . especially the synthesis of the required protein fragments in high purity and good yields demands for effi cient methods. fragment condensation of protected peptides can reduce deletions in the crude product and thus facilitates purifi cation. condensation of peptide fragments in cspps ( ) is however subject to epimerization upon cterminal carboxyl activation. to overcome this problem the use of cterminal gly or pro is usually performed. peptides containing c-terminal pseudoprolines can also be coupled without stereomutation because the serine or threonine residue has been reversibly protected as a prolinelike oxazolidine ( ). this concept facilitates the synthesis of long peptides by epimerization-free fragment condensation at c-terminal ser/ thr residues in addition to gly/pro residues thus increasing the number of safe condensation sites. after solving several problems associated with the generation of peptide acids with c-terminal pseudoprolines the synthesis of the glycosylated rnase fragment was carried out as a racemization-free fragment condensation. tuftsin is liberated from fc-domain of the heavy chain of igg by two specifi c enzymes. being the tetrapeptide of biological origin is extremely important product because it can activate a few elements of immune system such as granulocyte and macrophage. tuftsin indicates not only immunological stimulating factor but also antibacterial, antivirial and antitumor properties. in spite of its wide range of activity, the peptide is unstable in plasma and it has become the aim of the formation of novel analogues more resistant to proteolysis degradation. the introduction of the additional residue at -amino group of lysine caused that new bond became stronger than peptide bond in central chain [ ] [ ] [ ] . we synthesized linear tuftsin derivatives that were prepared on the solid phase using a fmoc/tbu procedure. the method of elongation of peptide chain was based on two-step procedure: deprotection and coupling step. segment coupling reaction was carried out with tbtu, hobt and in the presence of diea in dmf/dcm/nmp mixture. the introduction of the simple amino acid (ala, -ala, val, ile or gly) at -amino group of lysine let us obtain the isopeptide bond. the modifi cation was achived by introducing lysine residue, protected at -amino group with mtt. the selective removal of mtt group was caused by % tfa treatment. peptides were cleavage from resin and purifi ed. tuftsin derivatives were confi rmed by ms, amino acid analysis, elemental analysis and rp-hplc analysis. peptides were sent to assay their microbiological properties and the results will be described as a structure-activity relationship. the synthesis and structural study of iso-aß( - ) the aß( - ) peptide is diffi cult to synthesize, because of its high tendency for aggregation during the synthesis. both the couplings and the fmocremoval can be troublesome, due to the steric hindrance. the incorporation of the ester-bond disturbs the structure, thus ease the synthesis after the th residue. if the peptide would be synthesized using boc-chemistry the removal of the -amino protecting group would be less problematic, because the % tfa/dcm mixture solubilizes well the peptide chain. thus a boc-strategy was devised for the synthesis of iso-aß( - ). the purifi ed peptide was studied with cd-spectroscopy and dynamic light scattering. these structural studies revealed that the isopeptide has some ß-sheet content even in a ph solution. it was realized also that small aggregates are present in the precursor peptide. both techniques mentioned above showed, that after altering the ph to . , the ß-sheet content of the peptide increases and aggregation takes place. with the use of the isopeptide, aß oligomers and -with prolonged incubation -fi brillar structures can be formed for biological studies. continuing our program of syntheses of muramyl dipeptide (mdp) and nor-muramyl dipeptide (nor-mdp) conjugates as potential immunomodulators, we designed novel conjugates of mdp or nor-mdp with tuftsin derivatives containing isopeptide bond between -amino group of lysine and carboxylic group of simple amino acids such as alanine, glycine and valine. the synthesis of a greater number of conjugates will enable structure-activity relationship studies. tuftsin analogues containing isopeptide bond showed increased chemical resistance and activity in relation to tuftsin. the introduction of the additional residue at -amino group of lysine by nhco-formation caused that isopeptide bond became stronger than peptide bond in central chain. the protected pentapeptides (h-thr-lys(y)-pro-arg(no )-obn, y= ala,gly,val) were synthesized by the conventional chemical procedure using mixed anhydride method. acylation of the thr amino group of partially protected pentapeptides by -benzyl-mdp or -benzyl-nor-mdp was performed using the mixed anhydride method with isobutyl chloroformate and n-methyl-morpholine (nmm) in dry dmf. the protected conjugates were isolated and purifi ed with a preparative tlc. the identities of the protected products were confi rmed by high resolution h-nmr ( mhz, cosy, tocsy, roesy, ghsqc, ghmbc) spectroscopy. the fi nal products were hydrogenated with h / pd/c in % methanol-acetic acid and purifi ed with preparative tlc. the identities of the conjugates were confi rmed by tlc qualitative amino acid analysis, and elemental analyses. finally, the combined use of muramyl peptides with other immunomodulators, e.g. such as tuftsin, retro-tuftsin other chemotherapeutics is promising in the therapy of different infections, autoimmunological diseases and anticancer therapy. effi cient microwave-assisted synthesis of myelin epitopes mog - and mog - using cltr-cl resin fmoc/tbu methodology. unlike conventional heating, microwave energy directly activates any molecule with a dipole moment and allows for rapid heating at the molecular level. the protected peptides were synthesized using the cem liberty automated microwave peptide synthesizer in and . hours respectively, with a -minute fmoc deprotection ( % piperidine solution in dmf) and -minute coupling reactions using dic/hobt in dmf solution. the maximum temperature reached during both the deprotection and coupling reactions was °c except for the coupling of fmochis(trt)oh ( °c). the fi nal crude products were of high purity as identifi ed by analytical rp-hplc. siah up-regulates the hif- alpha hypoxic response pathway: siah binding peptides coupled to cpps inhibit siah activity and demonstrate proof of concept that siah is a viable anti-tumor drug target vegf, a secreted downstream component crucial for angiogenesis, has been successfully targeted clinically using antibody therapy (avastin). the hypoxic response, however, activates other pathways that stimulate tumor growth, suggesting that inhibitors of upstream components in the pathway may be useful to give a broader spectrum of inhibition. we have focused on the siah proteins that regulate hif- ƒÑ levels by ubiquitylation and degradation of the prolyl hydroxylases (phds) immediately upstream of hif- ƒÑ in the hypoxic response pathway. in a proof-of-principle study, we have shown that siah inhibition by expressed protein fragments (from a drosophila high affi nity interacting protein, phyllopod) can inhibit the stabilization of mammalian hif- ƒÑ during hypoxia and limit tumor growth in a mouse tumor model. a amino acid peptide sequence (phyl) from the phyllopod protein has been identifi ed as the interaction site with siah. the aim of this work was to show that a small peptide could mirror the activity of the transfected recombinant phyllopod protein. to achieve this, phyl was covalently attached to cell penetrating peptides (cpps) and tested for its ability to inhibit hif- ƒÑ stabilization and hypoxic response in human u os cells. both the tat sequence and penetratin were utilized as cpps and attachment was via disulfi de, maleimide or through a pro spacer sequence. the cpp¡vphyl constructs were found to have varying activities but a penetratin-pro -phyl was found to be inhibitory in the u os cell-line hif- ƒÑ stabilization assay. this result demonstrated proof of concept that siah inhibition could be attained by targeting a restricted and specifi c protein-protein interaction site. ( ) . the principal event in the development of prion disease is the transformation of the normal cellular prion protein (prpc), which is highly helical in nature, into the pathogenic isoform prpsc which is insoluble and has an extensive ƒÒ sheet structure. the events surrounding this transformation are very poorly understood, but the mechanism of the detailed steps involved in this process is fundamental to the understanding of prion disease pathogenesis. in an endeavour to study the structure of polypeptide component of the n-terminal section of prpc, synthesis of a range of prp polypeptides were assembled on a cem liberty microwave synthesiser. these fragments ranged in length from to amino acids and span the protein sequence from position to . standard cem synthetic coupling cycles were used, except when peptides were greater than amino acids in length, whereby longer coupling cycles were employed. a number of purifi cation strategies were employed, such as c and c rp-hplc at either c or c and size exclusion chromatography. the purifi ed peptides were characterised by rp-hplc and esi-ms. in addition, they were analysed for secondary structure by cd spectrometry, and evaluated for cell toxicity and fi bril forming ability with tht. the synthesis of a mer peptide for investigating the mechanism of action of the hiv fusion inhibitor t however, the mechanism of action of t is still in debate. it is believed that peptides derived from gp chr region may share a common mechanism, by binding to gp nhr coiled coil and preventing formation of the fusogenic gp core-six helix bundle ( hb), thereby inhibiting fusion between the virus and target cell membrane. we have synthesized a -mer nhr peptide-n -covering all the binding sites for any length of the chr-peptide. synthesis of the polypeptide was carried out using conventional as well as microwave assisted solid phase synthetic methods. details, including comparison of the synthetic approaches will be presented. using c , another anti-hiv chr peptide containing the pocket-binding domain, as a control, we analyzed the activity of t to interact with n- to form hb and to inhibit the -hb formation between n- and c by cd spectroscopy and elisa. we found that t- , unlike c could neither form a stable hb with n , nor inhibit the -hb formation of the fusogenic hb core. our results thus suggest that t- and c- peptides inhibit hiv fusion by different mechanisms of action. the oligomerisation equilibrium of ts is modulated by a fi ne tuning; shifting this equilibrium towards the monomeric form would cause ts inactivity and low translation, overcoming resistance mechanisms encountered for (co)substrate-like inhibitors as the inactive monomer regulates its own expression. our group designed some small ligands to interfere with thymidylate synthase dimerisation, including short peptides taken from the interface sequence that represent the natural ligand for this region, mimicking the other subunit without forming a functional dimer. interesting biological data are arising from the activity tests but a rapid screening assay is necessary to prove they are really interfering with ts oligomerisation. fret is a spectroscopic phenomenon whose intensity depends on the distance between two fl uorophores; when this value varies due to conformational changes of the protein the probes are linked to, consequent fret variation can be used to sense the state of the protein(s) ( stromal derived factor- (cxcl ) is a amino acid cxcchemokine with critical role in homing, migration and guiding of different cell types including hematopoietic progenitor cells (hpc), stem cells, tumour cells and neuronal cells during embryogenesis and in adults ( ). these various functions in physiological as well as pathophysiological processes make this small protein interesting for regenerative medicine. to apply this chemoattractant in medicine, it is needed to form spatially and temporally controllable concentration gradients of active sdf- in response to a non-tissue damaging trigger like visible or near uv-light. after irradiation of an inactive prodrug dramatic change in conformation or lack of sterical hindrance should then lead to fully biological activity under physiological conditions within a few minutes. to prove the principle and assess its potential in photodynamic therapy of neuronal injuries a water-soluble, photosensitive sdf- analogue has been developed. for this the expressed protein ligation (epl) approach has been used, in which one segment has been recombinantly expressed and purifi ed using the impact ® -system to yield the corresponding peptide thioester ( ) . the modifi ed peptide fragment has been chemically synthesized on solid phase using fmoc-strategy. the photocleavable moiety, the -nitroveratryloxycarbonyl (nvoc) protecting group, has been introduced at a side chain amino group. furthermore, the analogue has been characterised by physico-chemical methods and has also been tested in vitro on transfected cos- cells in order to determine its biological activity after activation. sdf- is a chemokine that plays a major role in traffi cking of hematopoietic stem cells (hsc). thus it enables the formation of bone marrow during embriogenesis and later in adult life it supports retention and homing of these cells in the bone marrow. furthermore it is involved in organogenesis and regeneration, respectively. due to these promising features the subform sdf- could serve as a therapeutic target. for studies on the small protein concerning its molecular properties as well as its therapeutic potentials, it needs to be modifi ed chemically. in order to reach these goals, the n-terminus sdf- - has been cloned and expressed recombinantly in e. coli er as a thioester, while the c-terminus sdf- - has been synthesized via solid phase peptide synthesis. modifi cations are thereby introduced at the c-terminus at lys . up to now carboxyfl uorescein has been coupled to the -amino group of the lysine residue. the two fragments then have been ligated via expressed protein ligation (epl), a subform of the native chemical ligation (ncl). activity studies and fl uorescence microscopy on hek cells transfected with the sdf -specifi c g-protein coupled receptor cxcr have been conducted. stueber, werner; lewandrowski, peter; frisch, juergen; weinschenk, toni; singh, harpreet immatics biotechnologies gmbh, germany ima is a multiple peptide vaccine for the treatment of renal cancer (rcc). the tumor-associated peptides (tumaps) contained in ima were identifi ed by immatics directly from primary renal cells (= primary rcc tumor tissue samples), selected regarding their over-expression in rcc and proven to be immunogenic using in vitro t-cell assays. ima consists of individual peptides ( tumaps) and nonactive ingredients which are used as excipients of the pharmaceutical presentation of ima . all peptides are synthesized by conventional fmoc chemistry. the sequences of the peptides will be presented and technical issues will be discussed. in the fi nal formulation of ima g of each peptide plus excipients are fi lled into glass vials and lyophilized. the challenges of the production of multi peptide drugs will be discussed. such challenges comprise the synthesis, the production of the formulation as well as the analyses of the fi nal presentation of ima . results of the phase trial in vaccinated rcc patients showed that ( ) ima was safe, ( ) multiple t-cell responses to vaccinated peptides correlated with favourable clinical outcome and ( ) patients with a lower percentage of regulatory t cells (tregs) were more likely to develop a vaccine-induced multiple t-cell response. novel peptides -msh analogs with high candidacidal activity in an attempt to improve the candidacidal activity of -msh and to better understand the peptide structure-antifungal activity relations, we designed and synthesized novel peptide analogs. because previous data suggested that the peptide [dnal- , phe- ]--msh( - ) has greater candidacidal activity than -msh and is the most potent of the analogs tested in the past, this compound has became our lead ( , ) . from this lead compound we have synthesized a new library of peptides where we have replaced the glycine in position with unconventional amino acids. here, we report new analogs with a strong antimicrobial and candidacidal activity. the obtained results are very encouraging in that they show the great potential of these peptides as a truly novel class of candidacidal compounds. derivative were modifi ed by the same amino acid to establish infl uence of the adjacent amino acid on antimicrobial activity of the compounds studied. the activity of all obtained peptides was screened against model gram-positive (bacillus subtilis) and gram-negative (escherichia coli) bacteria whereas antifungal activity was tested against yeast pichia pastoris. all tests were performed using antibiogram method whereas the minimal inhibitory concentrations were determined using two-fold serial dilution technique. huang, yen-hua ; colgrave, michelle l. the cyclotides are a family of naturally occurring macrocyclic peptides that combine the unique features of a head-to-tail cyclic backbone and a cystine knot motif, which impart extraordinary stability to this peptide family. a recent study demonstrated that the prototypic cyclotide kalata b possesses signifi cant activity against two economically important sheep nematodes haemonchus contortus and trichostrongylus colubriformis. an alanine scan of the molecule highlighted the residues critical for activity. in this work, we explore the relative importance of positively charged residues in different regions of the molecule to aid the understanding of the structural and biological basis of its nematocidal activity. a lysine scan has been conducted, in which each of the non-cys residues in this amino acid peptide has been successively replaced with lysine and the suite of peptides have been assayed against the two sheep nematodes. substitution of residues in loop , v , g , n , t , w , v , l , p , and v decreased or completely abolished the activity, suggesting that these residues are critical to the nematocidal activity of kalata b . on the other hand, incorporation of a positive charge in positions g , t , t , n , and g signifi cantly enhanced the anthelmintic activity of the grafted peptides, up to fourfold, compared to native kalata b . these increases in activity after lysine incorporation into the kb scaffold raise the possibility of being able to engineer greater anthelmintic activity into the peptides, further highlighting their potential as anthelmintic agents. ivanova, v.p. interaction of cells with extracellular matrix (ecm) affects many aspects of cell behavior including growth, morphology, migration and differentiation. integrins are known to mediate cell adhesion to proteins of ecm. ligand-binding properties of integrins depend not only on composition of ecm ligands and level of integrin expression in cell, but also on spectrum and activity of soluble factors indirectly infl uencing cell-matrix contacts. interaction of cells with substratum may be divided into cell adhesion and spreading. following an initial cell attachment event, cell may or not spread depending on cell type and the nature of the molecular signals they receive. oligopeptides released from different proteins during their proteolysis in or out of cells may act as the such short-time existing signals. in the present study we investigated the effect of multiply repeated peptide fragment in different collagen types on cell spreading. murine embryonic fi broblasts ( /ml) were allowed to adhere for min at ° c with or without peptide (in various concentrations) to plastic surface pre-coated or not with gelatin. it was shown that the synthetic peptide increased the number of spread cells and caused shape changes in cells spread on different substrata. a -min pretreatment of cells with the peptide (before conducting of cell spreading assay) resulted in inhibiting of cell spreading on gelatin. our results suggest that the peptide regulation of cell spreading could be related to its effect on re-distribution of integrin receptors in sites of cell contacts with substratum. bioactive peptides from cyanobacteria cyanobacteria are versatile source of small peptides from which vast majority are cyclic. cyanobacterial culture collection in university of helsinki contains over strains and this collection is used for screening of new bioactive compounds. cyclic heptapeptides, microcystins are the best known cyanobacterial peptide family. over structural variants have been described from which most are strong hepatotoxins. our research group have participated to the determination of the structure of many novel microcystins and other cyclic and linear cyanobacterial peptides. in recent years we have found highly toxic and novel microcystins from lichen associated cyanobacteria ( ) and from anabaena strains of baltic sea ( ). in the structural analysis of new peptides from the known peptide families we have used liquid chromatography ion trap mass spectrometry which have proven to be very effective method and is in many cases the only method needed in the verifi cation of a new structure. with this lc-itms method we have found many new structural variants from anabaenopeptin, anabaenopeptilide and spumigin peptide families. in collaboration with many research groups we have found cyanobacterial compounds/extracts which inhibit protein kinase c activity, inhibit/activate boar sperm motility, disintegrate cell membranes or are antidotes for microcystin toxicity. structures of the compounds are under study except the microcystin antidote which structural analysis showed that it belongs to a rare peptide family containing imino bond in cyclic skeleton. the results of n-terminal modifi cation of arginine vasopressin with cis- -amino- -phenyl-cyclohexane carboxylic acid. the highly potent oxytocin receptor antagonists arginine vasopressin (avp) is a cyclic nonapeptide with multiple functions. the main peripheral physiological roles of avp are the regulation of water balance, the control of blood pressure, and the release of adrenocorticotropin hormone (acth). moreover, avp also exhibits to some extent typical oxytocin (ot, a closely related neurohypophyseal peptide) activities such as the galactogogic and the uterotonic effects. all peptides were tested for the pressor, antidiuretic and uterotonic in vitro activities in the rat. cis-apc modifi cation at position of avp is suffi cient to change the pharmacological profi le of the peptides. analogues i -iv were moderately potent antidiuretic agonists with prolonged action. in regard to the uterotonic activity, all peptides with cis-apc were highly potent antagonists, except compound i. it supports our earlier hypothesis that an amino acid residue in position has signifi cant impact on pharmacological activities. the incorporation of unnatural non-proteinogenic -amino acids into peptides has emerged as a novel and promising approach in peptide modifi cation. the conformationally restricted amino acid derivatives are of particular interest. this thesis are also supported by our already -years research focused on the effects of steric restriction and the presence bulky substituent in the n-terminal part of avp molecule on biological properties of the resulting analogues. all peptides were tested for the pressor, antidiuretic and uterotonic in vitro activities in the rat. all the analogues were devoid of the pressor potency and exhibited only negligible antidiuretic activity. interestingly, in regard to the uterotonic activity, the new compounds exhibited moderate (i) or high (ii -iv) antioxytocic potency. it should be point out that single substitution e.g. replacement of tyr in avp molecule with igl results in moderately potent and highly selective antagonists of oxytocin (i). our new igl substituted peptides proved that the presence of the sterically restricted amino acid residue may result in high active and selective antiuterotonic agents. these in our opinion interesting fi nding demonstrates the usefulness of our approach in the design of new highly active and selective analogues with desired pharmacological properties. arginine vasopressin (avp), a neurohypophyseal hormone and neuromodulator, is a cyclic nonapeptide with a disulfi de bridge between cys residues at positions and . as a hormone avp exerts its biological effects upon binding to three receptor subtypes termed: v a , v b (v ), and v . furthermore, avp to some extent can interact with the oxytocin receptor (ot). biological activity of peptides is determined by their structure and conformation. conformational restriction of bioactive peptides is therefore a well-established strategy to change their pharmacological profi le. peptide fl exibility can be restricted by a local constraint imposed, e.g. by introducing amino acids with limited conformational freedom, that has an impact on specifi c orientations of the peptide backbone and the side chains. in this work, we decide to check the infl uence of the bulky ( design, synthesis and biological activities of temporin a and temporin l analogues. temporins a (ta) and l (tl) are antimicrobial peptides isolated from the skin of red european frog "rana temporaria". temporins are active against a broad spectrum of microrganism: ta (flpligrvlsgil-nh ) is preferentially active against gram-positive bacterial strains; tl (fvqwfskflgril-nh ) has the highest activity against fungi, and bacteria, including resistent gram-negative strains, but it shows haemolytic activity too. ta exerts its antimicrobial activity by its ability to form a transmembrane pore via a 'barrel-stave' mechanism or to form a 'carpet' on the membrane surface via the 'carpet-like' model. recently we investigated the preferential conformation of tl and ta in sds and dpc solutions which mimic bacterial and mammalian membranes, respectively. in sds, the peptides prefer a location at the micelle-water interface; in dpc, they prefer a perpendicular location to the micelle surface, with the n-terminus imbedded in the hydrophobic core. tl shows higher propensity, with respect to ta, in forminghelical structures in both membrane mimetic systems and the highest propensity to penetrate the micelles ( ). on these results we designed and synthesized new ta and tl analogues and found interesting differences in their effi cacy against microbial species, and fi nding a new potent antimicrobial agent without haemolytic activity. arginine vasopressin (avp), a neurohypophyseal nonapeptide hormone [cycle - (h-cys -tyr -phe -gln -asn -cys -pro -arg -gly -nh )], elicits a variety of responses both centrally and peripherally by acting on three distict g-protein coupled receptors: v a (vascular), v b (pituitary) and v (renal). it also binds to the oxytocin (ot) receptor. in addition to its well-known antidiuretic activity, avp has also complex cardiovascular actions and adrenocorticotropic hormone (acth) releasing activity. binding of avp to the v a receptor subtype also stimulates glycogenolysis in the liver and promotes platelet aggregation. it is generally accepted that the conformation of the n-terminal part of neurohypophyseal hormones analogues is important for their pharmacological activity. in continuing our work aimed at the design of selective avp analogues, we synthesized twelve new analogues of avp containing mercapto propionic acid ( we also studied the effect of modifi ed c-terminal amide on biological potency of the new avp analogues. the analogues were synthesized by fmoc/bu t solid phase methodology and were tested for their rat uterotonic in vitro activity, rat pressor activity and antidiuretic activity using conscious rats. the modifi cations performed had a signifi cant impact on pharmacological activities of the analogues. acknowledgements: we thank the european social fund (esf), operational program for educational and vocational training ii (epeaek ii), and particularly the program pythagoras i, for funding the above work. it was also supported by the research project no. z of the academy of sciences of the czech republic. three-dimensional structure and mechanism of action of an antifungal peptide generated from hemocyanin cleavage in a penaeid shrimp an antifungal peptide, pvhct, which corresponds to the amino acid c-terminal sequence of the shrimp respiratory protein hemocyanin, has been previously identifi ed in the plasma of the penaeid shrimp litopenaeus vannamei ( ) . it is generated by proteolytic cleavage in response to a microbial challenge. similarly, a c-terminal fragment of hemocyanin displaying antimicrobial activity has been isolated from crayfi sh plasma ( ) . these peptides are believed to contribute to the crustacean defence. the phenomenon of in vitro antimicrobial peptide generation from a respiratory pigment already observed with hemoglobin thus appears not to be restricted to mammals ( ). pvhct displays a broad spectrum of antifungal activity with minimum inhibitory concentrations (mics) in the range - m ( . m against the shrimp pathogen fusarium oxysporum). its activity would be based on the inhibition of spore germination ( ) . to contribute to the elucidation of the mechanism of pvhct antifungal activity, we determined its three-dimensional structure by circular dichroism (cd), nmr and molecular modelling and examined its effects on the f. oxysporum spore ultrastructure by transmission electron microscopy (tem). cd and nmr data indicate that pvhct is unfolded in an aqueous environment but adopts a similar helical structure in methanol solution and in dodecylphosphocholine (dpc) micelles used to mimic biological membranes. the structure consists of an amphipathic -helix spanning residues to . tem shows that pvhct induces structural changes of the plasma membrane accompanied by a disorganization of the cytoplasm and a signifi cant decrease of lipid bodies. glucagon-like peptide- (glp- ), glucagon (gcg), and oxyntomodulin (oxm) are highly homologous peptide hormones derived from posttranslational processing of the preproglucagon gene. the sequence of oxm in particular, is identical to the sequence of gcg, with an amino acid extension at the c-terminus. pharmacological doses of oxm activate both the glp- receptor (glp r) and the glucagon receptor (gcgr) albeit with lower affi nity compared to glp- and glucagon, respectively. in order to understand the origin of this dual specifi city, we synthesized a number of oxm analogs in which one or more of the native amino acids were replaced. first, a chimera was produced in which all the residues differing between glp- and gcg were grafted into the oxm native sequence, yielding an analog with the same pharmacologic profi le as glp- . further studies showed that surprisingly, the switch from glp r/gcgr co-agonism to glp r-selective agonism could be obtained by a single amino acid substitutions at position of oxm. in particular, the analog with gln substituted by glu (oxm-q e) showed the same activity as native oxm on glp r, but complete loss of activity on gcgr. oxm and oxm-q e were compared in a hyperglycemic clamp study performed in diet-induced obese (dio) mice. due to the short half-life of the peptides, both were infused intravenously at ~ g/kg/ min in chronically catheterized mice. the amount of exogenous glucose required to maintain the hyperglycemic level was -fold greater with the selective glp r agonist oxm-q e than with the glp r/gcgr coagonist oxm, showing that abolishment of gcgr activity signifi cantly improves the glucose-lowering effect. we believe that these fi ndings could be useful for the development of a peptide therapeutic for the treatment of type diabetes. hospitals across europe and north america have lately been plagued with infections caused by clostridium diffi cile, a diffi cult to treat bacterium due to its resistance to many commercially available antibiotics. recently, we isolated a two-component bacteriocin, thuricin, produced by bacillus thuringiensis that exhibits activity against c. diffi cile. we found that these peptides, called trn and trn , operate together in a synergistic fashion to inhibit bacterial growth. the peptide combination was found to be highly active against a wide range of c. diffi cile strains, including the virulent epidemic strain of the o ribotype, as well as most other clostridia and some bacilli and listeria species. maldi mass spectrometry revealed that both peptides have molecular weights that are lower than those predicted from their genetic sequences, indicating that they are post-translationally modifi ed. sequencing of the mature peptides through tandem mass spectrometry showed that each peptide has three modifi ed amino acid residues near its c-terminus. these residues were found to be two units lighter than their expected natural amino acid masses, suggesting a loss of two hydrogen atoms through dehydrogenation or oxidation. in order to confi rm these proposed modifi cations, we are investigating the production of c-and n-labeled trn and trn for structure elucidation by nmr. isolation of labeled peptides will be achieved by growing b. thuringiensis on defi ned media containing [u- c] glucose and ( nh ) so . subsequent nmr experiments will enable us to determine the three-dimensional solution structures of trn and trn , individually and bound together. by elucidating thuricin's structure, we aim to better understand its mechanism of action against c. diffi cile. the most potent peptidic human bradykinin (bk) b receptor antagonist is hoe- (icatibant). in this study we present the synthesis of nine new analogues of hoe- substituted at position , or with chosen d-amino acid residues and/or nonproteinogenic amino acid residues, e.g. n-cyclohexylglycine (nchg), -aminocyclohexane- -carboxylic acid (acc), octahydroindole- -carboxylic acid (oic), piperidine- -carboxylic acid (nip) and -phenylpiperidine- -carboxylic acid (ppc). in the next nine peptides we combined the above mentioned modifi cations with the placement of -adamantane acetic acid (aaa) at position . all new analogues were tested on human umbilical vein for their antagonistic potency. only three compounds containing nchg , nchg or ppc exhibited noticeable antagonistic activities on human bradykinin receptors thus still less potent then original hoe- sequence. each of them with aaa showed lower activity then parent analogues. peptides: d-arg-arg-pro-hyp-gly-thi-ser-d-phe-nip-arg and aaa-d-arg-arg-pro-hyp-gly-thi-ser-d-phe-nip-arg, although strongly potent antagonists against bk-induced blood pressure lowering responses in rats, did not show noticeable antagonistic activity on bk-induced contraction of the isolated human umbilical vein, a well-established b r bioassay system, suggesting a species dependent activity of the compounds. due to advances made in the peptide fi eld during the last few years, peptides as therapeutics have continued to gain more interest. the interest in peptide therapeutics generally comes from their high specifi city to targeted sites, their diversity and their usually low toxicity. pt- , also known as bremelanotide, and mt-ii are potential future drugs and both are heptapeptides with a -membered cyclic monomeric lactam bridge. they are melanocortin receptor agonist and an analog of alpha-melanocyte stimulating hormone (a-msh). the pt- molecule has a c-terminal acid group while mt-ii has a c-terminal amide function. both neuropeptides have been tested in treating male sexual and erectile dysfunction as well as female sexual arousal disorder. pt- , patented by palatin technologies, new jersey, usa, is the only known synthetic aphrodisiac and unlike other erectile enhancers like viagra, it does not act upon the vascular system. instead, it directly increases sexual desire and is used nasally as a spray. a scalable method for the synthesis of both peptides using fmoc-chemistry and the problems involved during the synthesis process will be discussed in details. tateaki, wakamiya; takahiro, nishimaru; kiyoe, mori; yoshihiro, yamaguchi kinki university, japan l-glutamate (glu) is known to be major excitatory neurotransmitter not only in the mammalian central nervous system but also in the ganglia of arthropods. binding of spider toxins to glutamate receptors (glurs) results in the inhibition of glu-mediated neurotransmission. in order to elucidate the mode of binding between glu and glurs, we focused on the visualization of glurs by complex formation with fl uorescentlabeled analogs of nptx- ( ), a spider toxin with the structure of n -( , -dihydroxyphenylacetyl-l-asparaginyl)-n -l-lysyl- , -diaza- , -dodecanediamine [dhpa-asn-dada( lys)]. in the present study, the modifi ed nptx- , i.e., dhpa-asn-dada( abg) ( ) in which the lys residue of was replaced with the n-( -aminobutyl)glycine (abg) residue, was employed as a template compound to create the fl uorescentlabeled analogs of nptx- , since the biological activity of is three times higher than that of . we thus carried out the modifi cation of dhpa in the analog , i.e., ) dhpa was replaced with the coumarintype acyl residues (type- ); ) the phenylacetyl residues having alkyne side chains that can be converted into suitable fl uorophores based on the click chemistry (type- ); and ) the coumarin-type acyl residues having the mercapto group to form disulfi de bond with the cys residue in glurs (type- ). this paper presents the synthesis and biological activity of various nptx- analogs to adopt as probes for visualization of glutamate receptors. an investigation of the functional requirements of apidaecin ib c-terminal fragment by means of peptoidpeptide hybrids by acting on one or more intracellular targets, without damaging the cytoplasmatic membrane. their particular killing mechanism and their low toxicity against mammalian cells make them attractive for the development of new antibiotics. structure-activity relationships studies have been carried out on short pro-arg rich antimicrobial peptides isolated from insects and the characterization of various natural isoforms of the -residues peptide apidaecin ib allowed to identify an evolutionary conserved region in the c-terminal part of the molecule. even a single point mutation in this region results in reduction or loss of antimicrobial activity. we recently described the synthesis of some apidaecin ib peptoid-peptide hybrids in which each arginine was replaced by the corresponding n-alkyl glycine residue ( ). the afforded modifi cation made the resulting peptoid-peptide hybrids more resistant to proteolysis but moving the [narg]residue from the n-to the c-terminal end of the molecule progressively reduced the antibacterial activity. here we report the synthesis of a series of novel analogues containing a n-homoarginine or n-norarginine residue in position , or . the effect of the size of the side-chain of the peptoid residue on the antimicrobial activity is also reported. in order to strengthen the peptide resistance to proteolysis and by considering that enzymic cleavage of the arg -leu peptide bond yields a fully inactive compound, we also prepared the [nleu ]-apidaecin analogue. the conformational properties of the resulting peptoid-peptide hybrid, which is devoid of any antimicrobial activity, will be compared to those of apidaecin ib and the other [narg]peptoid-peptide hybrids. a wide variety of organisms produce antimicrobial peptides as part of their fi rst line of defense. however, antimicrobial activity is not the main function of some of these peptides. in the cuttlefi sh sepia offi cinalis we observed an antibacterial activity for the neuropeptide : h-alsgdaflrf-nh ( ). this decapeptide belonging to the fmrfamide family involved in regulation of reproduction and chromatophore function and is able to inhibit the growth of marine bacteria. circular dichroism studies have revealed for this amphiphilic peptide a helical structuration in sds micelles and in % tfe. to improve this antimicrobial activity, we fi rst introduce a lysine residue instead of aspartic residue, the antimicrobial activity of this new peptide has been improved by augmentation of the positive net charge (+ to + ). moreover preliminary results have shown that the incorporation of aza- -amino acids analogues could create original hybrid pseudopeptide with superior activity . the natural peptide is not effi cient against staphylococcus aureus whereas the pseudopeptidic analogue revealed a minimum inhibiting concentration (mic) between - m. therefore some -amino acids were replaced by aza- -amino acids. we will show in this communication that depending on the substituted residue and on the structuration, these modifi cations could lead either to no activity or to a drastic enhancement of the antimicrobial activity, demonstrating that aza- -amino acids can facilitate the burying in lipidic bilayer of antimicrobial peptides that acts on bacterial membranes( ) references: isolation and structural characterization of capistruin, a lasso peptide predicted from the genome sequence of burkholderia thailandensis e the poor overall fragmentation behavior in ms n studies suggested a branched cyclic peptide with a rigid lasso structure. optimization of the fermentation conditions increased the production by -fold and subsequent nmr structural studies proved the lasso structure of the peptide that was named capistruin. heterologous production of the lasso peptide in e. coli showed that the identifi ed genes are suffi cient for the biosynthesis of capistruin, which exhibits antimicrobial activity against closely related burkholderia and pseudomonas strains. to our knowledge, this is the fi rst rational based identifi cation of a novel lasso peptide and the presented approach should be advantageous for the isolation of further lasso peptides in the future. endogenous antimicrobial peptides (amps) are the earliest molecular factors in the evolution of innate immunity. marine invertebrate animals have no acquired immunity with a system of antibodies diversifi cation. they are presumed to use an amps-based system as principal defense against potential pathogens. we have discovered a new family of small ( -residue) amps, termed arenicins, in coelomocytes of marine polychaeta lugworm arenicola marina. these amps exhibited activity against gram-positive, gram-negative bacteria and fungi. complete amino acid sequences were determined for each isoform. arenicins have one disulfi de bond (cys -cys ). arenicins have no structure similarity to any previously identifi ed antimicrobial peptides. a novel -residue antimicrobial peptide, aurelin, exhibiting activity against gram-positive and gram-negative bacteria, was purifi ed from mesoglea of a scyphoid jellyfi sh aurelia aurita. complete amino acid sequence of aurelin was determined. aurelin has cysteines forming three disulfi de bonds. the total rna was isolated from the lugworm coelomocytes and from the jellyfi sh mesoglea, rt-pcr and cloning were performed, and cdnas were sequenced. a -residue preproarenicin contains a putative signal peptide ( amino acids) and a long prodomain. a residue preproaurelin contains a putative signal peptide ( amino acids) and a propiece of the same size ( amino acids). aurelin reveals partial similarity both with defensins and k+ channel blocking toxins of sea anemones and belongs to shkt domain family. overlapping of biological properties of marine animal amps and toxins along with their sequence homology might be a consequence of divergent evolution from a common ancestor. antimicrobial peptides from marine organisms could afford design of new antibiotics manifesting broad-spectrum antibacterial activity. cyclic enkephalin analogs containing two alkylurea units we shall present some structure-activity results for enkephalin analogs, derived from the exhaustive combinations of d-lys and d-orn in position with lys, orn, dab and dap in position , both positions coupled ¦Ø-¦Ø¡¯ by means of the urea bridge. accordingly, they all are restrained by - -membered rings. in addition, we introduced the -nh-ethylurea unit instead of -nh of amide group in previously published analogs [ ] [ ] [ ] . their in vitro activities were determined in the gpi and mvd assays. the peptides are more active than enkephalin in the gpi while have similar activities to the latter in the mvd assay. the effect of the introduction of ethylurea unit at the c-terminus on the activities is also discussed. chemical shifts of the peptides in water were fully assigned and their sequences confi rmed. several cross-peaks between the protons of amidoalkylurea unit and preceding residues have been observed in each case, suggesting that the unit may be involved in specifi c interactions between residues and . understanding the structure/activity relationships of hepcidin iron is an essential element for nearly all living organisms and plays a key role in a range of processes including oxygen transport and storage, catalysis of redox reactions, production of metabolic intermediates and host-defence ( ). until recently, little was known about the regulatory elements involved in the control of iron uptake and distribution within the body. the recently discovered peptide hepcidin has been shown to be a key regulator of iron metabolism within the body in response to a range of conditions, including infl ammation, hypoxia and anaemia ( ) . this talk will focus on work towards elucidating structure/activity data for hepcidin with the aim of gaining a better undertstanding of the interaction between hepcidin and its receptor, ferroportin. we hope that this information will facilitate the design of synthetic agonists or antagonists of ferroportin to be used as potential drug leads. three-dimensional structures of investigated analogues were determined using two-dimensional nmr spectroscopy and molecular dynamics simulations with time-averaged restraints. the analysis of structural differences exhibited by different modifi cations provides the basis for understanding conformation -activity relationships and thereby the mechanism of interactions of the analogues with receptors. nmr structure of the micelle-bound rfa and rfa, two peptide ligands of the gpr receptor a novel rfamide peptide, named rfa and with no meaningful similarity with other members of this family, has been recently characterized. its precursor encompasses several potential cleavage sites and thus may generate various mature peptides including an nterminally extended form of rfa, termed rfa. both peptides act as endogenous ligands of the g-protein coupled receptor gpr . this receptor has been recently implicated in the bone metabolism regulation the determination of the d structure of such peptides is essential for the elucidation of their structure/function relationships and for the design of potent agonists or antagonists. although structure elucidation of a ligand in the absence of the target receptor can deliver limited insight into the bioactive conformation, there is emerging evidence that interactions with the cell membrane is a key step required for receptor recognition. in this context, we have investigated the solution conformation of rfa and rfa by cd, nmr and molecular modelling in different media, in particular in one miming the "free" form of the molecule (methanol or mixture tfe/water) and in a cell membrane mimetic medium (dpc micelles). in an organic solvent, both peptides adopt the same conformation, i.e. an amphipathic alpha-helical structure, fl anked by two n-and c-terminal disordered regions. when bound to dpc micelles, the n-terminus remains fl exible and an helix is present at the same position as in the "free" form for both molecules. in contrast, the cterminal extremity becomes structured adopting an inverse gamma-turn conformation. these data represent the fi rst step for the rational design of new molecules that might be used in the treatment of osteoporosis. acknowledgements: supports were obtained from inserm and ifrmp . the nmr spectrometers are supported by grants of the conseil régional de haute-normandie. nmr and molecular modelling facilities are provided by the centre de ressources informatiques de haute-normandie. antimicrobial activity of analogues of a peptide isolated from venom glands of social wasps polistes major major inhabiting the dominican republic recently we have described isolation and biological activities of several new peptides from the venom glands of social wasps polistes major major found in dominican republic. we have also reported the synthesis of their analogues in order to investigate structure-activity relationship with respect to the antimicrobial and hemolytic activities ( ). here we report the activities of a few further analogues of one of the peptides called pmm (h-ile-asn-trp-lys-lys-ile-ala-ser-ile-gly-lys-glu-val-leu-lys-ala-leu-nh ). the parent sequence or its truncated analogues were modifi ed on the n-terminus with -aminocaproic acid, glycolic acid, palmitoic acid, and -acridinyl and -( , , , -tetrahydro)acridinyl groups. the new analogues were tested for their antimicrobial activity (determination of minimal inhibitory concentration values -micusing broth dilution method) and hemolytic activity (determination of the ic value using suspension of rat erythrocytes). the palmitoylation unfortunately did not enhance antimicrobial activity against the tested microorganisms (bacillus subtilis, staphylococcus aureus, escherichia coli and pseudomonas aeruginosa). substitution of amino acids ser or glu subsequently for alanine, serine or lysine did not infl uence the activity of the peptides signifi cantly. gramicidin s, gs, c-(val-orn-leu-d-phe-pro) , was isolated from bacillus brevis. it forms a two-stranded antiparallel -sheet fl anked by two ii' -turns. it was found that the distribution of hydrophobic and hydrophilic residues on the opposite sides of the sheet is a structural feature required for gs antimicrobial (am) activity. despite its wide gram+ and gram-antimicrobial activity gs is useless in therapy because of its high hemotoxicity in humans. it was found, however, that the analogues of gs- (gs with lys-leu inserted into each strand) got more am selective, when their amphipatic moments were perturbed by swapping adjacent lys leu/val or confi guration reversal at lys ( ) . here, we report on effects of similar perturbations put on gs original c-decapeptide, using the following examples: c-(val-lys-leu-d-his-pro) , , . as the mother compound, and its three analogs, viz. c-(val-d-lys-leu-d-his-pro-val-lys-leu-d-his-pro) -lys converted to d, c-(lys-val-leu-d-his-pro-val-lys-leu-d-his-pro) -val -lys swapped, and c-(val-leu-lys-d-his-pro-val-lys-leu-d-his-pro) -lys -leu swapped, - ; all having reduced ring-sequence symmetry. the peptides were synthesized by solid-phase methods using -fl uorenylmetoxycarbonyl (fmoc) methodology. having solved their structures by d-nmr and having tested their activities/selectivities, we confi rmed that only had relatively favorable bio-profi le, as already published ( ) cereulide, a foodborne peptide highly toxic towards the insulin producing beta-cells of the pancreas cereulide is a heat stable cyclic, lipophilic (log kow . ) peptide ( g mol/ ) produced by certain strains of bacillus cereus, a bacterium connected to emetic food poisonings. it is insoluble in water, soluble in ethanol, methanol, dmso, food oils. cereulide exposure caused collapse of mitochondrial membrane potential in all tested human cells at low (ng/ml) exposure concentration (nk cells, t lymphocytes, caco , calu , neural paju, hela). toxicity is caused by its action as ion carrier with a selectivity of k + : na + = > : . in various foods connected to human illness, concentrations of . to g/g of cereulide were measured. in a case where the remains of a meal that had caused acute serious illness of two adult persons, were obtained for analysis, . g of cereulide was found /g of food. we undertook to explore the effects of purifi ed cereulide, and cereulide containing bacterial extracts, on porcine pancreatic islet cells in culture. foetal porcine islet cells were exposed to heat killed extracts from food-borne b. cereus strains producing or not producing cereulide and to purifi ed cereulide. effects were assayed using viability staining with fl uorochromes and cellular contents of dna and insulin. exposure to ng/ml of purifi ed cereulide caused necrotic cell death of the islet cells impairing their insulin content within days. cell extracts of cereulide positive b. cereus strains connected to food poisoning or isolated from food items were toxic, corresponding to their measured cereulide content. extracts of b. cereus strains producing or not producing the b. cereus diarrhoeal toxin, but not cereulide, were tolerated by the porcine islet cultures up to concentrations fold higher compared to extracts from strains containing cereulide, produced substance toxic towards porcine fetal langerhans islets and beta cells. application of non-sequential pharmacophore concept for design of antimicrobial peptide dendrimers unique structure of dendrimeric compounds consisting of a central core and several generations of branches provides opportunity of multiple practical solutions in the area of medicine. location of a high number of functional groups at the surface, allows to present multiple pharmacophoric units to the receptors, with immediate application in the design of a new generation drugs or vaccines, tools for studying autoimmune diseases or understanding gene delivery mechanism. here we present another possible application of dendrimeric compounds -preparation of drug molecules, which mimic active conformations of various macromolecular ligands. we focused on low molecular weight basic dendrimeric peptides, which mimic active conformations of recently discovered natural antimicrobial peptides. structurally, natural compounds are linear cationic peptides consisting of - amino acids that kill a broad spectrum of microbes destabilizing ordered structure of their cell membrane. it is generally accepted that positive charge and an induced amphipathic conformation are necessary for their antimicrobial activity. the project is related to multi-drug resistance of numerous bacteria against conventional antibiotics and involves de novo design of - generation dendrimeric peptides, which mimic sequencerelated active conformations of natural compounds (non-sequential pharmacophore concept). apparently, several groups of small peptide dendrimers were synthesized and structurally characterized (nmr, cd). they are potent antimicrobials, active against broad spectrum of species including mrsa and esbl strains. interactions between model membranes and peptide dendrimers of various structure will be discussed. seawater desalination is most commonly done today by reverse osmosis (ro) using thin-fi lm composite membranes. a major problem in ro desalination is biofouling, caused by adhesion and growth of bacteria to form biofi lm on the membrane surface. recently we proposed a new approach to reduce biofi lm formation on ro membranes that is based on immobilization of antimicrobial peptides (amps) onto the membrane surface. in this study we screen amps capable of reducing biofi lm growth under conditions simulating seawater desalination, and search for mode of binding to the membrane without affecting the peptides bioactivity. a specifi c bioassay was developed for screening peptides activity in high salinity conditions in order to evaluate the inhibition of biofi lm growth, based on growing biofi lmforming bacteria in a -wells microtiter plate. we prepared various amps known from the literature by solid phase peptide synthesis (spps) using fmoc-chemistry. the bactericidal activity of amps was examined in fresh water and compared to high salinity water. most amps lost their activity in high salinity conditions; yet, few peptides possessed their bactericidal activity and were used in subsequent experiments. searching for mode of linkage was performed by evaluating the bactericide activity of amps modifi ed with numerous types of linker molecules. based on literature studies that showed no decrease in bactericidal activity of amps upon n-terminal modifi cation, we prepared the corresponding peptides with modifi ed spacers on their amino-terminal and evaluated their antimicrobial activity in solution. indeed, we obtained gly -spacers that retained activity, which were used subsequently as linkers to ro membranes. the mode of binding, as well as bactericide activity of the peptides and of the membranes will be presented and discussed. this study will lay the bases for a novel approach to decrease biofi lm formation on the surface of ro membranes during ro desalination. bioactive tripeptides ile-pro-pro and val-pro-pro protect endothelial function in vitro in normotensive and hypertensive rats milk drink containing casein-derived bioactive tripeptides isoleucylprolyl-proline (ipp) and valyl-prolyl-proline (vpp) has been shown to decrease blood pressure both in animal models and clinical studies. this effect can be attributed to the tripeptides. it has been suggested that one possible blood pressure lowering mechanism of the tripeptides could be angiotensin-converting enzyme (ace) inhibition. however, not all studies support this fi nding. the effect of tripeptides ipp and vpp on vascular function was investigated in vitro using rat mesenteric arteries. superior mesenteric arteries isolated from male wistar-kyoto and spontaneously hypertensive (sh) rats were incubated in krebs solution containing mm of the peptide (either ipp or vpp) in + ºc for , , or h. after incubation mesenteric artery rings were mounted in an organ bath chamber and extensive vascular reactivity measurements were performed. acetylcholine-induced endothelium-dependent relaxation was better preserved (p < . ) in mesenteric arteries of both strains incubated with ipp or vpp compared to the control. clear differences were not observed in sodium nitroprusside-induced endotheliumindependent relaxation. the ace-inhibitory activity of ipp and vpp was studied by measuring the response to a single administration of angiotensin i and ii in organ chambers. proportioned to kcl-induced contraction, no clear reduction in angiotensin i -contraction was seen. thus, ace-inhibition may not be the main mechanism for the long-term effects of ipp and vpp in the protection of endothelial function. we suggest that the tripeptides do not affect smooth muscle but they protect endothelium during incubation indicated as preserved acetylcholine-induced endothelium-dependent relaxation. and directly and modulate other parts of host innate immunity. today, more than cationic peptides have been identifi ed. they all have certain conserved physical features including a net positive charge, contain approximately % hydrophobic amino acids and have sizes ranging from to amino acids. however, virtually any -sheet, loop including -helix, type of secondary structure can arise including -turn and extended. the multitude of cationic peptide sources, structures and a spectra of activity is matched by a number of complex and controversial models attempting to describe and explain their modes of action. little is known about the sequence requirements of short host defense peptides like bactenecin ( mer). with help of our novel technique using a artifi cially created luminescence producing gram negative bacteria and peptide synthesis on cellulose we can investigate the sequence requirements of such peptides. hundreds of peptides are tested for their ability to kill pseudomonas aeruginosa. complete substitutional analyses of different bactenecin variants as well as a semirandom peptide library with about members were measured. the complete substitutional analysis will give us information about the importance of each single position whereas the peptide library will give us broader information which composition of amino acids results in an active antimicrobial peptide. the data will be analyzed using the quantitative structureactivity relationship approach (qsar) to identify sequence patterns that discriminate between superior activity cf. equivalently active and inactive. this will give us mechanistic cues for a better understanding of the mode of action of the short antimicrobial peptides. the results of these measurements and analyses will be discussed in detail. here we propose a simple and rapid fl ow cytometric method to assess internalization of the peptides in bacteria. the method is based on the use of fl uorescently-labeled peptides and of the extracellular quencher trypan blue to discriminate between a cell surface and cytoplasmic localization of the tested molecules. to his aim, we used bodipylabeled peptides showing different modes of action. these included some fragments of bac , a proline-rich peptide known to penetrate bacterial and eukaryotic cells without membrane damage [ , ] , and polymyxin b, a peptide antibiotic that binds to lps and to the cell membranes. by using this approach coupled to fl ow cytometric analysis, we showed that the fl uorescence intensity of e. coli and s. typhimurium cells treated with sub-inhibitory bodipy-bac concentrations did not decrease despite extensive washing and addition of the quencher trypan blue. in contrast, the fl uorescence of cells treated with bodipy-polymyxin b, as well as that of bacteria treated with a fl uorescein-labeled anti lps antibody, were promptly and almost totally quenched by addition of trypan blue, indicating their accessibility on the bacterial surface. these results confi rm the suitability of this method to rapidly infer the localization of labelled molecules in an accessible or inaccessible compartment of the treated bacterial cells. hp ( - ) is an antimicrobial peptide derived from the n-terminus of helicobacter pylori ribosomal protein l (rpl ). in our previous study, several analogues of hp ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , with amino acid substitutions that increased or decreased net hydrophobicity, were designed and showed that an analogue, a designed by substituting gln and asp with trp at positions and , respectively, caused increased antibacterial activity in minimal inhibition concentration (mic) and minimal bactericidal concentration (mbc) without having hemolytic activity. the peptide a acted also synergistically with known antibiotics including chloramphenicol against bacterial cells. fluorescence activated fl ow cytometry showed that a -treated cells had higher fl uorescence intensity than untreated cells, similar to that of melittin-treated cells. the peptide a showed a strong antimicrobial activity against antibiotic-resistant pseudomonas aeruginosa from otitis media, clinically isolated mrsa and vrsa including biofi lm-forming bacteria in vitro and in vivo. the ototoxicity of a was studied in vivo by topical application to the middle ear in guinea pig model. twenty guinea pigs ( groups, each group n= ) were each injected by transtympanic approach with ul of ug/ml, ug/ml, ug/ml, ug/ml of a , and . % gentamicin sulfate was instillated as a control. auditory brainstem responses (abr) to click were measured between st and th days after injection. histologic investigation of cochlea was performed by scanning electron microscope and light microscope. the results showed that topical application of a to the middle ear is well tolerated without cochlear damage. the present study, therefore, demonstrates that the usefulness of antimicrobial peptides for multi-drug resistant bacteria including as a new ototopical agent for crpa otitis media. structure-activity relationship study of kiss- : identifi cation of an antagonist of gpr in the absence of ccda, ccdb inhibit the cell division and can kill bacteria by a mechanism that involves the dna gyrase. bacterial dna gyrase is unique among the type ii topoisomerase with ability to negatively supercoil dna. the enzyme consists of two subunits, a (gyra) and b (gyrb) and operates as an a b heterotetramer. the mechanism of the inhibition of the gyrase activity by ccdb is still an object of many debates, but is clear that r residue of gyra and the c-terminus of ccdb (w -i ) play a crucial role in the gyrase-ccdb interactions. as an approach for a better understanding of this mechanism as well as for development of new gyrase inhibitors, we have synthesized peptide analogues of the ccdb protein and studied its activity by supercoiling assays and bacterial growth. five fragments (ccdb et , ccdb et , ccdb et , ccdb et and ccdb ss ) of the natural ccdb were designed and synthesized by spps. for the design, we considered the residue c-terminal -helix (residues e to w ), the loop that connects two strands of the wing sheet (residues r to l ) and an n-terminal region that includes the fi rst of the fi vestranded antiparallel -sheet. all peptides, except ccdb et , showed inhibition of the supercoiling activity of the dna gyrase, especially ccdb et with a mic = m. free peptides not showed antimicrobial activity, but when encapsulated in liposome (suv) were able to inhibit the bacterial growth in liquid culture medium. the growth inhibition in vitro for ccdb et was about % for gram negative bacteria. our fi ndings revealed a novel synthetic inhibitor of dna gyrase and ccdb et analogue is a good starting point for the development of a new and specifi c class of antibacterial agents based in the dna gyrase inhibition. acknowledgements: support: fapesp and cnpq synthesis and neuroprotective properties of short peptides consisting solely from glycine and proline residues now appears more and more data on biological activity of short peptides consisting solely from glycine and proline residues. it is suppose, that these peptides, named as "glyprolines" (gps), can be formed from collagen, ÅÑÌ and related proteins. however an effect of these peptides on cns is not yet understood. the aims of this work were to improve a methodology of gps synthesis and to study cytoprotective properties of some new gps in culture of neuronal cells. gps with a common structure (gp)n, (pg)n, and (pgp)n (n= - ) were synthesised by consecutive growing of peptide chain and fragment's condensation. synthesised peptides were characterised by hplc, mass-spectrometry, element analysis etc. cytoprotective activity of gps was assessed on an increase of survival of cultivated ÐÑ cells after Í Î -induced oxidative stress. it was shown, that from all tested peptides only (gp)n and pgp reveal cytoprotective activity. at the concentration m these peptides reduced an amount of damaged cells . - . times in comparison to the control. thus preliminary data received show that some gps demonstrate a strong cytoprotective activity and therefore it is advisably to put them on further study on in vivo models. in preliminary investigations we found that all the peptides inhibited to a high degree the replication of hsv- in vero cells. moreover, these compounds did not show any cytotoxic activity against the vero cells. sexual dimorphism of hldf- peptide neuroprotective action in alzheimer's models in vivo and vitro we established that hldf- peptide, biologically active fragment of human leukemia differentiation factor (hldf), restores ability for learning, and also prevents loss of long-term memory and decrease in the exploratory behavior of wistar rat-males with experimental alzheimer's disease induced by intrahippocampal injection of betaamyloid peptide a ( - ) and ibotenic acid. the hldf- peptide was shown to render protective infl uence directly on primary culture of hippocampal and cerebellar neurones, isolated from newborn male brain, under conditions of the beta-amyloid toxicity. protective action of hldf- peptide on newborn rat-male neurons is connected with poster abstracts its ability to reduce -alpha reductase mrna expression in more than times, blocking testosterone metabolism into dihydrotestosterone (dht) and thus interfering hyper activation of nmda receptors in ca areas of hippocampus. the protective action of hldf- peptide was investigated also and on female-rats with experimental alzheimer's disease. it was shown, that in contrast to males at which both forms of memory are broken: and long-term and working ones, in the case of females, injections of a ( - ) + ibotenic acid result in infringement of only working memory, at safety of long-term memory. introduction of hldf- peptide restored the broken working memory at females as effectively, as at males. however the mechanism of protective action of peptide on primary culture of hippocampal and cerebellar neurones, isolated from newborns female-rat brain, is connected not with the decrease in hyper activation of nmda receptors, but with abad ( beta-hydroxysteroiddehydrohenase of the th type) mrna expression enhancement which is a target of beta-amyloid peptide action and blocking of progesterone conversion into alpha-dihydroprogesteron due to -alpha-reductase mrna expression inhibition. in vitro and in vivo studies of p- , an antimicrobial peptide active against multidrug resistant gram positive cocci we have designed octapeptide based on the sequence of sepecin b, an antibacterial protein of sacrophaga peregrina, effective against gram + bacteria. we systematically incorporated internal hydrophobic residues for a cationic, helical amphipathic structure effective for its high antimicrobial activity. it is also modifi ed c-terminally by a dehydro leucine residue effective for its structural stability. the synthesis of dehydro amino acid was done by solution phase and other amino acids were coupled by solid phase peptide synthesis method. anti-bacterial activity of the peptide was done by standard micro broth dilution technique against clinical isolates of gpc including methicillin resistant s. aureus (mrsa), methicillin sensitive s. aureus (mssa), hlar, group a and group b streptococci cultured from pus, wound and throat swab. all these isolates were known to be responsible for nosocomially acquired infections. s. aureus atcc was used as quality control strain. invivo effi cacy of this peptide was also tested in a mouse model with epidermal lesions caused by mrsa. the mic obtained for the peptide was g/ml (average) for the tested strains. the peptide showed no hemolytic activity against human red blood cell. in the invivo studies the healing was induced early (after hrs total healing was seen) in the experimental animals. this novel peptide has a potential to evolve as a therapeutic option in infections caused by resistant gpc. , an adhesive protein, is recognized by the activated platelet integrin á ÉÉb through the arg-gly-asp (rgd) sequence resulting to platelet aggregation and thrombus formation. inhibition of this process can be achieved by rgd peptide analogues that bind to á ÉÉb receptor. however, this class of inhibitors upon binding to receptor cause an outside-in signaling which induces a further activation of the platelet. in previous studies we presented cyclic (s,s)-cdc-containing compounds (ic ~ ìm) and á ÉÉb derived sequences (y mesradr , á ÉÉb - ) (ic ~ ìm ) that exhibit a non-rgd-like inhibitory activity [ , ] . this interesting aspect could be the basis for the design and development of a new class of anti-platelet agents that could overcame the drawback of platelet activation through the outside-in signaling. to this aim we designed, synthesized and tested for their inhibitory potency various á ÉÉb - hybrid analogues incorporating the (s,s)-cdc-motif. cyclization reduces the allowed conformations, of both the backbone and the side chains, and possibly induces a favourable for the biological activity orientation of the charged side chains. the inhibition assays on the adp induced platelet aggregation revealed that incorporation of the (s,s)-cdc-motif considerably increases the inhibitory activity of the á ÉÉb - analogue. the unique toxic effect of acrebol, a novel peptide from acremonium exuviarum a novel peptaibol, named acrebol, was isolated and purifi ed from the fungal strain bmb found in water-damaged wood-based indoor building material. the strain bmb was identifi ed as acremonium exuviarum based on morphology, its sequence, and cycloheximide resistance. ms/ms analysis showed that acrebol is a mixture of two almost identical peptaibols composed of - amino acid residues with masses and da, acephe-iva/val-gln-aib-ile-thr-leu-aib-pro-aib-gln-pro-aib and acephe-iva/val-gln-aib-ile-thr-leu-val-pro-aib-gln-pro-aib, respectively. the c-termini of the peptaibols was seroh however the sequence (mass of da) between b and seroh could not be interpreted. both isoforms of acrebol had a strong toxic effect on boar spermatozoa, feline fetus lung cells, murine neuroblastoma, and mouse insulinoma min cells. we found that, unlike other peptaibols, acrebol in toxic concentrations did not increase the ionic and solute permeability of membranes of isolated rat liver mitochondria. acrebol did not disturb the ionic homeostasis and osmotic balance of mitochondria and induced no release of apoptogenic proteins (cytochrome c) from the intermembrane space of mitochondria. acrebol strongly inhibited complex iii of the respiratory chain (ic ~ ng/ml), presumably, the outer quinone-binding center and, similarly to myxothiazol but in contrast to antimycin a, decreased the production of superoxide anion in the outer compartments of mitochondria. in the boar spermatozoa, acrebol, blocking the respiratory chain, caused the atp depletion due to the oligomycin-sensitive reversion of the reaction of atp synthesis, which resulted in the inhibition of progressive movement. acrebol induced necrosis-like death of mouse insulinoma min cells whose energetic metabolism is strongly dependent on oxidative phosphorylation in mitochondria. thus, acrebol is a unique peptaibol with a specifi c pattern of the toxic effect. delta sleep inducing peptide (dsip), its analogues and deltaran®: biological activity and mode of action for the last decade we have been engaged in studies on the endogenous neuromodulator dsip (waggdasge) and a large group of its derivatives in respect of both their physiological activity and mechanism of action. a wide range of evidences confi rmed benefi cial effects of dsip and some active analogues under experimental stress models. dsip has emerged as promising and potentially effective therapeutic agent due to strong and unique adaptive and stress protective activity revealed during the study. dsip related drug deltaran® registered in russia has also showed the signifi cant effi ciency in animal test models and clinic. the peptides of dsip family often do not demonstrate any effects under normal and comfortable conditions or even cause slight prooxidative and stress promoting effects. these properties and established wide profi le of biological activities of dsip complicate the work on dsip mode of action. cellular and subcellular effects of this peptide still remain poorly studied. previously we investigated some biochemical events underlying the stress protective effi ciency of dsip and its derivatives. in continuation of these studies we have attempted to evaluate the putative dsip infl uence on classical cellular processes utilizing stressprotective heat shock proteins (hsps) and apoptosis. effect of dsip on the level of hsp expression in human erythroleukemia cell line k was detected.. we have found that dsip down regulates the increase of intracellular hsp level during incubation of cells in high density cell culture. according to our preliminary data dsip increased hsp level in murine t-cell line ctll- similar to -and ß-adrenergic receptor agonists. in murine thymocytes dsip increased both apoptosis and hsp . we propose that registered effects of dsip are mediated through adrenergic receptors. cellular mechanisms of dsip and related peptides action are under way. identifi cation, chemical synthesis, and antimicrobial activity of tbd- -the fi rst -defensin isolated from reptiles antimicrobial peptides (amps) and proteins have been discovered in single and multicellular organisms indicating the importance of this peptide class for the innate immune system. amps are active against bacteria, fungi and viruses by affecting either the microbial cytoplasmic membrane, thus increasing its permeability or interacting with specifi c targets. defensins form one of the major subfamilies of amps, among which -and -defensins play a signifi cant role in bridging innate and adaptive immunity in mammals. the cationic -defensins are cysteine-rich and vary in length from to residues. the -stranded structure of -defensins is stabilized by a characteristic arrangement of three conserved disulfi de bonds between cysteines - , - , and - . although -defensins lyse the bacterial membrane at higher concentrations it has been shown that they modulate also the immune system, e.g. being chemotactic for t-cells. we have isolated a novel mer -defensin called tbd- from leukocytes of the european pond turtle and deduced its complete sequence de-novo by combining different tandem mass spectrometry techniques (maldi, esi; cid and etd) and edman degradation. it was also possible to identify the disulfi de pattern in a tryptic digest by maldi-mass spectrometry. the deduced peptide sequence was afterwards confi rmed by solid phase peptide synthesis. thus two fragments were synthesized by standard fmoc/ t bu chemistry using three orthogonal cysteine protecting groups (trt, acm, tbu) to selectively form the right disulfi de bridges. after native chemical ligation of the two peptide fragments the fi rst two cysteines were oxidized on air followed by iodide oxidation for the second and dmso oxidation for the third disulfi de bridge. in antimicrobial activity assays, tbd- synthesized in -conformation was active against gram-positve, gram-negative bacteria, and fungi similar to the native peptide. trichogin ga iv (trga), an antimicrobial peptide of the lipopeptaibol family, continues to reveal peculiar properties since the time of its former identifi cation by rebuffat and coworkers. x-ray diffraction studies showed that trga is folded in a mixed /a-helix conformation, while nmr, cd and ir absorption experiments proved that this structure is predominantly populated in solution, although more disordered structures contribute to the conformational landscape of trga. we have recently shown by time-resolved experiments, that an equilibrium between helical conformers and more compact, folded conformers takes place in solution, with interesting transition dynamics in the microsecond time scale. in our conformational studies on trga we realized that the d geometry of the peptide chain reproduces the structural environment of the ion coordination site of calcium-binding proteins. this fi nding urged us to investigate the binding properties of trga with respect to ca(ii), gd(iii) and tb(iii), because lanthanide ions have been widely employed in biochemical studies as best substitutes of ca(ii). the binding of ca(ii), gd(iii) and tb(iii) to trga gives rise to a conformational transition, monitored by cd spectra at different ionpeptide molar concentration ratios. at high r values, the cd curves of all the ion-peptide complexes show a positive maximum at ~ nm, typical of type ii turns, suggesting the population of bent structures. the quasi-isodichroic point found for all systems between and nm, indicates that an equilibrium between extended helical and bent conformations actually takes place. fluorescence experiments on the tb(iii)/trga adduct have shown that, upon ion binding, the fl uorescence quantum yield of tb(iii) markedly increases, due to the release of water molecules from the ion coordination inner shell. molecular dynamics calculations on the peptide/ca(ii) adduct were also performed to obtain structural and dynamical information. antibiotic resistant bacterial strains represent a global health problem with a strong social and economic impact. thus, there is an urgent need for the development of antibiotics with novel mechanisms of action. castros group isolated and determined the sequence of the peptide hy-a (ifgailplalgalknlik) of skin secretion from the frog hypsiboas albopunctatus which showed antimicrobial activity. the aim of the present work was evaluated analogues to supply information poster abstracts about the relationship structure-biological activity. the peptides were synthesized by spps using the fmoc chemical approach. the biological activities were assayed by measuring growth inhibition of two types of gram-positive bacteria and others two types of gram-negative. the synthesis and purifi cation of peptides by hplc was effi cient and a high purity level ( %) was obtained. the peptide containing trp in position (for fl uorescent studies) replacing leu presented mic values comparable to wild type sequence: um, um, um and um for e. coli, p. aeruginosa, s. aureus and b. subtilis, respectively. two peptides with this modifi cation but containing at the n-terminal region one group acetyl or a residue of asp showed mic values of um for e. coli and p. aeruginosa, although um for gram-positive bacteria. different results were observed when the residue added was lys. in this case, the activity against whole bacteria was sustained or increased. conformational properties were investigated by cd techniques in water, tfe and in zwitterionic micelles (lpc). the cd experiments demonstrated that in water, the peptides have a random structure, but in tfe and lpc solutions they acquired an ordered structure, composed mainly by -helix. however, these data there is no relationship between the structure and activity against bacteria gram-positive. these results showed that the n-terminal region of the peptide hy-a develop key roles in its antibacterial action different types of bacteria. pexiganan is an antimicrobial peptide remarkably effective against bacteria causing skin infections, and has been commercially developed as a topical cream to treat infected diabetic foot ulcers. [ ] [ ] [ ] as peptide drug-based therapy often suffers with low drug stability in vivo, suitable delivery systems must be developed. with this purpose in mind, we have designed a pexiganan-chitosan conjugate to combine the exceptional bioadhesion and tissue regenerating abilities of chitosan [ ] [ ] [ ] with the excellent antibiotic properties of pexiganan. we herein wish to report our fi rst results on the successful synthesis, ft-ir and amino acid analysis of a pexiganan-chitosan conjugate prepared by covalent attachment of a cys-containing pexiganan analogue to the chitosan's amino groups, by means of the heterobifunctional cross-linker sulfo-emcs. . overexpression of e. coli oligopeptidase b confers resistance to the proline-rich antibacterial peptides scocchi, marco; de gobba, cristian; mattiuzzo, maura; gennaro, renato university of trieste, italy the proline-rich antimicrobial peptides (pramps) are a large group of cationic peptides isolated from mammals and insects, which show a spectrum of antibacterial activity limited to gram-negative bacteria and a remarkably low cytotoxicity towards eukaryotic cells. pramps are thought to act in a permeabilization-independent manner, via energydependent internalization into bacteria followed by recognition and inactivation of internal molecular targets. with the aim of investigating their mode of action, we have isolated a number of e. coli clones showing increased resistance to the bovine proline-rich peptide bac after transformation with a dna library from bac -resistant mutants. among the recombinant plasmids responsible for resistance, some of them harboured the gene coding for the oligopeptidase b (opdb), a serine peptidase belonging to the prolyl oligopeptidase family (pop) broadly distributed among unicellular eukaryotes and gram-negative bacteria, which has emerged as an important virulence factor. opdb was cloned and expressed under the control of an inducible promoter and the transformants tested for susceptibility to a panel of antimicrobial peptides, including pramps. olipopeptidase activity of purifi ed opdb was then tested in vitro against the same peptides. the results indicate that the clones overexpressing opdb are more resistant to the pramps and that the degree of resistance correlates with the expression level of opdb. in addition, in vitro incubation of pramps with the purifi ed peptidase, followed by mass spectrometry analysis, showed that it promptly hydrolyzes all the peptides assayed to short, inactive fragments. these results suggest that opdb may contribute to cleavage and inactivation of antimicrobial peptides that are internalized into the target cells and support the notion that opdb is a novel virulence factor. guan, shuwen; huang, lei; li, pengfei; wang, liping; li, wei college of life science, jinlin university, china great progresses have been made in the research on identifying molecules of therapeutical potential for delaying aging. using caenorhabditis elegans, we had tested the effects on stress resistance and life span of treatment with dhhp- (deuterohaemin-alahisthrvalglulys), synthetic mimetics of the antioxidant peroxidases, which neutralizes peroxide. to further test the mechanisms of dhhp- on life span extension, exogenous protein sod and catalase levels were measured. we show that dhhp- is able to elevate in vivo sod and catalase activity levels after two days administration. treatment with exogenous dhhp- affected endogenous protein sod and catalase levels and elevated the expression of sod- ::gfp in the head and vulva. on the other hand, dhhp- can extend life span of c. elegans lacking sod- or ctl- gene expression by rnai. this suggested that the antioxidant enzyme in c. elegans may not the only target affected by dhhp- . du, haidong; yan, yumei; wang, liping; li, wei college of life science, jinlin university, china high concentration of hydrogen peroxide (h o ) induces nuclear dna fragmentation, lipid peroxidation and has been implicated in many diseases including heart failure, parkinson's disease, and cancer. in a systematic attempt to develop an effective scavenger of h o , we have successfully synthesized an artifi cial microperoxidase, deuterohaemin -alahisthrvalglulys (dhhp- ) as core catalytic center to which amino acid peptide was covalently attached. dhhp- exhibited potent peroxidase activity, favorable membrane permeability and thermal stability.two experimental models of oxidative injury were established in order to investigate the anti-oxidative effect of dhhp- : one was induced by hypoxia-reoxygenation in cultured heart-derived h c cells bioactive peptides and another was caused by h o in cultured neonatal rat ventricular myocytes. dhhp- could protect cells against oxidative injury by determining mtt cell proliferation assay, ldh leakage and ca +-atpase activity. furthermore, dhhp- repressed the apoptosis gene expression, such as p , hsp and heme oxygenase¢ñ, that proved dhhp- had protective effect in cultured neonatal rat ventricular myocytes . taken together, this small microperoxidase exhibited excellent ¡°druggable¡± properties, and could be a promising agent for ros-associated diseases with unmet needs. trichogin ga iv is the most extensively investigated member of the class of lipopeptaibols that are linear peptide antibiotics of fungal origin, characterized by the presence of a variable, but remarkable, number of aib residues, a fatty acyl group at the n-terminus, and a , -amino alcohol at the c-terminus. several analogues of trichogin ga iv with amino acid substitutions or deletions were designed which allowed determination of the minimal inhibition concentration against gram-positive and gram-negative bacteria and various pathogenic fungal cells. the natural peptide exhibits a specifi c activity against s. aureus and only a marginal hemolytic effect. interestingly, trichogin ga iv is active also against several methicillinresistant s. aureus strains. studies on synthetic analogues demonstrated that substitution of the c-terminal leucinol by leu-ome, or substitution of one aib residues by the epr label toac do not perturb signifi cantly the biological activity of the peptide. on the other hand, removal of or n-terminal residues eliminated any antibacterial activity. finally, studies of proteolytic degradation on trichogin ga iv and analogues where the aib residues are replaced by leu demonstrated that the presence of several non-coded aib residues endows the natural peptaibol with remarkable resistance to proteolysis. the present results indicate that trichogin ga iv is a promising lead compound for the development of new, selective and protease-resistant, antibacterial drugs. the siderophore microcin family: from the genetic systems to the antimicrobial peptides vassiliadis, gaëlle; peduzzi, jean; rebuffat, sylvie muséum national d'histoire naturelle-cnrs, france microcins are low molecular weight antimicrobial peptides secreted by enterobacteria and involved in microbial competitions within the intestinal tract. they are synthesized by the ribosomal pathway. we have isolated the fi rst siderophore peptide ( ), a post-translationally modifi ed form of the chromosomally encoded microcin e (mcce ) from klebsiella pneumoniae, having a potent bactericidal activity mainly directed against escherichia coli. the post-translational modifi cation consists of a glycosylated catechol-type siderophore linked to the c-terminus. we have recently identifi ed the genes responsible for the acquisition of this modifi cation and proposed a model for its biosynthesis ( ) . in order to identify novel siderophore peptides, we analyzed the genetic systems of several microcinogenic strains. based on the genetic organization of the microcin gene clusters, three microcins which had preparation of a close mimic of the n-terminal part of the c a receptor (c ar) by selective introduction of sulfated tyrosine residues ( ) and chips in complex with our sulfated c ar-model by multi-dimensional nmr techniques. based on these interaction data and the solution structure of the complex, chips-based c ar inhibitors for use in anti-infl ammatory therapy might be designed. insulin-like peptide (insl ) was fi rst identifi ed through a search of the expressed sequence tags (est) databases. primary sequence analysis showed it to be a prepropeptide that is predicted to be processed in vivo to yield a two-chain sequence (a and b) containing the insulin-like disulfi de crosslinks. the high affi nity interaction between insl and the receptor rxfp (gpcr ) coupled with their apparent co-evolution and partially overlapping tissue expression patterns strongly suggest that insl is an endogenous ligand for rxfp . given that the primary function of insl /rxfp pair remains unknown, an effective means of producing suffi cient quantities of this peptide and its analogues is needed in order to systematically investigate its structural and biological properties. a combination of solid phase peptide synthesis methods together with regioselective disulfi de bond formation were used to obtain insl . both chains were identifi ed as being ¡ §diffi cult sequences¡¨ and were unusually resistant to standard synthesis protocols including those mediated by microwaves. the a-chain was also prone to signifi cant aspartimide formation. the b-chain, in particular, required the use of the strong tertiary amidine, dbu, for more effective nƒÑ-deprotection during its assembly. following chain combination and sequential disulfi de bond formation, the resulting synthetic insl was obtained in good overall yield and shown to possess a similar secondary structure to human relaxin- (h relaxin). the peptide was able to inhibit camp activity in sk-n-mc cells expressing the human rxfp receptor with a similar activity to h relaxin. in contrast, it had no activity on the human rxfr receptor. novel cyclic bacteriocin-like peptides from strains of anabaena (cyanobacteria) leikoski ( ) and microcystis aeruginosa ( ) . the genome sequence of fi lamentous and diazotrophic cyanobacterium anabaena revealed a putative gene cluster (acy) encoding a bacteriocin-like peptide. one of the acy genes encoded a prepeptide, which showed n-terminal homology to the known cyanobacterial prepeptides but the mature peptide product in anabaena could not be predicted. we sequenced prepeptide genes from several anabaena strains to fi nd a prepeptide containing either cysteine or methionine, since sulphur containing amino acids enable the detection of the mature peptide product through s-labelling and lc-ms. the nucleotide sequence of the prepeptide genes revealed enormous variety in closely related anabaena strains. one strain, anabaena b contained a methionine in the prepeptide, and a cyclic heptapeptide product corresponding to the precursor was discovered in this strain. since the cleavage sites were found to be conserved in all anabaena strains the products could be predicted from the amino acid sequence and identifi ed by lc-ms. in addition, the biosynthesis of the decapeptide anacyclin in anabaena was verifi ed by heterologous expression of the acy genes in e. coli and the structure of the peptide was confi rmed with a synthetic reference peptide. altogether, new cyclic peptides were found in strains of anabaena with very little sequence conservation. cyanobacteria seem to be versatile producers of peptides using both ribosomal and non-ribosomal biosynthetic pathways. it is well established that n-terminal fl ank of corticotropin-releasing factor molecule is crucial for its biological activity. little, however, is known about in vivo effects of crf-derived peptides. this study was aimed to investigate possible actions of a tripeptide crf fragment - pro-pro-ile (ppi). tripeptide ppi was found to exert effects similar to fullsize crf molecule after central administration. the tripeptide induces behavioral activation in home cage, but inhibits behavioral activity under stressful conditions. ppi increases blood pressure and heart rate, blood glucose level and body temperature, decreases pain sensitivity, increases eeg amplitude and large doses of ppi induce seizures. ppi inhibits sexual motivation and performance in mating tests in males. crf antagonist -helical crf - abolishes ppi infl uence on circulatory system, glucose metabolism, thermoregulation and pain sensitivity. adrenalectomy does not interfere with hyperglycemic and hyperthermic actions of ppi, while pancreatectomy prevents hyperglycemic effect. nonselective beta-adrenergic blocker obsidan prevents hyperglycemic and decreases hyperthermic effects of the tripeptide and ganglionic blocker hexamethonium abolishes both effects. taken together effects of ppi correspond to stress-reactions, involving corticotropin-releasing factor and evidence that ppi is either ) a part of a crf molecule active site, or ) physiologically active crf derivative, realizing its effects through activation of crf receptors, or ) an independent regulator, affecting crf-ergic neurons. acknowledgements: the work was supported by rfbr grant - - - -à huggins, kelley; andersen, niels university, united states amyloid fi bril formation is associated with at least human diseases; among these, the human-amylin(ham)-derived deposits in type ii diabetes were the discovery system and ham aggregation is one of the more thoroughly studied systems. to date, inhibitors of beta-aggregate and fi bril formation have been polyphenols, mutants of ham, or short peptide related to the ham( - ) sequence, nfgailss. we now report that stable beta-hairpin scaffolds displaying trp and tyr residues are effective inhibitors, delaying the onset of both the cd changes associated with beta structure formation and the nucleation time and net enhancement of the fl uorescence observed with added thiofl avin-t (tht). under our test conditions ( microm ham, % hfip in mm phosphate buffer, ph ), ham begins to display an increase in beta structure by cd at min with a constant maximal value from - min. tht fl uorescence also indicates a circa min onset time with a rapid (< min) rise to the full response. inhibition (delayed onset and reduction in the maximal fl uorescence enhancement) has been observed with a number of hairpins; those with two trp residues on a single face of the hairpin are more potent. of these, our best inhibitor to date, kkltvwipgkwitvsa (p = d-pro), increases the onset time more than -fold at equimolar concentrations. at molar equiv., the onset time is greater than min and tht fl uorescence levels out at < % of the control value. in analogy to the report by prof. ghosh (jacs , p. ) that a beta-sheet protein with added tyr and trp residues inhibits fi bril formation by the alzheimer-related abeta peptide; we expect that designed beta-hairpin scaffolds will be more generally applicable and will afford new insights into the recognition phenomena of amyloidogenesis. baabur, hemda; ashkenasy, gonen ben gurion university, israel the main advantage in utilizing proteins for the development of sensors and receptors, over the more often used approach that exploits small molecules, is the ability to manipulate large recognition surfaces, which expose toward the bulk solution number of different functional groups. we search for stable artifi cial proteins that can be utilized as 'universal' recognition entities. to that end, modular chemical syntheses are exploited for the preparation of repeats-proteins. leucine rich repeat proteins (lrrs) are - residue sequence motifs present in several proteins with diverse functions. internalin b (inlb), a surface lrr protein of the human pathogen listeria monocytogenes, promotes invasion into various host cell types by inducing phagocytosis of the entire bacterium. consensus design uses statistical analysis of sequence alignments of families of homologous proteins for protein engineering. we show here that using consensus design, series of protein mutants that differ in the recognition surfaces are synthesized and will be probed for their ability to bind different natural and non-natural ligands. comparative structural studies of potent neuroprotective peptides of the humanin family although the rescue activity of hn peptides has been linked to a number of signaling pathways and receptors, their mechanism of action remains unknown. in this work cd and nmr data on the above peptides are presented and compared in an effort to defi ne structural characteristics related to their function. evaluation of our fi ndings in combination with existing structure-function relationship data for this class of peptides, brings forth fl exibility as an important structural feature that may facilitate interactions with functional counterparts of the neuroprotection pathway. the ability to adopt a partial helical conformation in the presence of low concentrations of tfe is another common structural feature that may be defi ning the interactions of these peptides in the environment of cell membranes. desleu-aga(c r)hng and cl display a more complex behavior shifting from -helical to -sheet conformations depending on ph, peptide concentration, and % of tfe present in solution. this fact may be related to their high potency since in hn literature evidence for self-association ability has been linked with neuroprotection. our cd and nmr experimental data combined with theoretical modeling will hopefully provide important clues for the elucidation of the mechanism of action of the hn family of peptide the process of molecular recycling describes a dynamic mechanism by which individual amyloid fi brils are continuously dissolving and reforming ( ) . the present work aims at the study of the dynamic properties of the -amyloid, a ( - ), amyloid fi brils. since the formation of a aggregates has been suggested as a key process in the pathology of alzheimer's disease, the study of the dynamic nature of a ( the coiled coil structure is widely distributed in natural proteins, such as transcription factors, receptor proteins and enzymes. this motif has been recently used by chemists for the design of functional synthetic proteins. specifi c coiled coil sequences can be utilized as templates for self replication processes ( , ) . the stability and the activity of these replicators depend on the characteristics of the amino acid residues in the recognition interface. it has been suggested that it is possible to control protein structure and the replication process by external triggers such as light, and that the light can also be used to monitor the conformational changes and/or to follow the protein functionality( ). we show here our ability to control the folding stability of coiled coil peptides and their reversible or irreversible self replication effi ciency by light, and we demonstrate the possibility to exploit fret couples to facilitate in-situ monitoring of the folding and reactivity. we use 'caged' mutants with a photo-switchable molecule in the recognition interface of the peptidewhich disrupts its coiled coil structure -as inactive species. deprotection of the caged proteins is used as a mechanism to restore the self replication process. the ligation is followed by monitoring the changes in fl uorescence of either the donor or acceptor of the fret couple. we will describe synthesis and structural characterization of caged and cage-free peptides and measurements that show, as expected, that the cage free peptides are more stable as coiled coils and better catalyst for their own formation than the caged analogs. moreover, we discuss the reversible replication process and how we can shift and control its equilibrium staes. , a member of the rfamide peptide family, has been reported to have pronociceptive and analgesic activities as well as pro-and anti-opioid effects. these contradictory effects seem to result from npff capacity to bind two different gprotein coupled receptors (npff and npff ). although the exact role of npff and npff receptor is still unclear, this complex system appears to play an important role in pain regulation. structure-activity relationship (sar) studies using analogs of npff and derived peptides have shown that the c-terminal part of the molecule (i.e. pqrf-nh ) is crucial for affi nity and activity. however, some of the essential features for ligand recognition by npff receptors are still missing to design highly selective molecules. in order to provide further insight into ligand recognition, we have investigated the solution conformation of npff in different media using circular dichroism and nmr spectroscopy. our results showed that ( ) in the presence of methanol or trifl uoroethanol, turn-like elements are present in npff structure, and ( ) are not yet established, although genetic and animal models have shown a causal role of amyloid -peptide (a ) in ad. however, recent debate has focused on whether amyloid fi brils or soluble oligomers of a are the main neurotoxic species contributing to neurodegeneration and dementia. one approach for preventing aggregation would be the conversion of the peptide conformation. prior investigations indicate that polymeric nanoparticles offer strong advantages in modulating the secondary structure of the peptide ( , ) . these results have encouraged us to extend our work on polymeric nanostructures for conformational transformations contributing to the development of new therapies for these diseases. complexes of polyampholytes and dodecanoic or perfl uorododecanoic acid were prepared ( ) resulting in nanoparticles with hydrodynamic diameters ranging from to nm. the fl uorinated nanoparticles induced -helix rich structures in a peptide, whereas their hydrogenated analogues were less effi cient leading in most cases to aggregation orsheet formation, as determined by circular dichroism spectroscopy. the degree of fl uorination, the hydrophilic balance and the charge density of the fl uoropolymers, as well as the size of the nanoparticles in aqueous solution, are decisive for the interactions. the impact of these structures on the a -induced toxicity in cultured neurons was studied. we report that the fl uorinated nanoparticles increased the a -mediated mtt ( -[ , -dimethylthiazol- -yl]- , diphenyltetrazoliumbromide) reduction, which indicates a higher cell viability. the anti-apoptotic effect of the complexes was evaluated by determining the activation of caspase- . this assay also confi rms the decrease of a -mediated cytotoxicity in the presence of fl uorinated biocompatible complexes. alternative strategy to investigate enzymatic activity using peptides containing toac spin probe ( ), the present work extended the investigation of the specifi city of angiotensin i-converting enzyme (ace, ec . . . ), a dipeptidyl carboxypeptidase which cleaves the c-terminal dipeptide from angiotensin i (angi, drvyihpfhl) to produce the potent vasoconstrictor angiotensin ii peptide (angii). the use of paramagnetically labeled ai analogues attaching the toac ( , , , tetramethylpiperidine- -oxyl- -amino- -carboxylic acid) probe ( ) advantageous allows the monitoring of their conformation and its enzymatic hydrolysis specifi city through the epr and fl uorescent methods, the latter due to the quenching effect induced by the stable free radical toac probe upon the tyr residue of angi. the study of toacattaching ai analogues at positions , , , , , and indicated that the fi rst four analogues are substrates for ace in the decreasing order ~ > > , thus confi rming that greater the proximity of the unnatural probe to the cleavage site ( - ) of the sequence, the smaller are the substrate specifi city of analogues. otherwise the quenching effect of tyr fl uorescence by toac decreased with increasing distance between both residues, thus suggesting overall fl exible structures for most of analogues. these fi ndings were also corroborated in a combined cd and epr studies although some differences were detected among the derivatives either in the variation of ph or amount of the structuring tfe studies. finally, differences between epr spectral lineshapes of some labeled analogues and their corresponding cleavage products seems to allow a real time monitoring of the enzymatic reaction. the effect of the so called " -sheet breakers" (bsbs) on a - aggregates inhibition of aggregation of amyloid -peptide seems to be a critical step in the therapeutic approach to prevent amyloidosis in alzheimer's disease. the therm " -sheet breaker" (bsb) had been introduced by soto c. et. al. as a -residue peptide in , that inhibits amyloidprotein fi brillogenesis. we synthetized several derivatives of the "soto peptide" as well as a big number of peptidomimetics. their mechanism of action has been studied by nmr spectroscopy, cd and transmission electron microscopy (tem). all of these methods showed that the soto peptide lpffd and the similar peptides can not prevent a aggregation. these compounds bind to the surface of a aggregates and decrease bioactive peptides the specifi c surface area of a which accessible for the cell-membranebound receptors, can lead to a decreased toxicity. the -sheet breaking effect does not work up to an a peptide-small peptide ratio of : . as a consequence, these compounds are not -sheet breakers, only they modify the surface of a fi brils and rather speed up the formation of -sheet structure. designing trehalose-conjugated peptides for the inhibition of alzheimer's a oligomerization and neurotoxicity. interactions between cationic or anionic porphyrins and polypeptide templates with charges that are opposite to those of porphyrins have been extensively investigated for their possible applications in biomedicine and photodynamic therapy (pdt). the infl uence of porphyrin on the conformation of the peptide part of the complexes is studied in this work. non-covalent interactions of cationic tripeptide l-lysyl-l-alanyl-lalanine (kaa) with anionic meso-tetrakis( -sulfonatophenyl)porph yrin (tpps) and its copper(ii) (without axial ligand), iron(iii) (one axial ligand), and manganese(iii) (two axial ligands) derivatives were investigated in aqueous solutions by vibrational (vcd) and electronic circular dichroism (ecd) spectroscopies. although both the cd spectroscopies are sensitive to conformation, particularly vcd is extremely sensitive to subtle conformational changes. the vcd spectra of pure kaa in the amide i' (c=o stretch vibration) region showed the spectral patterns typical for left-handed polyproline ii helical conformation (ppii). interaction of kaa with non-metallated tpps was accompanied by change of the amide i' vcd patterns -loss of vcd intensity and arising of a new negative band at ~ cm - -that was interpreted as a partial change of ppii into less compact conformation as extended helix or -sheet segment. in case of cu(ii)-and fe(iii)-tpps, the loss of the amide i' vcd intensity of kaa was observed only. for mn(iii)-tpps having two axial ligands, the vcd pattern was unchanged compared to the pure tripeptide indicating that this derivative is not able to change the conformation of kaa in peptide-porphyrin complex. suitable models of biologically important small-sized proteins -histons -located in the chromosomes of eucariotic cells. they are able to interact with negatively charged functional groups of many biologically important molecules as dna, polyuronic acids, and porphyrins and thus infl uence a broad range of biological functions. in this work, the solution conformation of synthetic oligotripeptides (llysyl-l-alanyl-l-alanine) n [n = , , ] was investigated at the different temperatures and ph using combination of vibrational (vcd) and electronic circular dichroism (ecd) spectroscopies. vcd spectra of all the oligopeptides measured at room temperature show a negative couplet (positive to lower frequency) in amide i' region. this spectral pattern is indicative of left-handed polyproline ii helical conformation (ppii), which is stable at wide range of ph. the temperature dependence of the vcd spectra indicates that ppii conformation of all the oligopeptides remains stable even at °c, independently on length of oligopeptide chain. these results were confi rmed by temperature dependent ecd experiments, where the characteristic negative and positive bands at about and nm, respectively, were observed. taken together, vcd and ecd results suggest that ppii conformation of (lys-ala-ala) n sequence is the dominant conformation within the range of temperature from to °c. all the results including spectral data analysis are discussed in detail. acknowledgment: the work was supported by the research grant / /p from the grant agency of the czech republic. we thank miroslava Žertová, msc (iocb prague) for the oligopeptide synthesis. -peptides are probably the most thoroughly investigated peptidomimetic oligomers. to extend the fi eld of -peptides towards the construction of possible new secondary structures, the replacement of the c and c atoms of the -amino acid with heteroatoms could be an attractive modifi cation, for example c -atom of -peptides by an nr moiety, leading to hydrazine peptides. in the literature, there are only a few studies about hydrazine peptides [ ] [ ] [ ] , and hydrazine peptides with cyclic side-chain have not been studied yet. in order to determine the secondary structure preference of h-[(cis or trans)-acpc- s-aza-acpc] -nh peptides (figure ), their potential energy hypersurface were probed at the ab initio b lyp/ - g** level. the cis ( r, s) formed / -helices, while the opposite acpc enantiomer resulted strand. the trans ( s, s) formed -strand, while the opposite acpc enantiomer resulted -helix. the hybrid-peptides in question were synthetized on solid support, and their high-resolution d assignments were made by using nmr, which was supported by ecd, vcd, qls and tem methods. the hydrazino modifi cation resulted in better water solubility than that of the acpc homooligomers. this result is very important concerning the further biological applicability. mazur, adam; katarzy ska, joanna; zabrocki, janusz; jankowski, stefan technical university of lodz, poland cyclolinopeptide a, (cla, ), a cyclic nonapeptide cyclo(-pro -pro -phe -phe -leu -ile -leu -val -), possesses strong immunossupresive and antimalarial activity as well as the ability to inhibit cholate uptake into hepatocytes [ , ] . the mechanism of cyclolinopeptide a activity is similar to that of cyclosporine a. the object of our investigations are six cla analogues with pro and pro residues replaced by -isoproline or -homoproline. the immunosuppressive activity of these new cla analogues was evaluated on the basis of the mouse splenocyte proliferation assay .. nmr spectra analysis (chemical shifts assignment and structural constraints) was based on d and d nmr experiments at mhz. long ( ns) molecular dynamics calculations were carried out using gromacs program (gromos g a force fi eld) for isomers of at least % content. in woolley, andrew university of toronto, canada thiol-reactive azobenzene-based cross-linkers provide a straightforward means for introducing a photo-isomerizable unit into a peptide or protein structure. we have shown that the conformational response of the peptide in such systems is critically dependent on the manner of attachment of the cross-linker as well as the cross-linker structure. cross-linkers can be introduced in which trans-to-cis photoisomerization leads of formation of helical structure, or conversely to loss of helical structure. these effects can be understood in a semi-quantitative manner by calculating the degree of mismatch between the steric requirements of the linker and the conformational ensemble of the peptide. kinetic studies reveal that in general photoisomerization of the linker is fast (several ps) whereas subsequent peptide folding or unfolding occurs over hundreds of microseconds. such cross-linkers are thus potentially useful phototriggers for studying protein folding processes, as well as for controlling the equilibrium stability of different conformational states. applications to photo-control of bioactive peptides and proteins will be discussed. short corticotropin-like peptides with stress-protective activity it was synthesized linear è cyclic corticotropin-like peptides, which contained from to amino acid residues. the ability of each of the synthesized peptides to inhibit the specifi c binding of tritium-labelled corticotropin ( - ) to the adrenal cortex membranes of rat in vitro was investigated. on the base of the obtained results peptides with the highest inhibitory activity were selected for in vivo tests. the infl uence of the selected peptides on the level of -oxicorticosretoids and catecholamines in the adrenals and blood of rats in the experiments on acute hemorrhage and hypobaric hypoxia, cold and heat shock and low doses of g-radiation were studied. it was established that intravenous injection of short peptides at the dose of g/kg could correct disturbance of -oxycorticosretoids-catecholamines system in the adrenals and plasma of rats that were subjected to hemorrhagic shock and hypoxia. the rest of investigated peptides possessed lower stress-protective activity. it was also shown that under cold or heat shock, thrice-repeated intranasal injection of these peptides into rats at the dose of - g/animal abolished temperature induced changes in the level of -oxycorticosretoids and catecholamines in the adrenals as well as the content of free histamine and the activity of diaminoxydase in the myocardium. it has been shown that the stress-protective activity of corticotropin-like peptides is mediated by the corticotropin receptor in cortex of the adrenals. a study of immunobiological activity of the wrnwdyyk octapeptide the evaluation of the effect of japanese herbal in many cases, japanese herbal (kampo) medicines have been used in the empirical treatment of chronic hypofunction. however, the kampo medicines consist of several herbs, whose pharmacological mechanism is not clear. in western medical science, medical doctors usually treat patients according to disease diagnosis. in eastern medical science, treatment is based on diagnostics called gsho h, which is a unique concept in kampo medicines and quite different from diagnosis. the concept of gsho h is diffi cult for non-professionals to understand, furthermore, there are responders and non-responders when nonprofessionals prescribe kampo medicines. the concept of gsho h focuses on individuals, not diseases, therefore, it is diffi cult to gain a given effect for everyone by general clinical trials. in this study, we investigated the effects of prokinetic kampo medicines on plasma levels of gut-regulatory peptides (somatostatin, motilin and gastrin) and compared with that of dopamine receptor antagonists, the western prokinetics. the fi ve kampo medicines, including pinelliae tuber and zingiberis rhizoma, three dopamine receptor antagonists or placebo was orally administered. venous blood samples were taken before and till min after administration. plasma peptide levels were measured using a sensitive enzyme immunoassay. the dopamine receptor antagonists and kampo medicines caused signifi cant increase of plasma gut-regulatory peptide levels compared with placebo group. in recent years, chronic hypofunction without mechanical problem, such as non-erosive refl ux disease and functional dyspepsia, is diffi cult to cure using western medicines. the preliminary study indicated the plasma somatostatin levels of patients with any symptoms are high and plasma motilin levels of them are low compared with those of healthy subjects. to evaluate kampo medicines using bioactive peptides as biomarkers, it may be possible to cure diseases that is diffi cult to treat by western medicines. bapst, jean-philippe; calame, martine; eberle, alex n.; tanner, heidi university hospital basel, switzerland radiolabeled -msh analogs are potential candidates for melanocortin- receptor (mc -r)-mediated melanoma targeting. several short -msh peptides carrying a dota ( , , , -tetraazacyclododecane- , , , tetraacetic acid) metal chelator were designed and evaluated as potential diagnostic (e.g. with in, , ga) or therapeutic (e.g. y, cu) radiopharmaceuticals. the analogs tested to date showed high affi nity for the mc -r in vitro, excellent internalization into the tumor cells, as well as a good incorporation in tumor xenografts and a low uptake in normal tissues in vivo, except the kidneys where considerable uptake is observed. our current studies attempt to infl uence the pharmacokinetic parameters in order to address specifi c uptake (i.e. by melanoma) versus non-specifi c uptake (i.e. by the kidneys). as glycosylation had been shown to improve tumor-to-kidney ratios in the case of somatostatin and to reduce peptide re-uptake by the tubular system of the kidneys in general, we investigated glycosylated analogs of [nle , asp , d-phe ]--msh - (napamide). carbohydrate moieties such as glucose, galactose and maltotriose were introduced at various positions on the msh peptide carrying the metal chelator dota for labeling with in. the peptides were evaluated in vitro in both murine and human cell lines for mc -r binding and cellular localization, and in vivo in b f tumor-bearing mice for tissue distribution. the tumor-to-kidney ratio for gal-napamide ( - h aucs) was superior to any of the previously published msh peptides. other glycopeptides showed very good binding affi nities but lower selectivity in vivo. in additional, a series of non-glycosylated dimeric derivatives, bearing one or two moieties of the chelator complex, were developed which displayed excellent receptor affi nity but tended to result in higher kidney accumulation. by contrast, at least one negatively charged dota-napamide showed excellent tumor-to-kidney ratios. there is a critical lack of validated early biomarkers for most conditions and diseases. early diagnosis does enable treatment of less severe disease states, the use of less invasive techniques and could potentially reduce the costs of healthcare systems. however, it is most likely that peptides in tissue or blood -or their modifi cations -can be specifi cally associated with different disease states. the discovery and verifi cation/ validation of such disease markers are two distinct workfl ows. during the discovery phase, a relatively small number of samples with a high number of potential biomarker candidates are screened. the highthroughput provided by the itraq™ reagent strategy allows for simultaneous analysis of such samples. once biomarker candidates have been identifi ed with initial statistical signifi cance, these have to be validated. this validation workfl ow involves analyzing a large number of samples with a relatively small number of candidates to establish the biological signifi cance of the biomarker candidates. rather than switching to immunological techniques for this validation step, we suggest a mass spectrometry based approach. this orthogonal strategy is a novel targeted, high throughput quantitative multiplexed multiple reaction monitoring (mrm) approach. the approach relies on assay development using a combination of mrms to target specifi c peptides identifi ed in discovery, followed by ms/ms to confi rm that the quantitative mrm signal results from the target peptide. in addition to being a quantitative method, this validation approach is extremely specifi c and sensitive. several published examples of the application of this mrm-based approach utilizing the actual or hypothetical physical properties of peptides in complex mixtures will be presented. infl uence of the charge on the in vivo behavior of radiolabeled bombesin analogues prostate and breast cancers are the second leading cause of cancer death in men and women, respectively. the side effects related to the treatments that are available today have a great infl uence on the patients' quality of life. therefore, the development of new diagnostic and therapeutic strategies may have an important impact in the outcome of these cancers. gastrin-releasing peptide (grp) receptors are present in high quantities in a variety of cancers, prostate and breast tumors among them. targeting of over-expressed grp receptors with radiolabeled bombesin (bbs) analogues would offer an interesting tool for tumor imaging and therapy, depending on the radionuclide used. some analogues of bbs, based on the fragment - , were functionalized with the (n his)chelator for labeling with the m tc-and re-tricarbonyl-core. despite an increased metabolic stability, these analogues showed very low tumor uptake. additional insertion of a ala-ala linker led to increased tumor uptake but still unfavorable in vivo properties. in order to further improve the biodistribution, novel polar linkers with different charge were introduced in the molecule. a positive charge resulted in increased kidney uptake, whereas one single negative charge led to a signifi cant increase in the tumor uptake and also signifi cantly higher tumor-totissue ratios. co-injection with natural bbs importantly inhibited the uptake in the tumors and receptor-expressing tissues, which confi rmed the specifi city of the in vivo uptake. moreover, imaging of the tumor xenografts by spect/ct was also much clearer with the analogues bearing one negative charge. additional negative charges, however, resulted in a loss of binding affi nity and internalization, and unfavorable biodistribution. in conclusion, bbs analogues with one single charge in the linker hold a greater potential for imaging and therapy of grp receptor-overexpressing tumors peptide-macrolide conjugates as novel instruments for protein biosynthesis study bacterial ribosomes are targets of numerous therapeutic agents. macrolide (ml) antibiotics prevent nascent polypeptide growth by blocking the ribosomal exit tunnel (rt). availability of high-resolution structures of complexes of ribosomal s subunits with ml enabled developing novel ideas concerning their inhibitory activity of translation. formerly we obtained peptide derivatives of ml in which the peptide part modelled a growing chain, while an antibiotic moiety served as an "anchor" for positioning the peptide in the rt. the goal of this study was to synthesise tryptophan-containing peptide derivatives of -omycaminosyltylonolide (omt) to monitor location of the specifi c trp binding site that modulate the activity of the peptidyl transferase centre of the ribosome. the peculiarity of compounds designed in this study lies in that of a trp containing peptide fragment, connected to the primary hydroxyl group in position c of omt, is oriented in the rt towards the hypothetical trp binding site. the following peptides were used: boc-l-trp-gly-oh, boc-l-trp-ala-oh, boc-l-trp-abu-oh, boc-l-trp-ape-oh. variable distance between trp residue and macrolide ring was achieved by introduction of glycine, -alanine, -aminobutyric acid and -aminovaleric acid as c-terminal amino acids of the dipeptides. the peptides were obtained from boc-l-trp-oh and ethyl esters of the corresponding amino acids by condensation with bop followed by saponifi cation of the peptide esters. the reactions between peptides and omt were produced using dcc and dmap as condensation agents. as a result peptide-macrolide derivatives were obtained. all compounds were purifi ed by column chromatography on silica gel and characterized by hplc, mass-spectrometry and nmr. it was shown by means of chemical probing that trp containing omt derivatives specifi cally bound to rt of the ribosome. moreover these compounds displayed an antibiotic activity in testes with several staphylococcus strains. zwanziger, denise ; neundorf, ines ; schatzschneider, ulrich ; beck-sickinger, annette g. university of leipzig, germany; university of bochum, germany npy is a amino acid peptide amide that belongs to the pancreatic polypeptide-hormone family ( ) . it is the most abundant neuropeptide in the brain and triggers a number of central activities, such as regulation of food intake as well as stress induced reactions [ ] [ ] [ ] . npy forms a selective interaction with at least three receptors, which are called y , y and y . in the past it was shown that y -receptors are overexpressed in > % of all breast tumors as well as in % of the derived metastases. normal breast tissue expresses y -receptors, while the neoplastic tissue expresses y -receptors .. as a result, the npy-y -receptor system can be used for tumor targeting and therapy. in the fi rst step we synthesized the truncated and modifi ed npy analogue [pro ,nle ,bpa ,leu ]npy( - ) (nle-norleucine; bpa-benzoyl-phenylalanine) by solid bioactive peptides phase peptide synthesis using fmoc/tbu strategy. the npy analogue was characterized with respect to in vitro binding affi nity and selectivity at y -receptor expressing human breast cancer mcf- cells and the metabolic stability in human blood plasma, respectively. then we coupled different potential chelators to [pro ,nle ,bpa ,leu ]np y( - ), which was n-terminally modifi ed by two ß-alanines as spacer between the peptide sequence and the chelator. next, we determined their ability of metal conjugation of diverse metals, as cu + and re +. metal conjugated peptides were characterized by maldi-tof-ms (matrix assisted laser desorption/ionization mass spectrometry), rp-hplc (reversed phase high performance liquid chromatography) and ir-spectroscopy (infrared). after optimization of the metal conjugation fi rst binding affi nity studies were performed. chain is mainly expressed in skeletal muscles and peripheral nerves, and interacts with cell surface receptors such as integrin, dystroglycan, and heparan sulfate proteoglycans. biological functions of the laminin chain n-terminus including integrin binding and heparin/heparan sulfate binding were found previously. here, we focused on the n-terminal region of the mouse laminin chain (position - ) and screened the biologically active sequences using peptides. the synthetic peptides were generally amino acids in length and overlapped with neighboring peptides by amino acids. cell attachment activity of the peptides was evaluated using a peptide-coated plastic plate assay and a peptide-conjugated sepharose bead assay using ht- human fi brosarcoma cells. eleven peptides showed cell attachment activity on plate assay and fi ve peptides showed cell attachment activity on beads assay. previously we screened active sites on the laminin chain and identifi ed several active sequences. when we compared with active sequences of the and chains, a - (yydetvasrnlsln) and a - (ggklkyaiyfea) are unique active peptides only within laminin chain. these results suggest that the biological activity of a - and a - are chain specifi c and are useful for investigating the chain specifi c functions of laminin chain. novel peptide biopharmaceuticals by using phage display technology Štrukelj, borut; lunder, mojca; bratkoviè, tomaž university of ljubljana, faculty of pharmacy, slovenia libraries of random peptides displayed on the surface of bacteria, mammal cells or bacteriophages are an essential tool that enables systematic study of target molecule interactions. the identifi cation of ligands from large biological libraries by phage display has now been used for almost years. in a last few years several improvements have led to numerous high affi nity peptide ligands that express various biological activities. phage-displayed peptide libraries have been used successfully to isolate peptide ligands directed to a functional site for which the natural ligand is or is not a protein or peptide. by using a modifi ed, in-house developed selection proctocol, we successfully selected several phage clones with high affi nity to pancreatic lipase, ghrelin and cysteine protease cathepsin k as target proteins. based on their deduced animoacid sequences, twenty heptapeptides with the highest affi nity to target proteins were synthesized and characterized for their capacity to inhibit enzyme or acceptor function. the most succesful peptide candidates inhibited pancreatic liapase with the apparent inhibition constant of um, and cathepsin k with the apparent inhibition constant of , um. a set of peptidomymetic compounds were sinthesyzed based on the aminoacid sequence of selected peptides and their inhibitory activity was determined. the most potent candidates were selected for further development of peptide biopharmaceuticals aganist obesity (inhibitors of pancreatic lipase and ghrelin) and in the prevention of osteoporosis (cathepsin k inhibitors). novel approach to non-specifi c elution in phage display using ultrasound phage display is used to select and optimize peptides or protein domains binding to virtually any protein and sometimes even non-protein targets. several rounds of screening are performed, until the increase in phage output, or binding assays performed with phage pools, indicate that the population of binding phages has been adequately enriched. in cases where ligands of particular target are not known or available, targetbound virions are released by non-specifi c elution for example with acidic buffer, or competitively with free target molecule, or by addition of bacterial host directly to the target-bound phages. we used streptavidin, immobilized by adsorption, as a model target protein for affi nity selection of peptides from phage display library. the effi ciencies of a number of well-known typically used non-specifi c elution strategies in selecting and retrieving a phage clone displaying the tripeptide biotin mimetic (hpq) from a streptavidin coated surface were compared. all the commonly used elution strategies have failed to elute and select high affi nity hpq-bearing phage clones bound to streptavidin. the failure was shown to be due to the inability of eluants to break the interaction of high affi nity clones with the target, which is thus likely to be the cause for failed selection with other targets also. to surmount this, we have introduced a new elution strategy, combining low ph elution buffer with sonication which, in addition to loosening the peptidetarget interaction, also serves to detach the target molecule from the immobilization surface. this ultrasound-based method enabled single step selection of a high affi nity peptide from a library of diversity greater than , and thus represents a dramatic improvement in searching for novel, specifi c peptide ligands. apoptosis to various tumor cells but not in normal cells. using fmoc solid phase synthesis, cellulose membrane-bound octameric peptide library of trail scan was prepared and cell viability assay was directly performed on peptide disk with jurkat cells. six peptide sequences that could induce cell death were found, and particularly rnscwskd (trail - ) peptide was shown the strongest effects. then, peptides of stronger effects were found through amino acid substitution, and the cnscwskd peptide induced > % cell death in treated cells. features of apoptosis, such as dna fragmentation, activation of caspase, phosphatidylserine externalization, chromatin condensation, and competition with trail for binding to the death receptor (dr) or dr were observed, suggesting that this peptide is a trail mimic. caspase- activation was observed in various tumor cells treated with this peptide as well as with trail, while no activation was observed in human normal fi broblasts. the cnscwskd peptide is a potential candidate for use in cancer therapy. the being an incretin mimetic, exendin- exerts the same action as glp- including the glucoregulatory and the insulinotropic effects through glp- receptor. exendin- may be preferable to glp- on the aspect of stability. however it was comprised with amino acids and was not suitable for developing as an oral drug. recently, there was an upsurge in the development of exendin- mimetic as potential therapy for type diabetes. in this study, a receptor-binding region on the surface of rinm f cell was used as the target to screen peptide ligands for the receptor in a phage -mer peptide library. dna sequencing revealed a group of closely related peptides from the fourth round of selection. through the activity of decreasing blood glucose assay in vivo, the cell proliferation assay and capability against dppiv in vitro, one highest bioactivity peptide (qpsvgmkpsprh, ex- pa) was acquired. the bioactivity of ex- pa was almost identical with exendin- on the promoting cell proliferation in a dose-dependent manner. the decreasing blood glucose effect of ex- pa was up to min after administration. the stability against dppiv of ex- pa maintained good performance that % parent peptide remained after h. in summary, ex- pa as a shorter glp- receptor agonist mimicked the action of exendin- and built a good foundation for designing the oral diabetes drug. key words: exendin- , mimetic, phage display peptide library, screen over the last decades we have witnessed the emergence of bacterial resistance to virtually all clinically important antibiotic types. therefore, continuous development of antibacterial agents with completely novel modes of action accompanied by rationalization of chemotherapeutic prescription is the key strategy to adopt in a long-run struggle against the growing problem of pathogen resistance. numerous indispensable antibiotics interfere with peptidoglycan cell wall biosynthesis making this unique metabolic pathway a well validated target for antimicrobials. while nearly all of these antibiotics inhibit late stages of murein synthesis occurring on the extracellular side of plasma membrane, initial cytoplasmic steps have not been extensively exploited as drug targets. we have performed affi nity selections from random linear and conformationally constrained (disulphide cyclized) peptide libraries displayed on bacteriophage particles against two essential bacterial enzymes murd and mure, involved in the cytoplasmic synthesis of peptidoglycan monomer precursor. selected peptides were found to inhibit respective targets in an in vitro assay with ic values of m to . mm. reported inhibitory peptides should be regarded as templates for design of low-molecular-weight peptidomimetics. resulting murd and mure inhibitors with improved potency and/or physicochemical characteristics (especially membrane permeability) have the potential to act as broad spectrum chemotherapeutics. fidan, zerrin; ljeskovica, nizama; portwich, michael; volkmer, rudolf charité-universitätsmedizin berlin, germany coiled coil (cc) sequence motifs are common structural motifs and versatile protein interaction modules. the cc is composed of at least two right-handed amphipathic a-helices that wrap around each other into a left-handed supercoil such that their hydrophobic surfaces are in contact. cc can associate up to heptamers, form homomeric and heteromeric complexes at different stoichiometries, and be aligned parallel and antiparallel. a characteristic of all coiled coils is the presence of heptad repeat sequences [abcdefg]i, where i denotes the heptad number. although cc motifs can be predicted with a high degree of confi dence, predicting the association states and topologies is still a great challenge. we will report on synthetic peptide arrays useful to study cc associations at the amino acid level. in contrast to rational design, which mostly depends on short model peptides, our approach relies on the full-length homodimeric gcn -and the heteromeric cjun/cfos coiled-coil domain formation. the infl uence of amino acid substitutions on association is tested without restrictions and presumptions and the stoichiometry is examined by biophysical methods. furthermore, we generate arrays comprising hundreds of (putative) cc sequences and subsequently probe them for association to a set of several native cc sequences. an interactome can be drawn for each of the investigated cc sequences, enabling one to interpret the biological functions. we will present: (i) association analyses of all single substitution variants of the gcn -, cfos and cjun leucine zipper probed with the associated native leucine zipper; (ii) the exposure of a heteromeric cc-network deduced from gcn variants and the determination of the networkstoichiometry; (iii) the cc-interactom of several native coiled coils like the cc sequences of akap , pqbp , orai and others. almost two decades ago we used the spatial screening technology to optimize activity and receptor subtype selectivity among rgd recognizing integrins. this culminated in the avb selective superactive pentapeptide c(-rgdfv-) which gave rise to a number of peptidomimetic modifi cations. among them we studied all retro-, inverso-and retroinverso peptides of this structure. only one retro-inverso peptide exhibits full activity: the peptide c(-dgrvf-) strongly inhibits avb /vitronectin binding ( nm) but not aiibb /fi brinogen binding (> nm). all investigated retro-sequences show very low affi nity for avb . recently it was discovered that the ngr sequence in the fi bronectin domain fi after rearrangement into iso-asp-g-r exhibits high binding affi nity for integrin avb . this surprising result stimulated us to investigate cyclic peptides containing the iso-asp-gly-arg sequence .. after optimisation we obtained peptides which bind with high activity to integrin a b and lower, but signifi cant activity for avb . in the ongoing work we investigate if receptor modelling can help to understand these results. selectivity and activities for these two integrins have been obtained recently in our group using two different peptidomimetic scaffolds bioactive peptides (tyrosine based and diacylhydracine based) using a homology model of the integrin a b for which no x-ray structure is known. binding of integrin ligands involves binding of a carboxyl group to the metal ion (ca or mn) in the so-called midas region of the integrin. so far all integrin ligands contain a carboxyl group and any attempt to substitute this group by an isoster failed. we recently found that hydroxamic acids (-conhoh) allow for the fi rst time a substitution of this carboxyl group. the activities and selectivities for integrin subtypes avb , a b and aiibb have been explored and give interesting results. development of gnrh-iii-antracycline conjugates as multifunctional drug delivery systems for targeted chemotherapy targeted chemotherapy based on the cell specifi c or overexpressed receptors on tumors might be an effi cient therapeutic approach for the treatment of cancer. expression of gonadotropin-releasing hormone (gnrh) receptor was identifi ed on different types of tumors. it has been shown that gnrh-iii (glp-his-trp-ser-his-asp-trp-lys-pro-gly-nh ) isolated from see lamprey has antiproliferative activity on numerous tumor cells, and signifi cantly less potency on releasing gonadotropin hormones (lh, fsh); therefore, it is a more selective antitumor agent than the human gnrh derivatives. in our work, gnrh-iii was used as targeting moiety for the preparation of multifunctional drug delivery systems for targeted cancer chemotherapy. daunomycin (dau) and doxorubicin (dox) as antineoplastic agents were attached to the side chain of lys of gnrh-iii through amide, oxime, hydrazone or ester bonds, either directly or by insertion of an enzyme cleavable tetrapeptide spacer. stability studies of the conjugates were performed in human serum, as well as in the presence of chymotrypsin. the effect of chemical structure of the conjugates on in vitro antitumor activity was studied using different cancer cell lines (mcf- human breast, ht- human colon and c murine colon cancer cells). oxime bond linked dau-gnrh-iii conjugate was selected for in vivo experiments using c colon tumor bearing mice. the conjugate showed similar antitumor activity as the free drug, but less toxic side effect and longer survival of the animals was determined in the case of the application of the conjugate. guillon, g. ( ) . it was also shown to be highly selective for the human v b receptor ( ) . we now report the synthesis and some pharmacological properties of three fl uorescent hv br ligands (a,b,c), based on modifi cations of the lys residue in d[leu ,lys ]vp, with alexa (a), alexa (b) and antraniloyl (atn) (c). the fl uorescent peptides a, b and c exhibit the following affi nities for the hv br: (a) ki = . nm, (b) ki = nm and (c) ki = . nm. peptides a and c exhibit moderate affi nities for the hotr and very weak affi nities for the hv ar and for the hv r. on v b-transfected att cells, they activate plc coupling as evaluated by stimulation of ip levels. they also induced receptor internalization visualized by accumulation of fl uorescence in endosomial vesicles. one or more of these new fl uorescent hv br ligands promise to be useful tools for studying human v b receptor localization or traffi cking. distribution of prolyl oligopeptidase in the mouse wholebody sections and peripheral tissues prolyl oligopeptidase (pop) is a serine endopeptidase that hydrolyses proline-containing peptides shorter than -mer, including many bioactive peptides. the distribution of pop in the brain has been studied but little is known about the distribution of peripheral pop. we used immunohistochemistry to localize pop in mouse whole-body sections and at the cellular level in peripheral tissues. furthermore, we used a pop activity assay to reveal the associations between pop protein and its enzymatic activity. the highest pop protein densities were found in brain, kidney, testis and thymus, but in the liver the amounts of pop protein were small. there were remarkable differences between the distribution of pop protein and activity. the highest pop activities were found in the liver and testis while kidney had the lowest activity. in peripheral tissues, pop was present in various cell types both in the cytoplasm and nucleus of the cells, in contrast to the brain where no nuclear localization was detected. these fi ndings support the proposed role of pop in cell proliferation in peripheral tissues. the dissociation of the distribution of pop protein and its enzymatic activity points to nonhydrolytic functions of pop and to strict endogenous regulation of pop activity. the rapid cytoplasmic entry of cationic cell-penetrating peptides in the past few years, our understanding of the cellular import of cellpenetrating peptides has evolved rapidly. the initial concept of cell entry by direct permeation of the plasma membrane, was followed by endocytosis as a major route of import at least for most cellpenetrating peptide-cargo conjugates. more recently, we and others observed a rapid cytoplasmic delivery of fl uorescein-labeled analogs of the cationic cpps nonaarginine and tat-peptide at subtoxic lower poster abstracts micromolar concentrations ( ). this import originates from spatially confi ned zones of the plasma membrane and depends on the presence of heparan sulfate proteoglycans. these molecules have been proposed to interact polyvalently with the guanidinium groups of the arginine residues. moreover, the threshold for the induction of this import could be lowered by inhibition of endocytic routes of entry. recently, it was reported that the absence of serum in the medium lowers the threshold for this uptake process ( ) . in summary, these observations suggest that the concentration of peptide associated with the plasma membrane is a decisive trigger for this import process. currently, it is not known, to which degree this cytoplasmic entry of labeled conjugates at higher concentrations is based on similar molecular mechanisms as the direct permeation of unlabeled conjugates that has also been observed at lower concentrations ( ). here we summarize the current knowledge on direct transport across the plasma membrane of cationic cpps and present new data on the molecular events involved in this uptake process. plasma membranes have numerous essential functions for maintaining cellular homeostasis. however, the membranes are also barriers to intracellular delivery of various therapeutic molecules. for improvement of their translocations, we developed a novel method using gala peptide/cationic lipid complexes. gala, a -residue amphipathic peptide with a repeat sequence of glutamic acid-alanine-leucine-alanine, was designed to mimic the function of viral fusion protein sequences that mediate escape of virus gene from acidic endosomes to cytosol ( ) . when attached with bioactive cargoes, the gala peptide may thus serve as intracellular vector bearing effi cient endosomal escape function. however, because of negative charges from glutamic acids ( -residues) in the gala sequences, access of the peptide on negatively charged cell surface would be not so effi cient. to overcome this problem, cationic lipid was employed as an gadhesive h for pasting the gala peptide onto cell surface to accomplish effi cient cellular uptake. we examined the ability of gala peptide as a delivery vector using fitc as a model of membrane-impermeable low-molecular weight drugs. when fitc-gala ( ƒÊm) was administrated to hela cells, co-addition of cationic lipid, lipofectamine (lf ), drastically increased the effi ciency in uptake of fitc-gala. in a time-dependent manner, the fitc-gala escaped from endosomes, and diffuse fl uorescent signals were observed in both cytosol and nucleus. also the gala/cationic lipid system was applied for the intracellular delivery of fitc-avidin protein ( kda). when fitc-avidin was mixed with biotinylated-gala/ lf complexes, fitc-avidin ( nm) was internalized into cells effectively. in the absence of these complexes, internalization of fitcavidin was poor. these results suggest the usefulness of our approach for intracellular delivery using gala peptide and cationic lipid. the membrane repair response masks membrane disturbances caused by cell penetrating peptide uptake even though cell-penetrating peptides are able to deliver cargos of different sizes into cells, their uptake mechanism is still not fully understood and needs to be elucidated in order to improve their delivery effi ciency. recent studies have suggested that there might be a direct penetration of peptides in parallel with different forms of endocytosis. however, the direct penetration of hydrophilic peptides through the hydrophobic plasma membrane is highly controversial. three proteins involved in target cell apoptosis -perforin, granulysin and granzymes share many features common in uptake of cell-penetrating peptides e.g. they bind proteoglycans on the plasma membrane. the uptake of perforin activates the membrane repair response, a resealing mechanism triggered in cells with injured plasma membrane, due to extracellular calcium infl ux. upon activation of the membrane repair response, internal vesicles are mobilized to the site of the disrupted plasma membrane resealing it within seconds. in this study we present evidence that the membrane repair response is able to mask damages caused by cell-penetrating peptides when they internalize cells, thus preventing leakage of endogenous molecules out of the cell. the hypothalamic decapeptide gonadotropin-releasing hormone (gnrh-i; gnrh symmetric dimer derivatives ([ in vitro (cellular uptake and antiproliferative effect of the dimer derivatives) and in vivo (comparison of per os and intraperitoneal administration) antitumor effect of these symmetric dimers were investigated. the cellular uptake of dimer derivatives were studied by fl ow cytometry (bd lsr ii) on mcf- (human breast cancer) and ht- (human colon carcinoma) cell lines using carboxifl uorescein labeled symmetric dimer derivative. the cells were treated with different concentration of synthetic dimers and after the treatment were analysed by fl ow cytometry in order to investigate their cellular uptake. the antiproliferative effect of symmetric gnrh-iii dimers were studied on mcf- , ht- and t -d (human breast cancer) cell lines. ht- xenograft was applied for studying in vivo antitumor effect of gnrh-iii dimers in different administration routes. the cellular uptake and the antiproliferative effect are cell type dependent as it can be found in the literature. we found that symmetric dimer derivatives of gnrh-iii signifi cantly decreased the tumor volume in vivo ( %). iib receives intracellular signals (inside-out signaling) that allow cytoplasmic proteins to interact with the cytoplasmic domains of iib subunits, resulting in platelet aggregation. the aim of this work is to inhibit platelet thrombus formation by specifi cally disrupting the insideout signalling pathway using synthetic peptides based on the cytoplasmic region of subunit, residues - . peptide analogues derived from - region ( - , - (ptyr ), - , - and - ) were synthesized in their free, palmitoylated and/or tagged with the tat( - ) signaling sequence and carboxyfl uoresceinlabeled in order to investigate their membrane permeability, as well as their inhibition potency on the platelet aggregation. from the biological assays in prp and washed platelets we concluded that the modifi ed peptides that carry either palmitoyl-group or the tat( - ) signaling sequence penetrate platelet membrane and inhibit human platelet aggregation, in contrast to the corresponding free peptide analogues. acknowledgements: the gsrt (epan yb/ ) and e.u. for the fi nancial support. ; aib = -aminoisobutyric acid) is a potent mitochondriotoxic analogue of mp that demonstrates a signifi cant intracellular co-localization with mitochondria. through co-operation with a protein of the mitochondrial permeability transition pore vdac, mitoparan specifi cally promotes apoptosis. in contrast, cytc - , a cryptic fragment of human cytochrome c, is a moderately potent apoptogenic cpp that demonstrates a strong propensity for colocalization within the endoplasmic reticulum. using a sychnologic modular design, we have synthesized and evaluated a broad range of chimeric constructs combining apoptogenic cpp (message) and peptidyl address motifs. the overriding objective of these studies was to enhance cytotoxicity and/or develop target-selective drug delivery vectors. address motifs that target both plasma membrane and nuclear envelope protein structures included i) the integrin-specifi c rgd sequence, ii) a fas ligand mimetic wewt, iii) heptagastrin (aygwmdf) that targets a novel membrane binding site on high grade astrocytoma and, iv) a mimetic of fg nucleoporins (nups). incorporation of peptidyl address motifs, by simple n-terminal acylation or via a fl exible aminohexanoic acid (ahx) linker, produced chimeric constructs with enhanced cytotoxic potencies. most signifi cantly, ld values readily achievable in vivo were demonstrated by the modular apoptogenic cpp z-gly-rgdf-mitoparan, (ld = . m) and ac-nfkfglss(ahx)cytc - (ld = . m). in conclusion, targetspecifi c modular design of apoptogenic cpp is a promising strategy for the study and therapeutic induction of apoptosis. phage display screening of geminin-binding peptide and validation of its therapeutic use in tumors yoshida, kenichi meiji university, japan dna replication is controlled by the stepwise assembly of a pre-replicative complex (pre-rc). geminin is a component of the pre-rc and plays a role in preventing incorporation of the minichromosome maintenance protein complex into the pre-rc via binding and inhibiting cdt function. it may be possible to design low-molecular-weight chemicals that would bind to geminin to suppress the aberrant cellular proliferation in tumor cells. a random -mer peptide phage library was used for the selections. phage was selected by panning on immunotubes coated with recombinant geminin. positive clones were identifi ed by a screening phage produced from single colonies for specifi c binding to gst-geminin in elisa. fluorescent-labeled peptide linked to the sv nuclear localizing signal was synthesized by solid-phase synthesis, using fmoc chemistry. by detecting fl uorescein fl uorescence to mark specifi c transfected cells, we examined the effects of the peptide transfection on the synthesis of new dna in hct cells. by screening a random peptide phage library, we identifi ed a certain peptide sequence bound to geminin. using a series of mutant geminin, elisa test revealed the amino acid residues - are responsible for the peptide binding. we found fewer brdu positive cells following transfection of the geminin-binding peptide than following that of the control peptide. this study suggests that the chemical peptidomimetics of this peptide might form the basis for the development of drugs that could be used to prevent tumor progression. acknowledgements: this study was supported by kakenhi. it has been previously demonstrated that n -( -metoxyfumaroyl)-l- , diaminopropanoic acid (fmdp) is a strong inhibitor of glucosamine- phosphate synthase, a potential target for antimicrobial chemotherapy. fmdp is transported into the cells when incorporated in a peptide chain and hydrolyzed by intracellular peptidase releasing free inhibitor as the "warhead" component, which can react with the target enzyme. the concept of utilizing of peptide transport system for delivery of toxic amino acids into the microbial cells is characterized by authors as "illicit transport". unfortunately, peptides are not stable in physiological fl uids, due to activity of peptidases. analogs with modifi ed amide backbone are more resistant towards enzymatic degradation. it has been shown that enzymatic hydrolysis of endothiopeptides is often signifi cantly slower than natural peptides. in our studies we synthesized nva[csnh]-fmdp, an analog of nva-fmdp, which is a strong antimicrobial peptide. because of the fact that the "warhead" component in this peptide contains an amide backbone, fi rstly we decided to synthesize free fmdp with thioamide backbone. the results of enzymatic kinetics studies showed that in this way modifi ed compound is much weaker inhibitor of glucosamine- -phosphate synthase than fmdp. it was the reason why we planned and carried out a multi-step synthesis in order to obtain a potential more resistant towards enzymatic degradation, antimicrobial peptide containing a thioamide backbone only between nva and fmdp. activity of nva[csnh]-fmdp nva-fmdp against selected microorganism in medium containing blood serum was determined. neuroprotective effects of cortexin and cortagen in rats with focal brain ischemia and brain trauma there is a growing body of evidences that natural peptides and their smaller synthetic analogues have good outlooks for medical use. three compounds were studied in this research -cortexin, cortagen and nîleylcortagen. cortexin is a balanced combination of oligo-peptides isolated from calf/porcine cortex. the number of non-clinical and clinical studies confi rmed cortexin effi ciency as nootropic and neuroprotective drug. in spite of benefi cial results the natural source of the drug implies some diffi culties for it general use. to overcome this issue a number of putative cortexin synthetic analogues were developed. among them is tetrapeptide cortagen. in order to improve the peptide transport to the brain and lipophility the hydrophobic oleyl radical was added to the peptide chain. the aim of research was to study the effects of these peptides on recovery of conditional refl ex in active avoidance paradigm after heavy brain trauma and neurologic defi cit dynamics induced by focal brain ischemia in rats. males albino rats were involved in experiment. closed craniocerebral trauma was modeled by weight-drop method. focal cerebral ischemia was induced by cortical phototrombosis. experimental therapy started in . hours after mechanical action and continued for days. synthetic peptides were administrated i.p. once-a-day in dose of - mcg/kg. cortexin was used at the same schedule in dose of mcg/kg. cortagen and cortexin administration in dose of and mcg/kg respectively resulted in pronounced neuroprotective effect in rats with focal cerebral ischemia. the observed effects were proved by limb-placing test. cortexin experimental therapy resulted in earlier restoration of conditional refl ex starting from the rd day after brain trauma. nîleylcortagen had weaker benefi cial effect with signifi cant improvement in conditioned response recovery by the th day. probiotics such as lactobacillus rhamnosus gg provide health benefi ts beyond their mere nutritive value, and are useful in the treatment and prevention of several diseases. isolation and use of the anti-apoptotic factor(s) would ultimately culminate in the development of novel therapeutic agents in enteropathy resulting from chemo-and radio-therapy in the treatment of cancer patients, which could in turn be administered in a consistent and pharmacologically sound manner. in present study, intestinal epithelial cells were isolated from wistrar rats (n= ), and were grown in dmem medium supplemented with % foetal bovine serum at °c in a humidifi ed % co atmosphere. after h, these cells were treated with bioactive peptides isolated from lgg ( - ìmol/ lit) for h, and were incubated with camptothecin ( ìmol/ lit) for and h, and cell viability was measured by mtt assay. caspase activity assay was carried out to determine caspase and caspase activity. moreover, cell lysates were also pretreated with caspase inhibitor ( ìm). dna fragmentation assay was carried out to determine the increase in dna fragmentation. bioactive peptides signifi cantly prevented camptothecin induced apoptosis in rat intestinal epithelial cells. the treatment of intestinal epithelial cells with camptothecin for h resulted in fi ve-fold increase in dna fragmentation, and resulted in three-fold increase in caspase activity, compared with controls. pre-treatment with bioactive peptides ( ìmol/ lit) signifi cantly attenuated the camptothecin-induced dna fragmentation by % (p< . ), and signifi cantly inhibited caspase and caspase activity by % and %, respectively, after h of camptothecin exposure (p< . ). hence, it was concluded that novel bioactive peptide from lgg were found to be potential anti-apoptotic agents and hence could be administered as therapeutic molecule for treatment of enteropathy resulting from chemo-and radiaton-therapy. cisplatin belongs to the most powerful and useful anticancer agents. it binds strongly to dna in regions containing several guanine units, forming pt-dna links within strands. through disrupting base-pairing guanine to cytosine cross-links lead to unwinding of the dna. as a result cisplatin works against both types of cells, destroying cancer and normal type ones. therefore more selective delivery system of platinum to cancer cells is still needed. based on the evidence of the presence of -and -opioid receptor types in carcinoma cells we proposed to use opioid peptides as selective carriers for delivering platinum ions to the cancer cells. we designed hybride molecules which combine two fragments. one part of the molecule contains the opioid pharmacophore and the other fragment is designed to form a complex with platinum ion. such molecule can serve not only as carrier for platinum, but also give a strong analgesic effect. as a result such hybride molecule should express analgesic properties and provide anticancer activity. we will present synthesis of a few opioid peptide-platinum(ii) complexes, the binding affi nity at the opioid receptors and effect on the proliferation of the human glioblastoma cells. peptides derived from the c-terminal heptad repeat region of the hiv fusogenic protein gp are potent inhibitors of viral infection, and one of them, t (enfuvirtide), is used for the treatment of therapy-experienced aids patients. the mechanism of action of these peptides is to interfere bioactive peptides with the fusion of the viral and target cell membranes, and it is known that hiv entry takes place in membrane microdomains ('lipid rafts') enriched in cholesterol and sphingolipids. therefore we explored the advantage of targeting a peptide fusion inhibitor to membranes by addition of a lipid moiety, specifi cally a cholesterol group. since derivatization with lipids is also known to improve the half-life of peptide therapeutics, we chose the antiviral peptide c , which is more potent than t in cell-based assays, but unlike t has a very short half-life in vivo. we show here that attachment of cholesterol to c (c -chol) dramatically increases its antiviral potency against a panel of hiv strains, including primary isolates: for strain hxb , ic = pm for c -chol, versus pm for c , and pm for enfuvirtide). consistent with the anticipated mechanism of action, c -chol accumulates at the site of action, since washing of the target cells after incubation with the peptide, but prior to triggering fusion, increases ic only -fold, relative to -fold for c . moreover, cholesterol must be strictly positioned at the c-terminus of c , in line with the need of an antiparallel orientation of the c-peptide relative to n-peptide trimeric coiled-coil, present in the fusion-active conformation of gp . in addition to boosting antiviral potency, derivatization with cholesterol has the expected benefi cial effect on the peptide pharmacokinetics. we believe that these fi ndings may be of general utility for viruses, which share with hiv the dependence on a type i fusogenic machinery. multiple sclerosis (ms) is an autoimmune demyelinating disease mediated primarily by cd + t cells of the th subset. the design of peptide mutants of disease-associated myelin epitopes to alter immune responses offers a promising avenue for the treatment of ms. we designed and synthesized a number of peptide analogues by mutating the principal tcr contact residue based on mbp - epitope. the synthesis of the linear peptide agonist mbp - , as well as of the cyclic analogue was carried out by the fmoc/tbu methodology, utilizing the -chlorotrityl chloride resin. cyclization was achieved using obenzotriazol- -yl-n,n,n',n'-tetramethyluronium tetrafl uoroborate (tbtu) and -hydroxy- -azabenzotriazole, , , collidine allowing fast reaction and high yield cyclization product. the purifi cation was achieved using hplc reversed-phase chromatography and the peptide purity was assessed by analytical hplc and by mass spectrometry (esi-ms). agonist and antagonist (linear and cyclic) peptides were conjugated to reduced mannan. immune responses were diverted from th to th in sjl/j mice and generated antibodies which did not cross react with native mbp protein. ( ) . due to its high effi cacy and specifi city, hn represents a potential lead to new therapeutic approaches of ad. however, the molecular mechanism(s) of hn function(s) are not yet fully elucidated. wild-type hn and hn-derivatives were synthesized by spps and amino acid sequences and homogeneities of the rp-hplc purifi ed peptides ascertained by esi-and maldi-mass spectrometry (ms). the complex formation between hn and a ( - ) was studied by affi nity-chromatography and high resolution ms (fticr-ms), as well as by immunoanalytical (elisa) techniques. the binding sites between the two peptides were identifi ed by applying proteolytic epitope extraction/excision procedures [ , ] integramide a, an effi cient inhibitor of the coupled reaction of hiv- integrase, is a -mer linear peptide characterized by c -methylated -amino acids ( iva, isovaline, and aib, -aminoisobutyric acid, residues) that was isolated from fungal extracts of dendrodochium sp. the amino acid sequence was fully elucidated by the merck group a few years ago (s. b. singh et al., org. lett. , , - ) . on the other hand, the chiral sequence was only partially determined. in particular, the precise stereochemistry of the iva -iva dipeptide (known to contain one d-and one l-residue) near the c-terminus was not reported. to solve this unsettled issue and to assess integramide a primary structure-bioactivity relationship we performed by solution methods the total chemical independent syntheses of both l-d and d-l -mer diastereomers and compared their properties with those of the natural inhibitor. for an unambiguous, complete stereochemical assignment of integramide a we relied heavily on hplc and nmr techniques. our results clearly indicate that the chirality sequence of the iva -iva dipeptide of the natural product is l-d. the two integramide a diastereomers were also evaluated as inhibitors of hiv- integrase in the coupled reaction of proviral dna into the host cell dna. . a prototype peptide, r - , spanning rantes residues to , contains two hydrophobic clusters of fundamental biological importance connected by a nonessential hydrophilic linker. r - has been rationally modifi ed to improve its ccr binding and its antiviral potency. nmr studies on the modifi ed peptides revealed important similarities and differences with the three-dimensional organization of rantes. although these peptides are consistently more active as dimers, an increase in anti-hiv- activity of the monomeric forms was observed in parallel with their molecular evolution. in addition, no tertiary interactions could be detected by nmr, indicating an autonomous folding of the monomers also in the context of dimeric peptides. the most potent peptide designed so far, rmax, shows anti-hiv- activity in the low nanomolar range (vangelista et al, in preparation). strikingly, a similar potency was observed in the same assay with t , an hiv- gp -derived peptide currently licensed for use in aids therapy. these results provide a rationale for the use of rantes-derived peptides as new candidates for treatment and prevention of hiv- infection. % of the population older than years and % exceeding the age years are affected by alzheimer's disease (ad).( ) a factor in the pathogenesis of ad is the cerebral deposition of amyloid fi brils as senile plaque. bace initiates the pathogenic processing of app by cleaving at the n-terminus and the resulting c membrane bound c-terminal peptide can then be hydrolyzed by -secretase to form apeptide (a or a ). bace is not only a promising target cause it is a key player in formation of a but also because bace knock-out mice are viable and free of the gross phenotypic changes. our group was recently able to shown that the phosphino dipeptide (pdp) isostere is a suitable replacement of the hydroxyl ethylene isostere in om - ( ) resulting in pseudo peptidic inhibitors of about same potency. [ , ] in our search for conformationally restrained pdp isostere bace inhibitors we speculated that the p and p cyclization would lock the active conformation of the cyclic linear pseudo peptidic inhibitor in the n-terminal region. first the detail analysis of the crystal structure of om - bound to bace ( ), revealed that the ideal macrocyclus should consist of a -membered heterocycle. on this basis we developed a macrocyclic inhibitor with p and p cyclized side chains containing a pdp isostere with improved serum stability. specifi c tumor-targeting, via tumor-associated antigens (taa), selectively expressed or over expressed on tumor cells, is the goal of modern cancer therapy aimed at overcoming non-specifi c toxicity of most anticancer drugs. the expression of taa varies among different tumors and patients, resulting in highly variable response to tumor targeted therapies. therefore, diagnosis should provide information on the expression of the targeted antigen in each patient, thus allowing to predict possible effi ciency of a therapy mediated by targeting agents directed to the same tumor antigen. in this approach, the molecule used for tumor cell tracing should be as close as possible to that used for therapy. the use of peptides as tumor targeting agents was envisaged years ago with the fi nding that receptors for different endogenous regulatory peptides are over-expressed in several primary and metastatic human tumors, and can be used as tumor antigens (reubi jc. j nucl med ; : - ). in previous works, we demonstrated that peptides synthesized in a branched form, result in molecules that are resistant to proteolytic activity and can retain (or even increase, through multivalent binding) peptide biological activity. a branched peptide that targets neurotensin (nt) receptors, known to be over-expressed in a number of tumors, including colon, pancreas and prostate carcinoma (reubi jc. endocr rev ; : - ) and was conjugated to effector units and proved to be stable and active in vivo (falciani c, et al. mol cancer ther. ; : - ). here, we created new nt-based molecular tools conjugated to different fl uorophores and chemotherapeutics and demonstrated that branched peptides can be modulated either as tracers for measuring the presence of the specifi c target in primary tumor or metastasis on human surgical resections, or as specifi c drug-carriers, which use the same target to enter and eventually kill tumor cells in vitro and in vivo. aquaculture has become an increasingly important activity, as an immediate source of animal protein required for several countries growing population. vaccination of fi sh against bacterial and viral diseases is one of the best strategy for controlling certain infectious bioactive peptides diseases in aquaculture worldwide, thus decreasing the need for antibiotics, and increasing cost-effectiveness and net profi ts. infectious pancreatic necrosis virus (ipnv) is a pathogen of farm fi sh with a worldwide distribution. peptides derived from ipnv protein (vp ), a major structural protein, could be used for development of effective vaccine against ipnv, according to a search of vp antigenicity we selected two peptide sequences, k pyvrledetpqg (vp - - ) and n fslaeqpanetk (vp - - ), that are expected to be highly immunogenic. these peptide sequences were synthesized in their n-terminus iodoacetylated form and conjugated to ac-(lys-aib-cys) -nh , a cell penetrating sequential carrier (cpsc). conjugation of four copies to cys side chains of the carrier was realised by the chemoselective ligation method. the resulted constructs were purifi ed by hplc and characterized by esi-ms. immunizations are in progress in order to test their immunoprotective potency. acknowledgements to the gsrt (greece-egypt project -å) for the fi nancial support. for more then two decades, the rgd sequence is known to bind integrins, e.g., , v , and iib among many others. it is present in the natural ligands for these integrin receptors, as in fibronectin, vibronectin, or fibrinogen. recently, it was shown that fibronectin in which the rgd sequence has been mutated into an rge sequence (which is known not to be recognized by integrins) still has the ability to interact with its receptor. however, it forms fn fi brils only with a slightly different phenotype than wild-type fibronectin ( ). a hypothetical model for these unexpected results was proposed by curnis et al. ( ) . the ngr (asn-gly-arg) sequence, present at four positions in the fi bronectin molecule, is able to undergo a rearrangement to isodgr (isoasp-gly-arg), which shows activity on v and -with less potency -on . the mechanism of asn-deamination is already known for a long time and has widely been considered to be a process of degradation, acting as a biochemical clock that limits protein lifetimes in vivo ( ). curnis et al. were the fi rst to show that the deamination process increases protein function instead ( ). these fi ndings stimulated us to create a library of cyclic peptides containing the isodgr sequence. a screening of this library showed various new peptides with high activity and selectivity towards the integrin receptor . the correlation between the intensities of muramyl peptides adjuvant effect and nod activation. it is well known that essential activity of muramyl peptides -minimal structures of bacterial cell wall -are adjuvanticity. the comparison of the adjuvant effect of these compounds with the ability to activate nf-kb pathway through nod was examined. the adjuvant activity of di, tetrasaccharide peptides and stearoyl containing derivatives has at least two peaks in dose-response curves and greater of them correlates with respective dose-response data for nf-kb stimulation through nod . introduction of stearoyl moiety, with the aim of improving muramyl peptide interaction with the cell membrane and subsequent intracellular delivery, infl uenced the corresponding activities in vitro, but did not correlate with improved effects in vivo experiments. the comparison of the adjuvanticity in vivo and the nod activation in vitro revealed clear correlation between two responses. these fi ndings confi rm the view that nod pathway activation should account, at least in part, for the adjuvant effect of these compouds. the correlations of the nf-kb pathway induced by muramyl peptides with the aim of enhancing their adjuvant activity were investigated. the thymus gland plays an important role in overall immunomodulation. the studies in early s by several authors have established that thymus is necessary for the normal development of immune response. it is thought to be responsible for the development and regulation of tcell immunity, acting through endocrine mechanism ( ). it is known that thymus peptides play an important role in the development, maturation, differentiation, and activation of t-lymphocytes. investigation of the function and properties of this gland shows that the thymus contains pharmacologically active components with immunological properties. when the immune system is challenged, thymic peptides seem to regulate the expression of various cytokine and monokine receptors on t-cells and induce secretion of il- , interferon alpha, and interferon gamma. up to date several thymic extracts have been isolated using different biochemical methods of extraction, homogenization and purifi cation. these are, usually, semipurifi ed aqueous and lipid calf thymus extracts. the goal of this study was to determine the biological activity of peptide components, isolated from the calf thymus. extract of calf thymus was prepared and fractioned into lipid and nonlipid (peptides) fractions. the nonlipid fraction was isolated and characterised by biuret, ir , nmr and hplc methods. analyses of ir and nmr spectra indicated the presence of charasteristic bands and peaks for peptides. results estimated from hplc analyse showed that molecular masses of isolated peptides were below daltons. biological activity was accesed by using proliferation of thymocites and splenocites in vitro in the presence of mitogen, and obtained results showed signifi cant imunomodulatory effect. keywords: thymus, peptides, immunomodulators, thymocites, splenocytes, proliferation according to contemporary knowledge about proteins, every protein can play the role of a precursor of biologically active peptides. bioactive peptides isolated from food proteins display various activities, for example: antihypertensive, antithrombotic, immunomodulating, opioid and antibacterial. the database of protein and bioactive peptide sequences designed in chair of food biochemistry (www.uwm.edu.pl/biochemia) contains information on proteins which are precursors of bioactive peptides. the database, biopep, enables an evaluation of food proteins according to the following criteria: the frequency of the occurrence of fragments with the given activity in a protein chain, a potential activity of protein fragments, and profi les of a potential biological protein activity i. e. the type and location of a bioactive fragment in the protein chain. the biopep database was used to determine the profi les of potential biological activity of food proteins and classify them into families and subfamilies. results that we obtained indicate that the greatest number of bioactive peptides can be released from milk proteins. we also analysed structural properties of bioactive fragments encrypted in protein chains which are predicted to be accessible for endopeptidases. the application of biopep and msblast software enabled us selection of a fragments with high degrees of identity to the celiac-toxic peptides in food proteins sequences. based on the biopep, we are able to design the processes of the release of bioactive peptides from protein sequence and with the use of mass spectrometry -to identify released peptides. the detailed results of authors in silico food proteins analysis will be presented. the computational methods and the above-mentioned tools can be applied for designing food with the special designed and desired properties (functional food) as well as production of nutraceuticals i. e. food with therapeutic properties. neurotensin analogs with high affi nity and selectivity at human neurotensin receptor . neurotensin (nt), pglu -leu -tyr -glu -asn -lys -pro -arg -arg -pro -tyr -ile -leu -oh, exerts numerous physiological actions in the central nervous system and in the periphery. studies in animal models have suggested its involvement in the modulation of dopamine transmission, hypothermia, analgesia, locomotor activity, cardiovascular function, and others. at present, three receptors are known to bind nt; two of them are g-protein coupled-receptors (ntsr and ntsr ). the physiological effects of ntsr have been extensively studied but the functions associated with the nt binding to ntsr are less well defi ned. the c-terminal fragment of nt, arg -arg -pro -tyr -ile -leu -oh, has been long recognized as an essential and suffi cient segment for the effective interactions of nt with the receptors; a short peptide, arg -arg -pro -tyr -ile -leu -oh, designated nt( - ), displays binding affi nity similar to that of the full-length nt at both ntsr and ntsr . in this study, the role of arg in the interactions of nt( - ) with the human ntsr and ntsr was examined through ligand structure-function studies. the side chain of arg was determined to not be critical for binding to hntsr but essential for binding to hntsr . several analogs of nt( - ) are reported which are high affi nity hntsr ligands (ic = . to nm) and more than -fold selective versus hntsr . human umbilical vein endothelial cells (huvecs) form tubular networks when cultured on top of the matrigel basement membrane preparation, refl ecting the ability of the cells to form blood vessels. using this model, we have previously shown that psa (also known as klk ) inhibits endothelial cell tube formation( ), indicating reduced angiogenic potential. furthermore, we have shown that the antiangiogenic activity of psa is related to its enzymatic activity [ , ] . we have developed peptides that stimulate the enzymatic activity of psa towards a small chromogenic substrate. these peptides also enhance the anti-angiogenic activity of psa both in vitro and in vivo. this supports our hypothesis that enhanced psa-activity by our peptides could be used to reduce tumor angiogenesis and, thus, to reduce tumor growth. the most extensively studied tads are those that contain acidic tads and one extremely important protein is the human tumour suppressor protein p . given the presence of repetitive stretches of acidic amino acids tads are generally disordered in the free state and this has also been demonstrated for p tad. using a combination of nmr spectroscopy, isothermal titration calorimetry and site-directed mutagenesis studies we have recently characterized the interaction of the p tad with the pleckstrin homology (ph) domain of the tfb / p (yeast/human) subunit of tfiih. the nmr structure of the tfb / p tad complex demonstrates that p forms a short alpha-helix in complex with tfb ( ). this structure is an important step towards developing a sequence code for acidic tad binding to the ph domain of tfb /p . such detailed structural information is essential to design molecules that modulate transcription activators such as p . we will present the structure of the p /tfb complex, structural and biophysical characterization studies with peptides designed to mimic acidic tads and compete with p for binding to tfiih. integrins are a family of transmembrane cell surface receptors, which mediate cell-cell and cell-matrix adhesion. the binding of integrins to their natural ligands is the molecular basis of physiological processes such as cell adhesion, migration and signal transduction of cells, as well as of patho-physiological processes. thus, small molecules capable of interfering with this integrin-natural ligand binding process have pharmacological potential in the therapy of cancer and infl ammatory diseases. the amino acid sequence rgd, present on many of the natural ligands, is a prominent recognition motif of integrin ligands. synthetic rgd-containing peptides are an excellent starting point for the identifi cation, synthesis and development of selective integrin ligands. the affi nity and selectivity of the peptide ligands towards different integrins depend strongly on the secondary structure of the sequence and the overall three-dimensional shape. cyclization is frequently used as a method to reduce the accessible conformational space. additionally, the incorporation of non-natural conformationally constrained amino acids can greatly affect the secondary structure of the peptide, in such a way that the synthetic ligands prefer to adopt a particular conformation. the aim of this investigation are small cyclic peptides containing the rgd motif and constrained amino acids (such as -methylated amino acids, -amino acids or dehydroamino acids) that exhibit well-defi ned conformational properties. the present communication describes the synthesis of different cyclic rgd peptides with the general sequence c-(-arg-gly-asp-xaa-yaa-) and the evaluation of their activity as ligands for the v and integrins, present on human cells. acknowledgments: this work is supported by a marie curie intra-european fellowship from the th framework programme. in recent years there has been increasing interest in the design of chemical agents capable of inducing dna interstrand crosslinking (isc), which, shutting down the dna replication process, represents by far the most cytotoxic of all the alkylation events. quinone methides (qms) are interesting compounds that have been proposed as intermediates in a large number of chemical and biological processes. the asymmetry introduced by the presence of two electronically different substituents, carbonyl and methylidene, on the cyclohexadiene ring imparts a strong dipolar character to quinone methides not found in benzoquinones and quinodimethanes. qms have been successfully used to accomplish nucleoside alkylation and dna isc by photochemical and fl uorideinduced activation. apart from their involvement in biological processes, o-qms react with a variety of nucleophiles (michael addition), resulting in substituted hydroquinones. they also function as effi cient heterodienes in diels-alder reactions and undergo a variety of self dimerization reactions. here we present the synthesis of a new series of synthetic o-qms (bis-napthalene type) conjugated with rgd analogue peptides. the biological activity of these compounds is under investigation. aknowledgements: we thank the european social fund (esf), operational program for educational and vocational training ii (epeaek ii) and particularly the program pythagoras ii for funding the above work. platelets aggregation causes clotting in the blood vessels during the blood circulation due to several reasons. factor viii (fviii), a blood coagulation glycoprotein, is a key component of the blood coagulation system. fviii in its activated form (fviiia) acts as a cofactor to the serine protease fixa in the conversion of the zymogen fx to the active enzyme (fxa). the role of fviiia is to increase the catalytic effi ciency of fixa in the activation of factor x (fx). the target of this research is the synthesis of biologically active peptides, which are expected to inhibit selectively the maximisation of thrombin production depended on factor ix (fix) and accordingly the additional activation of platelets. these peptides are based on the regions in which the fviii interacts with fix. glycoprotein fviii is composed of three distinct domain types in the arrangement, a -a -b-a -c -c . the sequence - of a subunit is the following: ser -val-asp-gln-arg-gly-asn-gln this work covers the synthesis and biological evaluation of linear and cyclic head to tail peptides, analogs of the loop sequence - of the a subunit, aiming at the inhibition of interaction of fviiia with fixa. substitutions have been taken place at asn , which are related to the pharmaceutical groups at the side-chain, like asp(r) . all the synthesized analogs are purifi ed (rp-hplc) and identifi ed (esi-ms). the synthesized peptides analogs were investigated for their inhibitory activity and tested for clotting defi ciency by measuring their activated partial thromboplastin time (aptt) in vitro. these results will be discussed in relation with their biological activity against the fviiia factor of blood coagulation. nerve growth factor (ngf) is an homodimeric protein that binds two different cellular receptors: ) the transmembrane tyrosine kinase receptor trka, a member of tyrosine kinase family; and ) p , a member of the tumor-necrosis-factor receptor superfamily. both receptors are involved in the activation of different intracellular signal transduction cascades. small peptides retaining the most essential elements of ngf may be useful either as agonist or competitive antagonist in the treatment of several neurodegenerative desease and nerve injuries. the n-terminal fragment of ngf was previously demonstrated to be an important determinant for affi nity and specifi city in the binding to trka. crystal structure of ngf-trka complex, site-directed mutagenesis studies and substitution of individual amino acids, contributed to identify within the ngf molecule the most relevant domains for its biological activity in the loop- ( - ), loop- ( - ) and in the nterminal region ( - ). we synthesized twenty peptides mimicking the loop and linked together with aminoacid-based spacers (n glycines) or with two unit of -ammino- , -dioxaottanoic acid with or without the n-terminal region. l l and l l homodimeric loops, moreover l and l were also synthesized, as single loop, and their biological properties were compared to l l and n-l l activity . the n-l l (hpifhrgefsvadsvsvwvgdctdikgkctgacdgkqc) and l l (ctdikgkctgacdgkqc) peptides showed a good ngf agonist activity at concentration as low as um. both were able to induce differentiation of chick dorsal root ganglia (drg) and to stimulate the tyrosine phosphorylation of trka but not of trkb receptor. in addition the l l peptide was able to induce the pc cells differentiation into sympathetic-like neurons. moreover the l l peptide was shown to reduce neuropathic behaviour and restore neuronal function in a rat model of peripherical neuropathic pain, thereby suggesting a potential therapeutic role for this peptide. short proline-rich peptides interact with sh domains, exhibiting little or no secondary structure before their binding to the cognate proteintargets. under these conditions the binding process of a proline-rich peptide with the sh domain shows unfavorable binding entropy, likely resulting from a loss of rotational freedom on the formation of the ppii helix. with the aim of stabilizing the ppii helix conformation in sh binding motifs, in the previous years, we replaced the proline residues of the hpk proline-rich decapeptide, ppplppkpkf (p ), either with -r-(fp) or with -s-(fp) fl uoro-l-proline at different i, i+ positions. the interactions of the fl uoroproline-peptides with the sh domain of cortactin protein were analyzed quantitatively by non-immobilized ligand interactions assay by circular dichroism (nilia-cd), whereas cd thermal transitions were measured to correlate their propensity to adopt ppii helix with their affi nity for sh . results show that although the introduction of the fp residue stabilizes the ppii helix conformation of peptides in a position-dependent manner, the induction of a stable peptide conformation does not increase the ligand affi nity towards the sh domain of cortactin. to explore the effect of electron-withdrawing substituent in the pro residue, the fp residues of p were replaced by the natural amino acid -r-hydroxy-proline (hyp). unexpectedly, nilia-cd and cd thermal transitions results showed that the hyp-containing peptides exhibit a stable conformation in aqueous buffer and k d values lower than the corresponding fp peptide-analogues. in particular, hyp peptide containing hyp residues at i, i+ and i+ positions adopts the greater percentage of ppii helix conformation over the entire studied temperature range and shows a k d value comparable to that of the parent p peptide. we are now searching for inhibitory peptides able to reduce survivin function in breast cancer cells. we take advantage of the observation that survivin forms homodimers in vivo and have derived short peptides representing the dimerization domain. to enable intracellular expression and visualization of these short peptides, they will be fused to a fl uorescent carrier protein. alternatively, the peptides comprising the dimerization domain will be inserted into a scaffold protein. this allows the presentation of the peptides in a constrained and stabilized conformation. we will analyze the effects of lentiviral expression of these fusion peptides in cancer cells expressing high levels of survivin. the peptides will be analyzed for their ability to inhibit proliferation, cell-cycle progression and/or to induce apoptosis in cancer cells. cd c/cd ( x ) is a member of the leukocyte integrin (cd or integrin) subfamily of adhesion molecules and it is expressed on monocytes, macrophages and granulocytes. its ligands include fi brinogen, ic b, lps, collagen type i and denatured proteins as well as intercellular adhesion molecules icam- and icam- . icam- is a red cell specifi c membrane glycoprotein that was fi rst described as anerythrocyte blood group antigen lw (landsteiner-wiener) and later it was found to belong to the family of intercellular adhesion molecules. the fi rst reported receptors of icam- were leukocyte integrins cd a/cd and cd b/cd . later it has been reported to bind to several other integrins as well. latest reported interaction is to cd c/cd . the binding of icam- to integrins expressed in macrophages might clarify some of the controversy concerning the recognition and uptake of senescent red blood cells in spleen. another function of icam- / macrophage integrin interactions could be in retaining the maturing red cells in bone marrow until they are ready to be released in the circulation. adhesion between icam- and integrins have also been found to be important in different pathological situations and inhibition of these adhesion events could be of important therapeutic value. we have studied the interaction between icam- and cd c/cd using peptides derived from both binding partners using pepspot method and synthetised peptides. the inhibitory or activating role of these peptides could beof clinical importance in pathological conditions where abnormal red cell adhesion is observed. the regulatory effects of relaxin in mammals realize via receptors of the serpentine type. in this work we studied the relaxin activating effect on the adenylyl cyclase (ac) activity in the rat tissues and muscle tissues of invertebrates using a peptide strategy. the strategy involved the synthesis of the peptides - and - , as well as the palmitatemodifi ed peptide - , all corresponding to the c-terminal region of the third intracellular loop (c-icl ) of the human type relaxin receptor lgr . the peptides - and - -lys(palm) had a dose-dependent activating effect on the ac activity and gtp-binding in myocardium, brain and, to a smaller extent, skeletal muscles of rat. the - -lys(palm) had a stronger ac effect than the longer peptide - , because of the possibility to be anchored in the membrane by the hydrophobic radical and, hence, to interact with the g protein more effectively. in mollusks and earthworm muscles, the stimulatory effects of both peptides were weak. competitive inhibition of the stimulating effects of relaxin by the lgr -derived peptides indicates that the relaxin signal in myocardium and brain is transmitted to ac via lgr receptor. in skeletal muscles of rat and muscle tissues of invertebrates, the inhibition of ac and gtp-binding stimulating effects of relaxin by peptides was almost indiscernible. it can be concluded that relaxin controls the ac of these tissues via the receptor different from the relaxin receptor lgr . the stimulating effects of lgr -derived peptides also decreased in brain and myocardium in the presence of Ñterminal peptide - of mammalian g s -subunit and after cholera toxin treatment. thus, relaxin stimulates ac via lgr receptor and g s protein in myocardium and brain, and the coupling between receptor and g s protein is mediated by the interaction of receptor Ñ-icl and Ñ-terminal segment of g s . acknowledgements: supported by rfbi (grant - - ) and «russian science support foundation». normal tissue function depends on adequate supply of oxygen through blood vessels. angiogenesis is a fundamental process by which new blood vessels are formed and which is highly regulated in healthy individuals. however, many diseases are driven by unregulated angiogenesis. excessive angiogenesis is associated with cancer, rheumatoid arthritis, psoriasis, while insuffi cient angiogenesis results in ischemia or artherosclerosis. many new angiogenic modulators have been developed in the last years, mostly to inhibit angiogenesis but only few peptide-based angiogenic stimulators have been reported. there are data in the literature suggesting roles of small peptides or basichexa peptides as angiogenic modulators like ringseis et al. reporting effects on endothelial cell function (such as ec proliferation) and fazekas et al. describing effects for the latter. endostatin, an endogenous inhibitor of angiogenesis, among its proteolitic fragments contains both, an inhibitor and a stimulator of angiogenesis. we considered it possible that fragments of basic heptapeptide d-phe-cys(-)-tyr-d-trp-lys-cys(-)-thr-nh (tt- ), a strong antitumor agent, could also act as angiogenic modulators. the heptapeptide was developed in our laboratory and has been recently in a clinical trial (phase ii). we synthesised, characterized and tested partially protected di-and tripeptide fragments of it such as boc-d-phe-cys(acm)-tyr-ome, d-phe-cys(acm)-tyr-ome, boc-tyr-d-trp-cyclohexilamide and h-tyr-d-trp- -adamanthylamide. the biological activity of the compounds was tested in vitro using an immortalized kaposi sarcoma cell line to determine their pro-anti-angiogenic character. to test the angiogenic potential of the best compound, the aorta ring assay was used, which by using intact vascular explants reproduces more accurately the environment in which angiogenesis occurs. in this study we report the synthesis and angiogenesis modulating effects of the peptides. somatostatin is a neuropeptide that regulates several functions of the endocrine and exocrine systems. it also affects cell proliferation and neurogenic infl ammation through a family of g protein-coupled receptors. neurogenic infl ammation plays signifi cant role in the pathogenesis of numerous infl ammatory diseases (such as asthma, arthritis, allergy and migraine). inhibitory effect of somatostatin on infl ammation is well known but the pharmaceutical use of the native peptide is limited due to its broad spectrum of anti-secretory effects and short plasma half-life time. tt- , a heptapeptide analogue of somatostatin (developed by our research group and it is in clinical phase ii trials) has selective antitumour and anti-infl ammatory effect without regulating other endocrine or exocrine processes. receptor-ligand binding experiments with various analogues of the somatostatin as well as tt- [d-phe-c(cys-tyr-d-trp-lys-cys)-thr-nh ] verifi ed that the side chains of tyr, d-trp and lys are important pharmacophoric groups for somatostatin-like biological activity. linear, cyclic and branching derivatives were designed and synthesised using above amino acids to selectively inhibit infl ammatory actions. unnatural moieties also were applied to enhance their enzyme resistance. linear and cyclic peptides consist tyr and d-trp, while ring closed ones contain lys too. branching peptidomimetics have the same, fl exible core [tris( aminoethyl)amine] and three protected or unprotected amino acids situated in equal positions. the biological activity of the compounds was evaluated by in vitro assay of substance p release and in vivo assay of plasma protein extravasation. the most potent agents strongly inhibited substance p release by - % and one of them showed strong anti-infl ammatory activity when administered orally. the structure of the novel compounds and the relationship between their structure and biological activity will be discussed. urotensin ii (u-ii), a potent vasoconstrictor, is found in diverse species, including human. several biological studies indicate that u-ii is the most potent mammalian peptide vasoconstrictor reported to date, and it appears to be involved in the regulation of cardiovascular homeostasis and pathology. in order to elucidate the importance of trp residue for receptor interaction and biological activity recently we have designed, synthesized new analogues where trp was replaced with constrained analogues ltpi or dtpi ( , , , -tetrahydronorharman- -carboxylic acid). the tpi residue was replaced in both agonist p u and antagonist urantide sequences. on these new ligands we performed biological and nmr conformational studies. the new ligands will be used in further biological investigations of the ut receptor. gnrh analogs as carriers for targeted suicide gene delivery suicide gene therapy represents one of the promising approaches to the cancer treatment. an application of herpes simplex virus thymidine kinase (hsvtk)/ganciclovir system possesses additional advantage due to bystander effect on neighboring cancer cells. overexpression of gnrh receptors in the case of most adenocarcinomas creates the basis of gnrh analogs use as carriers for targeted suicide gene delivery. we investigated different manners of gnrh molecule modifi cation; their infl uence on peptide/dna complex formation and its penetration into cancer cells. analogs, containing nls from large antigen of sv were synthesized using combination of boc-and fmoc-chemistry. depending on peptide structure (agonist or antagonist) nls was attached via position or of the natural molecule. the competition experiments demonstrated that internalization of peptide/ dna complex into hepg cells is mediated by specifi c receptor binding. moreover, nls/dna complexes, lacking gnrh moiety were unable penetrate cellular membrane. subsequent studies permit to identify the infl uence of analog structure on in vitro effi ciency of suicide gene therapy, followed by acyclovir treatment. it was shown that application of reference peptide gene delivery system damaged about % of tumor cells. use of cationic peptide conjugated with rgdf sequence provides high effi ciency of treatment, however can not ensure selective action on cancer cells. gnrh analogs completely suppress tumor growth in vitro due to specifi c interaction of peptide/dna complex with correspondent receptor. the effi ciency of suicide gene therapy depends on peptide structure and is in favor of agonists as compared to antagonists. preliminary data of experiments in vivo on laboratory animals demonstrated practical utility of tested peptides in the course of intravenous administration. thus, it was shown that gnrh analogs containing nls moiety represent promising candidates for the delivery of hsvtk gene into the cancer cells. the structural-functional study of putative grape (vitis vinifera) uncharacterized protein sequences was performed. we developed a special method of computer analysis ( ) for this. this method has allowed to reveal new potentially active regulatory oligopeptide sequences yet not investigated experimentally. information on grape amino acid sequences of public databases [ , ] , computer database erop-moscow (endogenous regulatory oligopeptides) . containing the information on structure and functions of known natural oligopeptides and specially created computer programs were used for this. protein amino acid sequences were compared with all known oligopeptide sequences in this method. as a result several tens of grape oligopeptide sequences were elucidated. the similarity of their sequences with the known oligopeptide structures of other biological species was the basis for the prediction their potential functional properties. it has been shown that grape contain putative regulatory oligopeptides possessing functions of antibacterial and antifungal agents, enzyme inhibitors, calmodulin binding structures, rapid alkalinization factors, etc. the primary structure similarity of grape sequences was found not only with plant species but with bacteria, fungi, and animals also. cyclotides are plant derived mini-proteins with compact folded structures and exceptional stability. their stability derives from a headto-tail cyclised backbone coupled with a cystine knot arrangement of the three-disulfi de bonds. taking advantage of this stable framework we developed novel vegf-a antagonists by grafting a peptide epitope involved in vegf-a antagonism onto the stable cyclotide framework. antagonists of this kind have potential therapeutic applications in diseases where angiogenesis is an important component of disease progression, including cancer and rheumatoid arthritis. a grafted analogue showed biological activity in an in vitro vegf-a antagonism assay at low micromolar concentration and importantly the in vitro stability of the linear epitope was markedly increased using this approach. in general, the stabilization of bioactive peptide epitopes is a signifi cant problem in medicinal chemistry and in the current study we have shown the cyclotide framework is ideally suited for such stabilization. cystine rich scaffolds are emerging as valuable templates in drug design and in the current study we have shown that the cyclotide scaffold, with the advantageous features of a knotted disulfi de core and a cyclic backbone, has signifi cant potential in stabilizing a wide range of bioactive peptide epitopes. gonadotropin releasing hormone (pglu-his-trp-ser-tyr-gly-leu-arg-pro-gly-nh , gnrh) plays a signifi cant role in the controlling of gonadotropins and steroids hormones. a large number of linear gnrh analogues has been synthesized and tested for several medical uses. leuprolide acetate (pglu-his-trp-ser-tyr-(d)leu-leu-arg-pro-nhet, lpa) is a potent gnrh agonist and is used to treat a wide range of sex hormone related disorders, including prostatic cancer, endometriosis and precocious puberty. despite its widespread use, only limited information based on spectroscopic evidence regarding the solution conformation of leuprolide are known. moreover, non crystallographic data is available for the receptor of gnrh (g protein-coupled receptor). the aim of this study was to characterize the conformation of leuprolide and its modifi ed linear analogue (pglu-his-trp-ser-tyr(ome)-(d)leu-leu-arg-aze-nhet) in dmso solution (which simulates better the receptor environment) using nuclear magnetic resonance (nmr) and molecular modeling techniques. by using both nmr and molecular modeling we have characterized the secondary structural preferences of these gnrh analogues. structural determinants of binding to the mu-opioid receptor -an important target in analgesia -attracts great scientifi c attention. many natural and synthetic peptides and peptidomimetics were shown previously to bind to the mu-opioid receptor selectively but there is no consensus about what structure is responsible for such biological activity. no high resolution structure of this receptor is available and the binding site of ligands is not exactly known despite numerous site-directed mutagenesis studies. this suggests that the determination of structural aspects of mu-opioid activity should focus on the ligands. mu-opioid ligands with similar affi nity and selectivity should possess at least one common structural feature in which they differ from other ligands of different affi nity and selectivity. comparative structural analysis of such ligands, considering adequate representation of binding conditions may reveal key features of bioactivity. in this study ten mu-opioid receptor ligands, damgo, tyr-w-mif- , morphiceptin, endomorphin- and and their analogues, possessing different affi nity and selectivity were examined using molecular dynamics. conformational preference of these molecules was determined in aqueous and dmso media which were meant to model different possible binding environments. no structural trend, correlating with previously measured bioactivities was observed in aqueous media. in dmso it was found, that a preference for trans orientation of the tyr side chain and gauche (-) orientation of the third aromatic side chain, the free rotation of the phe side chain and a high propensity of bent backbone structure is favorable for high affi nity binding to the mu-opioid receptor, while deviations from these criteria results in variable loss of bioactivity. constellation of these four key conformational parameters may be a guiding principle in the future for the design novel mu-opioid receptor ligands. in cui-catalyzed azide-alkyne , -dipolar huisgen's cycloaddition -prototypic "click reaction"-is a recently developed synthetic procedure to obtain cyclopeptides carrying, as a rigid linking unit, the lactam bioisoster, , -disubstituted [ , , ] triazolyl ring ( ). we have recently reported the synthesis and conformational analysis of the , -disubstituted- [ , , ] triazolyl containing cyclopeptide derived from the sequence of the potent i-to-i+ side chain-to-side chain lactam-containing antagonist of parathyroid hormone-related peptide (pthrp) .. the conformational properties of triazolyl-containg peptide were compared to those of the corresponding lactam analog. cd and nmr studies revealed that, despite a slight difference of the backbone arrangement, triazolyl containing cyclopeptide and lactam-containing cyclopeptide share a common orientation of the side chains ( ). here we present the structural study of a new library of disubstituted- [ , , ] triazolyl containing cyclopeptides designed to obtain different cycle dimensions and different triazolyl-ring positioning. cd and nmr analysis in different solvent systems shows that both, the dimension of the cycle and the specifi c positioning of [ , , ] triazolyl ring are critical to fully resemble the conformational properties of the potent lactamcontaining antagonist of parathyroid hormone-related peptide (pthrp). the somatostatin (srif) is a cyclic tetradecapeptide, which exerts inhibitory effects on the secretory processes in the endocrine and exocrine systems, and the cell proliferation through somatostatin receptors (sstr - ). sstrs are distributed throughout human body, not only in normal cells but also in tumor cells. most of the somatostatin analogues developed for clinical use, such as octreotide which act longer than somatostatin are being used in the diagnosis and treatment of endocrine tumors. the use of these analogues as antitumor agents has been limited because of their antisecretory effects and poor oral bioavailability. tt- [d-phe-c(cys-tyr-d-trp-lys-cys)-thr-nh ], was reported by keri et al. to have potent antiproliferative activity without antisecretory action .. based on the above, we aimed to design and synthesize somatostatin analogues with more potent antiproliferative activity and high oral bioavailability. we synthesized pyrazinone ring containing cyclic peptides ( ) and linear peptides which are substituted at the c-terminus lys with hydrophobic and rigid groups. our focus was on the active sequence: tyr-d-trp-lys. we also examined their antiproliferative activity and found that boc/h-tyr-d-trp- -adamantylamide exhibited the most potent antiproliferative activity higher than that of tt- . furthermore, on the best analogues we studied dna fragmentation by facs analysis and cellular morphology. the results demonstrated that these somatostatin analogues induced cell death by apoptosis. kobe gakuin universit, japan background and aims: endomorphin- (em- : h-tyr-pro-phe-phe-nh ) has high affi nity and selectivity for the mu opiod receptor ( ). we focus on the pro residue, which imposes strong restraints on the conformation of the peptide chain or induces cis-trans isomerization of x-pro bonds. we substituted the pro with -azetidinecarboxylic acid (aze) or piperidinecarboxylic acids (pip) to increase or decrease the conformational fl exibility. in this paper, we deal with the synthesis of em- analogues containing aze and pip and the evaluation of the biological functions of the analogues on the opioid receptors. methods: the synthesis of peptides was achieved according to the procedure of okada y. et al. ( ) the fi nal products were identifi ed by maldi-tof mass spectrometry and elemental analyses. the receptor binding activity of peptides was assessed by radio-ligand receptor binding assay using mu and delta opioid receptors from cos- cell membranes expressing each opioid receptors. for the evaluation of the biological function of peptides, the gpi and the mvd tests were performed. the new analogue of csf (glc) ., [pro ,asn (glc),thr ]csf is characterized by a type i -turn around the minimal epitope asn(glc), and it was demonstrated to show the highest antibody affi nity in competitive elisa in multiple sclerosis (ms) patients' sera and thus it appears as a promising tool for the detection in patients' sera of specifi c autoantibodies. in previous studies, we synthesized and tested different fragments of this new glucosylated peptide, [pro ,asn (glc), thr ]csf , identifying the shortest sequence [pro ,asn (glc),thr ] csf ( - ) able to detect antibodies by elisa in ms patients' sera. this heptapeptide is characterized by thr in position generating a characteristic n-glycosylation consensus sequence. according to the results obtained with the linear heptapeptide in ms, we applied the backbone cyclicization method to develop two backbone cyclic libraries of glycopeptides based on the sequence of the linear hepta active peptide. backbone cyclization is a method that allows obtaining cyclic peptides without changing the natural sequence or the chemical character of the amino acid residues, in order to enhance activity, stability to metabolic degradation, selectivity and bioavailability ( ) . the fi rst library contains a gly building unit at the c-terminus and is connected to the n-terminus by linker of various lengths (n= , , , ) . in the second library, his of the heptapeptide is replaced by gly building units with various alkyl chains (n= , , , ) [fmoc-n (n alloc(n-alkyl))gly-oh] that is connected to the n-terminus by linker of various lengths. twenty cycloglycopeptides were synthesized, and screened by competitive elisa s on ms patients' sera to select the most bioactive cyclic glycopeptide. peptide nucleic acids have become, arguably, one of the most interesting of dna mimics. owing to their high chemical stability and resistance towards nucleases and proteases, they are very attractive as antigene/ antisense agents, molecular biological tools and for genetic diagnosis. the lack of charge and polar groups in the backbone decrease their solubility in aqueous environment and their ability to cross cell membranes, reducing their performance in in vivo applications. in order to overcome these problems -to improve solubility, increase affi nity and specifi city of binding, a number of analogues were synthesized. this study describes the synthesis of pna-monomers on the base of non-protein amino acids analogues of basic amino acids lys and arg. nucleobases are the second residue we have selected. studies will include replacement for example of the uracil, with the fl uorinated analogue. growing evidences indicate that n-glycosylation is a co-translational modifi cation that, either native or aberrant, may play a fundamental role in a large number of biological events. in particular among postand co-traslational modifi cations glycosylation plays a crucial role in the immune system. in fact, almost most of all the key molecules involved in the immune response are glycoproteins. there are growing evidences of defects in glycosylation with diseases that assets to pathway oligosaccharides as code words. in previous studies, we demonstrated that the presence of a -dglucopyranosyl moiety on an asn residue at position of csf (glc) is fundamental for auto-ab recognition resulting the fi rst multiple antigenic synthetic probe (msap) able to detect autoantibodies in ms patients' sera. up to now we have investigated the carbohydrates infl uence and specifi city in ms antibody recognition introducing several glycosilated building blocks in the msap sequence (i.e. glc, man, glc glc, gal, glcnac on the side chain of ser, thr, asp, glu, hypro) .. due to microheterogeneity and the extremely high specifi city of carbohydrateprotein interaction we included in our library screening, the ribose. we report the synthesis of new asn-derivatives bearing on the side chain ribose rib and rib linked by an n-glycosidic bond and protected for spps.these building blocks introduced in the csf -turn scaffold lead to the new ribosylated peptides contributing to the library of glycopeptides to fi shing out families of autoantibodies specifi c for different autoimmune diseases. fraczyk, justyna; kujawska, nina; kaminski, zbigniew, j. we designed and prepared supramolecular structures formed from nlipidated oligopeptides immobilized in the regular pattern on the cellulose surface which are able to specifi c binding of ligand molecule. due to the conformational fl exibility of the fragments forming the supramolecular structure, the shape and prosperities the binding cavities are adjusted the most effectively to requirements of the guest molecules. the previous studies documented that process of binding guest molecules is highly selective, reversible and competitive. therefore, we supposed that under favorable circumstances the structures could operate as catalysts if suitable molecular fragment are included inside the binding pocket. in order to verify this hypothesis we prepared library of supramolecular hosts with catalytic triade: his asp(glu) ser, incorporated into the binding pocket .. for the fi rst generation library the rate of hydrolysis of p-nitophenyl esters of n-protected amino acids was measured by spectrophotometric determination of liberated p-nitrophenole in buffered, aqueous methanol and compared with appropriate data obtained in the absence of catalytic structures. the most active catalyst were selected from the library and their stability, selectivity and ability for re-use was studied. for the second generation of library the stereoselectivity of artifi cial esterase we present here a new technique for identifying very small quantity of peptide mixtures that are selected out from a peptide library in solution, by using multi-component fl uorescence labeling. the technique is basically a modifi cation of positional screening method associated with a : correspondence between the amino acid at the i-th position and the type of the fl uorescence lebel at the n-terminal. -dimensional fl uorescence spectroscopy was employed for identifying the fl uorescence labels in the mixture of peptides that bound to target cells or target proteins. the results of the new screening method will be presented for peptides that specifi cally bind to human cancer cells. show increased biological activity in a dimeric form . in recent years there have been signifi cant efforts to obtain minimized versions of naturally occurring proteins such as dimeric dna binding proteins which retain their function. the gcn basic region peptides were connected trough a disulfi de bond to give a dimer which specifi cally bound the ap -dna sequence. dimeric peptides and proteins were obtained also by non covalent interactions. in this work we propose a strategy for obtaining by expressed protein ligation (epl), one pot protein homodimers covalently connected at the c-terminus. the synthetic strategy was extended also to the synthesis of heterodimers. epl is a protein engineering tool for the chemo and region-selective modifi cation of proteins based on the use of intein containing constructs. in this work dimers were obtained by reacting a new bi-functional linker with carboxyl-activated polypeptides. we synthesized a linker containing two cysteines in a n-terminal-like position, separated by an ethylendiamine spacer, and obtained thioester proteins by intein mediated splicing reactions. this strategy affords chemically stable dimeric proteins. the linker can be easily modifi ed at need, changing the lenght and rigidity of the spacer between the cysteines. this strategy has potential in biochemical and bioorganic applications, for obtaining minimized and/ or modifi ed natural proteins and for joining two different proteins at the c-terminus position. this technique will be extended to the synthesis of dimeric proteins mimicking the transcription factor ap- . previous studies showed that csf (glc), , a designed glycopeptide characterized by a -d-glucopyranosyl moiety, can detect and isolate specifi c autoabs in sera of a signifi cant number of ms patients. this synthetic ag could be considered a mimetic of aberrantly glycosylated myelin proteins triggering autoimmunity in ms. myelin oligodendrocyte glycoprotein (mog) is considered a putative autoag in ms. our aim is to obtain mog properly glycosylated to characterize the molecular mechanisms of ab-mediated ms and to design new antigenic probes to detect autoabs as biomarkers. production of specifi c glycoproteins may benefi t from a chemical approach, such as expressed protein ligation (epl) a protein engineering strategy useful to introduce noncanonical amino acids and biological probes into proteins. epl allows synthetic and recombinant polypeptides to be chemoselectively and regioselectively joined together. the recombinant rmog ed ( - ), obtained as c-terminal thioesther by protein splicing, will be ligated to the peptide fragment [gly ,a sn (glc)]mog ed ( - ) bearing a cys residue at the n-terminus. an alternative strategy exploits the selective reaction between a glucosyl iodoacetamide derivative and the cys free thiol of a protein. a site directed mutation has been performed on rmog to introduce a cys residue at its native site of glycosylation. the semi-synthetic proteins will be tested by elisa using ms patients' sera. automation is an identifi ed goal in the peptides r&d at lonza. this approach would facilitate and accelerate peptide production. this is highly desired within peptides r&d, where the need exists for rapid synthesis of peptides to fulfi l iso and gmp projects requirements. automated peptide synthesisers are available on the market but many have limitations which make them inappropriate for lonza (e.g., low scale, coupling systems limitation, pre-activation procedure at low temperature). furthermore, there is no obvious standard instrument for scalable spps equipped with pat. ge healthcare provides scalable and complete solutions for automated solid-phase synthesis of oligonucleotides from small research amounts to full commercial production ( mol- mol) based on the Äkta, oligopilot and oligoprocess™ platforms. the unicorn™ software provides control and monitoring of the processes. ge healthcare synthesisers are designed around fl ow-through column reactors, giving faster kinetics and lower solvent and reagent peptide biotechnology and diagnostics consumption compared to batch synthesisers. based on experience with scale-up of oligonucleotides, ge healthcare and lonza are confi dent that the fl ow through-column technology can be scaled up for peptides to a cost effi cient process. this is a strong argument that the potential of the Äkta platform as a peptide synthesiser should be explored. ge healthcare was approached by lonza to develop a peptide synthesiser based on the Äkta platform instrument to compete with the state of the art in the peptide fi eld. the aim is to develop the fl uidics, programming and chemistry methods of the Äkta system to lonza's needs for routine production of . g to g of crude peptide, and later scale up to kg. this synthesiser, controlled by the unicorntm software, has the capability to perform synthesis using on-line mixing or pre-activation at different times and temperatures of amino acids, coupling reagents, solvents and additives. results from peptides will be presented. neuropeptides are produced from precursor proteins by selective cleavage at specifi c sites. classical biosynthetic cleavage occurs at basic residues due to the activity of a small number of well-known proteases. however, with the discovery and characterization of new neuropeptides, a new non-classical pathway has been described with cleavage occurring at tryptophane, leucine and other residues. neuropeptide-processing peptidases involved in this new non-classical pathway are completely unknown but essential for correct processing of certain neuropeptides. therefore, we are interested in identifying proteases involved in the non-classical pathway using activity-based proteomics. for this purpose, we fi rst designed and synthesized some peptides based on the sequence the mature neuropeptides described in the literature but with an active functional group able to bind the active site of the proteases of interest. bound peptides were labelled with biotin or rhodamine by click chemistry, and the brain and pituitary proteome was then characterized by in-gel analysis and multidimensional nlc-ms/ms. several proteases that may be involved in neuropeptide processing have been identifi ed in this experiments. further studies concerning cloning, protein expression and activity assays will confi rm their role in biological tissues. the incidence of autoimmune diseases continues to increase. although an enormous research effort has been directed in understanding this increment, etiology of human autoimmunity remains enigmatic. primary biliary cirrhosis (pbc) is a chronic cholestatic liver disease characterized by destruction of bile ducts and the presence in the serum of antimitochondrial antibodies (ama positive in - % of patients), directed against the e , which is one of the three component enzymes of the -oxo acid dehydrogenase multienzyme complex family chiefl y pyruvate dehydrogenase complex (pdc-e ). environmentally induced co-or post-translational modifi cations of autoantigens are hypothesized to break immune tolerance leading to self reactivity in pbc .. in this context it is possible to take advantage of a unique technology that allows the monitoring of peptide epitope modifi cations that will lead to the identifi cation of altered autoantigens that the human host will recognize as foreign, similarly to what recently reported in multiple sclerosis ( ) . our approach will lead to new diagnostic tools that can be used in earlier stage patients and possibly to monitor disease activity. herein we report the synthesis of lipoamide and glyco-peptides characterized by a -hairpin structure with the modifi cation on the tip of the -turn as peptidomimetic of native antigens involved in pbc. in order to identify the best synthetic antigens and to clarify the role of the -turn in exposing the modifi cation we compared the antibody recognition in pbc sera by elisa using the modifi ed peptides in comparison with the proposed autoepitope of pdc-e [pdh ( - ) ]. the synthetic antigens were able to detect by elisa autoantibodies in % of ama negative sera of pbc patients confi rming that ptm-peptides are useful diagnostic/prognostic tools. alzheimer fs disease is characterized by the abnormal accumulation of amyloid peptide (a ) into extracellular fi brillar deposits known as amyloid plaques. a can self-assemble into soluble oligomers, protofi brils, and amyloid fi brils, and all of these aggregated forms contain signifi cant -sheet structure. the core of a ( - residues) containing hydrophobic region is a key to promoting a aggregation. in the previous study, we constructed green fl uorescent protein (gfp) variants which have the core part of a sequence on the surface -sheets. it has been demonstrated that the gfp variants inhibit a aggregation .. in this study, we have utilized a small protein, insulin-like growth factor ‡u receptor domain (igf r-d ) as a scaffold, and a part of a sequence was incorporated into the -sheet surface of igf r-d . igf r-kk and igf r-ka were designed by substituting two a derived sequences for some amino acids in igf r-d as parallel and anti-parallel -sheet models, respectively, of the a aggregates. these insoluble proteins expressed by e coli. were solubilized by denaturing buffer, and the denatured proteins were refolded in conditions permitting formation of native proteins. after refolding, the proteins were purifi ed as monomers by size exclusion chromatography. we have investigated the interaction between a and the designed protein variants by surface plasmon resonance studies. as a result, igf r-kk bound tightly to a more than igf r-ka. inhibitory activities of a fi brillization in the presence of igf r-kk, igf r-ka, or wild type igf r-d were evaluated by thiofl avin t (tht) binding assay. it was demonstrated that igf r-kk inhibited a fi brillization effectively. because of their high structural fl exibility. on the other hand, a stranded -structure is presumed to be more stable than a single strand as a fi bril forming intermediate. therefore, the aggregation mechanism of prion protein remains to be further studied on their precursor structures. recently, we have established a novel strategy to determine the amino acid sequence for amyloid formation, based on their essential interactions. a number of fi bril forming peptides at positions from to in the amino acid sequence were obtained by our calculation method. in order to confi rm whether aggregates of the candidate peptides consist of the -stranded -structure, a series of peptides consisting of g residues-turn- residues h were prepared. several candidate peptides, of which regions are - , - , - , and - , showed the typical enhanced fl uorescence intensities in the thiofl avin t-binding assay, suggesting the amyloid formation. ir spectra of these peptides showed the typical bands corresponding to the -structure. in addition, the peptide ( - ) exhibited a shoulder band at cm - in ir spectrum, refl ecting a turn structure. these results revealed that the amyloids of the peptide ( - ) are constructed with the -stranded -structure, which may be formed by intra-molecular hydrogen bonds. in conclusion, the results obtained here indicate that the sequence from to could be a key region for the transition from a normal to an abnormal structural state. single-molecule force spectroscopy provides a powerful tool to investigate biomolecular interactions. a different approach measures forces required for breaking a bond in a differential format by comparison with a known reference bond of dsdna ( ). here we apply this molecular force balance to the integrin v , which is over expressed in ovmz- cells. this protein interacts with ligands containing an -arg-gly-asp-(rgd) motive. ligands containing the rgd sequence were made by peptide synthesis and linked to a gcn -peptide using a peg-spacer. for detection carboxyfl uoresceine was introduced to a lysine side chain between both peptides. the gcn -peptide can be recognized by specifi c anti-body fragments ( ) and can act as a reference bond. these force balances was linked to a pdms-surface, whereas the reference bond is attached to the surface and the rgd-peptide is accessible to the integrin. when the pdms-stamp gets in contact with the ovmz-cells, the interaction of ligand and receptor can occur. by applying a force at the stamp the force balance is stretched and the weaker bond brakes. investigation of the fl uorescence-level on stamp and cells enables the localization of the balance construct and thus an estimation of the bond force. first stamp-experiments on living cells showed that the gcn anti body interactions are too strong to act as a reference system. since the binding forces of dna are well investigated by afm and small dna-molecules were already used as force sensor, dna can act as a more sensitive reference. thus in a further approach the rgd-peptide was linked to a biotinylated peg. this enables the application of short double-stranded dna as a reference bond via a fl uorescence-labelled streptavidin. using this system the force balance could be transferred to the v integrines located in the cell membrane of ovmz-cells. nanogaps, which allow making electrical contact to structures on the nanoscale, are increasingly used for the preparation of biosensors. the positioning of the synthetic or biological species inside the nanogap must be controlled to reach optimal electrical or detection properties. ( ) ( ) ( ) in this context, the chemical properties of the layer between the electrodes is of prime importance, since they will impose the imbibition of the nanogap and permit the formation of chemical bonds between the molecules of interest and the substrate. thin fi lms can be characterized by a variety of physical or chemical methods. however, the characterization of the chemical properties of nanogaps is complicated because of the small size of the substrate delimited by the electrodes. in this context, novel experimental tools are needed for probing rapidly the chemical reactivity of nanogaps. we show here for the fi rst time that a specifi c functional group in a - nm nanogap can be detected by combining peptidecapped gold nanoparticles and electrical detection.( ) a semicarbazide layer and semicarbazone chemoselective ligation was used in this proofof-concept study, which thus required the preparation of stable peptidecapped gold nanoparticles modifi ed by aldehyde groups and control gnps derivatized by amide groups. the chemoselective insertion of gold colloids into the nanogaps led to current increases from to orders of magnitude, in accord with the number of gold nanoparticles in the nanogaps detected by scanning electron microscopy. we present the electrical detection of immunoglobulin g (iggs) from human serum using a nanogap-based biosensor. the detection method is based on the capture of iggs by a probe immobilized between gold nanoelectrodes of to nm spacing. the captured iggs are further reacted with secondary antibodies labelled with gold nanoparticles (gnps). insertion of gnps into the nanogap resulted in increasing the conductance through the nanogap. the use of a chip with ninety nanogaps enabled the calculation of a quality factor for the detection which, coupled with a non-linear regression analysis of the data, easily discriminated specifi c and differential capture of human antibodies by arrayed probes. we obtained a -fold higher quality factor with protein a compared to goat anti-murine antibodies. this method can be applied, through these proof-of-concept experiments, to the detection of protein-protein interactions in biological samples. in the past several decades, hundreds of peptides have been identifi ed which have specifi c biological activity and are highly potent in in vitro assays but lack the prerequisite pharmacokinetics to become effi cacious human therapeutics. we have developed a novel antibody-based platform technology that provides an improved pharmacokinetic profi le for biologically active peptides, resulting in a long duration of action. one such mimetibody™, cnto , is a novel erythropoietin (epo) receptor agonist. although cnto bears no sequence homology to erythropoietin, it is a potent erythropoietin receptor agonist, rescuing epo dependent cells from apoptosis in vitro and stimulating erythropoiesis in vivo. studies were done in normal rats to explore the pharmacodynamics and pharmacokinetics of cnto in normal rats and to demonstrate its effi cacy in rat models of anemia. in vitro, cnto was approximately fold less potent than rhepo in stimulating the growth of ut- epo cells. despite this lower in vitro potency, when compared to rhepo and darbepoietin in normal rats, a single subcutaneous dose of cnto resulted in a longer-lived reticulocytosis and longer-lived increase in hemoglobin. also, cnto caused only minor changes in red cell distribution width (rdw) or mean cell volume (mcv) and led to the release of mature reticulocytes. we have also shown that cnto was effi cacious in rat models of anemia and in a rat model of pure red cell aplasia. taken together, our data show that cnto is a novel stimulant of erythropoiesis in rats. this platform has been applied to other biologically active peptides as well and has proven to be a robust platform for enhancing the pharmacokinetics of peptides that would otherwise be rapidly cleared. cell penetrating peptides (cpps) have been recognized as promising tools for the delivery of different therapeutic molecules. previous studies in our laboratory have shown that the s ( )-pv peptide accumulates inside cells very effi ciently through a rapid, dose-dependent and nontoxic process. formulations based on the s ( )-pv cell penetrating peptide presented great potential for the delivery of plasmid dna, which may prove useful for gene-based therapies. in the present work, we aim to ) investigate the relevance of the dermaseptin-derived sequence and of the nuclear localization signal to the effi ciency of the overall process of plasmid dna delivery by the s ( )-pv and related peptides; ) compare the potential of the s ( )-pv peptide to mediate plasmid dna delivery with that of the extensively studied tat cpp. a comparative analysis of the transfection effi ciency mediated by the systems based on the s ( )-pv, reverse nls and scrambled peptides was performed in tsa and hela cells. in general, for both cell lines, the reverse nls peptide mediated transfection at effi ciencies comparable to those observed for the s ( )-pv peptide. however, transfection mediated by the scrambled peptide was signifi cantly less effi cient than that obtained for the s ( )-pv and reverse nls peptides. to compare the biological activity of the s ( )-pv with that of the tat peptide, we transfected tsa cells with various s ( )-pv-and tatbased formulations. our results have shown that both peptides enhanced the activity of cationic liposome-based systems. above a threshold peptide/cationic liposome/pdna charge ratio ( / / ), the enhancing effect was independent of the peptide used, although for the lowest charge ratios, this effect seemed to be more relevant in the case of the s ( )-pv peptide. higashi, nobuyuki; kawamura, yoko; koga, tomoyuki doshisha university, japan the aim of tissue engineering is to replace failed organs with new functional tissue and organs. to realize this, materials are needed, which can direct the growth of cells to generate new tissue. to control and direct cell behavior, a defi ned biomimetic environments is needed, which surrounds the cells and promotes specifi c cell interactions. the rgds sequence has been recognized as the cell attachment site of the natural extracellular matrix. the purpose of this study is to fabricate rgdscarrying nanoscaffords towards cell adhesion using a self-assembling technique. here we synthsize two types of rgds-based materials, one of which is an amphiphilic triblock peptide composed of leu and lys (rgds-l k l ) that has been revealed to self-assemble into ƒÀ-sheet nanofi ber under specifi c conditions ., and another one is a rgdsended polystyrene (pst-rgds) that is a typical artifi cial polymer. these peptide and peptidomimetic were prepared by solid phase synthesis using fmoc-chemistry and by coupling fmoc-chemistry and atom-transfer radical polymerization (atrp) method, respectively. rgds-l k l was found to self-assemble into ƒÀ-sheet-based nanofi ber at ph . , and by lowering ph the conformational change from ƒÀ-sheet to random coil structure was induced, giving no aggregation of the peptide. when mouse nih/ t cells were seeded on the rgds-l k l nanofi bercoated plate, successful cell adhesion and spreading were observed, but not for the rgds-l k l random coil-coated plate, indicating the importance of the rgds sequences densely and regularly located at the nanofi ber surface. the utility of pst-rgds nanofi lms will be discussed, in comparison with that of nanofi bers. the role of deiminated protein antigens in the diagnosis of rheumatoid arthritis autoantibodies directed against citrulline-containing peptide (acpa) have high specifi city of rheumatoid arthritis (ra). citrullinated proteins are formed by posttranslational modifi cation, namely by deimination of arginine residues in protein sequences by peptidylarginine deiminase enzymes (padi). autoantibodies to deiminated (citrullinated) proteins are the most specifi c serological markers of rheumatoid arthritis. rheumatoid arthritis symptoms develop gradually, and it is diffi cult to precisely date the beginning of the disease. these antibodies are detectable already years before the fi rst clinical symptoms of the disease. the aim of our study was to identify the epitopes of vimentin and fi laggrin derived-peptides targeted by ra specifi c antibodies to provide further information about the nature of the initial autoantigenic substance. we used conventional solid-phase peptide synthesis (fmoc strategy) carried out on "multipin ncp" (chiron mimotopes peptide system) non-cleavable kit. identifi cation of epitope structure of antigenic proteins represents one of the major applications of these technologies. the peptides were prepared in duplicates. citrullinated peptides and the nonmodifi ed counterparts containing arginine instead of citrulline residues were synthesized in order to compare their respective reactivities. in the "indirect" elisa experiments the presence of acpa was determined using serum samples of ra patients and healthy blood donors. this series of experiments effi ciently identifi es citrullinated epitopes of fi laggrin and vimentin as a potential antigenic target for ra specifi c antibodies. the determination of epitopes of these proteins could be important for the development of appropriate diagnostics in this most frequent human systemic autoimmune disease. at the present time the human lysosomal enzymes cathepsins b and l, cysteine proteinases of c family, attract great attention. they not only take part in protein degradation but also appear to play role in other important physiological processes, for example, antigen presentation, caspase-independent cell death and involved in different diseases such as osteosarcoma, acute pancreatitis, tumor invasion and metastasis. but selective detection of these enzymes is diffi cult because determination of their enzymatic activity is carried out using substrates also correspond to specifi city of trypsin-like enzymes. the chemo-enzymatic synthesis of selective substrates of cysteine proteinases of c family was developed. these compounds are chromogenic glp-phe-ala-pna (i), glp-val-ala-pna (ii) and fl uorogenic substrates abz-phe-ala-pna (iii), glp-phe-ala-amc (iv). peptides were obtained in preparative quantities and were characterized by the data of amino acids analysis, hplc, mass-spectrometry, spectrophotometry (i and ii) and fl uorimetry (iii and iv). the specifi c activities of cathepsins b, l and similar enzymes of plants papain, bromelain and fi cain were determined using synthesized substrates and commercial available substrates z-phe-arg-pna, z-arg-arg-pna and bzl-arg-pna. the specifi c activities of enzymes of other classes -serine (chymotrypsin, trypsin and subtilisin), aspartic (pepsin) and metalloproteinases (thermolysin) -were also defi ned with these substrates. it was shown, that glp-phe-ala-pna, glp-val-ala-pna and glp-phe-ala-amc are not detectable cleaved by enzymes of other classes. acknowledgements: this work has been supported by rfbr grant ¹ - - à. to implement a new electrochemical biosensor for autoantibody detection in ms we used csf (glc) analogues, properly modifi ed at n-terminus with ferrocenyl and ferrocenyl-thiophosphine derivatives, as "electrochemical probes" in cyclic voltammetry ( ) . the electrochemical properties of ferrocene, coupled to thiophosphine ability to build simple monolayers on gold surfaces, allow peptides to be anchored on the working electrode used for detection. in particular, -fcphp(s)abu organometallic amino acid was specifi cally designed to be used directly in solid phase peptide synthesis. the organometallic moiety introduced in the new glycopeptides did not affect autoantibody recognition as demonstrated both in sp-elisa and in inhibition experiments. an electrochemical monitoring was able to detect interactions of the modifi ed glycopeptides with isolated antibodies from ms patients' sera. we demonstrated a detection sensitivity comparable to elisa method. therefore, the new electrochemical probes can be proposed to characterize autoantibodies as biomarkers of multiple sclerosis by a simple, rapid, and reproducible cyclic voltammetry-based diagnostic methodology. troponin is a structural protein complex, located on the thin fi lament of the contractile apparatus. it is composed of three protein subunits: troponin i ( kda), troponin c ( kda), and troponin t ( kda) and exists as isoforms specifi c to the cardiac and skeletal muscle cells, respectively. cardiac troponins are released in the peripheral blood during irreversible cardiac muscle damage in a time-specifi c manner. rapid troponin elisa assays based on the production of specifi c antibodies against the whole complex or individual subunits have been shown to possess suffi cient sensitivity and specifi city for use in the emergency departments. however, their usefulness sometimes is limited by various factors, such as the selection of epitopes for antibodies production, derived from cardiac troponins representing high homology against the skeletal isoforms, interfering blood factors etc. aiming to contribute in the fi eld of developing highly sensitive and specifi c reagents for the detection and isolation of cardiac troponins in the sera of patients with cardiovascular diseases, we selected epitopes derived from the cardiac isoforms for production of antibodies, mainly based on their predicted immunogenicity, the minimum homology against the skeletal isoforms and the lack of interferences of the produced antibodies with various blood factors. the selected sequences were conjugated to the tetrameric sequential oligopeptide carrier (soc ), either by the classic solid phase step-by-step methodology or by chemoselective ligation reactions and the resulted conjugates were used as immunogens for releasing anti-troponin specifi c antibodies. the performed elisa experiments revealed the high affi nity and specifi city of the produced anti-troponin antibodies against the native protein. a chemical reverse approach for autoimmune diseases diagnosis alcaro an increasing number of individuals throughout the world is affected by autoimmune diseases, a large and diverse group of disorders that are categorized by tissue injury or pathology. although the incidence and prevalence of individual autoimmune diseases are not high, the population burdens of the disease are large and underestimated. thus, reliable diagnostic/prognostic tools are necessary for an early diagnosis and for monitoring disease activity. in this scenario, we proposed a 'chemical reverse approach' based on the use of patients' sera to screen synthetic modifi ed peptides. we showed that this approach could lead to the effective identifi cation of specifi c probes able to characterize highly specifi c autoantibodies as disease biomarkers of highly relevant autoimmune diseases such as multiple sclerosis and rheumatoid arthritis [ , ] . toscana biomarkers is a r&d company involved in the application of the "chemical reverse approach" to different autoimmune conditions for the development of innovative diagnostic/prognostic tests. as a number of autoimmune diseases have been associated with posttranslational modifi cations, which alter the function and immunogenicity of protein/peptide antigens, we are synthesizing and screening focused peptide libraries based on post-translational modifi ed amino acid, conformational and minimal epitope diversity. lead compounds can be thus used as antigenic probes for specifi c recognition of autoantibodies as biomarkers of diseases in the set up of diagnostic/prognostic assays ( ). autoimmune diseases are considered now as a plague. in fact some autoimmune diseases previously considered rare are actually increasing their frequency because of an earlier diagnosis (e.g. celiac disease). possibly also an environmental factor (bacterial and/or viral infection) should contribute to autoimmune diseases. the idea we have been investigating for years and more recently proposed by others, is that aberrant post-translational modifi cations, i.e. glycosylation, deimination etc., create neoantigens triggering autoantibodies. this could explain why proteins (both recombinant and isolated), components of target organs or tissues, are failing. anyway, it is evident that one single biomarker will never enable to reach successful diagnostic & prognostic tools. on the contrary, synthetic peptides specifi cally modifi ed (with sugars, citrulline, lipoyl moieties, etc.) are interesting tools to fi shing out of patients' sera these autoantibodies. we have recently reported that this can be effi ciently done following a "chemical reverse approach" .. we successfully applied this strategy in the development of the fi rst multiple sclerosis antigenic probe [msap] : an n-glucosylated peptide characterised by a -hairpin structure exposing at the best the minimal epitope asn( -glc) involved in antibody recognition ( ) . a wider application of our sap is obtained by its citrullinatation and/or galactosylation ( ) useful for rheumatoid arthritis or lipoylation for investigating primary biliary cirrhosis. in addition to being able to show the viral infection from a serum sample within a few minutes, at the point of care, the poc assays are inexpensive, easy-to-perform and do not require special equipment or laboratory. as antigen in the poc assay we used a -amino acid peptide in four branches of a lysine core, with the kyvtgin sequence in the middle. the peptide antigen was conjugated to gold particles and absorbed to a fabric ribbon and dried. in the test, a serum sample is added together with buffer, and the solution dissolves the conjugate. a positive result is obtained when the antibodies together with the gold conjugate bind to anti-human-igg on the nitrocellulose, and form a specifi c coloured line which can be detected in the test window. serum samples were collected from patients with acute b infection, and control samples were drawn many years after infection; additional control sera came from subjects devoid of b antibodies. the assay was shown to be stable in accelerated stability study. the conditions for industrial scale manufacturing were evaluated and the sensitivity and specifi city were addressed, whereby the assay proved to be highly specifi c for acute b infection. alzheimer's disease (ad) is a chronic neurodegenerative disease characterized by a progressive loss of memory and cognitive decline, for which the aggregation and plaque-formation by the -amyoild (a ) polypeptide has been identifi ed as a key event. recently, unpaired variable domains of llama single chain antibody (vhh) fragments against a have been found to exert considerable therapeutic potential for ad. vhh represents the smallest antigen-binding unit with a molecular size of ~ kda, compared to ig-heavy and light chain variable domains, fab fragments and complete igg antibodies. human cystatin c (hcc) is a cystein protease inhibitor present in all human body fl uids which has a propensity to co-associate with a -plaques/fi brils, and has plaque-inhibitory properties. using proteolytic extraction and excision of the llama-vhh-a ( - ) immune complex (e.g., trypsin, glu-c protease) in combination with electrospray ionization (esi)-and maldi-mass spectrometry, the a -binding epitope was identifi ed at the middle-carboxyterminal domain of a , a ( - ). an analogous mass spectrometric approach was employed for the identifi cation of the binding epitopes of the hcc-a -complex, using immobilized hcc and a -affi nity matrices. an almost identical minimal epitope to that of the vhh-anti-a -antibody ( a ( - ) ) was found, which binds to a specifi c c-terminal domain of hcc, hcc( - ) .. the identifi ed hcc epitope peptide was found to specifi cally inhibit a -oligomerization in vitro, in agreement with the a -epitope domain interefering with the a -aggregation. the identifi ed a and hcc epitopes represent new lead structures for designing neuroprotective inhibitors of the a -aggregation process, and for molecular ad diagnostics. using peptide arrays to reveal mechanisms of apoptosis aspp is a pro-apoptotic protein that stimulates the p -mediated apoptotic response. the c-terminus of aspp contains ankyrin repeats and an sh domain (aspp ank-sh ), which mediate its interactions with apoptosis-related proteins such as p and bcl- . we have used a combination of membrane-bound peptide arrays and biophysical methods to study the protein-protein interactions of aspp at the molecular level in order to reveal their possible role in apoptosis. using peptide arrays, we have mapped the binding interfaces of aspp with its partner proteins such as bcl- , nf-b and other proteins that are involved in apoptosis. we then applied the peptide array results to study profoundly the interactions of aspp with proteins from the anti-apoptotic bcl- family. we found that aspp ank-sh binds to bcl- , bcl-xl and bcl-w at two homologous sites in all three bcl proteins tested: (i) the conserved bh motif (ii) a binding site for pro-apoptotic regulators. quantitative biophysical analysis of the interaction with the free peptides revealed that the binding was selective, and the bh domain of bcl- binds tightest to aspp . we propose a mechanism in which aspp induces apoptosis by inhibiting functional sites of the anti apoptotic bcl- proteins. the array screening results also served as a basis for docking studies that resulted in binding model for the complex between the full length protein bcl- and aspp ank-sh . we conclude that the use of combinatorial methods such as peptide arrays, combined with quantitative biophysical techniques, can signifi cantly contribute to better understanding of biological pathways. micron-sized monodispersed polystyrene for synthesis, tagging and biological screening of small molecules peptide arrays are useful tools to characterize antibodies, to determine sequence specifi cities of enzymes (e.g. kinases) or to fi nd interaction partners to given peptide sequences. one popular format for such arrays is a cellulose sheet with hundreds of synthetic peptides bound to it. these spot-arrays have been used successfully in a broad range of applications since their invention years ago .. a drawback is the use of large reagent volumes and the limited throughput with only one copy of the library. celluspots™ represent a new method [ , ] that allows the production of hundreds identical peptide arrays from a single synthesis run on individual membrane disks. the peptides are synthesized on a modifi ed cellulose support which is dissolved in a cleavage-mixture after the synthesis. resulting solutions of peptide-cellulose-conjugates are then spotted onto coated microscope slides by conventional spotting techniques. the identical arrays are useful tools for large, parallel sera screening projects. there is a great importance in biological macromolecules integration within electronic devices. this is since these molecules are expected to be suitable both as the active components of electronic devices, or as guides for bottom-up assembly of hybrid structures. the goal of this research is to study the assembly and electronic properties of novel devices that exploit synthetic protein molecules. here we show the fabrication of de-novo protein based diode-like devices. we have chosen coiled coil protein structures, which can adopt two conformational states that differ in their internal molecular dipole. the proteins have been equipped with surface binding groups, typically cysteine thiols, on both ends. dithiol bridges at one side facilitated self assembly on gold surfaces. protected cys residues were placed on the other end that after deprotection will allow successive binding processes in order to complete the device assembly process. characterization of the correct folding in solution has been achieved by hplc and cd. the dependence of the dipole in the dimeric protein has been shown using large scale kelvin probe and high resolution kelvin force microscopy (kfm) measurements. we believe that this work will contribute to the understanding of electronic processes in proteins and may serve as foundation for their exploitation in device confi gurations by making use of the ability to trigger conformational changes in order to control device activity. synthesis of new tetracyclic rgd peptides for specifi c tumor cell targeting. the integrin family of adhesion molecules participate in important cell-cell and cell-extracellular matrix interactions in a diverse range of biological processes. the avb integrin is overexpressed in several types of cancer cells and play an important role in angiogenesis as well as in tumour cell migration by interacting with vitronectin on the extracellular matrix mainly through the recognition of the tripeptide sequence rgd. the search for highly selective ligands to target the endothelial cell integrin avb is currently the focus of many research groups as they may represent new therapeutics or valuable diagnosis in a number of areas such as metastasis, angiogenesis, arthritis and retinopathy. to date, the cyclic (-rgdfx) peptides and related n-methylated analogues developed by kessler's group are among the most active and selective compounds for the avb integrin receptors. for instance, the use of c(-rgdf(nme)v-) is actually evaluated in several clinical trials. therefore, we designed a new class of cyclic tetrapeptides. rgdk peptides were fi rst synthesized by solid phase synthesis then cyclized in solution through a urea bond using n,n'-carbonyldiimidazole. using skmel and a cells, expressing and non-expressing avb respectively, we demonstrate that one of our peptide showed a better internalization than the reference peptide crgdfe. our strategy allowed the coupling of the best cyclic recognition peptide through its extracyclic acidic group to any molecular moieties containing an amino group that makes him a great cadidate for easy multimerization. along this line, different applications are currently developped in our group. design in this study, we described a simple method for aminocylation of unprotected saccharides with mildly activated amino acids esters as potential ace inhibitory activity. as a model reaction, we investigated esterifi cation of sucrose -" royal carbohydrate" with cyanomethyl ester of benzyloxycarbonyl-phenylalanine.the difference of reactivity between the eight hydroxyl groups is a key factor in chemical exploration. in polar solvent as dmf or dmso the primary positions might react faster than secondary ones if the reaction is essentially sensitive to steric interactions. on the other hand, the reaction might be more sensitive to the activity of the alcohol functions. in this respects, g-oh has been shown to be the most reactive among eight hydroxyl groups of sucrose. after removal of benzyloxycarbonyl group, the products possess groups which can accommolated in the hydrophobic s and s subsites of angiotensin i converting enzyme. the free amino group in the amino acid-carbohydrate esters can also serve as good ligands for zn + in the ace active site. carbohydrates possess both hydrophobic and hydrophilic groups in their structure and could also bind with enzyme subsites. the measurements of ic value of newly amino acyl esters of carbohydrates are in progress. synthetic polyelectrolytes (pe) have been widely used to modify proteins via complex formation and covalent attachment, increasing (or reducing) the immunoreactivity and/or immunogenecity of originally antigenic proteins and improving their in vivo stability with prolonged clearance times. such conjugates seem to be great importance for medicine and immunobiotechnology in particular with respect to drug delivery and vaccine innovation. synthetic peptides are promising candidate vaccines for the control of viral diseases. previous studies with foot-and-mouth disease virus (fmdv) have identifi ed fragments of isolated vp protein and synthetic peptides from vpi which stimulate antibody production, albeit of poor neutralizing activity, or are recognized by antivirus antibodies. fmdv is an attractive model with which to study the potential of peptide-based synthetic vaccines. in this study, we sought to evaluate the immunogenecity of a candidate containing - synthetic peptide epitops of vp capsid protein of fmdv. the immunogenic properties of the conjugates were also investigated and the relationship between immunogeneticity and structure formation in the solutions is analyzed. a new high immunogenic protein-polymer complex and conjugates with antibody production, processing relatively prolonged times was obtained. it was obtained that a single immunization of mice with pepeptide bioconjugates without classical adjuvant increased the primary and secondary peptide-specifi c immune response to fmdv. polyacrylic acid (paa) is a well known bioactive polymer (bioadhesive nano-and microparticles, ph-sensitive paa grafted poly(vinylidene fl uoride) membrane bags, ultrafi ne cellulose fi ber surfaces grafted with paa for enzyme immobilization, paa-polyvinylpyrrolidone biopolymeric systems for the treatment of the dry eye, etc. paa, their alkyl-esters and non-toxic copolymers with vinyl pyrrolidones are strong adjuvants for primary and secondary responses and that they are promising alternatives to the mineral oil-based adjuvants presently used in various veterinary vaccines. in this study, the interaction between peptide epitops of vp protein of foot-and mouth disease (fmdv) and polyacrylic acid (paa) will be discussed on the basis of the experimental results obtained by the size-exclusion chromatography (sec) with online quadruple detection system: uv absorption (uv), refractive index (ri), right angle light scattering (ls) and viscosity (vis) detectors and the binding coeffi cient, i.e., the binding ratio of peptide to polyacrylic acid. human igg-specifi c binding peptides to distinguish normal and abnormal conformers: applications for igg purifi cation and detection in recent years, human immunoglobulin g (igg) attracts attention as protein of the main format of the antibody medicine. in the purifi cation of igg, protein a originated from bacteria is frequently used as a affi nity ligand, but several problems such as the contamination of endotoxin and the deterioration of protein a by the repeated use were pointed-out in the use of protein a. on this account, the development of low molecular ligands and mimic peptides which can be used instead of protein a has been performed. we have searched for the peptides which specifi cally bind to human igg using a t random peptide phage library and, as a result, succeeded in isolation of two kinds of peptides called type i and type ii. type i peptide recognize normal structure of human igg and can be used for the purifi cation and the detection of igg. however, it not only binds to human igg but also to mouse and rabbit igg with comparatively high affi nity. on the other hand, type ii peptide is extremely specifi c only for human igg. a more important thing is that type ii peptide does not bind to igg in serum, but get possible to bind to igg antibody purifi ed by a protein a column. from this result, we elucidated the abnormal structure of igg is generated by acid condition used in the elution from a protein a column and type ii peptide recognizes this specifi c structure. in this presentation, we report the results that the type i peptides functioning as an affi nity ligand instead of protein a can be applied for the purifi cation system of the antibody by improvement by amino acid substitutions and chemical conversion. furthermore, we also report the generation condition of an abnormal conformer of igg structure by ph and the temperature and the effective removal of the generated specifi c conformer by the type ii peptideimmobilized column. acknowledgements: this research was partially supported by a grant of practical application research from japan science and technology agency. the isolation and detection of low abundant enzymes and proteins is one of the most challenging tasks in bioanalytical and pharmacological fi elds, especially if information about their state of activity is required. for this purpose tailored solutions for addressing the members of a protein family are required. peptide chemistry provides established methods to assemble building blocks to construct such molecular probes. we chose matrix metalloproteinases (mmp) for validation of such an approach. this protein family processes and degrades various extracellular matrix proteins and possesses a highly conserved catalytic site with a zinc ion in its centre. most of the known and potent inhibitors are peptidomimetics containing zinc chelating hydroxamate groups (e. g. marimastat). although it binds reversibly, it is potent and active against a wide range of metalloproteinases. it was chosen as synthetically available binding group to target the protein in its active state. it was modifi ed by peptide chemistry in order to introduce multiple functions. depending on the purpose, different reporter groups, photoreactive or cleavage sites were chosen. the probes were tested positively to inhibit various human recombinant mmps using activity assays. mmp were isolated using streptavidin coated magnetic beads and a biotinylated probe. furthermore a photoreactive group was introduced to enhance the binding to the target protein. a covalent interaction was achieved by irradiation of a probe protein mixture, isolation with magnetic beads and cleavage from the beads. pyclock superior coupling reagent for biosensors construction based on peptides coupling onto solid phase polymer atias, danit; abu-rabeah, khalil; marks, robert s. electrochemically biosensors are valuable analytical tools for the monitoring of various biological/ chemical molecule levels, and tremendous research effort has been put into the development of such accurate analytical devices. one crucial aspect in the fabrication of a biosensor is the deposition of biological macromolecules in high amounts with retention of their specifi c activity. among the various deposition methods of biomolecules, such as direct adsorption onto the surface, cross-linking, covalent attachment by carbodiimide chemistry is one of the few methods that allows a stable deposition into a biocompatible environment. hence the urgent need for proteins coupling with high yield to solid surface in the aim of biosensors construction. however the use of carbodiimide as peptide coupling agents was found to be limited in various aspects. thus, during the activation of hindered carboxylic components, such as those involved in bulky protein coupling reactions to solid phase fi bers pyclock was found to be very effi cient for slow coupling reactions and it can be used in excess to assure a complete activation of the carboxylic function. in this study -chloro-benzotriazole- -yl-oxy-tris-pyrrolidino-phosphonium hexafl uorophosphate coupling reagent (pyclock) was shown to have better performance than -ethyl-[ -(dimethyl¬amino)propyl]- ethylcarbodiimide (edac) and hydroxysulfosuccinimide (nhss) in peptides large sequence coupling to solid phase fi bers. cantel, sonia; valmalle, charlene; subra, gilles; enjalbal, christine; martinez, jean general approaches used in protein identifi cation and characterization involve consecutive purifi cation steps. then the desired protein extract is submitted to mass spectrometry analysis (lc-ms/ms) after enzymatic digestion. technical diffi culties are involved in determining the pmf of a protein particularly in relative low abundance. a typical protein will give rise to at least twenty to thirty peptides after trypsin digestion. not all of these peptides will appear in maldi analysis. one factor that is believed to cause incomplete detection is competition for protonation during the ionisation process inducing ion discrimination. we have recently developed a new technology allowing specifi c labeling of lysine residues in proteins and easy maldi-ms detection and identifi cation of labeled peptides following protein hydrolysis ( ) . nhydroxysuccinimide ester of -cyano- -hydroxycinnamic acid (chca) was used as a labeling reagent to increase maldi signal of lysinecontaining peptides in cytochrome c proteolytic mixture. this original approach enables to discriminate labeled peptides of interest among other abundant peptides. herein, we report the optimization process to investigate the limits of this tool. a defi ned receptor-binding domain (rbd) on the viral spike protein (s) mediates the attachment of sars-coronavirus to its cellular receptor, angiotensin-converting enzyme (ace ). we have synthesized peptide libraries for identifi cation of rbd binding epitopes by surface plasmon resonance (spr) and saturation transfer difference (std) nmr spectroscopy. three dodecapeptides were identifi ed to have signifi cant affi nity to ace ; the best of them had a kd = m . further refi nement yielded a hexapeptide from y to l (ykyryl) of s that bound ace at kd= m. std nmr spectroscopy reveals close contacts of the aromatic tyrosine residues to the receptor. the peptide was also analyzed for antiviral activity using an in vitro assay that measures sars-cov infection of vero cells. at a concentration of mm virus replication was reduced about fold. no replication occurred at peptide concentrations above mm. the peptide blocks the binding site on ace that is necessary for the virus to infect the cells. it can be used to design peptidomimetic compounds as entry inhibitors for sars-cov. to exclude other effects that were due to unspecifi c inhibition of viral replication, the peptide was tested during an alpha virus infection of vero cells. no signifi cant inhibition was observed. however, for the human corona virus nl that causes severe colds in humans and that uses the same receptor a clear inhibition could be observed comparable to the results obtained for sars-cov. additionally, we have synthesized a hexapeptide library with amino acid substitutions of the chemical lead ykyryl and measured their binding affi nity to ace via spr to analyze the importance of the individual amino acids. spr studies indicate an important role of r . prostate cancer is one of the most common malignancies in men and is responsible for more deaths than any other cancer, except for lung cancer. in the last decade we have developed bradykinin antagonist peptides (b , b ), peptide dimer (b , suim-(d-arg-arg-pro-hyp-gly-igl-ser-d-igl-oic-arg) , suim: suberimidyl; hyp: trans- -hydroxyproline; igl: -( -indanyl)glycine; oic: s, as, asoctahydro- h-indole- -carboxylic acid) and four generations of our small molecule, bkm- which showed high inhibition against lung cancer in vitro and in vivo. some of the compounds also showed inhibition against prostate cancer. it is well known that prostate cancer metastasizes into bones. while prostate cancer itself has many treatment options, no drugs currently exist for the treatment of bone metastatic prostate cancer. we modifi ed our potent anti-cancer small molecules by incorporating an aminobisphosphonate group to target these compounds to bone. bkm- , f c-oc y-atmp (f c: , , , , pentafl uorocinnamoyl; oc y: (o- , -dichlorobenzyl)-tyrosine; atmp: -amino- , , , -tetramethylpiperidine) is the fi rst generation of our small molecules. this compound consists of three parts: a, an acyl group; b, a tyrosine amino acid residue and c, an amide group. we introduced aminobisphosphonate or aminobisphosphonate derivatives at position c, and the new n-(bis-phosphonatoalkyl)amide small molecules had anti-cancer activity against prostate cancer metastases in mice. one of these compounds has been shown to be effective against the growth of pre-established human prostate tumors in mouse skeleton through the induction of apoptosis and blockade of survivin expression. the synthesis of these aminobisphosphonate small molecules, the structure relationship studies and the biological activity of these compounds in cultured human prostate cancer cells and animal models will be presented. hayouka novel, potent and selective angiotensin iv short analogues the hexapeptide angiotensin iv: h-val-tyr-ile-his-pro-phe-oh (ang iv) mediates a wide range of physiological actions, including control of blood fl ow and cognitive enhancement. it exerts its effects by binding to at receptors, which are widely distributed across tissues. the at receptor has been identifi ed as the insulin-regulated aminopeptidase or irap. it has been proposed that ang iv exerts its action by inhibiting the catalytic activity of this enzyme. we have reported that the -homo amino acid containing analog h- hval-tyr-ile-his-pro- phe-oh (al- ) is a potent, selective and stable ang iv antagonist, in which the hval is responsible for stability and the hphe for selectivity. in this study we report the new ang iv short analogues. the incorporation of erythro-mephe or tic resulted in analogues which have great ability to inhibit the hydrolysis of leu-p-nitroanilide by irap or by ap-n. our data showed that the full ang iv sequence is not necessary for high potency. peptides with pro deletion containing tic or erythro-mephe were even more potent than full ang iv sequence. moreover a peptide design and synthesis of a non -peptide par thrombin receptor antagonist, using cyclohexane as template receptors that mediate thrombin action are attractive drug discovery targets because of their involvement in cardiovascular pathophysiology (dysregulation of platelet aggregation and endothelial cell function). the cellular actions of thrombin are, in large part, caused by the activation of proteinase-activated receptors (pars) , and (pharm. rev. : ). the serine proteinase thrombin cleaves and activates cellular par in many pathophysiological settings associated with hemostasis, tissue injury and the proliferation of vascular smooth muscle and tumor cells. in the present study, we synthesized a novel non-peptide par mimetic, based on a conformational analysis of the s fllr tethered ligand sequence of par in order to inhibit the cellular actions of thrombin. the rational design, based on nmr constraints and molecular dymamics, led to compound (fig. ) containing the spatially closed key pharmacophoric guanidyl and phenyl groups, attached to cyclohexane as a template. compound , inhibited both tfllr-amide ( m) and thrombin ( . and u/ml)-mediated calcium signaling in a cultured human hek cell assay (j pharm. exp ther. : ). aboye, teshome leta ; clark, richard j ; craik, david j. ; göransson, ulf biomedical centre, sweden; institute for molecular bioscience, australia the cyclic cystine knot motif, as defi ned by the cyclotide peptide family, is an attractive scaffold for protein engineering ( ). however, to date the utilization of this scaffold has been limited by the inability to synthesize members of the most diverse and biologically active subfamily, the bracelet cyclotides. here we describe the synthesis and fi rst direct oxidative folding of a bracelet cyclotide, cycloviolacin o , and thus provide an effi cient method of exploring the most potent cyclic cystine knot peptides ( ) . the linear chain of cycloviolacin o was assembled using fmoc solid phase peptide synthesis and cyclized by thioester-mediated native chemical ligation, and the inherent diffi culties of folding bracelet cyclotides were successfully overcome in a single step reaction. the folding pathway was characterized and included predominating fully oxidized intermediates that slowly converted to the native peptide structure. angiogenesis, the process of new blood vessel formation, is important in both physiologic and pathologic situations, and its inhibition can be useful in the fi ght against several diseases, such as tumor development, diabetic retinopathy, etc. one of the key factors in promoting angiogenesis is the vascular endothelial growth factor (vegf), which exerts its biological activity through interaction with specifi c receptors, vegfr- (flt- ), vegfr- (kdr, flk- ) and vegfr- (flt- ) . vegf is over-expressed in all examples of pathologic angiogenesis, and its effects on tumor growth are mainly mediated by kdr. our approach to novel anti-angiogenic drugs is centered on the search for inhibitors of vegf-kdr interaction. directed mutagenesis studies allowed the identifi cation of a surface of vegf recognized by kdr, with residues arg , ile , lys , his and glu , located in a -hairpin of loop , identifi ed as essential for the interaction. most residues in this region are also located in the interface of interaction between vegf and some none-humanized phage-displayed antibodies with potent antiangiogenic activity, suggesting a binding to vegf similar to that of vegf receptors. starting from vegf - fragment (mrikphqgqhi), we have designed different hydrocarbon-bridged analogues to preserve the -hairpin native structure. this communication will describe the solid-phase synthesis of olefi n-bridged (c=c) peptides and their saturated (c-c) analogues. considering that the -turn of native vegf - is slightly distorted, due to the presence of an extra amino acid residue, in addition to the bridged undecapeptides, two series of decapeptide analogues have also been prepared, just by removing the residues glu or gly . the conformational behavior of linear and bridged-peptides has been analyzed by nmr (noe, h and c chemical shifts). the ability of these peptides to adopt the native vegf -hairpin structure will be compared with their anti-angiogenic activities. integrins constitute a family of heterodimeric, transmembrane cell adhesion receptors which connect cells to the scaffolding proteins of the extracellular matrix. the pioneering observation that integrins -especially v and -are hallmarks of metastatic cancer and seriously involved in the process of tumor angiogenesis turned them into attractive targets for cancer therapy. out of that, the inhibition of integrin function is a major challenge in medicinal chemistry. potent ligands are currently in different stages of clinical trials for the antiangiogenic therapy of cancer and age-related macula degeneration (amd). especially the subtype has recently been drawn into the focus of research due to its genuine role in angiogenesis.( ) in here, we describe the rational design and the synthesis of high affi nity binders and the optimization of their activity and selectivity against v by means of extensive sar-studies and docking experiments.( ) starting from a tyrosine scaffold( ) we succeeded in getting compounds with affi nities in the low and even sub-nanomolar range and selectivities of fold against v . the insights about the structure-activity-relationship gained from the tyrosine based ligands could then be successfully transferred to ligands bearing an aza-glycine( ) scaffold to yield ligands with affi nities of even sub-nanomolar range and selektivites exceeding . fold. modeling studies and biological activities of a nonpeptide at receptor angiotensin ii antagonist the octapeptide angiotensin ii (h-asp-arg-val-tyr-ile-his-pro-phe-oh) is the major factor of the renin-angiotensin system (ras) and plays a signifi cant role in the regulation of arterial blood pressure. in the present study, we have modeled a losartan analogue, the non-peptide angiotensin ii at antagonist, -butyl- -hydroxymethyl- -{[ '-( htetrazol- -yl)biphenyl- -yl]methyl} imidazole (v ). structure activity relationship (sar) and molecular modeling studies indicate close proximity of hydroxymethyl and tetrazole pharmacophoric groups of antagonist v and losartan. conformational analysis was performed using a grid scan search in order to derive all the possible conformations from which six energy local minima (syn and anti) were extracted after a cluster analysis. furthermore, these different conformations were superimposed with losartan, where a spatial correlation among the pharmacophoric groups is observed. antagonist v showed similar potency with losartan in our in vivo model with anesthetized rabbits and in vitro binding studies to at receptor. at specifi c concentrations compound v showed higher affi nity compared to losartan indicating that reorientation of butyl and hydroxymethyl groups on imidazole template allows a better binding to at receptor. h-benzotriazolium -[bis(dimethylamino)methylene]- -chloro-,hexafl uorophosphate ( -), -oxide (hctu) is a non-toxic, non-irritating and non-corrosive coupling reagent [ ] [ ] . seven biologically active peptides (ghrp- , - acp, oxytocin, g-lhrh, c-peptide, hamylin - , and -amyloid - ) were synthesized with reaction times reduced to deprotection times of minutes or less and coupling times of minutes or less using hctu as the coupling reagent. no expensive coupling reagents or special techniques were used. total peptide synthesis times were dramatically reduced as much as . hr ( . days) without reducing the crude peptide purities. it was shown that hctu can be used as an affordable, effi cient coupling reagent for fast fmoc solidphase peptide synthesis. human sdf- contains sixty-eight amino acids and is a member of the chemokine family of peptides. this long peptide was synthesized step-wise using our quality control conditions in hours. the reaction times were then reduced to deprotection times of x min and coupling times of x . min, resulting in a total synthesis time of hours. the effect of different resins, resin substitutions and deprotection reagents on the crude peptide purities were compared. a small portion of crude peptide was purifi ed using an rp-hplc column and the mass of the fi nal product was confi rmed with maldi-tof mass spectrometry. references: . steven l. kunkel and nuria godessart, chemokines in autoimmunity: from pathology to therapeutics, autoimmunity reviews, , - ( ). pedersen, søren l.; sørensen, kasper k.; jensen, knud j. despite the development of new coupling reagents and solid supports, spps is still often faced with diffi culties in the assembly of long or "diffi cult" sequences, e.g. due to aggregation and steric hindrance giving rise to incomplete reactions. the use of convenient and precise heating with microwaves for spps has gained in popularity as it for many syntheses has provided signifi cant improvement in terms of speed, purity, and yields, maybe especially in the synthesis of long and "diffi cult" peptides. thus, precise microwave heating has emerged as one new parameter for spps, in addition to coupling reagents, resins, solvents etc. we have previously reported on microwave heating to promote a range of solid-phase reactions in spps. here we present a new, fl exible semi-automated instrument for the application of precise microwave heating in solid-phase synthesis. it combines a slightly modifi ed biotage initiator microwave instrument, which is available in many laboratories, with a modifi ed semi-automated peptide synthesizer from multisyntech. a custom-made reaction vessel is placed permanently in the microwave oven, thus the reactor does not have to be moved between steps. mixing is achieved by nitrogen bubling. washing steps are automated, however the activated amino acid derivatives have to be added manually. first, we developed optimized protocols for short cycle times in semi-automated spps with general fmoc chemistry. we utilized a microwave-compatible temperature probe for exact temperature measurements during microwave heating. then we developed protocols for on-resin reductive amination for anchoring of the fi rst amino acids to a bal handle. finally, we used the new instrument and the optimized protocols to assist in the synthesis of a range of diffi cult and long sequences. we believe that these successful syntheses demonstrate that this semi-automated instrument and the methods developed for it, can be an effi cient starting point for spps with microwave heating. investigation of polyelectrolyte-antigenic peptide conjugates by fl uorescence spectroscopy in proteins or peptides, it is possible to localize the interaction between protein and pe at certain protein domains. we investigate covalent binding mechanism of synthetic peptides which include tryptophan residue in the peptide sequence with copolymers of acrylic acid and n-vinylpyrolidone (vp\aa) depending upon the weight concentration ratio of components by fl uorescence method. antigenic peptides are synthesized by microwave assisted solid phase peptide synthesis method. characterization of these peptides are performed with lc-ms. subsequently these crude peptides are purifi ed by preparative hplc system. peptide-polymer covalent conjugation are performed in organic and pbs media. covalent conjugation are carried out by carbodiimide method. carbodiimide is used for the activation of carbonile groups of synthetic polymer which is the binding area of peptides that includes free amino groups. from the analysis of peptide-pe conjugates, it is possible to discuss the mechanism of the conjugate formation and structure of forming particles. according to the fl uorescence analysis results, free peptide which containing trp residue, gives fl uorescence spectrum. however, after the conjugation reaction it is observed that products max decreased and it is characterized as blue shift of max. this indicates that when conjugates formed, trp is completely isolated from aqueous solution. antiangiogenic thrombospondin type repeat analogs: synthesis, structure, and biological activity the thrombospondin type repeat (tsr) has been shown to inhibit angiogenesis and tumor growth in a number of in vivo models of cancer, but the details of this activity, including structure-function relationships, are not well understood. we explore these tsr elements in further detail via structural and biological evaluation of a series of tsr analogs. the tsr domain has a characteristic fold consisting of a two-stranded -sheet and a third 'rippled' strand. three tryptophans on the 'rippled' strand form cation-stacking interactions with two arginines on the adjacent sheet, forming an extended cation-network. in addition its structural importance, it has been postulated that the surface formed by this interaction is a key site for protein-protein recognition. a synthetic approach to the generation of novel tsr analogs, utilizing spps and native chemical ligation, has allowed us to rationally introduce unnatural amino acids in an effort to probe the structural and functional landscape of this clinically relevant protein domain. we have synthesized analogs of the second tsr domain of human thrombospondin- (tsr ) designed to probe the importance of the cation-stack. the arginine residues have been replaced by ornithine and citrulline, and thermal denaturation experiments by circular dichroism have been used to estimate stability differences between the mutant tsr and the native. taken as a set, these thermodynamic measurements provide insight into the stability afforded by a cationinteraction in the context of a native protein fold. synthetic access to the tsr domain also provides the opportunity for the introduction of various modifi cations, including the introduction of aminooxy groups as chemical handles, pegylation for in vivo stability, and biotinylation to create an affi nity caputure reagent for identifi cation of protein-protein interaction partners. the tetradecapeptide bombesin (bbs) has a high affi nity for the gastrin-releasing peptide (grp) receptor. these receptors are overexpressed in human tumors such as breast and prostate cancers. therefore they can serve as targets for in vivo imaging and therapy of these tumors with m tc-and re-radiolabeled bbs analogues, respectively. for the radiolabeling, a chelator is attached to the n-terminus of the bbs analogues. our group developed the (n his)ac chelator, which is easy to synthesize and has a very high affi nity for the m tc-and retricarbonyl complexes. however, an important part of the injected radiolabeled bbs analogues accumulated in healthy organs, such as liver and kidneys. to improve the pharmacokinetic properties, a bbs analogue was glycated via the lys side chain of the spacer using the maillard reaction. unfortunately, during glycation, overalkylation on the secundary amine of the chelator was observed. therefore, two alternatives for this glycation were examined. the fi rst one was based on the chemoselective reaction between a hydroxylamine (incorporated into the spacer) and an aldehyde (glucose). the second alternative was the cu(i) catalyzed cycloaddition between an alkyne (incorporated into the spacer) and an azide (azido-glucose). these approaches for carbohydration circumvented the problem encountered during glycation via the maillard reaction. glycation by the cu(i) catalyzed cycloaddition was, by far, the easiest way to incorporate the glucose moiety. moreover, after m tc labeling, this analogue showed the best biodistribution and the best diagnostic properties of all glycated analogues. an alternate approach to improve the pharmacokinetics consisted of including different polar spacers such as ³hglu, ³hasp, ³hlys, ³hser and -nh(ch ch o) co-between the (n his)ac chelator and the bombesin sequence. in particular the analogues with a negative charge ( ³hglu, ³hasp) in the spacer showed a favorable effect on the pharmacokinetics. determination of binding ratio of hydrofobic peptidepolymer conjugates by using fl uorescamine assay budama battal, yasemin; derman, serap; mansuroðlu, banu; mustafaeva, zeynep yildiz technical university, bioengineering department, turkey non-fl uorescent -phenylspiro-[furan- ( h), -phthalan]- , '-dione (fl uorescamine) reacts readily with primary amines in amino acids, peptides and proteins to form stable, highly fl uorescent compounds (fl uorophors). fluorescamine has been used to detect free amino groups on peptides or completion of coupling reactions in solid phase peptide synthesis and not only in aqueous solution but also in organic solvents and on solids. in this study, peptide epitops of vp capsid protein of foot-and-mouth-diseases virus - amino acid residues (val-lys-ile-asn-asn-thr-ser-pro-the-his-val-ile-asp-leu-met-gln-thr-his-gln-his-gly) were synthesized by sigma. we synthesized the covalent conjugate of - amino acid residues with copolymers of acrylic acids and nvinylpyrolidone (vp\aa) at different ratio of components (npeptide/ npolymer) and investigated the mechanism of condensation reaction by using different physicochemical analyses as hplc, fluorescence spectroscopy and fluorescamine assay. for determination of binding ratio of hidrofobic peptide-polymer congutages, fl uorescent measurements were performed in the presence of fl uorescamine at the wavelengts of excitation nm and emission nm. when conjugation reaction completed, the amount of free amino groups had decreased and observed that fluorescence intensity had decreased and the estimated degree of primary amino group after conjugation, calculated from fl uorescence spectrums. ( -phenylspiro-[furan- ( h), -phthalan]- , '-dione) which heterocyclic dione is a reagent for the detection of primary amines on peptides or completion of coupling reactions in the picomole range. its reaction with amines is almost instantaneous at room temperature in aqueous media, organic solvents ect . fluorescamine reacts with primary amines (in peptide, protein ect.) to form highly fl uorescent product (fl uorophors) whereas the reagent and its degradation products are nonfl uorescent ( ). this is the basis of a fl uorescent protein assay [ , ] . fluorescamine is used in many sensitive detection methods, e,g., characterization of poly-l-lysine (pll)/dna complexes post-modifi ed with a multivalent hydrophilic polymer ( ), or synyhetic peptide-polymer conjugates. in this study, foot-and-mouth-diseases virus vp capsid protein's synthetic peptide epitope which containing tryptophane (trp), - (p ) amino acid residues (try-ser-lys-tyr-ser-thr-thr-gly-glu-arg-thr-arg-thr-arg-gly-asp-leu-gly-ala-leu-ala-ala-arg-val-ala-thr-gln-leu-pro-ala) were synthesized by using the solid-phase methods. we synthesized the covalent conjugate of copolymers of acrylic acids and n-vinylpyrolidone (vp\aa) with - amino acid residues at different npeptide/npolymer ratio and investigated the mechanism of condensation reaction by using fluorescence spectroscopy and fluorescamine assay. in the fl uorescamine assay the conjugate solution was measured on a pti qm- steady state fluorescence spectrometer with an excitation wavelength of nm and emission at nm. after the conjugation reaction, the amount of free amino groups had decreased because of binding carboxyl group and observed that fluorescence intensity had decreased. determination of free amino group degree after conjugation, calculated from fl uorescence spectrums. newly developed high strength and chemically stable silica gel based preparative reversed phase packing materials kuriyama, naohiro; shoji, noriko; morishita, kiyoshi; omote, masakatsu ymc co., ltd., japan a new high strength silica gel and a bonding technology based on preparative bulk packing materials for hplc have been developed to provide improved recovery, selectivity, and longer life time for the preparative peptide separations. the novel preparative silica particle was successfully prepared by the new generation process, which allows the higher gel density than typical silica gel and the particle size distribution would be practically mono-dispersed character. for the effective reversed phase peptide separations, pore size and pore volume of these new particle were optimized depending on the molecular weight of peptides. to enhance chemical stability and selectivity under the typical peptide purifi cation conditions, the combination of chemical bonding method and functional group density was optimized for maximum performance. by repeated packing and unpacking of this synthesized gel with large dynamic axial compression column, it was demonstrated that no fi ne has appeared and no back pressure increasing has occurred comparing to commercially available packing materials. also cost effective peptide purifi cation with high loadability, productivity, and recovery was achieved with signifi cant small and large peptides. calcitonin gene-related peptide (cgrp) is a amino acid neuropeptide produced by tissue-specifi c alternative mrna splicing of the calcitonin gene. the cgrp peptide signals through a seven transmembrane g protein-coupled receptor (gpcr) belonging to the secretin receptor family. co-expression of the calcitonin-like receptor (clr) with receptor activity modifying proteins- (ramp ) forms a mature cgrp receptor on the cell membrane surface. cgrp is widely distributed in the peripheral and central nervous systems. in the later, cgrp is expressed in trigeminal ganglia nerves and when it is released has potent dilator effects on cerebal and dural vessels. through this mechanism, cgrp is involved in the regulation of blood fl ow to the brain and painsensitive meninges. the pathology of migraine has been associated with the vasodilation effects of cgrp on cerebal circulation. it has been reported that the cgrp peptide consist of an alpha-helical n-terminal region, a fl exible center region, and two putative c-terminal beta-turns. truncation of the fi rst seven residues in cgrp results in antagonists of the cgrp receptor (ic nm), however, cgrp( - ) is rapidly degraded in plasma. here, we report the iterative design and synthesis of new high affi nity cgrp antagonists with signifi cantly increased plasma stability, and higher affi nity for the human cgrp receptor. cordopatis, paul; pappa, eleni; zompra, aikaterini; magafa, vassiliki; diamantopoulou, zoi; lamari, fotini; katsoris, panagiotis university, greece mammalian gonadotropin releasing hormone (gnrh-i) and the sea lamprey gonadotropin releasing hormone type iii (gnrh-iii), belong to the class of conserved gonadotropin releasing hormone peptides. in addition to the classic hypophysiotropic action of gnrh-i, it has been shown that many malignant cells, such as prostate cancer cells, secrete gnrh-i and express the gnrh-i receptor/s. gnrh-iii has no endocrine activity in mammals even at high doses, but has been shown to suppress directly the growth of breast and prostatic cancer cells in vitro. in a continuation of our previous work and in order to study the effect of modifi cations in positions and of leuprolide, an agonist of gnrh-i, on prostate cancer cell proliferation, we synthesized ten new conformationally restricted analogues. d-leu of leuprolide was substituted by d-lys, d-or l-glu and ser by n-me-ser. we also synthesized fi ve new analogues of gnrh-iii and studied their effect on prostate cancer cell proliferation. asp in position of gnrh-iii was substituted by asn and glu, pro was substituted by á-aminoisobutyric acid (aib) and trp and/or trp by d-trp. peptides were synthesized by the solid phase methodology and fmoc/but chemistry in high yields. results show that the inhibitory effect of gnrh-i analogues on the proliferation of human prostate cancer cells depends on the nature of the substituted amino acid in position . incorporation of d-trp in positions and of gnrh-iii preserved its antiproliferative activity, whereas all other modifi cations reduced its activity. mastoparan (mp) is an antimicrobial cationic tetradecapeptide with following primary structure inlkalaalakkil. this amphiphilic -helical peptide was originally isolated from the venom of wasp paravespula lewisii. however some mp analogues (tetradecapeptides with different amino acids sequence) have been also isolated from the venom of hornets, yellow jackets and paper or solitary wasps. they are rich in hydrophobic amino acids such as leu, ile, ala and commonly posses lys residues. mp shows a variety of biological activities such as inhibition of the growth of gram-positive bacteria, activation of mast cell degradation and histamine release, activation of phospholipase a and c or erythrocyte lysis. mp is also turned out to enhance the permeability of artifi cial and biological membranes and activate gtp-binding regulatory proteins by a mechanism analogous to that of g protein-coupled receptors. nowadays many mastoparan analogues have been synthesized. some of them contains in primary structure the mastoparan sequence but they loss some of the properties of mp, such as transportan (a cell penetrating peptide, consisting n-terminal fragment - of galanin and mastoparan at the c-terminus linked with lys residue) or its truncated analogue, transportan- . in present study we have designed and synthesized several new chimeric mastoparan analogues composed of mp and other biologically active peptides (e.g. galanin, rnaiii inhibiting peptide) and containing natural or unnatural structures. next we examined each of these hybrid constructs as well as natural peptides for they antimicrobial activities. acknowledgement: this work was supported by the university of gdansk grand bw - - - cinnamic acid derivatives as esters, amides and glycosides are known to have antibacterial, antiviral, antiinfl ammatory, antiproliferative, immunostimulatory etc. properties. some of them (amide and ester analogues of caffeic acid with natural amino acid esters) exibit stronger antioxidative activity. in particular thiazole, oxazole and imidazole amino acids that may play a key role in biological activities of unusual peptides are also important intermediates for natural product synthesis and peptidomimetics. here we report the synthesis of novel cinnamic acid amides with oxazole containing glycyl-methyl ester as potential antioxidants. the amides were synthesized from sinapic, coumaric, ferulic acids and the corresponding glycyl-methyl ester hydrochloride form using n-ethyl-n-( -dimethylaminopropyl) carbodiimide hydrochloride (edc) and -n,n-(dimethylamino)-pyridine (dmap). their antioxidant activity was evaluated by , -diphenyl- -picrylhydrazyl dpph ( , diphenyl- -pycrylhydrazyl) test. the venoms of marine cone snails contain a complex mixture of peptide neurotoxins known as conotoxins ( ) . they are a diverse class of biomolecules that exhibit exquisite selectivity and potency for a wide range of pharmacological targets in the central nervous system, making them valuable tools for studying the mechanisms of neurotransmission and pain. despite their diversity, conotoxins have evolved from a relatively small number of rigid disulfi de bonded frameworks that give rise to a series of intervening loops of amino acids that project outwards from the framework and interact with the receptor binding site. receptor targets are generally determined by the shape of the framework, with the amino acids within the framework infl uencing receptor subtype specifi city. this work aims to use positional scan libraries ( ) based on / -conotoxin frameworks to study the interactions of amino acids with the binding site of neuronal nicotinic acetylcholine receptors, and to discover new ligands that possess greater potency and selectivity for different subtypes these receptors. the disulfi de bond frameworks were formed by oxidizing each mixture in dilute aqueous buffer and their correct formation monitored by cd spectroscopy. an á nachr functional assay was used to screen each mixture to determine the activity of each mixture and the results of this assay were used to design a series of individual candidates for use in further structure activity relationship studies. melittin effect on the sensory neurons prelevated from double transgenic vs. normal mice melittin is a aa peptide. it is the active compound of bee venom and a powerful stimulator of phospholipase a . its antimicrobial effect was extensively studied in the literature, but little is known about its neuroactive properties. melittin increases the cell membrane permeability in excitable and non-excitable tissues, as a result of its interaction with the negatively charged phospholipids. our interest was focused on the algesic properties of melittin and on its potency against the electrophysiological parameters of sensory neurons from dorsal root ganglia. in our study, we have performed patch-clamp studies, by the whole-cell confi guration. primary cell cultures were obtained from double transgenic mice tcr-ha+/ins-ha+ with type i diabetes. this animal model is well-suited for the study of neuropathic pain and it enables us to test the analgesic effects of melittin ( . - m). our recordings indicate that the active-peptide modifi es the algesic profi le of the sensory neurons, in particular by acting against the capsaicinactivated ionic currents. in addition, we have monitored the action of melittin on the i h currents, na + voltage -dependent currents, delayed rectifi er k + currents. a particular interest was focused on the recordings of ca + voltage-dependent currents. the maximal effect was obtained at a dose of m melittin, and at higher doses the active-peptide strongly disturbs the lipid membrane order. in conclusion, the algesic profi le of the sensory neurons from double transgenic mice is signifi cantly modifi ed by the neuroactive melittin in comparison with the profi le of neurons from balb/c mice. these electrophysiological data are important, and are very well corroborated with the increased thermal and mechanical hypersensitivity induced by melittin itself and the bee venom extract, that have been proved their effi ciency in behaviour tests . the toxic effects of amps tested on mammalian cell cultures the amps (antimicrobial peptides) is a class of molecules that belongs to the innate immunity. the ability of some of these peptides to penetrate the microorganism's external membrane and to induce their death suggests that this class of molecules may represent a new generation of antibiotic pharmaceuticals. in our work, we focused on the amps side effects on mammalian cells. the target was to characterize the toxicity of several amps evaluated on in vitro cellular cultures. the following amps were used in our experiments: melittin, magainin i and ii, cecropin a and mastoparan. the viability of cells in cultures was assessed by mts test on chinese hamster lung fi broblasts v and on human lymphocytes primary cultures. concentrations in the range of . - m were used. the ic value for each type of amp was evaluated from the viability versus concentration curves. haemolysis assays were also performed for these active-peptides. based on ic values, melittin has a stronger citotoxicity than magainin i and magainin ii. on the other hand, magainins appears to posses higher haemolytic properties than melittin. the less cytotoxic peptide seems to be mastoparan. these types of results are very useful to characterize the ability of amps to induce toxic effects in human body during the treatment. acknowledgements: financial support by the cnmp research grant pnii number - / . the melanocyte-inhibiting factor (mif- ), which is the tripeptide pro-leu-gly-nh , was isolated in the conventional way from bovine hypothalamus tissue. mif- represents a class of naturally occurring opiate antagonists with varying activities in independent situations. the purpose of the present study was to investigate the analgesic activity of mif- and its analogues modifi ed at position with unnatural amino acids cav, slys, sleu, sile and snle. the experiments were carried out on male wistar rats ( - g). the changes in the mechanical nociceptive threshold were measured by the paw-pressure test using an analgesiameter (ugo basile). mif- and analogues (all in dose mg/ kg) were administered intraperitoneally (i.p.). mif- exhibits a weak analgesic effect and selective affi nity for the -opioid receptor. mifanalogues -mif-cav, mif-sleu, mif-ile and mif-nle were found to have a naloxone-reversible analgesic effects. pacap (pituitary adenylate cyclase-activating polypeptide) exists in two biological isoforms, i.e a -amino acid peptide and a shorter form of residues corresponding to the n-terminal part of pacap . previous structure-activity relationships studies revealed that the n-terminal segment is essential for the activation of the specifi c and selective type i pacap receptor (pac ). in order to clarify the molecular requirements for pac activation, we initiated structure-activity investigations of the n-terminal part of both pacap isoforms. in particular, an important library of analogs focusing on the fi rst seven residues (his -ser -asp -gly -ile -phe -thr ) was rationally developed and pharmacologically evaluated for their capacity to bind the pac receptor and to induce ca + mobilization in chinese hamster ovary (cho) cells stably expressing the human pac receptor. ala scan demonstrated that the carboxylic acid function of residue asp and the benzyl group of phe are essential feature for proper pac receptor activation. the inversion of chirality of residues ile , phe and thr caused a loss of affi nity whereas the incorporation of d-amino acids in positions , or did not affect the binding properties of pacap. moreover, using a n-methyl scan, we showed that the n-terminal domain of pacap is not tolerant to back-bone modifi cations. however, replacement of ser by pro did not decrease signifi cantly the potency of the peptide while substitution of this same residue by d-pro totally inhibited the biological activity of pacap. furthermore, other structural constraints reduced the potency of pacap, suggesting that the n-terminal domain needs fl exibility to bind and activate the pac receptor. finally, residues - of c-terminally extended peptides played a favorable role for the binding affi nity towards the pac receptor as pacap analogs demonstrated highest binding affi nity compared to their pacap counterparts. neutrophil elastase-dependent synthetic host defence pro-peptides for the treatment of bacterial infections in cystic fi brosis patients chronic infection and infl ammation play a major role in the pathophysiology of lung disease in cystic fi brosis (cf). besides pseudomonas aeruginosa, there is increasing evidence that other multidrugresistant organisms, such as methicillin-resistant staphylococcus aureus (mrsa) are implicated in the activation of the infl ammatory response in cf patients. cationic host defence peptides, multifunctional mediators of the innate immune system, have been recognised as promising candidates for the development of novel antimicrobial agents ( ). most of these macromolecules are produced as inactive prepropeptides and are proteolytically activated to release a c-terminal cationic peptide with antimicrobial activity. we thought to mimic this natural mechanism to target cf pathogens with synthetic host defence propeptides. saltresistant, all-d cationic antimicrobial peptides ( ) were conjugated at their n-termini to a polyglutamic acid sequence to compensate the net positive charge of the parent peptide, through a linker selectively degraded by a disease-associated enzyme (neutrophil elastase). in vitro effects of the all-d p peptide candidate on planktonic and biofi lm forms of pseudomonas aeruginosa and staphylococcus aureus clinical isolates were assessed. reactivation studies of the propeptide were performed in presence of purifi ed neutrophil elastase and in physiologically relevant conditions, using bronchoalveolar lavage fl uids from cf patients. the nmr structures of the propeptide and active p sequences were also compared. these studies confi rmed that the propeptide remains inactive in the absence of neutrophil elastase and that the latter enzyme can potentiate its antimicrobial activity. synthesis and biological activity of a series of aza pseudopeptides related to rfa, the endogenous ligand of gpr rfa, a novel neuropeptide of the rfamide family, is the natural ligand of the previously orphan receptor gpr . both rfa and gpr mrnas are highly expressed in hypothalamic nuclei of rodents, and icv injection of rfa induces a potent orexigenic effect in mice. recently, it has been shown that gpr -defi cient mice suffer from osteopenia. analysis of the rfa precursor reveals that it may generate several additionnal rfa-peptides including an n-terminally extended form ( rfa) and a truncated form ( rfa ( - ) ). rfa and rfa increase dose-dependently [ca + ]i in gpr -transfected cells while rfa( - ) is about times less potent than rfa. molecular modeling under nmr constraints of rfa shows that the n-terminal region encompasses an -helix and the c-terminal region adopts a -turn in dpc micelles. the c-terminal peptide rfa( - ) exhibits major distortions of this turn that may be responsible for its weak potency. the aim of this work was to introduce an aza residue in rfa( - ) in order to stabilize the -turn. sequential aza -counterpart substitution of amino acids at positions and enhanced by and folds the potency of rfa( - ), respectively. the aza -phe analog was times less potent than rfa( - ). replacement of the native ser by the aza surrogate of (ho)homothr (aza -hth) to favor hydrogen bonding, generated an analog that was folds more potent than rfa( - ). in contrast, substitution of residues to led to analogs totally devoid of effect on calcium mobilization. as the -turn is centered on the ser of rfa, we can assume that the aza -hth moiety partially mimics the -turn in the rfa( - ) sequence. these data constitute the fi rst step towards the development of new gpr analogs that could prove useful for the treatment of feeding disorders and/or osteoporosis. acknowledgements: supported by inserm (u and pnr-re) and the région haute-normandie. olm is recipient of a fellowship from mesr. it is well known that microtubule targeting agents (mtas) are used in clinical treatments as anticancer agents. recently, it has become clear that some mtas also function as gvascular disrupting agents (vdas), which induce tumor-selective vascular collapse. as one of such candidates, a natural cyclicdipeptide, phenylahistin (plh) exhibiting colchicine-like anti-microtubule activity, has been our focus. we have succeeded in synthesizing plh, and performed the structure activity relationship study of its derivatives. from the biological evaluations, according to these results, a highly potent derivative npi- (ic = nm, ht- ) was selected, which is now in phase i clinical trial as an anticancer drug in the us. furthermore, we have established the synthetic route of npi- and its derivatives. about analogs were prepared so far and screened by ht- citotoxicity assay. as a result, several highly potent derivatives ware developed. although npi- and its derivatives are believed to be recognized around the colchicine binding site on tubulin, the three-dimensional structure of npi- could not be superimposed over that of colchicine. in order to understand the precise binding mode of npi- , we developed a highly potent cytotoxic derivative, kpu- (ic = . nm, ht- cells) with a benzophenone structure. because benzophenone is recognized as a photo-reactive group, we synthesized biotin-tagged kpu- derivative as a photoaffi nity probe. since this probe has biological activity enough to function to tubulin, photoaffi nity labeling was performed to analyze the binding site. as a result, irradiation-time-dependent labeling towards tubulin was observed. the labeling was also dose-dependently inhibited by colchicines addition, suggesting that the probe specifi cally recognizes around the colchicine binding site on tubulin. chemical biology study for determining the precise binding site is now in progress. conformational analysis of the new temporin analogues: gln ta and pro tl. temporins are antimicrobial peptides (amp €™s) isolated from the skin of red european frog rana temporaria. they are active particularly against gram-positive bacteria, candida species, fungi. they have the ability to bind and permeate both artifi cial and biological membranes. we have recently investigated by spectroscopic means two members of this amp family temporin l (fvqwfskflgril-nh ) and temporin a (flpligrvlsgil-nh ) ( ). based on the nmr results, we developed two new analogues of these peptides named pro tl (fvpwfskflgril-nh ) and gln ta (flqligrvlsgil-nh ). biological data indicate that pro tl has a higher antimicrobial activity and a lower hemolytic activity than the native peptide tl. in contrast, gln ta more hemolytic than the parent peptide ta. the conformational behavior of the new analogues was investigated in membrane mimetic environment (sds and dpc micelles) by spectroscopic and computational methods. diagnostic nmr parameters observed for glu ta and pro tl indicate a conformational propensity toward helical structure. for glu ta, bturn structures are also observed along the n-terminal fragment of the peptide. we are currently studying the conformational behavior of the four peptides also by molecular dynamics simulation in explicit solvated dodecylphosphocholine and sodium dodecylsulfate systems. these simulations would provide a realistic picture of the interactions between the peptide and models of bacterial and mammalian membranes. where xaa was selected form a collection of cyclic and acyclic nonaromatic amino acids. the peptides were prepared by standard spps methods using either the fmoc or boc strategy and tested in vitro for their agonistic potency and effi cacy at the v a and the related receptors. unlike avp, which has aromatic amino acids in positions and , these analogues contain only non aromatic residues, but are still potent v a agonists. compounds with cyclic aliphatic residues (e.g. cha) in position were found to be generally more potent v a agonists than the ones containing acyclic residues. the ala analogue ([ala ,ile ,dab ]vp) was totally inactive (no signifi cant v ar agonism up to nm) as pharmacology and medical peptide chemistry reported previously for [ala ]avp ( ). the cha containing peptides are slightly less potent as v ar agonists than their phe counterparts, but the overall in vitro pharmacological profi le of the two classes of compounds is similar. selected new analogues were also tested in vivo in a rat pressor model. the compounds were found to be effective in raising arterial blood pressure and appeared to be longer acting in vivo when compared to avp and related compounds, as demonstrated by slower disappearance of their pressor effect at equieffective doses. detailed experimental procedures, the results of biological testing, and sar discussion will be presented. the impact of lithium cations on the peptide bond lithium ions play an important role in some biological processes, e.g. in the treatment of neuronal dysfunctions and some metabolic pathways. however, the mechanisms for these actions are not known up to now. recently it has been shown that lithium cations infl uence the isomerisation state of peptide bonds which is essentially pronounced in the case of the amino acid proline. such an isomerisation goes hand in hand with conformational changes possibly resulting in altered folding and structuring. thus, lithium cations might essentially infl uence the biological function of peptides and proteins. to shed light on the underlying mechanism we carried out detailed studies on several model peptides. thus, the isomerisation as well as the kinetic properties of the peptides have been determined employing various techniques of nmr spectroscopy. besides, quantum chemical studies on li + -peptide complexes were performed to get insight into the structural aspects of the cation-peptide interaction. enhanced screening and understanding of hypolipidemic peptides assisted by informatics hypolipidemic peptides targeting the bile acid to inhibit cholesterol absorption in intestine has been indicated by several naturally obtained short peptides. these bile acid-binding peptides are known to disrupt the micellar formation of bile acid for capturing intestinal cholesterol, and lead the aggregates pass the intestine without absorbance. such effect also disrupts the hepatic circulation of bile acids, which accelerates the conversion of stored cholesterol into lacking bile acids. however, traditional extraction procedure of peptides form natural products is time-consuming. therefore, to enhance the screening of such functional short peptides, we have combined the array-based screening strategy with bioinformatics strategy, to design effective experiments by prediction of physicochemical peptide structures. as a result, we resulted in signifi cantly higher screening effi ciency and high bile acid affi nity compared to random screening. we hare report the introduction of one of supervised learning algorithms, fuzzy neural network, and unsupervised algorithm, hierarchical clustering scheme for such functional peptide screening. analogues of the kinin b receptor antagonist r- bearing n-terminal lipid moieties. adrenomedullin (am) is a -amino acid peptide that shares structural similarities with regulatory peptides belonging to the calcitonin family (calcitonin, cgrp and amylin). am is known to produce a potent vasodilation via the coupling to the calcitonin receptor-like receptor (crlr) associated with a chaperone protein, the receptor-activity modifying-protein (ramp). binding studies performed in mammalian tissues revealed that the largest distribution of am binding sites was found in the heart and lungs. in fact, the pulmonary blood vessels display a vast abundance of am binding sites that act as clearance receptors. pulmonary arterial hypertension (pah) is a condition associated with obliteration of pulmonary arterioles which carries a very poor prognosis, in part because of delayed diagnosis due to the lack of specifi c noninvasive diagnostic tools. likewise, there currently exists no molecular imaging agent to diagnose pulmonary embolism, a pathological condition that occurs when a blood clot blocks a lung artery. therefore, due to the high density and incidence of am receptors in the cardiorespiratory system, am could represent a key-target for diagnosis with radiolabeled am-related drugs. in this study, we looked at structureactivity relationships and synthesized various am fragments exhibiting high binding specifi city for am receptors but reduced biological activity. moreover, we introduced different chelating moieties into these am peptides to evaluate the possibility of labelling those molecules for imaging purposes. using cell binding assays, we observed that a cyclic moiety combined with the am( - ) sequence is able to maintain a good affi nity for am receptors. furthermore, we showed that the incorporation of a -amino acid sequence into the peptide chain was the best chelating moiety that allows high effi ciency labelling with mtc. hence, our study strongly suggests that am derivatives are promising leads for lung specifi c imaging. m tc-labeled analogs with pansomatostatin properties may lead to clinically useful radiotracers and further search for analogs displaying improved biological profi le is warranted. and for the rat (r) sst by competition binding assays in ar - j cell membranes with [ i-tyr ]octreotide as radioligand. after m tc-labeling, radiopeptide internalization was studied in ar - j cells. biodistribution of [ m tc]demotates was studied in healthy swiss albino mice and for [ m tc]demotate , , and in ar - j tumor bearing mice. results: demotate showed the highest affi nity binding for the hsst and the rsst , while introduction of one asp linker(s) at the n-terminus diminished the affi nity for the hsst and the rsst and led to lower internalization rates. substitution of thr by asp in demotate resulted in good affi nity for the rsst and lower affi nity for the hsst . single (d)phe -substitution/ or thr substitution by asp combined with asp introduction at the nterminus/ led to inferior binding affi nity, poor internalization capacity and poor uptake of the respective radiopeptides in target-organs and, in the case of [ m tc]demotate , , and , in ar - j tumors in mice. conclusion: introduction of asp residues in the original [ m tc]demotate chain exerted a negative effect on receptor affi nity, internalization capacity and targeting of somatostatin binding sites in mice precluding their use as hydrophilic pharmacokinetic modifi ers. the family of secreted aspartic proteinases (saps), which are encoded by ten distinct genes, is an important virulence factor of the human pathogen candida albicans.( ) due to an increase in the number of candida strains that are resistant to the drugs currently used in therapy, these proteinases are highly promising new drug target candidates. based on the knowledge of sap -substrate specifi cities( )and x-ray structural studies of sap in complex with inhibitors,( ) we designed and synthesized a series of sap inhibitors by modifying the structure of the pentapeptide pepstatin a, which is a potent yet non-selective aspartic proteinase inhibitor. these inhibitors were synthesized manually via a spps methodology using a -cl-tritylchloride resin and fmoc-protected amino acids. in addition, the key residue of the designed peptide inhibitors, the -amino acid statine (sta), which was prepared according to a slightly modifi ed literature procedure, . was further protected at its hydroxyl group as a silyl ether for the purpose of avoiding competitive side reactions during amino acid couplings. we prepared new inhibitors by varying the pepstatin a structure at the p , p , or p ' position. all variants showed effi cient inhibition when screened against the isoenzymes sap , sap , and sap , and some of them exhibit ic values similar to or even lower than that observed for pepstatin a. peptide library is a general technique for screening specifi c peptides that bind to a target protein. but, in a standard screening method, peptide libraries need to be fi xed on large carriers like phage, beads and so on. the large carriers would affect the binding between peptides and the target protein. in this work, we develop a new method for screening peptide libraries without carriers. we attached a variety of fl uorescent amino acids to peptide libraries and the peptides that bind to target proteins were identifi ed through -dimensional fl uorescence spectroscopy in aqueous solution. in this presentation, -different fl uorescent amino acids were synthesized or purchased. of those amino acids, we newly synthesized fl uorescent amino acids. -d fl uorescence spectra of those amino acids were measured. they were different in fl uorescence excitation/emission wavelengths. excluding those of weak fl uorescence intensities, fl uorescent amino acids are selected. those fl uorescent amino acids are mixed in methanol/water (ph . ) (= / (v/v)) and the -d fl uorescence spectrum was measured. then, concentrations of the component amino acids were evaluated by a linear least-squares method. the results suggested that all fl uorescent amino acids can be quantifi ed. the set of fl uorescent amino acids combined with the -d fl uorescence spectroscopy technique will be a powerful tool for screening peptides and other drug compounds without using any carriers. n-methyl phenylalanine-rich peptides as potential blood brain barrier shuttles malakoutikhah, morteza; teixidó, meritxell; giralt, ernest institute for research in biomedicine, barcelona, spain several peptide families containing n-methylated amino acids were designed and synthesized using solid-phase peptide synthesis (spps). the permeability of these compounds was studied by parallel artifi cial membrane permeability assay (pampa) and immobilized artifi cial membrane chromatography (iamc) so as to select the best peptides in terms of length, terminal groups and amino acid replacement to be used as carriers that pass through a model of blood-brain barrier (bbb) by passive diffusion for non-permeating agents. furthermore, their enzymatic stability in human serum and their cell viability were tested by mtt assay. these peptide families showed great stability and nontoxicity. the three best peptides were coupled to levodopa and assessed. these peptides transferred levodopa through an artifi cial membrane and transformed it from a non-passive permeating drug into a compound able to cross the membrane by passive diffusion. the structure-activity study of insulin-like peptide (insl ) requires the design and synthesis of various analogues. in order to test these for their receptor binding affi nity, a high-throughput receptor binding assay is needed. we have therefore developed an effi cient solid phase synthesis protocol to prepare specifi cally mono-labeled human insulin-like peptide (insl ) for the study of its interaction with its g-protein-coupled receptor, rxfp . a commercially available chelator, diethylene triamine pentaacetic acid (dtpa), was coupled to the n-terminus of solid-phase bound insl a-chain and then a coordination complex between eu + and dtpa chelator was formed. after combination of the purifi ed aand b-chains together with sequential formation of the three insulinlike disulfi de bonds, the labeled peptide was purifi ed at high yield using high-performance liquid chromatography with high ph buffer to prevent the liberation of europium from chelator. saturation binding assays were undertaken to determine the binding affi nity (pk d ) of labeled insl for rxfp in hek stable cell line expressing rxfp . the binding affi nity of dtpa-labeled insl ( . ± . ) was comparable to that of i-labelled insl ( . ± . ). the effi cient solid-phase synthesis has provided a novel lanthanide-coordinated, dtpa-labeled insl with excellent sensitivity, stability and high specifi c activity and which is superior to the traditional i-insl . this labeled peptide can be used in a high-throughput screening of insl analogues in structure-activity studies. multimodal tumor imaging using mri and pet/spect provides comprehensive diagnostic information as it combines anatomical information (by mri) and functional information (by pet or spect). development of dota-derived imaging probes is advantageous for multimodal imaging as it accommodates many metal ions employed in different imaging modalities including gd(iii) for mri. however, high relaxivity and enhanced specifi c up-take at the targeted site is important for the gd-based mri contrast agents. the dota-based prochelators required for the multivalent vectorization of targeting ligands were synthesized by functionalizing cyclen or do a-tertbutyl ester with modifi ed glutamic acid. multivalent conjugation of bombesin peptides, which specifi cally target tumors expressing gastrinreleasing peptide (grp) receptors, yielded the corresponding mono-, di-, and tetravalent bombesin analogues. the conjugates showed excellent chelating properties with gd(iii) (for mri applications) and also with in, lu and ga (for radiopharmaceutical applications). the in and lu labeled divalent conjugates showed rapid internalization and slower externalization rate compared to their corresponding monovalent analogues as studied with prostate tumor cell lines. the gadolinium complexes of monovalent and divalent conjugates showed signifi cant relaxivities at mhz ranging from . to . mm- s- at °c. the values represent the to fold enhancement of relaxivity compare to clinically employed dotarem® (gd-dota). further, the relaxivities increased signifi cantly from monovalent to divalent conjugates. this is highly promising as we would expect much higher relaxivities in case of tetravalent conjugates, which are currently under investigation. in conclusion, the work demonstrates the development of high relaxivity multivalent bombesin analogues, which could be employed for the multimodal imaging such as pet/mri or spect/mri with improved tumor targeting capabilities. a new highly potent dota-conjugated bombesin antagonist for grpr-positive tumor targeted imaging peptide receptors are very promising targets for tumor imaging. the somatostatin receptors were successfully targeted with peptides labeled to -emitters ( in, ga and m tc) and positron emitters ( f, ga, y) for diagnostic imaging. for tumor targeting, radiolabeled agonists were developed as they usually trigger the internalization of the peptidereceptor complex. we have recently shown that somatostatin-based radiolabeled antagonists may not only have a higher tumor uptake than equipotent agonists but also a longer lasting tumor uptake. among the most promising receptors to be targeted, the bombesin receptors are of great interest as they are overexpressed in major human tumors such as prostate and breast. the aim of this study was to develop a radiolabeled bombesin-based antagonist conjugated to the macrocyclic chelator dota which allows effi cient and stable labeling with a variety of two-and three-plus charged radiometals for imaging (pet, spect). we compared its in vitro pharmacologic properties and biodistribution in the pc- mouse model side-by-side with the potent agonist [ nat, in]-do a-ch co-g- -aminobenzoyl-q-w-a-v-g-h-l-m-nh (amba). in(iii)-amba was shown to be a potent agonist by ca + fl ux and immunofl uorescence studies and in(iii)-rm was effi ciently antagonizing the activity of in(iii)-amba. the pharmacokinetics showed a distinct superiority of the radioantagonist with regard to the high tumor uptake as well as to all tumor to normal tissue ratios. [ ga]-rm showed similar pharmacokinetic than [ in]-rm with a lower initial kidney uptake and a faster wash out from the kidney. the pet/ ct scintigraphic studies of [ ga]-rm in the animal model confi rm the signifi cant high tumor uptake and tumor to background ratio. as we found for somatostatin receptor-targeting radiopeptides,bombesin-based radioantagonists also appear to be superior to radioagonists for in vivo imaging and potentially also for targeted radiotherapy of grpr-positive tumors cell penetrating peptides (cpp) are a special class of peptides that possess the property to traverse the formidable barrier of the plasma membrane and deliver cargos into cells. using cpp as vectors and dna, mrna or proteins/enzymes as potential intracellular targets, a new generation of intracellular contrast agents (cas) can be developed. these agents have prospective use for molecular imaging (both optical and magnetic resonance imaging) by targeted labeling of cells. aiming to image the presence of specifi c mrnas or enzymes, two mrna targeting (contains a pna sequence antisense or non-sense to the target mrna of dsred) and one enzyme targeted (contains a unit cleavable by -galactosidase) cas were tested for their activity in the presence and absence of respective targets. the antisense targeting ca, their nonsense derivative and the enzyme targeted ca were taken up effi ciently into cells by an exclusively endosomal mechanism as observed by fl uorescence microscopy. cell free binding assays proved a specifi c interaction with a synthetic target for the antisense but not for non-sense ca. magnetic resonance studies showed a higher uptake in transgenic dsred expressing cells than the parent cells. however, no difference was observable for antisense versus non-sense ca in dsred cells, due to the vesicular entrapment which is preventing the specifi c interaction between ca and cytosolic target. since a comparable cellular distribution was visible for the enzyme targeted agent, a specifi c accumulation in -galactosidase containing cells is also unlikely. the results show that even though the designed cas were effi ciently taken up into cells, they can interact specifi cally with the target only if colocalization is achieved. however, a lack of specifi city is caused by the endosomal entrapment. further modifi cations are required to achieve the release from endosomes or a direct uptake into the cytosol. the infl uence of pegylation on the tumor accumulation of frop- , a tumor specifi c peptide identifi ed by phage display the pool of natural peptides suitable for the tumor targeting is limited and therefore novel lead structures identifi ed by screening of phagedisplayed libraries are highly warranted. however, transfer of the novel peptide sequences to clinical application is often diffi cult. we had shown that the coupling of dota to frop- , a peptide identifi ed in phage display libraries resulted in a fundamentally improved in vitro binding capacity. however, a biodistribution study revealed that the slow binding kinetics frop-dota (h-glu-asn-tyr-glu-leu-met-asp-leu-leu-ala-tyr-leu-lys(dota)-cys-nh ) allowed the excretion to forestall a signifi cant tumor accumulation. the aim of this study was to investigate whether the conjugation of peg to frop-dota results in a derivative with a prolonged residence time in the blood. frop- bearing a c-terminal dota residue to allow labeling with in- and a cysteine to attach a maleimido-modifi ed da peg oligomer was obtained by solid phase synthesis. the conjugate was purifi ed by size exclusion chromatography. several attempts to couple different peg derivatives on the solid support did not proceed satisfactorily and therefore the peg residue was attached in solution. the breast cancer cell line mcf- showed a relative low accumulation of the pegylated peptide in the cells. in contrast, biodistribution studies of the labeled conjugate in mice bearing human fro - showed a time dependent increasing uptake of the pegylated peptide with a high retention (at h p.i. % of the maximal activity concentration persisted in the tumor). the highest uptake values were determined at min. p.i. reaching . %id/g tumor as compared to . % %id/g observed for the non-pegylated derivative at min p.i. apparently pegylation provides a substantially improved stabilization in the circulation which allows a stable tumor accumulation. in vivo spect/ct imaging of intracranial human glioblastoma xenografts with indium-labeled homing peptide. huhtala, tuulia* ; enbäck, julia* ; rautsi, outi ; weisell, janne ; laakkonen, pirjo* ; närvänen, ale* *equal contribution. university of kuopio, finland; university of helsinki, finland phage display libraries provide a powerful tool to identify new biologically active peptides that bind specifi cally to their target molecules. different tumors express a distinct range of molecular markers on their vasculature providing a possibility to use peptides for targeting. tumor-homing peptides offer an opportunity to target, image and destruct the target tissue. glioblastomas represent the most aggressive form of brain tumors. we have identifi ed a novel peptide, coop, using an ex vivo / in vivo phage display screen. this peptide homes specifi cally to the early stage astrocytoma model. in addition, the peptide homes to the u human glioblastoma xenografts (enbäck and laakkonen, unpublished data). we have studied the biodistribution and tumor homing properties of indiumlabelled peptide in mice bearing intracranial human u glioblastoma tumors using spect/ct imaging. coop peptide was conjugated with the dtpa (diethylenetriamine pentaacetic acid) via amino terminus and labeled with indium. imaging was performed in four different time points ( minutes, hour, hours & hours), and the organ and tissue specifi c radioactivity was measured after the scarifi cation of the animals. intravenously injected in-labeled coop peptide accumulated in the u braintumors. the amount of the peptide increased with time and a clear radioactive accumulation was seen in / animals at hour and in / animals at hours after injection. due to the small size of the dtpa-peptide conjugate the majority of the molecules were secreted via the kidneys during the fi rst minutes. at h timepoint the tumorto-brain tissue ratio was , . we report here that the coop peptide, which specifi cally homes to the brain tumor vasculature and tumor cells, strongly accumulated in vivo to the xenografted human glioblastoma tumors in mice. this is the fi rst time when a synthetic peptide has been used in spect imaging of brain tumors in animal mode. failure of the development of a novel peptide tracer for molecular imaging of cancer therapy response - ( ) have shown that fl uorescently labeled derivatives of the hvggssv motif accumulated in tumors undergoing therapy. this peptide motif had been identifi ed by the in vivo screening of phage-displayed peptide libraries. the ability of the peptide to differentiate between responding and nonresponding cancers after treatment could be utilized for the development of promising tracers to monitor cancer therapy. the goal of this study was to investigate whether radiolabeled derivatives of the hvggssv motif can be used for the noninvasive determination of therapy response in vivo. therefore three different conjugates were synthesized by solid phase synthesis using fmoc chemistry. the fi rst peptide was n-terminally modifi ed with the chelator dota (dota-ggghvggssv-conh ) to allow radiolabeling with metallic radioisotopes such as in or ga. a glycine linker was placed at the n-terminus to separate the peptide motif from the chelator. in order to obtain derivatives accessible to radioiodination a tyrosine was introduced at the n-terminus (h n-yggghvggssv-conh ) of the second peptide. the third peptide was biotinylated at the n-terminal amino group and bound to radioiodinated streptavidin (sa-biotin-ggghvggssv-conh ). the biodistribution was monitored by scintigraphy in mice bearing a -tumors treated with a vegf receptor-specifi c inhibitor (su ). this study revealed that the in labeled hydrophilic dota conjugate shows a rapid renal clearance, the iodinated peptide accumulates mainly in the liver and kidneys. all of the peptide derivatives do not specifi cally accumulate in treated tumors after i.v. injection. in contrast to the promising results shown by han z. et al. our study shows that the hvggssv motif can not be easily adapted to radiolabeling approaches with clinical relevance. in conclusion, the peptide motif is not attractive for further clinical evaluation as new diagnostics for the imaging of cancer response. a novel approach to improve cellular delivery of -aminolaevulinic acid: new ala-containing peptide prodrugs for photodynamic therapy. photodynamic therapy (pdt) is a binary therapeutic modality which is currently under investigation for the treatment of several kinds of malignancies. it relies on the interaction of two individually harmless components: a photosensitiser and an external radiation. the interaction of the photosensitiser with light of the appropriate wavelength and molecular oxygen results in the generation of cytotoxic species, namely singlet oxygen and/or radicals, and localized destruction of tumours or infected tissue. in -aminolaevulinic acid photodynamic therapy (ala-pdt), exogenous administration of ala is employed to generate elevated intracellular levels of the natural photosensitiser protoporphyrin ix (ppix), via metabolism through the haem biosynthetic pathway. however this approach suffers from several drawbacks associated with ala's lack of stability at physiological ph and its highly hydrophilic nature, which prevent it from crossing biological membranes and hence limit tissue penetration. ala delivery with synthetic peptide prodrugs is a promising way to address these problems. in this work we report the synthesis and characterisation of a series of peptide prodrugs of general structure ac-xaa-ala-or, where xaa is an alpha amino acid, chosen to provide a prodrug with appropriate lipophilicity and water solubility. the uptake of the compounds and metabolism to ppix in pam keratinocytes, relative to ala is evaluated by fl uorescence spectroscopy, and further quantifi ed by recovery and chemical derivatisation of intact/ partially metabolised prodrugs. in a parallel study, we have also explored the possibility of coupling of ala and ala prodrugs to cell penetrating peptides (cpp). the conjugation of one or more molecules of ala to a cpp sequence represents an interesting approach to enhanced topical delivery of ala. preliminary results of these studies are reported herein. acknowledgements: thanks are due to biotechnology and biological sciences research council (grant bbd ) bioshuttle as a carrier for temozolomide transport into prostate cancer cells if metastatic prostate cancer gets resistant to antiandrogen therapy, there are few treatment options, because prostate cancer is not very sensitive to cytostatic agents. temozolomide (tmz) as an oral applicable chemotherapeutic substance has been proven to be effective and well tolerated with toxicity especially for brain tumors. unfortunately tmz was ineffi cient in the treatment of symptomatic progressive hormone-refractory prostate cancer. this may have different reasons like the short plasma half-life of tmz, a non adapted application schema and as a result, an insuffi cient bioavailability. to improve the specifi city, we built our so called tmz-bioshuttle-construct with cathepsin b (ctsb) mrna specifi city. this complex combines, a transmembrane transporter molecule connected via a disulfi de bridge to an antisensepeptide nucleic acid (pna) against a docking site in exon of the ctsb mrna. furthermore this part is connected via a ctsb cleavable peptide substrate to a nuclear localization sequence coupled with tmz. inside the target-cell, the pna recognizes the cytoplasmic ctsb mrna and after annealing (the pna/rna hybride is not a substrate for rnase h) results in a cell-specifi c retention of the tmz in the cytosol especially of ctsb-expressing cells. then, after the cathepsin b-mediated cleavage in the cytoplasm, the nls-sequence is separated and activated for an ran/importin-mediated transport of the tmz-cargo into the nucleus of the target cells. this bioshuttle-mediated tmz transfer could be a step forward to a successful therapy with higher specifi city, avoiding adverse reactions and circumventing the previous therapy limiting situation. the intracellular delivery of protein segments and other bioactive molecules using membrane -permeable peptides has been investigated in multiple aspects. most of the currently recognized cell penetrating peptides (cpp) are of cationic nature and derived from viral, insect or mammalian proteins endowed with membrane translocation properties. these peptides enter the cell by a receptor independent mechanism, which is poorly understood and carry only one epitope. our target was to achieve a cell-penetrating carrier able to transport more than one bioactive molecule. to this aim we designed a cell penetrating sequential carrier (cpsc) formed by the repetitive -lys-aib-cysmoiety, which incorporates the cysteine residue for anchoring the bioactive molecules through a thioether bond. the lysine free side chain possesses the cationic nature of the construct while the á-amino isobutyric moiety could induce a helicoid peptide backbone with amphipathic characteristics. to test the ability of the cpsc to penetrate the cell membrane we synthesized the carboxyfl uorescein -labelled cpsc, cf-[lys-aib-cys(-ch conh )] -nh , while the cf-tat-cys(-ch conh )-nh analogue, which is a small basic peptide that has been shown to deliver a large variety of cargoes into the cells, was used as a positive control. the results indicated that cpsc had a homogeneous distribution into the cytoplasm suggesting that cpsc may provide a new powerful biological tool for drug transportation. acknowledgements: the gsrt and eu (pened ÅÄ ) for the fi nancial support. amphipathic pro-rich peptides as intracellular drug delivery systems pujals, silvia; fernandez-carneado, jimena; giralt, ernest institute for research in biomedicine, spain shared among many of the known cpps. in our laboratory described a novel group of cpps: amphipathic proline-rich peptides. the principal advantages of these compounds are non-cytotoxicity, nonviral origin and high solubility in aqueous media. the best candidate for cpp among the amphipathic pro-rich peptides was sap (sweet arrow peptide), (vrlppp) .( ) derivatives with hydrophobic moieties, such as fatty acids or silaproline, have shown highly improved internalisation effi ciency; an all d-amino acid version of the cpp sap was shown to be completely protease resistant and was evaluated in a preliminary in vivo study. cd and tem studies regarding the self-assembly properties of this family of peptides highlight the possible role of aggregated species in the internalisation process. finally, these cpps have shown to be internalised via caveolae or lipid-rafts mediated endocytosis, which circumvents the lysosomal route of degradation. in order to test a challenging cargo for sap, gold nanoparticles were conjugated to sap. thereafter, the transport of au np by sap in hela cells has been studied by tem. while np alone are not internalised, np-c-(vrlppp) are clearly uptaken. studies with qds (quantum dots) and egfp as sap cargo are currently being done in our laboratory. . pujals, sílvia; giralt, ernest. proline-rich, amphipathic cell-penetratingpeptides. addr ( ), , - . novel cysteine-rich cell penetrating peptide: effi cient uptake and cytosolic localization introduction: crossing the plasma membrane is a prerequisite for intracellular targeted drug delivery. cell penetrating peptides are actively used as the delivery tool for intracellular delivery of various cargos. however, confi nement of biomolecules into endosomes limits their use for intracellular targeting. therefore, there is a need for vectors capable of transferring cargo molecules directly into the cytoplasm. herein, we focus on the development of a novel cpp (derived from polypeptide crotamine .) which shows an effi cient uptake at low concentrations ( . m) and cytosolic distribution along with vesicular uptake. methods: series of peptides were synthesized by fmoc strategy, introducing mutations in cro ( - ) (proposed cpp sequence in crotamine). all were n-terminally labeled with fl uorescein isothiocyanate for optical imaging. structure activity relationship (sar) studies were done by substitution and/or deletion of amino acid residues in the sequence observing the uptake behaviour by fl uorescence spectroscopy and microscopy. results: amongst synthesized peptides, one of shorter length showed the best intracellular delivery and cytosolic distribution at lower concentration ( . m) when compared to other cpp. replacing or deleting cysteines had negative impact on internalization. results also displayed the involvement of tryptophans in cellular uptake indicating, along with cationic amino acids, the importance of each residue in this optimized sequence. conclusions: sar studies identifi ed a novel cell penetrating peptide showing, besides of endosomal uptake, also an effi cient delivery into the cytoplasm at low concentrations. thus, this peptide might prove useful for effi cient transmembrane delivery of agents directed to cytosolic targets. peptide nucleic acid (pna) is a dna mimic consisting of the four common bases of dna on a pseudopeptide backbone that makes it extremely stable in biological fl uids. antisense pna is targeted against mrna in cytoplasm in a sequence specifi c manner. however, the main hindrance to the effective use of pnas has been their relatively poor uptake by cells. endosomal release or direct uptake into cytosol of agents is mandatory for attaining mrna based targeting. there are reports on the cell penetrating peptide (cpp) based delivery system. it has also been reported that conjugates of cholesterol and sirnas facilitate cellular import ( ) . the aim of this study was to synthesize different sequences of cholesterol coupled antisense pna and to compare its uptake characteristics with a cpp-pna conjugate. the synthesis of pna (anti-dsred pna (agcgcctgtacc), specifi cally targeted to mrna of dsred, a red fl uorescent protein) conjugated to cpp (d-tat) or cholesterol was performed in fully automated synthesizer (prelude, protein technologies, inc.) using continuous solid phase chemistry. to increase the solubility in water, linkers (aeea) and additional charged amino acids were coupled or the sequence of peptide, pna and cholesterol was changed. all compounds were labelled with fitc to confi rm the cellular uptake by fl uorescence microscopy and spectroscopy. cell uptake studies showed that the cpp bound pna was located predominantly in vesicles indicating an endosomal uptake mechanism and subsequent entrapment in vesicles. cholesterol bound pna was also effi ciently internalized. however, it was also located inside vesicles without detectable cytosolic distribution. pna-cholesterol has fewer synthetic steps than pna-cpp. however, it was also located inside vesicles restricting its applicability for mrna targeting. the effi cient uptake might make it a promising cellular delivery agent after further improvements. opioids are gold standards in pain treatments. unfortunately, fast development of tolerance and dependence creates limitations in application of these drugs in chronic pain treatment. over twenty years ago we have proposed development of multitarget medicines as a new avenue of drug discovery. identifi cation of numerous endogenous components that participate in the formation, transmission, modulation and perception of pain signals offers various strategies for the development of new analgesics. neurotensin is an endogenous neuropeptide that play important regulatory role of pain transmission. therefore, it was interesting to develop chimeric analogues that hybridize opioid and neurotensin active components. in such chimeric compounds the possible structural interference may signifi cantly infl uence on interaction of both components with target receptors and/or biological barriers transport systems. in this communication we present synthesis, pharmacological profi le and structural analysis of new potent chimeric compounds. acknowledgements: presented studies have been supported in part with eu grant normolife (lshc-ct- - ) artifi cial ribonucleases based on short peptides. the synthesis of molecules that are capable of nonrandom rna cleavage has found a variety of important applications in molecular biology. for instance, such molecules are used as structural probes for nucleic acids in solution, or in rational design of novel anti-infectives, since rna is the genetic material of many pathogenic viruses. here we represent design and synthesis of peptide-like molecules mimicking the catalytic site of natural ribonucleases (a and t ): series f aa -aa -aa -phe -oalkyl; aa -glu/lys, aa -thr/ser/lys, aa -glu/ lys/thr/arg; series r glu -x -arg -gly -oalkyl; series k glu -x -lys -gly -oalkyl; -gly, -ala, -aminobutyric acid, aminohexanoic acid, p-aminobenzoic acid; series l aa -aa -aa -l (l )-aa -aa -aa , l - , -dioxa- , -dodecanediamine, l - , -diaminododecane; aa -aa -glu, lys, ser, arg. ability of artifi cial rnases to rna cleavage was shown in experiments with mer rna hiv- . the effi cacy of rna cleavage depends on artrnases structure (for example, on location of negative and positive charged amino acids (glu, arg lys) in peptide). the effi cacy of the most active compounds was - % at h incubation. anti-infl uenza activity in vitro of such artrnases was investigated by inhibition of reproduction virus a/hong kong/ / (h n ) in tissue cultures of chorioallantoic membranes (ccm) of - days age chick embryos. acknowledgements: this work was supported by rfbr ( - - a, - - -bel_a), pharmamed ruxo- -n - , the grant of novosibirsk region government for the best young scientists. (ziagen)-( s,cis)- -[ -amino- -(cyclopropylamino)- hpurin- -yl]- -cyclopentene - -methanol is a synthetic carbocylcilic nucleoside analogue with inhibitory activity against hiv. serious and sometimes fatal hypersensitivity reactions have been associated with abacavir. a possible way to increase the side effects is by modifying the known antiviral drugs with various amino acids. the aim of this study was to design and to synthesize of new amino acids and peptide (gly, gly -gly) and thiazole containing (gly, gly -gly) esters prodrugs of abacavir and to explore their activity on the hiv. chemical stability of some purine analogues in the search of new prodrugs effective against herpes simplex virus, series of acyclovir- -[( -hydroxyethoxy)methyl]guanine (acv) esters with peptidomimetics, as well as abacavir-( s,cis)- -[ -amino- -(cyclopropylamino)- h-purin- -yl]- -cyclopentene- -methanol derivatives have been synthesized and tested for antiviral activity. the chemical stability of some of them is studied at ph and . and temperature of °c. a high-performance liquid-chromatografi c (hplc) method was developed for quantifi cation of the unchanged ester concentration. the promelittin represents a readymade cancer pro-toxin; the prodomain inhibits cytolytic activity and the sequence of the prodomain could be manipulated into a substrate for activating proteases. here we present the development of a novel promelittin pro-toxin that can be activated by the serine protease fi broblast activation protein (fap). fap is over expressed on the surface of reactive stromal fi broblasts present in the stroma of human epithelial tumors. fap is not expressed by tumor epithelial cells or by fi broblasts in other tissues, thus making fap a pantumor target for pro-toxin development. peptides containing truncated pro-domain sequences were tested on erythrocytes to determine the optimal prodomain length for inhibiting cytolytic activity. once the length was optimized, modifi ed promelittin peptides were generated that contained previously identifi ed fap substrate sequences in the prodomain. these peptides were digested with fap and tested in vitro against fap+ and fap-cell lines in order to determine their fap cleavage potential and toxicity. our lead promelittin peptide was found to be effi ciently activated by fap and selectively toxic to fap+ cell lines with an ic value in the low micromolar range that is similar to that of melittin. pharmacology and medical peptide chemistry region of highly hydrophobic and the antigenic part of hbsag by using microwave assisted solid phase peptide synthesis (spps). tryptophan at the n terminus of the sequence was added by our research group for fluorescence dedection analysis. this peptide was conjugated with synthetic polyanions. different conjugates comparised each other. the synthesized bioconjugates analysized by choromatographic and fl uorometric methods. the fi nal product peptide was characterized by lc-ms and purifi ed by rp-hplc. the bioconjugates were synthesized using different activation mechanisms and the different initial molar ratios (npeptide/npolymer = , , , , ) of the polyanions and the peptide. also physicochemical properties of the bioconjugates were investigated. the structure and characterization of the synthesized conjugates was analyzed by various choromatographic and fl uorometric methods such as hplc, gpc and fluorescence spectrometer. the reaction of high temperature solid-state catalytic isotope exchange (hscie)( ) between insulin and spillover tritium was studied. hscie reaction with the solid mixture containing mg recombinant human insulin was performed for min at o c using % pd/baso catalyst. the product of the reaction was purifi ed by hplc on waters delta-pak c a . x mm column. tritium labeled insulin with specifi c radioactivity ci/mmol and radiochemical purity of more than % was obtained. performic acid oxidation of cysteine residues and subsequent acid hydrolysis were used to analyze the distribution of tritium. it was demonstrated that specifi c radioactivity of À and b chains amounted to . and . ci/mmol respectively. all the amino acids contained tritium and its content in his residues of the b chain was equal to %. experiments for the evaluation of the biological activity of tritium labeled insulin were performed on cd- - -week-old awake male mice. activity of labeled insulin was compared with the activity of standard insulin. insulin was injected subcutaneously at a dose of . u/kg. twofold statistically signifi cant lowering of glucose level was observed min after injection ( , + , and , + , mmol/l for standard and tritium labeled insulin, respectively). lowering of the glucose level in animals obtaining labeled insulin was the same, as in animals obtaining standard insulin. thus, hscie reaction of insulin with tritium doesn't change its hypoglycemic activity. cortexin is polypeptide (up to kda) brain extract accepted for clinical use in several countries including russia due to its positive effects on memory, attention, and cortical processes. a synthetic peptide analog of cortexin, cortagen (ala-glu-asp-pro), was recently developed. the heptapeptide semax (met-glu-his-phe-pro-gly-pro) is an analogue of the acth( - ) fragment, which is completely devoid of any hormonal activity associated with the full-length acth molecule. both cortexin and semax are currently effectively used for treatment of brain hypoxia and ischemia with comparable clinical benefi ts. however, the cellular and molecular mechanisms underlying the action in the brain are mainly unknown. in present work we studied effects of cortagen, cortexin, semax, its analogue pro-glu-pro (pgp) on glutamate-induced neuronal death and intracellular ca + homeostasis deterioration in cultured cerebellar granule cells. peptide drugs gain increased interest as a new supra-group of therapeutics between the classic-organic, small-molecule drugs and the large biotechnology-derived bio-drugs. they are used in different therapeutic areas like allergy, anti-infection, oncology, obesity, etc… due to their particular structure and biochemical origin, pharmaceutical development of a peptide drug poses special challenges which will be discussed here, exemplifi ed by own research as well as literature data. currently, there are about a dozen classical peptides described in pharmacopoeia, excluding the derived peptidomimetics like ace-inhibitors. these pharmacopoeial peptide-drugs, together with the approved marketing authorisations of new peptidic entities, are a good starting point to look at the desired characteristics. last, the regulatory developmental guidelines are in a general way seldom taken peptide drugs into account, leaving an interpretational gap between the two current main supragroups of therapeutics. after the initial active pharmaceutical ingredient (api) synthesis, analytical characterisation is aimed at integrity and purity evaluation of the api, which is also required for biomedical experiments. in this analytical characterisation, sample treatment issues like solubility and adsorption are considered as well. the chemical and plasma-metabolic stability, a critical parameter for peptides, is to be assessed to obtain kinetic and mechanistic information. functionality is tested in vitro using cell-and organ-based protocols, including ligand binding studies, as well as in vivo encompassing adme and target-organ confi rmation like brain. the pharmaceutical drugability information thus obtained allows further development decisions including required api modifi cations and proof-of-principle drug delivery formulations. lysine dendrimers and starburst copolymers as new carriers of anticancer drugs based on the complexes of platinum and gold. though the complexes of metals such as platinum and gold are very promising compounds for the treatment of different types of cancer, the low solubility complicates their application as medicines. in this work a series of lysine dendrimers of different structure and starburst copolymers of lysine and glutamic acid was studied as solubilizing carriers of anticancer drugs based on the complexes of platinum (cisplatin) and gold. cd analysis showed the small changes in the structure of the polymers upon metal binding. the esem study revealed that the presence of the amino groups on the surface of the carriers is crucial for the metal ligation. the highest content of metal bound was found in the samples with high amount of lysine residues. while the content of gold was rather low in all the samples (about %), the copolymer of lysine and glutamic acid with the ratio : as well as lysine dendrimer of fourth generation had to % of platinum linked to the carrier. the absence of the bromine in the samples in the case of gold complex allowed to assume the covalent bond between metal and polymer. in the same time all the conjugates were highly soluble in water. the peculiarities of the synthesis and the anticancer activity in different cell lines will be discussed. acknowledgements: the work was supported by the "russian science support foundation". prospects for the development of synthetic peptide vaccines against hepatitis c no vaccinoprophylaxis of this disease or cardinal treatment means has been developed till now because of the problems produced by high genetic variability and heterogeneity of hepatitis c virus (hcv) isolates, lack of both hcv infection models and effi cient expression systems for the virus and its components. the problem of anti-hcv vaccine development can be solved via a principally new approach, the creation of artifi cial synthetic immunogenic constructs with the help of bioinformatics and high-throughput screening technologies. two approaches exist towards the development of anti-hcv peptide-based vaccines: one is to develop vaccines that stimulate cytotoxic t-cell response (peptides loading dendritic cells; the so-called "cell vaccines") and the second is to construct immunogenic peptides raising protective antibodies. however, the fi rst approach may lead to the massive hepatocyte death and the development of the heavy hepatic injury during hcv infection. in order to develop peptide immunogenic constructs able to raise antibodies against whole native hcv envelope proteins analysis of amino acid sequences of these proteins belonging to different hcv genetic variants has been performed. this analysis has allowed to choose the most conserved and presumably functionally important protein fragments. immunogenicity of these fragments in the whole protein molecules has been determined and interaction sites for one of the putative hcv receptor, heparansulfate, have been revealed with the help of peptide scanning. despite of the low immunogenicity of the most fragments inside the whole protein, antipeptide antibodies against them have been obtained via immunizing by conjugates of the peptides with certain carriers. two synthetic peptide immunogenic constructs have been developed that have raised antibodies against whole hcv envelope proteins. synthetic immunoactive fragments of endogenous proteins: selection and application for diagnostics and immunotherapy we have selected and synthesized the immunoactive fragments of four endogenous proteins: nucleophosmin, survivin, prion and alpha- subunit of acetylcholine receptor (achr). nucleophosmin and survivin are overexpressed in tumor cells and exhibit an antiapoptotic activity. their detection in tumor cells could be used for tumor differential diagnostics and selection of optimal chemotherapy scheme. accumulation of pathogenic isoform of prion protein in brain causes the neurodegenerative prion diseases and antiprion antibodies as known could be used for immunotherapy of prion disorders. alzheimer's disease (ad) development is accompanied by an accumulation ofamyloid peptides ( a) in brain and destruction of neurons . one of the ad development hypotheses assumed that a when binding to the alpha- achr forms a complex that penetrates into the cells, resulting in the neurons death. we have proposed that alpha- achr could be a new target in the ad therapy and antibodies against alpha- subunit could prevent its binding to a. selected synthetic fragments of these four proteins were able in a free nonconjugated to a protein carriers state induce antibody response in experimental animals. obtained rabbit antibodies against fragments of nucleophosmin and survivin were affi nity purifi ed. antibodies against nucleophosmin detected monoand oligomeric forms of this protein in immunoblotting of hela cells lysates. antibodies against survivin were able to detect this protein in immunohistochemical test of the breast cancer tumor samples. rabbit antiprion antibodies interfered with patigenic prion isoform in scn a neuroblastoma cell culture. it was shown that synthetic alpha- achr fragments induced the antibodies formation in mice with symptoms of ad which penetrated the blood brain barrier, regenerated the spatial memory and normalized the beta-amyloid level in animal's brain. acknowledgements: supported by ras program mcb and rfbr grants - - , - - . investigation peptide-based cyclin a inhibitors: new tools to understand and to regulate the cell cyle-apoptosis signalling pathway the knowledge of the aetiology of cancer has increased considerably in the last two decades. the objectives have moved from unspecifi c cytotoxic chemotherapeutics to the identifi cation of small molecules (and antibodies) with a well defi ned molecular target. proliferation of eukaryotic cells is under control of a series of concerted molecular mechanisms defi ned as the cell division cycle whose progression is tightly governed by members of the cyclin-dependent kinase family. the protein-protein complexes formed between different cyclins and cdks (cdks) are central to cell cycle regulation. these complexes have been object of extensive research in cancer programs. considerable effort has been focused on the development of atp-competitive small molecule inhibitors of cdks. however, in general, these compounds have several alternative protein targets that compromise their demanded selectivity. cdk- was recently suggested to be dispensable for cell proliferation, although this does not appear to be the case for cyclin a, which therefore is defi ned as a more appropriate target for drug design. we have identifi ed an hexapeptide (nbi ) that inhibits the kinase activity of the cdk -cyclin a complex through selective binding to cyclin a ( ). the characterization of the inhibitory mechanism revealed that the hexapeptide does bind neither to the atp site nor to the cyclin recruitment site. a cell permeable derivative of the nbi peptide induces apoptosis and inhibits proliferation of tumor cell lines. we believe that the current structural and mechanisms of action studies on nbi will be useful for characterizing and controlling the important relation between cell cycle and apoptosis signalling pathways. the average size and size distributions of protein, polymer and peptideprotein or polymer-protein mixtures (complexes) and conjugates was investigated using photon correlation spectroscopy with a zetasizer nano zs instrument. the zeta potential measurements of this complex and conjugates were carried out in the folded capillary cell of the zetasizer nano zs instrument the same as used for dynamic light scattering measurements. in this study we investigate that size and zeta potential of synthetic peptide-carrier protein covalent conjugates. synthetic peptides are small antigenic molecules and not strong immunogens because of their small size for this reason, synthetic peptide must be coupling to a suitable carrier proteins or polymers (bsa, klh, ova) for increasing their immunogenity. most of the vaccine development studies are focused on the preparation of a good effective adjuvant. in this study we prepare carrier-protein (bovine serum albumin protein)-synthetic peptide conjugates whit carbodiimide method by using -ethyl - -( dimethylaminopropyl) carbodiimide hydrochloride (edc). for one protein molecule we studied with different ratios of peptides (npeptid/ nbsa) and synthesized the conjugates at these ratios. conjugates are purifi ed by using different column systems. purifi ed conjugates are characterized and the mechanism of the binding process was investigated comparatively with synthetic peptide, bsa and bsa-synthetic peptide physical mixture by using zeta-sizer nano zs depent on the time. an analysis of deletion mutants of the pld d domain defi nes short regions within the pld interacting with ped/pea : implications for the development of peptides-specifi c antagonist. ( ) . several studies have revealed that it regulates cellular functions by binding components of intracellular transduction pathways. recent reports also evidenced that it binds to phospholipase d (pld ) and enhances its stability, resulting in increased intracellular levels of diacylglycerol( ), deregulating protein kinase c signalling and generating resistance to insulin action on glucose transport ( ) . thus, disrupting the interaction between these proteins by a cell-penetrating compound represents a novel strategy for improving insulin sensitivity in target cells. the expression of d domain, (the shortest ped-interacting region with pld ) in l skeletal muscle cells stably overexpressing ped, reduces the interaction of this protein with pld ( ), suggesting that this region could bind ped preventing its interaction with the whole pld and restoring insulin action. aim of this work is the identifi cation of d crucial residues for ped interaction and the development of specifi c antagonists. we expressed three soluble truncated d domains, determining whether binds to ped like the d wild type, by carrying out dose-response elisa. only one of these regions exhibited an effi cacy similar to d -wt and functional cellular data support this evidence. to further investigate the d residues involved in ped binding, we prepared a set of overlapping peptides covering the d -region identifi ed. our results suggest that the n-terminus region of d encompassing residues - is involved in ped/pea recognition and, currently, cellular experiments on peptides are underway. ew-peptide family. one family, two drugs. two dipeptide analogs -l-glu-l-trp ( ) and -d-glu-d-trp ( ) -had been registered in russia as immunomodulator thymogen ( ) and immunosuppressor thymodepressin ( ). these two drugs possessed reciprocal activities in vivo [ , ] . the individual peptides were initially separated by preparative hplc from the crude thymus homogenate and sequenced. dipeptide l-glu-l-trp was the most active in the majority of in vitro and in vivo tests. in the process of sar studies, the "signal" role of l-glu-l-trp in the immune response was discovered, as well as the critical role of manifestation of in vitro effects as well as the infl uence of precise chemical and optical structures on biological activity of different ewpeptide analogs will be discussed. the experiments were conducted on blood neutrophiles, monocytes, thymic epithelial cells, lymphocytes, thymocytes, endothelial cells of human blood vessels and newborn blood cord. we evaluated the peptide effects on the phagocyte activity of cells, on the expression of functionally important membrane molecules and cell activation markers, and on the capacity of cells for mutual adhesion. the dose dependence data of and on such activities as cytokine production, the processes of stimulation and suppression of apoptosis and proliferation of immunocompetent cells will be presented. ion exchange chromatography (iec) is widely used for analysis and purifi cation of biomolecules. we have newly developed polymer-based iec column, named ymc-biopro, specially designed for separation of proteins, peptides and nucleic acids. ymc-biopro iec columns are based on micron @porous and non-porous hydrophilic polymer beads with low nonspecifi c adsorption, and they show higher binding capacity and higher recovery of biomolecules compared to conventional iec columns. the completely spherical and monodispersed beads, with optimal packing technology, provide high theoretical plate number and symmetrical peak shape. excellent resolution is achieved from the high column effi ciency coupled with the excellent selectivity of qa and sp ion exchangers. in this poster, we will show benefi ts of ymc-biopro iec columns and some example cases of superior separation of important biomolecules, such as monoclonal antibody and dna. the analysis of proteins and peptides by rp-hplc is important in the development of well-characterized biotechnology pharmaceuticals. since polypeptides interact only slightly with the stationary phase after desorption, short columns are suited for fast separations. also, by decreasing the particle size of hplc packings, column effi ciency is increased. we demonstrate the use of short hplc columns packed with . um, large pore c silica for ultra-fast biomolecule analysis. due to the short column format, high fl ow rates and optimal separations are possible, with moderate backpressures (< psi), using a conventional high-pressure gradient, binary pump system. narrow-bore lc and lc-ms analyses were performed on a binary or a quaternary hplc system, with detection by uv or ms (ab/mds sciex q trap). the solvent system comprised of acn/water or n-propanol/water plus one or two ion pairing agents (formic acid, tfa, hfba) at . to . %. a fl ow rate of . ml/min ( cm/h), x higher than typical for a . mm id column, allowed for ultra-fast one minute separations of proteins with broad physicochemical characteristics. closely related insulin variants (bovine, sheep, human) may be separated in under . minutes. while the typical hplc run time for the separation of hemoglobin chains on conventional columns ranges from to minutes, runs under two minutes may be realized with the large pore, sub-two micron column. accordingly, fi ve different hemoglobin samples (from different animal species) may be compared after a total of only minutes (includes run and equilibration time). highly reproducible runs of a synthetic peptide mixture can be completed in < minutes, including equilibration with column volumes between runs. intact igg in sheep serum may be separated from albumin in < min. using a n-propanol/water solvent system containing . % tfa. new peptides and triterpene derivatives as dimerization inhibitors of hiv- protease protein-oligopeptide fragmentomics zamyatnin the substantiation and defi nition of the term "fragmentomics" is given. within the framework of this scientifi c direction the theoretical structural-functional analysis of all possible fragments of protein molecule can be performed with the purpose of determination of its sites which could be potential sources of the regulatory oligopeptides. the data on the primary structure of proteins from public protein databases, information from the erop-moscow database ( ) that contains data on the structures and functions of the natural oligopeptides, and special computer program complex were used for it. as a result the natural regulatory oligopeptides both representing exact structures of protein fragments and containing protein fragments were found. the method has allowed also to reveal new potentially active sites of protein amino acid sequence yet not investigated experimentally. it was shown that different fragments of the food protein molecules are involved in amino acid sequences of many natural antimicrobial oligopeptides, toxins, neuropeptides, and hormones. these results confi rmed deep relationship between the basic regulatory systems. in connection with the obtained data, the process of oligopeptide biogenesis, the possibility of natural formation of regulatory oligopeptides from different protein molecules, formation of exogenous oligopeptides pool, and correspondence of the obtained results with the conception of oligopeptide continuum are discussed. a possible practical importance of active fragments of proteins in regulatory processes of a living organism is noted. the binding of peptides containing tyrosine residue to -cyclodextrin cyclodextrins form inclusion complexes with various organic molecules. aromatic amino acid residues bind to -cyclodextrin with deep penetration of the cyclodextrin cavity. in case of oligopeptides binding depends on the peptide conformation. the formation ofcyclodextrin inclusion complexes with the tyrosine residues within three peptides was investigated using steady-state fl uorescence spectroscopy and molecular dynamic simulations. the free energy along the reaction pathway delineating the inclusion of tyrosine's aromatic ring into -cyclodextrin was computed using umbrella sampling molecular dynamic simulation. the association constant and the corresponding association free energy were derived by integrating the potential of mean force over a representative ordering parameter. the three peptides studied consist of eighteen amino acids and the tyrosine residues are located at the position , or . selected sequences are fragments of notch receptors. (notch =ykieavqsetveppppaq, notch =tlsyplvsvvsesltper, notch =pyplrdvrgepleppeps). out of three peptides only notch binds strongly to -cyclodextrin with binding constant similar to that of actyrnhme. the binding of cyclodextrins with phenolic compounds involves nonspecifi c van der waals and hydrophobic interactions and depends on accessibility of tyrosine sidechain. role of carboxyl groups in the secondary structure and function of sturgeon gonadotropin free negatively charged carboxyl groups were selectively modifi ed (neutralized) in sturgeon (acipenser güldenstädti br.) gonadotropic hormone (gth) and subunits. carboxyl groups, in and in subunit, were neutralized by the reaction with glycine ethyl ester. investigation of re-associated -dimers (recombinants) comprising one or both modifi ed subunits showed that specifi c hormonal activity was completely lost while immunoreactivity was lowered in comparison with that of the standard -dimer. cd-spectroscopy of the modifi ed subunits did not indicate considerable changes in their spatial structure. conclusion was made that free cooh-groups of gth are important as bearers of the negative charge necessary for the hormone activity on the level of the hormone-specifi c membrane receptors. wagner, nathaniel; dadon, zehavit; yishay, eliya; ashkenasy, gonen ben gurion university of the negev, israel living cells can process rapidly and simultaneously multiple extracellular input signals through the complex networks of evolutionary selected biomolecular interactions and chemical transformations. recent approaches to molecular computation have sought to mimic or exploit various aspects of biology. a number of studies have adapted nucleic acids and proteins to the design of molecular logic gates and computational systems, while other works have affected computation in living cells via biochemical pathway engineering. we described recently the graph structure and experimental analysis of a self-organized synthetic peptide network ( - ). the system was designed rationally to operate in neutral aqueous solutions based on sequence selective auto-and cross-catalytic template-directed coiled-coil peptide fragment condensation reactions. here we show that such de novo designed synthetic networks can also mimic some of the basic logic functions of the more complex biological networks. consequently, we describe experimental and simulation studies that highlight the possibility of segments of the networks to express all sixteen two-input boolean logic functions ( , ) . many studies deal with the folding of double helices in nucleic acids. in contrast, much lesser attention is given to double helices in peptides. nevertheless, the investigation of peptides with alternating l-and d--amino acids, like gramicidin a, shows various double helix patterns with antiparallel and parallel arrangements of the strands. in this study, we want to give a systematic overview on the possibilities of double helix formation in -peptides on the basis of ab initio mo theory. according to general principles, double helices with characteristic intermolecular hydrogen bonding patterns were generated and verifi ed as minimum conformations at the hartree-fock level employing the - g* basis set. the calculations show several types of antiparallel and parallel double helices with different stability, which is strongly infl uenced by backbone substituents. the dengue virus causes a spectrum of clinical symptoms, ranging from mild dengue fever to the severe forms of dengue hemorrhagic fever and dengue shock syndrome. there are four dengue virus serotypes (den - ). the dengue virus ns protease is an attractive target for development therapeutics against dengue. the aim of the study was to investigate the prime side specifi city of dengue virus ns protease substrates using proteochemometrics, a new technology for drug target interaction analysis. a set of internally quenched peptides were designed using statistical molecular design (smd), synthesized and assayed with proteases of four subtypes of dengue virus for michaelis (k m ) and cleavage rate (k cat ) constants. the obtained data were subjected to proteochemometrics analysis, concomitantly modeling all peptides on all the four dengue proteases, which yielded highly predictive models for both activities. the interpretation of the models suggested that considerably differing physico-chemical properties of amino acids (aa) contribute to k m and k cat activities. it was found that for k cat activity only p ' and p ' prime side residues played important role, while for k m all four prime side residues, p '-p ', were important. high cleavage rate (characterized by k cat ) was obtained for peptides having small aa (ser, gly, ala) at the p ' position and small or fl exible aa at the p ' position. for high affi nity (low k m ), at the p ' position aa should be small and moderately hydrophilic, while acidic residue is highly unfavorable. for the p ' position, the most favorable was trp; however, this position allowed a broader diversity of aa. for the p ' position, cys was more benefi cial than the aa present in native cleavage sites of the dengue polyproteins. for the p ' position, the best affi nity would be obtained with ala, gly or cys. the models may be used to modify each p' substrate position to optimize separately substrate affi nity and cleavage rate for den - proteases. identifi cation of novel bioactive peptide sequences from human proteins for the development of potential therapeutics many protein-protein interactions are facilitated by the binding of peptides recognition modules to short linear motifs within the target proteins's primary sequence. therefore, synthetic peptides based on parent sequences of human proteins containing functional motifs are useful tools to uncover protein signaling and interactions. to date, peptide studies typically derive sequences from a single identifi ed protein or (at the other extreme) screen random combinatorial peptides, often without knowledge of the signaling pathways targeted. the objective of the novel bioinformatic approach presented here was to determine whether rational design of oligopeptides specifi cally targeted to potentially signaling-rich juxtamembrane regions could identify modulators of human platelet function ( ) . the aim of this project was to identify peptides that span residues strongly conserved in the corresponding proteins in other species (orthologues), but that differ from those in related human proteins (paralogues). synthetic selection rules to avoid unfavorable amino acid combinations and excessively hydrophobic peptides were devised to reduce the risk of by-products during synthesis, postsynthetic degradation and solubility problems. high-throughput in vitro platelet function assays of fatty acid-modifi ed cell-permeable peptides corresponding to these regions identifi ed many agonists and antagonists of platelet function. the combined bioinformatic and experimental screens of human protein subsequences can therefore be used to validate functions of candidate proteins and provide templates for the development of potential therapeutics. denessiouk, konstantin; denesyuk, alexander; johnson, mark s. Åbo akademi university, finland many small molecule ligands (or parts thereof) with important roles in the biochemistry of the cell are recognized by different proteins with different cellular functions. these proteins do not necessarily share a common fold since their three-dimensional structures can differ completely such that they are considered to be unrelated proteins. surprisingly, we see on numerous occasions that similar ligands are often recognized in a very similar way by means of a common structural motif formed from main-chain elements of several consecutive amino acids within the ligand-binding site of the proteins. these short polypeptide segments can be found in many protein folds, and variations in their structure can often explain different elements of protein function. in particular, we have characterized the presence of a nucleotide binding motif present in more than protein folds that function in part to recognize the adenine ring system in many essential biological ligands (e.g. atp, nad(p), fad, fmn, coa, etc.). furthermore, a study of unrelated folds among pyridoxal phosphate dependent enzymes led to the characterization of a c nn structural motif for protein recognition of phosphate ions common to fold-representative protein structures that belong to different folds. interestingly, amino acid side chains play little or no role in ligand recognition, explaining the evolutionary independence of such binding mechanisms (i.e. the motifs are found in unrelated folds), while possibly refl ecting the types of interactions that were characteristic of the earliest protein-ligand interactions during early stages of molecular evolution. bioinformatics meets medicine: structure-based peptide design as a basis for drug development here, we present different structure-based approaches on the design of peptides mimicking the whole surface, a specifi c region of a protein or even tumor markers. the potential of such methods stretches from the development of peptide-derived drug candidates to immunological screenings. mimicry of binding sites: we have developed superficial, a program that proposes peptide libraries representing the entire surface or just regions of proteins. these peptides can be synthesised, e.g. using the spot-synthesis technique, and tested for their ability to mimic linear as well as non-linear binding sites. as a proof of principle, a library of peptides representing the surface of lysozyme was generated starting from a crystal structure of a lysozyme-hyhel- complex. adjacent sequence segments were linked by spacers to conserve local conformations. disulfi de bonds were generated to tether the peptides. binding assays against the hyhel- antibody identifi ed several peptides representing the non-linear recognition site of the lysozyme. translation of a tumor marker into peptides: the tumor-associated carbohydrate antigen gd is an established target for immunotherapy in neuroblastoma. we proposed peptidic mimics of this ganglioside for active immunisation against the tumor marker gd using molecular modelling. the ability of these peptides to mimic the carbohydrate moiety was verifi ed with in silico docking experiments. in a next step they were successfully tested as mimotopes in a mouse model. design of switchable peptides: photo-switchable compounds are becoming increasingly popular for a series of biological applications. goal is the reversible photo-control of structure and function of biomolecules. a required death domain for the binding of proapoptotic proteins (e.g. bak) to the hydrophobic groove of anti-apoptotic proteins is the bh helix. inserting the photo-reactive compound hemithioindigo into this short peptide, stabilization towards proteolytic degradation is achieved. the identifi cation of peptides that bind to antibodies is an important step in characterizing antibody specifi city in order to study molecular recognition. numerous approaches have been developed to select peptides that bind with desired specifi city and affi nity to antibodies of interest. in our study peptide arrays produced by spot technology were used to develop and optimize binders to antibody derived by mutations from the anti-p (hiv- ) single chain fv antibody, cb - . three mutations were introduced in the complementarity determining region of the light chain being in close proximity with epitope-homologous peptide. this mutated antibody had completely lost its affi nity for the epitope-homologous peptide. a substitutional analysis of epitopehomologous peptide was performed on cellulose, in which all positions of the peptide sequence were substituted by each of the naturally occurring amino acids. the peptides representing the binding activity in spot membranes were synthesized using solid phase synthesis and their binding activity was confi rmed by fl uorescent polarization method. a substitutional analysis indicated that binding to mutated antibody could be restored and improved by combining favourable substitutions at the n-and c-terminal residues of the epitope-homologous peptide. this approach has allowed us to generate binders from non-binders to mutated antibody in high micromolar range. it is well known that physiological regulatory peptides that act as hormones and neurotransmitters are produced by the specifi c cleavage of their precursor proteins that per se have no biological functions. during these processes, many fragmented peptides are also produced from the same precursor proteins. it is expected that they may have various unexpected biological activities, but their biological functions have not been investigated in depth. the neutrophil-activating peptides we recently purifi ed turned out to be the peptides that are cleaved from mitochondrial proteins by proteolysis, suggesting that fragmented peptides produced by maturation and degradation of functional proteins may also have various biological functions [ , ] . therefore, we named such functional cryptic peptides hidden in protein sequences cryptides, and those cryptides that are derived from mitochondrial proteins "mitocryptides" in particular ( ). we then identifi ed many mitocryptides by the combined investigation with "dry" and "wet" experiments, i.e., the sequences of mitocryptides that activate g proteins were predicted based on the distribution of charged and hydrophobic residues and their activities were examined on granulocytic cells. receptors for these peptides were also characterized by the direct cross-linking experiments between peptides and their targeted proteins. moreover, it is demonstrated the presence of a novel signaling mechanism involving mitocryptides. the comprehensive identifi cation of cryptides is expected to lead to the elucidation of various cryptic signaling mechanisms. the -benzoylphenylalanine (bpa) residue is widely used as a photoaffi nity label for the study of intermolecular (peptide) ligand-protein (receptor) interactions, where it is thought to function by hydrogenabstraction from met residues followed by covalent c-c bond formation of the resulting radical pair. we are carrying out detailed studies of this reaction in a series of fi ve, structurally rigid, hexapeptides of general sequences boc-u x bu y mu z -ome and boc-u x mu y bmu z -ome, where b = l-bpa, u = aib, m = l-met, and u x +u y +u z = , with a view to determining the effects of spacer length (u y = - ) on the rate of the intramolecular excited state reaction and the chemical and d-structures of the resulting products. the triplet state lifetimes of the bpa residues in the compounds, determined in a deoxygenated, dilute, acetonitrile solution by laser fl ash photolysis, vary in the following order: ubu mu, = ns; umu bu, = ns; ubumu , = ns; ubmu , = ns; and ubu m, = ns. in addition to the information these data provide on the structural requirements for intramolecular excited state quenching in the molecules, they also serve to defi ne the conditions necessary for optimal intramolecular reaction in preparative photolysis experiments. accordingly, the products resulting from ubu mu and ubumu have been prepared in high yields, isolated, and structurally characterized by hplc, mass spectrometry, nmr, and cd techniques. for each of the two photoinduced macrocyclizations, two diastereomers, arising exclusively from the bpa diradical regioselective attack on the met s-methyl group, were found. gattin, zrinka; van gunsteren, wilfred eth, switzerland during the past decades, the dependence of the secondary structure of peptides on their amino-acid sequence has been the focus of numerous investigations. in addition to naturally occurring peptides based onamino acids, peptides based on -amino acids, have raised particular interest because of their potential for pharmaceutical use. these peptides often have high folding propensities and it has been found that peptides with as few as four residue may fold into a stable secondary structure. -peptides offer the possibility to investigate the folding-unfolding equilibrium in the context of very small systems, therefore they have also been the subject of a number of investigations based on atomistic molecular dynamics (md) simulations which revealed that the foldingunfolding equilibrium typically occurs on time scale of nanosecond to tens of nanoseconds. how the backbone bound heteroatoms (oh, nh ,f) infl uence the structure of a peptide is little known by now, both experimentally as well as theoretically. we performed several md studies on the conformational behavior of centrally placed hala( -met) fl uoro and hydroxy analogues, hala( -f) and hala( -oh), as function of chirality and level of substitution to investigate the infl uence of these factors on secondary structure formation and stability. all -peptides were simulated in methanol solution at temperature of k and a pressure of atm using the gromos biomolecular simulation software and the gromos a force fi eld. the ensembles of trajectory structures were analyzed in terms of conformational space sampled by the peptide, folding behavior, structural properties such as hydrogen-bonding and in terms of the level of agreement with the available experimental nmr data, noe's and j-coupling constants. the nitroxide spin-labelled , -cyclic amino acid poac was synthesized, resolved and its absolute confi guration assigned (k . wright et al., tetrahedron, , , - ) in order to be used as a spin-probe to evaluate the -helicalpeptide secondary structure. a series of -hexapeptides, b o c -a c p c -p o a c -p o a c -a c p c -a c p c -a c p c -o m e , b o c -a c p c -p o a c -a c p c -p o a c -a c p c -a c p c -o m e , boc-acpc-poac-acpc-acpc-poac-acpc-ome, and b o c -a c p c -p o a c -a c p c -a c p c -a c p c -p o a c -o m e , based on the ( r, r)-poac enantiomer, combined with ( s, s)-acpc for confi gurational homogeneity of the amino acid components, was designed. in these hexapeptides two poac residues are incorporated at positions i, i+n (n = - ) to observe conformation-related spin-spin interactions. the peptides were synthesized by n-to -c chain elongation of n -boc protected peptide segments in solution. their conformational analyses, performed by cd, ft-ir absorption and esr spectroscopic techniques, will be also described. computational design has been successful in creating new proteins. we focus on the design of a -sandwich protein which is challenged by general problems as h-bonds between neighboring strands being dependend on a tightly packed hydrophobic core and aggregation. an improved version of our program propac was tested to repack the backbone of several natural protein structures with high reproducibility. starting with backbone coordinates we found amino acid sequences by packing amino acid side chains with defi ned conformations (rotamers) into the core of -sandwich proteins. in a fi rst approach we have assembled four synthetic -hairpins on a cyclic peptide template (tasp) to form a -sandwich with two identical four-stranded antiparallelsheets. improvement of surface residues reduced the aggregation of the proteins. spectroscopic analysis shows a fold with a free energy near - kj/mol and confi rms the -structure by cd and ftir. we improved the design to a partial asymmetric -sandwich assembled from three different -hairpins. the synthesized molecule was the fi rst -sandwich molecule showing a defi ned fold in d-nmr. this data and results from the symmetric betabellins ( ) suggest that symmetrical sheets do not fold into defi ned structures. to synthesize completely asymmetric sheets we simplifi ed the synthesis of -sandwiches by directed coupling of two four-stranded antiparallel -sheets. the computed proteins were selected for tight packing of the hydrophobic core and analyzed for a stable fold during dynamic simulations. one of our goals is to fi nd a correlation between the experimentally determined protein stability and the parameters used for the selection of the residues in the hydrophobic core and at the surface. biocatalyst-catalyzed peptide synthesis using inverse substrates as acyl donor sekizaki previously we reported that the p-amidinophenyl esters behave as specifi c substrates for trypsin and trypsin-like enzymes. in these esters the site-specifi c group (charged amidinium) for the enzyme is included in the leaving group portion instead of being in the acyl moiety. such a substrate is termed an inverse substrate ( ) . inverse substrates allow the specifi c introduction of an acyl group carrying a non-specifi c residue into the trypsin active site without recourse to a cationic acyl moiety characteristic of conventional substrates. we also showed in an earlier study that inverse substrates were applicable to enzymatic peptide synthesis. therefore, a general method for the preparation of a variety of inverse substrates would be valuable. we designed two series of new type inverse substrates, p-(amidinomethyl)phenyl and m-(amidinomethyl)phenyl esters derived from n-(tert-butyloxycarbonyl)aminoisobutylic acid, were prepared. we also analyzed the kinetic behavior of trypsin towards these synthetic esters ( ) . they were found to be readily coupled with an acyl acceptor such as l-alanine p-nitroanilide to produce dipeptide. the optimum condition for the coupling reaction was studied by changing the organic solvent, ph, and acyl acceptor concentration. the optimum standard condition was selected as follow: acyl donor (inverse substrate), mm; acyl acceptor (l-alanine p-nitroanilide), mm; enzyme, m; % dmso-mops ( mm, ph . , containing mm cacl ); Ž. an -aminobutyric acid containing dipeptide was obtained in high yield. streptomyces griseus trypsin was a more effi cient catalyst than the bovine trypsin. it was found that the enzymatic hydrolysis of the resulting product was negligible. peptides derived from nk- selective for phosphatidylserine as novel cancer agents chemotherapeutic agents, commonly used as anti-cancer drugs, have severe side effects, also affecting healthy human cells. natural antimicrobial peptides, and their derivatives, have gained interest as potential anti-cancer agents. under normal conditions, due to the asymmetric distribution of plasma membrane lipids across the bilayer, mammalian cells comprise phosphatidylserine (ps) only in the inner leafl et. in the case of malignant transformation inner leafl et ps can move to the outer leafl et and act as a surface marker. the surface exposure of negatively charged ps on various tumour cells makes these cells susceptible to killing by cationic membranolytic peptides such as nk- ( ).the aim of this study is to develop short peptide sequences still acting selectively towards ps exposed on cancer cells without damaging healthy cells. in order to use this new strategy with ps-specifi c peptides it is necessary to analyze the lipid composition of mammalian cancer and non cancer cell membranes. further as a basis for peptide activity studies the biophysical characteristics of cancer cell membranes and healthy counterparts with respect to lipid composition were determined by investigation of liposomal mimics composed of phosphatidylcholine and/or phosphatidylserine by dsc and x-ray. these model systems were also used to screen the activity of a series of peptides derived from nk- . fluorescence spectroscopy was applied to test the release of fl uorescence marker molecules from liposomes composed of solely ps, pc or pc/ps mixtures in the presence of various concentrations of peptides. data revealed that some nk- derived peptides have a high affi nity towards ps causing signifi cant leakage of liposomal content, whereas healthy mammalian cell mimicking pc liposomes were not affected. optimized peptides resulting from these experiments will be used for in-vitro studies on prostate cancer cell lines. in most moths, the sex pheromone production is regulated by pheromone biosynthesis-activating neuropeptide (pban), a - amino acid neuropeptide that is released from the subesophageal ganglion into the hemolymph in response to physiological and environmental cues. pban exerts its pheromonotropic effects by binding to pbanr, a member of the rhodopsin-like family of g protein-coupled receptors (gpcr) that is predominantly expressed in the pheromone-producing cells of the female pheromone gland. the structure-activity relationship studies for bombyx mori pban have revealed that the shortest peptide with pheromonotropic activity is the c-terminal pentapeptide-amide, pban( - )-nh (fsprl-nh ), and that the c-terminal amide group is required for the pheromonotropic activity of pban. in this study, we have analyzed the solution structures of the c-terminal decapeptides derived from bombyx mori pban with an amidated and a free c-termini by two-dimensional nmr. the pheromonotropically active decapeptide-amide, pban( - )-nh (srtryfsprl-nh ), and its inactive counterpart, pban( - )-oh (srtryfsprl-oh), were dissolved in three kinds of solvents: ( ) mm sodium phosphate buffer (ph . )/ mm nacl/ . % nan in %(v/v) h o/ %(v/ v) d o (buffer a), ( ) mm dodecyl phosphocholine (dpc)-d in buffer a, and ( ) %(v/v) , , -trifl uoroethanol (tfe)-d in buffer a. the nmr data indicated that these decapeptides did not adopt specifi c conformations in buffer a, but they adopted specifi c conformations in the presence of dpc micelles or tfe. these peptides exhibited different chemical shifts for some protons in all the solvents used, and they took similar but different conformations in the presence of dpc micelles. the conformational difference between these peptides may refl ect their difference in pheromonotropic activity. structuring and membrane interactions of the human antimicrobial cathelicidin ll cathelicidins are a family of vertebrate host defence peptides with both a direct capacity to inactivate microbes and to modulate components of the innate and adaptive immune systems. they are characterized by a conserved pro-region carrying individual, highly variable antimicrobial sequences, that become active only after proteolytic release, and with quite different structural and aggregational features that lead to differential biological effects on prokaryotic or eukaryotic cells. ll- is the only human cathelicidin, and displays a broad-spectrum, medium sensitive antimicrobial activity in vitro that is accompanied by some cytotoxicty towards eukariotic cells at antimicrobial concentrations, and a strong capacity to modulate host immune and healing processes at lower concentrations. these activities likely all involve interaction with biological membranes at some point, and are related to its amphipathic, helical structure and strong tendency to aggregate in specifi c conditions. the latter is an evolved feature absent in some primate orthologues, whose activities appear more limited to a direct antimicrobial action. the structural and aggregational behaviours of ll and selected primate orthologues were systematically investigated by biophysical and biochemical methods. these included cd spectroscopy in different buffers, sds micelles or anionic or zwitterionic luvs, transmission ftir spectroscopy, dye release from liposomes, and atr-ftir spectroscopy on supported lipid monolayers, followed by atomic force microscopy to probe morphological effects on lipid order. data was also collected from antimicrobial, cell lysis and cytofl uorimetric assays, giving a more complete picture of the possible mode of action of these helical peptides, and highlighting the importance of biochemical parameters such as structuring and dimerization/oligomerization processes in the selective interaction with biological membranes, leading to cytotoxic or immunostimulatory effects. oligomeric structure of fowlicidin- , an antimicrobial/ anti-endotoxic peptide from the family of cathelicidin, in lipopolysaccharide bilayer a recurring theme in the structure-function correlation studies of the cationic antimicrobial peptides (amps) is that the formation of oligomeric structures in lipid environments by amps. self-assembly of amps may result disruption of the membrane (outer/inner) structures of the microorganisms by forming pores or ion channels. however, high-resolution oligomeric structures of amps in lipid environments are diffi cult to obtain. to-date, oligomeric structure at atomic resolution of any antimicrobial peptide has not been reported. secondary structures of amps are usually obtained in perdeuterated sds or dpc micelles, as a mimic to the inner cytoplasmic membrane, by nmr spectroscopy. cathelicidins comprise a major family of host-defense antimicrobial peptides in vertebrates. these peptides are synthesized as a part of large precursor proteins containing a well conserved cathelin domain and an extremely variable, in terms of length and amino acid compositions, cterminal region. the c-terminal part of the cathelicidins is bestowed with antimicrobial and lps neutralizing activities. here, we report a tetrameric structure of a -residue active fragment of fowlicidin- or vk , one of the fi ve cathelicidins found in chicken, in lipopolysaccharide by nmr spectroscopy. the tetrameric structure of the vk determined from trnoe is highly helical. a large number of trnoe cross-peaks were observed connecting residues far apart in the sequence. calculated structures reveal an anti-parallel arrangement of four helices. the interface of the tetramer appears to be rich in polar or charged residues delineating a plausible pore or channel. most of the non-polar residues are found to be exposed indicating plausible interactions with non-polar fatty acyl chains of lps or with membrane in general. the oligomeric structure of vk peptide may be useful to understand structure/activity relationship, in particular pore formation, by the helical antimicrobial peptides. the interaction of the antimicrobial peptide nk- with different lipid systems antimicrobial peptides are important components of the natural defense system of most living organisms against invading pathogens. these are relatively small (below kda), cationic and amphipathic peptides of variable length, sequence and structure. in this study, the antimicrobial peptide nk- , which is a amino-acid residue derivative of the cationic core region of nk-lysin, has been used. the used membrane mimetic model systems have different dimensionality: two-dimensional (monolayers at the air-liquid interface) as well as three-dimensional (vesicles, micelles) systems of different phospholipids. the aim of this study was to investigate the infl uence of peptide-lipid interactions on the lipid structure and vice versa on the secondary structure of the peptide. the peptide secondary structure was measured by cd (circular dichroism spectroscopy) in bulk and irras (infrared refl ection-absorption spectroscopy) at the air-liquid interface. the structure of lipid langmuir monolayers has been examined by numerous techniques such as pressure-area isotherm measurements, fl uorescence and brewster angle microscopy and x-ray techniques. charged as well as zwitterionic phospholipids have been used to study the adsorption of nk- . the peptide adsorbs at the air-buffer interface due to its amphiphilic character. it also inserts into uncompressed phospholipid monolayers. the peptide reorients from random coil in bulk to -helix lying fl at at the interface. the incorporation of nk- into a dppg monolayer leads to a different orientation (either -helix with an oblique orientation or random coil) and to the fl uidization of the aliphatic chains. nk- infl uences also the structure of ordered dppg domains. to assess the location of the peptide nk- in the lipid matrix, specular x-ray refl ectivity studies were carried out. wadhwani, parvesh; reichert, johannes; buerck, jochen; ulrich, anne s. karlsruhe institute of technology, germany antimicrobial peptides (amps) constitute an essential part of innate immunity and act against an external microbial invasion where as cell-penetrating peptides (cpps) can carry a biologically functional molecule through the cell membrane and are relevant in drug delivery developments. fusogenic peptides (fps) are active on membraneinterfaces and are instrumental in fusion of membranes which is vital for various fertilization and viral processes. membrane fusion properties of short amps and cpps have been seldom tested and have therefore eluded the attention of most investigators, instead only their cytotoxicity is investigated. there is general lack of understanding if these peptides are fusogenically active. we have investigated the ability of amps and cpps to trigger membrane fusion. as an example, hiv- -fusion peptide fp is used as benchmark to compare fusion activity of various amps and cpps. most fps are believed to be unstructured or conformationally fl exible ( -helix or -sheet); therefore the secondary structure of the peptides before and after the fusion reaction is investigated and correlated to their respective ability to execute membrane fusion. our results show that various amps and cpps which are capable to switch their secondary structure are also able to promote fusion to an extent that is even higher than the known hiv- fusion peptide fp . these results will be presented in the poster. interaction of the minimal active peptide sequence of human growth hormone releasing factor with negatively charged liposomes zschörnig, olaf; thomas, lars; weigelt, heiko; köhler, guido university of leipzig, germany the most bioactive peptides such as hormones and neurotransmitters do not exist in an ordered structure in aqueous solution. the conformation of the peptide changes from this fl exible unordered structure into an inherent one, only when it reaches a biomembrane. that's why it is important to investigate the such peptides like growth hormonereleasing factor (ghrf) in the presence of lipid bilayers. human ghrf is an amidated peptide consisting of amino acids residues. a lot of studies has show that the residues n-terminal part are the active core of the peptide. we studied the interaction of human ghrf ( - ) with phospholipid membranes. the interaction of ghrf ( - ) with the liposomes was investigated fl uorescence spectroscopy. to detect a fl uorescence signal the peptide were labelled at fi rst, th, th and th position with dansyl. the fl uorescence intensity of ghrf ( - ) at different lipid-peptide-ratios were analysed using a model, which includes the hydrophobic and the electrostatic interaction between the peptide and the phospholipid membrane. the hydrophobic binding constant of ghrf( - ) and its effective charge were determined using this model. further the peptides position in the membrane were investigated using spin labelled phospholipids, which are able to quench fl uorescence over large wavelength arrays. the quenching effi ciency will decrease by increasing the distance between fl ourophore and spin probe. the use of mol% tempo-pc, -doxyl-pc, -doxyl-pc and -doxyl-pc in the phospholipid composition of the liposomes allows the determination of the peptides membrane penetration depth. all results indicate, that the binding of ghrf (aa - ) to phospholipid membranes is increased strongly by increase of the surface charge density of the liposomes. the peptide is arranged parallel to the membrane surface and is localized in membrane-water-interface of the liposomes. folding propensity and biological activity of selected antimicrobial peptides bozzi, argante ; di giulio, antonio ; aschi, massimiliano ; rinaldi, andrea c. university of l'aquila, italy; university of cagliari, italy the innate immunity of multicellular organisms relies in large part on the action of antimicrobial peptides (amps) to resist microbial invasion. crafted by evolution into an extremely diversifi ed array of sequences and folds, amps do share a common amphiphilic -d arrangement. this feature is directly linked with a common mechanism of action that predominantly (although not exclusively) develops upon interaction of peptides with cell membranes of target cells. it is generally agreed that amps are essentially unstructured in the aqueous phase and fold upon contact with the membrane, adopting an amphiphilic fold. this favours absorption of peptides onto lipid bilayer and their subsequent integration into the membrane with expansion of the outer leafl et, which in turn leads to membrane thinning and permeabilization. however, recent observation suggest that things might be more complicated than previously believed. indeed, md simulations coupled to cd spectroscopy, studies with model membranes, and antimicrobial assays, have shown that, at least for some peptides, a signifi cant correlation exists between the conformation adopted by the peptide in solution, i.e. before the interaction with membranes, and its antimicrobial activity. the linear peptides we have studied following this approach include two members of the frog skin-derived temporin family, namely temporin a and temporin l, and two members of amphibian bombinins h, i.e. bombinins h and h . we observed that the presence of a partially folded structure in water solution may facilitate, both thermodynamically and kinetically, the peptide folding in the microbial membrane, and thus favour biological activity. looking for such built-in conformational characteristics could well help to rationalize the different spectrum and level of activity recorded for cationic alpha-helical amps on membraneenveloped targets, and assist the design of improved analogs and biomimetic synthetic peptides with antibiotic properties. investigation and computational modelling of antimicrobial peptides linser the uprising resistance of pathogenic bacteria against treatments with conventional antibiotics emerged an acute search for alternatives. one class of promising alternatives are naturally occurring antimicrobial peptides. we present a comparative study of computational modelling of peptide properties with structural characterization of the interaction and antibacterial or haemolytic activity of three peptides (nk-cs, nkcs-[lp] and nkcs-[aa]). we compared computational interaction models with measurements of antibacterial and haemolytic activity, small angle x-ray and surface plasmon resonance data, and structure predictions by circular dichroism (cd). all peptides were active against escherichia coli (gram negative) and staphylococcus carnosus (gram positive) bacterial cultures, but the haemolytic properties against human red blood cells were found to be poor and indicated the peptides' selectivity. cd studies of the peptide secondary structure confi rmed the prediction of peptide helicity. the antibacterial activity can be correlated with a change of the hexagonal phase transition temperature of -palmitoyl- -oleoyl-snglycero- -phosphoethanolamine (pope) as determined by small angle x-ray scattering (saxs). the calculated peptide membrane affi nity is not related in linear way with the antibacterial activity. the reason for this might be aggregation as shown by surface plasmon resonance. the inverse hexagonal phase transition temperature was increased by the peptides and this promotes a positive curvature of the membranes. we assume that this curvature fi nally leads to the disruption of the model membranes. in summary an over all helical structure, electrostatic and hydrophobic parameters as well as strong amphipaticity are good measures to describe antibacterial peptide interaction. kier, brandon; andersen, niels university, united states over the last decade, -hairpins have emerged as excellent model systems for probing the intrinsic stabilities of portions of -sheet structure and for studying the sequence dependence of the folding dynamics of -strand association and alignment. for many decades, it has been established that peptide helices can be substantially stabilized ( - kj/mol) by starting the sequence with an n-cap (acetyl, asp or sede). no comparable strategy has appeared for reducing the end-fraying of -hairpins. fully folded models of hairpins have typically been constructed by conversion to a cyclic system: inserting another turn favoring sequence (e.g. d-pro-gly, pg) at the end of the hairpin strands. we now report a set of favorable through-space interactions that can serve to cap -hairpins. the discovery followed from nmr studies of ac-w-ixgk-wtg (x = p, n). these studies indicated a favorable face-to-edge (fte) w /w interaction which also allows for a g -hn to w indole ring h-bond and the sequestration of the thr hydroxyl by h-bonding to the acetyl. these interactions produce spectroscopic diagnostics: a cd exciton couplet together with extreme upfi eld shifts for he of the c-terminal trp ( . - . ) and hn of the c-terminal gly ( . - . ppm). these feature disappear when x = l-pro and the gly-hn shift returns to its coil value (as in ac-wtg) upon removal of the n-terminal ac. in its most favorable application, ch ch co-wipglwtgps, we were able to establish that dgu at k was . kj/mol ( . % folded) by backbone hn h/ d exchange protection measures. we now report that these interactions are retained in longer hairpins. peptides of the general formula, ac-w-(zz)ningk(zz)n-wtg (n = , , or ) display enhanced fold stability. in each case, the ac is essential to prevent end-fraying and to elicit the full set of chemical shift diagnostics of the " -cap". the unique structural features of polyproline peptides to adopt an extended, relatively rigid polyproline ii (ppii) backbone conformation in aqueous solution, has led to their widespread application as a "molecular ruler" in biological and biophysical investigations. in the left handed ppii helix the peptide bonds show the all-trans conformation resulting in an interterminal distance, which increases by . to . Å per proline residue. the same backbone conformation has been identifi ed in important proline rich protein-protein recognition motifs involved in the regulation of physiological processes like transcription, cell growth, cytoskeleton rearrangement and postsynaptic signal transduction via modular sh , ww or evh domains. to investigate the structural features of polyproline and proline rich sequences by fl uorescence resonance energy transfer (fret) and other spectroscopic methods, these peptides were labelled n-and c-terminally with different fl uorescent dyes. for synthesis of homopolymeric polyproline peptides with a sequence length of more than amino acids a fragment condensation strategy has been applied to prevent the occurrence of shorter side products which can perturb distance measurement by fret. spectroscopic analysis of these peptides indicated that even for short polyproline sequences (< residues) there is a remarkable deviation from the expected ppii conformation. fret measurements revealed that polyproline peptides show an increase of the mean interterminal distance of about . Å per proline residue. single molecule fret measurements of polyproline peptides with a length of amino acids point to a heterogeneous ensemble of different conformations caused by randomly distributed cis conformations resulting in diverse species with different interterminal distances. similarly the structural consequences of the integration of non-proline amino acids in a polyproline sequence have been investigated by fret. the unusual helix stability of a vegf mimetic peptide understanding helix stability and formation is a prerogative to elucidate mechanism of protein folding and design helix peptide with specifi c activity. peptide helix is a simple model system in which various contributions to helix formation can be dissected and understood qualitatively. many strategies have been pursued to design peptide helices and notable results have been achieved even with very short sequences, but mainly these methods rely on the use of non natural amino acids or introducing constraints. in this communication, we report the stability characterization, via cd, nmr and md studies, of a designed, -helical, -mer peptide, composed only of natural amino acids, which activates the vegf-dependent angiogenic response ( ). this peptide shows an unusual thermal stability whose structural determinants have been determined. two factors, the n-terminal region and an hydrophobic interaction i, i+ , are found as playing a mayor role of this remarkable stability ( ) . these results could have implication in the fi eld of protein folding and in the design of helical structured scaffolds for the realization of peptides to be applied in chemical biology. adiponectin, a cytokine secreted by adipose tissue, which has been shown to affect lipid and glucose metabolism, attracts interest because it is a target for therapeutics in the metabolic syndrome. the molecule has a tendency to associate to form various multimers. although this multimer formation could modulate its biological function, the details of this mechanism are still unclear. thus studies on this system from the aspect of molecular assembly are interesting. the molecule consists of three domains, i.e. a variable (v), a collagen-like (c) and a globular (g) domain. the structure of the g domain determined by x-ray crystallography showed that it exists as a trimer but the remaining c and v domains have not been well characterized. we synthesized various parts of the molecule, c, g, vc, cg, and full length vcg in e. coli. the cd spectra of cg and vcg showed a positive peak around nm which is the hallmark of collagen structure. this may be attributed to the repeats of typical collagen type triplet, x-y-gly. the temperature dependency of these cd spectra showed the three states conformational changes of these peptides. the lower transition, where the positive peak disappeared corresponds to the melting of collagen like structure and the higher one corresponds to that of globular structure of the g domain. the apparent molecular weights determined by ultra-centrifugal analysis showed that these peptides exist in trimeric form at the intermediate state. however, neither c nor vc showed such transitions. these results showed that, contrary to our expectations, the contribution of the triplet of x-y-gly is not the dominant one for stabilizing the trimeric state. in order to explore the stabilizing factor, we are carrying out various experiments, such as mutation analysis, titration of ca ++ , redox reaction of disulfi de bonds and so on. one result is that ca ++ was shown to be a factor for increasing the thermal stability of the trimer. are v mutants of amyloidogenic protein -human cystatin c more or less resistant to denaturation conditions? jankowska, elzbieta; orlikowska, marta; radulska, adrianna; szymanska, aneta university of gdansk / faculty of chemistry, poland cystatins are natural inhibitors of cysteine proteases -enzymes widely distributed in animals, plants and microorganisms. human cystatin c (hcc) has been also recognized as an amyloidogenic protein directly involved in formation of pathological fi brillar aggregates which deposit in the brain arteries of elderly persons causing cerebral amyloid angiopathy ( ). our studies were performed to explore possibilities of preventing this lethal disease. the overall d architecture of monomeric human cystatin c is not known but its fold could be anticipated from the structure of the dimer which has been already determined ( ) . the dimeric cystatin c is created through the exchange of 'subdomains' between two molecules ( d domain swapping) and consists of two identical subunits in great extent reconstituting the fold of the monomeric chicken analogue of hcc. it is possible that similar mechanism is involved in cystatin c oligomerization and fi brilization process. the most signifi cant structural changes during dimerization process are observed in the l loop ( - , qivag). this loop occurred to be a hinge region in the d domain swapping event. with the aim to check implications of greater or decreased stability of this loop for dimerization and aggregation propensity of human cystatin c, we designed and construct hcc l mutants with val residue replaced by asp, asn or pro, respectively. the structural studies of these mutants and their thermal denaturation process have been performed by means of cd and ftir spectroscopies. results of these studies will be presented. azobenzene-mediated photomodulation of a collagen triple helix monitored by ir spectroscopy ( ), our most recent efforts were addressed to the design and synthesis of a photoswitchable collagen triple helix. by replacing in suitable positions of the ac-(gly-pro-hyp) -nh a pro and hyp residue with ( s)mercaptoproline, respectively, a side chain-to-side chain crosslinking of the collagen peptide was afforded with a purposely designed azobenzene derivative. as expected from modeling studies, self-association of the modifi ed collagen model peptide into a stable triple helix was observed with the trans-azobenzene clamp, while its photoisomerization to the cis isomeric state leads to unfolding processes as well assessed by nmr structural analysis ( ) . unfolding pathways can be studied by comparing ftir difference spectra induced by temperature or light. we found the photomodulation of the triple helix to be reversible and the effi ciency of the photoisomerization increased with temperature reaching a maximum value shortly below the melting point. ir spectroscopy was thus used to identify the optimal temperatures required for structure destabilization at suffi cient extents to enable unfolding by the weak driving force of the azobenzene clamp. the results confi rmed the correctness of the design and the ability of the azobenzene switch to photocontrol this complex tertiary structure, thus allowing for time-resolved monitoring of the triple-helix unfolding process. studies on various collagen model peptides (x-y-gly) where x and y are often imino acids, pro or hyp r ( (r)-hydroxyproline), have shown that hyp r at the y position plays an important role in stabilizing the collagen triple helix. however substitution of non-natural (s)-hyp (hyp s ) at both positions decreases the thermal stability of triple helix as (pro-hyp r -gly) exists in a stable triple helical state, whereas (hyp s -pro-gly) and (pro-hyp s -gly) are in a single coil state. similar effects on the stabilities of triple helices are provided by the substitution of fl uoroproline. however there is an exception that (fpro s -pro-gly) takes a triple helix at c whereas the counterpart, (hyp s -pro-gly) , is in a single coil state. although the difference could be explained by the steric hindrance of hydroxyl group in s confi guration, it is still controversial how much this hindrance could obstruct the triple helix formation. therefore, even though apparently the tripeptide with the hyp s -pro-gly sequence is not capable of triple helix formation, we could expected that (hyp s -pro-gly) n forms a triple helix as n increases. the transition temperature of (pro-pro-gly) n was shown to have the chain length dependency. here, we synthesized (pro-pro-gly) n and (hyp s -pro-gly) n (n= , ) and investigated their thermal stabilities. the cd spectra of (hyp s -pro-gly) showed the conformational transition from collagenlike triple helix to random coil with the melting temperature at c. the apparent molecular weight, determined by the sedimentation equilibrium method, showed that (hyp s -pro-gly) exists as trimer at c and as monomer at c. the melting profi le was also clearly detected by dsc. @thus, it is concluded that the existence of hyp s at the x position is not essential to interfere the triple helix formation. we are on course to investigate the stabilizing mechanism of the collagen structure. the infl uence of o-glycosylation on the folding and stability of -helical coiled coil peptides glycosylated proteins have been shown to play a key role in many biological events like cell-cell communication, immune response, cell adhesion, intracellular targeting, protease resistance, and many other processes. recently, there is a growing interest in the effect of glycosylation on the secondary structure of proteins, because of the association with the so called conformational diseases that arise from the dysfunctional aggregation of proteins in not native conformations. in this study we used -helical coiled coil based peptides as model systems to investigate the effects of serine-linked -galactose on both the secondary structure and amyloid formation tendency. thehelical coiled coil structural motif consists of two to seven -helices which are wrapped around each other with a slight superhelical twist. its simplicity and regularity have made it an attractive system to explore fundamentals of protein folding.( ) the importance of the coiled coil motif is obvious with the amount of % of all amino acid residues in the protein data bank being part of a coiled coil motif. at fi rst we examined to which extent and at which positions a glycosylation is possible without destroying the coiled coil structure. therefore, we systematically incorporated one to six serine-linkedgalactose units into several solvent exposed positions of a amino acid long coiled coil peptide. the hydrophobic dimerization face was not modifi ed. the preformed l-ser(ac --d-gal)-oh building blocks were introduced by convenient solid-phase synthesis following the fmocstrategy. folding and stability of the glycopeptides were monitored by cd spectroscopy. the amyloid formation tendency was investigated by a tht fl uorescence assay. aging, associated with decreasing protein homeostasis (proteostasis) capacity and increasing oxidative stress, is a prominent risk factor for amyloid diseases. alzheimer fs disease (ad), which is one of the most common amyloid diseases, involves intra-and extracellular amyloid formation by the amyloid bata peptide (abeta). however, how abeta can aggregate in vivo even though its physiological concentration (pc) is much lower than its critical concentration (cc) for aggregation is a mystery. we have proposed that covalent modifi cation of abeta by small molecule oxidation products can explain how abeta can form amyloid at pc. the aldehyde-bearing cholesterol oxidation product ( ), which can modify abeta by schiif-base formation, is an example of the products that could affect ad onset. however, signifi cant questions about the modifi cation of abeta by ( ) persist, including: does modifi cation by ( ) lower the cc of abeta aggregation into the pc range? and, is abeta modifi ed by ( ) able to aggregate at low concentrations on a biologically relevant time scale? in this study, these questions are answered by studying chemically synthesized analogs of abeta that are site-specifi cally modifi ed by ( ) at asp , lys , or lys . modifi cation at the different sites has a similar effect on the thermodynamic propensity for aggregation. in contrast, the effect of metabolite modifi cation on aggregation kinetics depended strongly on the modifi cation site. abeta modifi ed at lys formed amorphous aggregates fastest and at the lowest concentrations. in contrast, the appearance of thiofl avin-t positive aggregates at higher concentration was fastest for abeta modifi ed at asp( ). the infl uence of modifi cation site on the nature of the aggregates suggests that amorphous aggregation and fi brillization place different conformational demands on abeta. furthermore, these studies may partially explain how abeta can aggregate at nm pcs when the cc of unmodifi ed abeta is in the ƒÊm range. code, christian; domanov, yegor; kinnunen, paavo k.j. antimicrobial peptides are ubiquitous in nature and consist of several families of cationic amphiphilic peptides. they partition into lipid membranes and promote the segregation of anionic phospholipids ( , ) . we have shown several antimicrobial peptides, e.g. temporin b and l, indolicidin, and magainin to activate secretory phospholipase a (pla ) ( , ) and we concluded that this could represent synergistic action of these peptides/proteins in defense against microbes. the fact that the sequences of these amps are very different suggest rather non-specifi c mechanism to be involved. to pursue the latter in more detail we used förster-type resonance energy transfer (fret) between labeled pla and temporin b. interestingly, fret coincides with concentration dependant activation of pla hydrolysis of dipalmitoylphosphatidylcholine (dppc) liposomes. accordingly, temporin b and pla interact forming a supermolecular complex terminating into amyloid-like fi brils ( ). homo-oligomeric fi bers are formed on a slower time scale when pla interacts alone on dppc liposomes ( ) . a general mechanism of peptide induced pla activation forming heterooligomeric cofi brils is suggested. human islet amyloid polypeptide forms lipid-encased amyloid fi brils on supported lipid membranes domanov, yegor; kinnunen, paavo university of helsinki, finland islet amyloid polypeptide (iapp) forms fi brillar amyloid deposits in the pancreatic islets of langerhans of the patients with type diabetes mellitus and its misfolding and aggregation are thought to contribute to -cell death. increasing evidence suggests that iapp fi brillization is strongly infl uenced by lipid membranes and, vice versa, the membrane architecture and integrity is severely affected by amyloid growth. we performed direct fl uorescence microscopic observations of the morphological transformations accompanying iapp fi brillization on the surface of supported lipid membranes. within minutes of application in submicromolar concentrations, iapp caused extensive remodelling of the membrane including formation of defects, vesiculation, and tubulation. the effects of iapp concentration, ionic strength, and the presence of amyloid seeds on the bilayer perturbation and peptide aggregation were examined. growth of amyloid fi brils was visualized using fl uorescently labelled iapp or thiofl avin t staining. two-colour imaging of the peptide and membranes revealed that the fi brils were initially composed of the peptide only, and vesiculation occurred in the points where growing fi bres touched the lipid membrane. interestingly, after - hours of incubation iapp fi bres became "wrapped" by lipid membranes derived from the supported membrane. progressive increase in molecular level association between amyloid and membranes in the maturing fi bres was confi rmed by förster resonance energy transfer (fret) spectroscopy. the possible role of lipid wrapping in stabilization of iapp amyloid in vivo is discussed. fibrinogen-derived peptides that mediate cell adhesion: structure and activity studies interaction of human islet amyloid poly peptide with phospholipid membrane vesicles dannehl, claudia; zschörnig, olaf university of leipzig, germany amylin, also known as human islet amyloid polypeptide (hiapp), is a -residue peptide, suspected to play a major role in the malfunction of insulin secretion in diabetes mellitus type ii. co-secreted with insulin in the beta-cells, hiapp, in higher rates destroys the barrier function of the beta-cells, leading to a failure in insulin production. because of its amyloidogenity, aggregates of fi brils can be observed in the islands of langerhans to indicate its overexpression. we studied the physico chemical properties of hiapp by observing changes in its structure depending on time and the surrounding media using maldi-tof-ms, atr ft-ir-spectroscopy. to understand the process of penetration and toxicity to cells, we performed leakage measurements of carboxyfl uoresceine containing phospholipid large unilamellar vesicles by means of fl uorescence spectroscopy. moreover we determined membrane binding of dansyl-labeled hiapp. at physiological ph value, hiapp is positively charged and thus negative charges at the phospholipid membrane surface accelerate the process of peptide folding. being random coil as initial state, a mixture of anti-parallel betasheet and alpha-helices emerges in time. in the presence of negatively charged phospholipids, hiapp aggregates can be seen within a few minutes after titration. also in absence of any free charges, as seen in water, fi brils grow and after an incubation for hours at °c, some alpha-helices are twisted and after two weeks, no random coil is detected anymore. titration of hiapp to carboxyfl uoresceine fi lled liposomes, showed different results concerning equilibrium time and maximal extent depending on age and preparation of the peptide. in particular the composition of the vesicles seems to determine their stability in the presence of hiapp. nanoparticle induced folding and fi bril formation of a coiled coil peptide nanoparticles present large surface areas and are capable to catalyze fi bril formation of peptides. ( ) . one assumes that the nature of surface controls which of the peptides will interact with the nanoparticles and that an enhanced local peptide concentration reduces the lag-time for aggregation. in previous studies, we reported the design of a coiled coil peptide that can adopt a random coil, -helical structure, and -sheet folding in dependence of ph and peptide concentration. ( ) here, we expose this peptide to au nanoparticles that are either negatively or positively charged. the interaction of peptide and nanoparticle was investigated by cd and uv/vis spectroscopy, gel electrophoresis, and transmission electron microscopy. we found that electrostatic interactions with au nanoparticles can affect the peptide folding resulting in more than one secondary structure, namely, a competition between the -helical and -sheet structures. the latter folding is very likely a consequence of the high local peptide concentration on the surface of nanoparticle that facilitates a -sheet fi brillation of peptide. moreover, several factors such as ph, peptide concentration, and size of the nanoparticle have a strong infl uence on the nanoparticle-mediated folding. these results provide valuable information on pharmaceutical applications of nanoparticles and will help to reduce possible adverse effects. antimicrobial peptides are synthesized by all living organisms as a part of their innate immune system. those molecules are endowed with a broad spectrum of activity against pathogens. they can be divided into two classes differing in the mechanism of killing: membrane disruptive antibiotics cause a dysfunction of the membrane and subsequent cell lysis; non-membrane disruptive peptides are focused on the intracellular targets. in both situations the initial stage consists in the interactions with the cytoplasmic membrane, in most cases without the exploitation of any receptors. a non-receptor type of interactions decreases the possibility of development of microbial resistance and makes peptides a very potent alternative to conventional antibiotics. in the face of the reduced effi ciency of traditional drugs there is an urgent need for the design of new therapeutic agents with the optimized activity. monte carlo simulations of peptide-membrane interactions can be one of the strategies. that method takes in the account biophysical properties of a membrane as well as structural and physicochemical nature of peptides. in our work we are focused on the development of new analogues of nkcs, a derivative of potent antibiotic peptide nk- . in the present study the outcome of monte carlo simulations is compared to experimental results of antibacterial tests performed against escherichia coli. the insight into the molecular mechanism of peptides activity is obtained in vitro using saxs method and artifi cial systems mimicking a bacterial cytoplasmic membrane. the results indicate that monte carlo modelling is a good tool to predict the peptide -membrane interactions and can be very useful for the design of novel antibiotics. a - peptide: structural features in membrane mimicking systems amyloid (a ) peptides, the hallmark of alzheimer disease, depending upon conditions, undergo conformational transition from soluble monomers to the highly toxic -sheet oligomers which form the mature fi brils. conformational and biological analyses were carried out on several different a fragments, to understand the role of the single residues in the fi brillation process. a - , is considered the shortest fragment exhibiting large -sheet aggregates and retaining the toxicity of the full-length peptide. we recently reported the conformational analysis of the synthetic a - under several solution conditions, and analyzed the modulation of the conformational behaviour of a - peptide, by interaction with nicotine-like molecules. a - is the -mer a peptide, composed of the a - fragment, and additional n-terminal residues, known to be endowed with fi bril disaggregating activity. a - encompasses part of hydrophobic ( - ) and hydrophilic ( - ) a -regions. due to the amphipathic character of this fragment, common to the full a - and a - , a tossicological mechanism involving a -peptide-membrane interaction may be hypothesized. on these bases we decided to perform the structural investigation of the fragment a - in membrane mimicking systems including micelle and vesicles aggregates, differing for composition and structural complexity. high resolution three dimensional structure was solved by nmr spectroscopy in negatively charged sds and in dpc/ sds mixed micelles. fluorescence and epr spectroscopies were used to monitor the peptide lipid interaction. the data agree on the critical role played by the membrane composition on the stabilization of different conformers, potentially driving to different oligomeric and/or polimeric toxic species. machan, radek; jurkiewicz, piotr; benda, ales; hof, martin j.heyrovsky institute of physical chemistry, czech republic charge and hydrophobicity are important determinants of membrane activity of antimicrobial peptides. the model peptide lah whose charge can be easily controlled by ph of the medium ( ) is a very convenient tool to study the relationship between physical properties of peptide molecules and their membrane activity. it was shown that the changes in the charge of lah molecules result in changes in their orientation with respect to phospholipid bilayers ( ). in the present study, effects of lah on the properties of phospholipid bilayers at different ph values are studied by several methods. its infl uence on the lateral mobility of lipids within an spb and on the stability of suspensions of large unilamellar vesicles were characterized by fl uorescence correlation spectroscopy, showing evidence of peptide induced aggregation of lipids at basic ph. changes of hydration and local viscosity within the bilayer following changes in orientation of peptide molecules were measured by solvent relaxation technique. the effect of phospholipid charge was also taken into account in each case. several bioactive peptides, such as antimicrobial, cell-penetrating or fusogenic peptides, exert their biological function by interacting with cellular membranes. therefore, structural data on the location of these molecules inside lipid bilayers are very important for a detailed understanding of their mechanism of action. fluorescence spectroscopic methods are particularly suited to the study of peptide-membrane association, but give only low-resolution information on peptide position in the lipid bilayer. molecular dynamics simulations, on the other hand, can provide a very detailed picture of the peptide-membrane interaction, but need to be validated by quantitative comparison with experimental data. we applied several fl uorescence approaches, together with md simulations, to the investigation of two antimicrobial peptides: the lipopeptaibol trichogin ga iv, and pmap- , a member of the cathelicidin family. to perform the spectroscopic studies, a variety of peptide analogues containing a single fl uorophore were synthesized. fluorescence spectra, depth-dependent quenching experiments, and peptide-translocation assays were employed to determine the location of the two peptides inside lipid bilayers, in particular as a function of peptide/lipid ratio. molecular dynamics simulations were performed by a "minimum bias" approach, starting from a random mixture of water, lipid and peptide, and following the spontaneous self-assembling of the lipid bilayer. the fi nal membrane-bound d-structure is in quantitative agreement with the position of the fl uorescent labels determined by depth-dependent quenching experiments. for both peptides investigated, the atomic details of md simulations provide new insights on the mechanism of membrane destabilization. acknowledgements: with the support of the ministry of education, university and research, and of foreign affairs of italy. cavaco-paulo, artur university of minho, portugal surfactant proteins (sp-) are found in lungs of mammalians. most surfactant proteins have a carbohydrate binding domain (crd) and neck domain. crd have defense function in mammalian mucosa's, by eliminating microbes, due to strong binding to sugars in the cell walls. neck domains are found to order phospholipids allowing the exchange of oxygen between the alveoli and blood. x-ray structures indicate that neck domains are alpha-helixes, but circular dicroism indicates that in the presence of phospholipids, those peptides present a disordered structure. neck domains with residues from natural sequences of sp-a, sp-b, sp-c, sp-d have been tested to be delivered in lipophilic media. only disordered structures would have the ability to cross the lipid barriers. the results obtained here indicated that neck domains possibly can be used as carriers to deliver molecules across skin and hair. fraternal twins ! -peptides and oligoureas are isosteric, isostructural foldamers endowed with yet distinct biomolecular recognition properties. in the fi eld of peptidomimetics, there has been a sustained interest towards the design of non-natural oligomeric backbones with new folding patterns. over the past years, the amide linkage has become the quintessential motif to elaborate folding oligomers. aliphatic and aromatic oligoamides (peptoids, -, -peptides) have provided numerous helical-folded structures, many of which have shown interesting biological activities ( ). interestingly, substituting urea for the ch -co-nh units in the -peptide backbone represent a spectacular case of isosteric and iso-structural replacement. detailed nmr studies of the resulting oligoureas revealed a helical fold very similar to that reported for the cognate -peptides. defi nitive confi rmation of this isostructural relationship came with the recent x-ray structure determination of the canonical . -helix of oligoureas. how such isosteric and isostructural oligoamide and oligourea backbones compare in biomolecular recognition events is an interesting question that we attempted to address in the present work. notably the two systems were compared for their antimicrobial activity, membrane interaction and disruption properties. both -peptides and oligoureas designed to mimic globally amphiphilic alpha-helical host-defense peptides have been synthesized and tested. the results showed a surprising dichotomy in bactericidal activity between the two isostructural systems and enlightened the unique antibacterial of amphiphilic oligourea helices. to question whether this functional difference results from differential membrane disruption activities, we have undertaken detailed physicochemical investigations using negatively charged phospholipid membranes as model systems. understanding of the cellular uptake of an amphipathic cell penetrating peptides complexed with sirna konate, karidia; divita, gilles; heitz, frédéric crbm umr -cnrs, france the effi ciency of delivery of macromolecules into living cells is very important for therapeutic purposes. the discovery of a class of peptides, known as cell-penetrating-peptides (cpps), with the ability to mediate translocation of various cargoes both in vitro and in vivo has provided new perspectives in the fi eld of delivery. we have designed a new secondary amphipathic cpp, caddy, which can transfect sirna. many studies have tried to elucidate cpp internalization mechanisms and there is currently still much controversy between endocytosis phenomena and direct interaction with membranes. however, elucidating the interactions between peptides and lipid membranes is essential to understand how caddy delivers cargoes in cells. to highlight these interactions, we have applied biophysical method to phospholipids monolayer and bilayer as model plasmic membranes. regarding the increase of the phospholipids monolayer surface pressure in presence of caddy, it is obvious that this peptide have high affi nity with lipids. while cholesterol presence does not induce anything in peptide insertion on a phospholipids monolayer, the cargo and a widely studied partner of the internalization: heparan sulfate of the gag' s family, do not induce the same behaviour. the intrinsic probe of caddy, tryptophan residues, and the fitc probe bound to the cargo allowed to follow and identify interactions between these two entities by fl uorescence spectroscopy. adding a bilayer vesicular solution unsettle these interactions, tryptophan residues interact with phospholipids while fitc probe are less embarrassed by peptides. caddy alone in solution is not structured but cd measurements show a typical spectrum of helical conformation in presence of phospholipids vesicles. and when the peptide complex its cargo, cd spectrum show a contribution of the sirna relevant of a conformational change inside both partners interacting together. high membrane coverage as the basis of antimicrobial peptide activity the interaction of the antimicrobial peptides (amps) omiganan (h-ilrwpwwpwrrk-nh ) and bp (h-kklfkkilkyl-nh ) with model bilayers was characterized. the activity and selectivity of these peptides could be attributed to a strong preference towards anionic membrane model systems, which mimetize bacterial membranes. regarding the interactions with bacterial membrane models, there were marked differences in the interaction patterns, as well as in functional properties of the peptides at high peptide:lipid ratios. these differences occurred for both peptides, despite their being unrelated in sequence and in occurrence in nature. such events at high membrane coverage could represent the equivalent at the molecular scale of the conditions at which the antimicrobial activity of the peptides is triggered. although the lipid: peptide ratios at these transitions are lower than phospholipids per peptide molecule, the plausibility of this hypothesis was demonstrated taking into account an estimate of the amount of lipid per bacterium, and the bacterial concentration in minimum inhibitory concentration (mic) assays. according to the partition constants obtained towards bacterial membrane models, these peptides are expected to reach, at the mic, precisely those high concentrations in the membrane. in addition, surface charge neutralization was shown to occur in these conditions. activity at high membrane coverage is thus likely not only for these peptides but also for any peptide displaying high membrane affi nity and micromolar mics, which is common amongst amps. biosensors based on artifi cial membrane system that permits the functional reconstitution of transmembrane receptors have been studied for the past decade. immobilized liposome encapsulates intracellular chemicals in the internal water cavity and provides a potential advantage over supported planar lipid membrane. to prepare a stable liposome adlayer, we have proposed an immobilizing method based on small-peptide modifi cation of solid surface. in the present study, we investigated kinetics of liposome adsorption on the surface-bound synthetic peptides which have several alanine, lysine and tryptophan residues as amphiphilic segment with a cysteine at terminus. the quartz crystal microbalance studies showed that the peptide sequences can be divided into two groups according to liposome-size dependence of langmuir adsorption constant. while the initial adsorption processes could be satisfactorily described by simple langmuir adsorption kinetics, the amounts of liposome adsorbed irreversibly were increased as time advances. the results from afm reveal that most of the liposomes are bound to peptidic surfaces as single fl attened particles without fusion together for several hours. we next performed the detection of ganglioside gm -lectin interaction on the liposome surface. all the binding constants and binding amounts, as observed by qcm, were fairly consistent with each other and and showed the effectiveness of our approach. herce, henry rensselaer polytechnic institute, united states recently we have proposed theoretically an energy independent pathway for the uptake of cell penetrating peptides (cpps) that challenges fundamental concepts associated with protein membrane interactions, h. d. herce and a. e. garcia, pnas, , ( ) . this mechanism involves strong interactions between cpps peptides and the phosphate groups on both sides of the lipid bilayer, the insertion of charged side chains that nucleate the formation of a transient pore, followed by the translocation of cpps peptides by diffusing on the pore surface. this mechanism explains how key ingredients, such as the cooperativity among the peptides, the large positive charge, and specifi cally the arginine amino acids, contribute to the uptake. we will describe the details of this mechanism and present novel experimental results that directly validate the model. a comparative studies on lipid affi nity of cell penetrating peptides in presence or absence of cargo a growing number of natural and /or synthetic peptides with cell membrane penetrating capability have been identifi ed and described in the past years. these molecules have been considered as targeting structures for the delivery of bioactive compounds into various cell types. although the mechanism of uptake is still unclear, it is reasonable to assume that the relative contribute of each proposed mechanism could differ for the same peptide, depending on experimental protocol and cargo molecule composition. in this work we try to connect the capability to interact with model lipid membrane of cpp and their structural and chemical characteristics in order to obtain a biophysical classifi cation that predicts the behavior of cpp-cargo molecule in cell system. indeed, the interaction with cell membrane is one of the primary step in the interaction of cpp with cells, and consequently the studies on model membrane could become important for understanding peptide-membrane interaction on a molecular level, explaining how cpps may translocate a membrane without destroying it and how this interactions come into play in shuttling cpps via different routes with different effi ciency. we analyzed by fl uorescence spectroscopy the binding properties of six different cpps (kfgf, antp and tat derived peptides, and oligoarginine peptides containing , or residues) in absence or presence of the same cargo peptide (the [ - ]ptyr fragment of hs protein). the binding properties were correlated to the conformational and chemical characteristic of peptides, as well as to the cell penetrating properties of the cpp-cargo conjugate. results show that even if certain physico-chemical properties (conformation, positive charge) govern cpp capability to interact with the model membrane, these cannot fully explain cell-permeability properties. great deals of data show that alzheimer's pathology lead to the loss of neuronal functionality and synaptic plasticity. these effects seem to be the consequence of a toxic effect of a peptides related to their ability to modify cell membrane homeostasis. in particular, a peptides, as full length or in fragments, being in oligomeric or polymeric form, could alter membrane fl uidity and compromise its functionality. we have previously demonstrated that the - fragment of a peptide a ( - ) is able to penetrate into the outer leafl ets of the membrane. furthermore, it is able to alter membrane fl uidity changing membrane response to cholesterol, and thus affecting its ability to form low fl uid membrane regions named lipid rafts. a ( - ) is considered the shortest fragment exhibiting large -sheet aggregates and retaining the toxicity of the full-length peptide. flavonoids are a group of naturally occurring, benzo--pyrone derivatives, ubiquitous in plants. they are endowed with tumor prevent activity and they act as antioxidants through a membrane mediated molecular mechanism involving the membrane ion transport. on the basis of the common ability of fl avonoids and a peptide of altering membrane fl uidity, we carried out an epr spectroscopic analysis of several fl avonoids -specifi cally quercetin, naringenin, rutin and naringin -in , -dioleoyl-sn-glycero- -phosphocholine (dopc) vesicles. the modifi cations of the dopc physio-chemical environment were analyzed in presence of fl avonoids and beta-amyloid fragment a ( - ). our results indicate that the addition of fl avonoids induces a decrease in membrane fl uidity. this effect is retained in presence of a ( - ). thus fl avonoid compounds could be able to antagonize the effect induced by amyloid peptide on membrane fl uidity and in this respect they could be therapeutically useful as neuroprotective agents. analyse lipid peptide interactions of p and lipo-p with membrane mimicking represented by micelles and vesicles. nmr structures of p and lipo-p in mixed sds/dpc micelles are reported; the positioning of p and lipo-p peptides with respect the lipidic surface is analyzed using -doxyl stearic and -doxyl stearic acid. epr analysis is carried out in the zwitterionic dimyristoyl phosphatidylcholine and the anionic dimyristoyl phosphatidylglycerol vesicles using several different lipid spin labels. both peptides bind to lipid bilayers and trp residues and lipidic chain of lipo-p show a important role in the process of peptide adsorption onto the membrane, to exert its antiviral activity. studies on human cell membranes were necessary to further establish the role of membranes in these peptides mode of action. this interaction was assessed by evaluating the effects that these peptides have on the membrane dipole potential of human erythrocytes, using the fl uorescence probe di- -anepps. in the presence of enfuvirtide or t- , a decrease in the di- -anepps fl uorescence excitation ratio dependent of peptide concentration was observed. these results show that t- has ten times more affi nity to the erythrocyte membrane than enfuvirtide, a factor that can be associated with the adsorption of t- on cholesterol rich membranes observed on the studies with membrane model systems. moreover, as a fraction of hiv associates with erythrocytes in vivo, these cells can have a role in delivering these peptides to the viral surface. the improved clinical effi ciency of t- relative to enfuvirtide may be related to its higher partition coeffi cient and ability to adsorb to rigid lipid areas on the cell surface, where most receptors are located upon membrane fusion. moreover, adsorption to the sterol-rich viral membrane helps to increase the local concentration of the inhibitor peptide at the fusion site. the platelet receptor iib plays a critical role in the process of platelet aggregation and thrombus formation. upon platelet activation its conformation changes leading to an increased affi nity for fi brinogen, which forms bridges between adjacent platelets and assembles them into an aggregate. the iib activation is regulated by "outside-in" and "inside-out" signaling. among the protein-protein interactions, which contribute to «inside-out» signaling, that of talin with the cytoplasmic tail is the most important. it has been recently suggested that talinmediated iib activation relies on the cooperative interaction of the membrane proximal (mp) and the membrane distal (md) regions with talin f domain and that the n ply motif of , which can be phosphorylated at y , plays a critical role in this process. to evaluate the interaction of talin with the tail of integrin we designed and synthesized two peptides corresponding to the md and mp parts of in their carboxyfl uorescein-labeled form (md: cf-r akwdtannplyke and mp: cf-k llitihdrke ). emission and anisotropy fl uorescence spectroscopy was used to quantitatively assess the affi nities of these peptides for talin. furthermore, to challenge the role of the y phosphorylation in talin-iib interaction we also studied the binding of talin to the modifi ed analogue of md, cf-r akwdtannpl(ptyr) ke . our experiments revealed that the md and mp parts of bind tightly to talin and that y phosphorylation has an inhibitory effect on this binding. finally, circular dichroism studies of all peptides in aqueous solutions and mixtures with tfe were also performed in order to characterize structure-affi nity relationships. acknowledgements: gsrt and eu (epan yb/ ) for fi nancial support. the infection of human cells with hiv requires two initial steps: binding of the viral glycoprotein gp to the receptor cd followed by the interaction between the v loop of gp and a seven helix transmembrane coreceptor (ccr on macrophages or cxcr on t cells). the n-type glycosylation at asn of the v loop is assumed to play a crucial role in this process. in order to understand the interaction in detail, it is important to analyze the binding of v -glycopeptides to the membrane integrated coreceptors on an atomic scale. here, we present binding studies between multiple v -peptides and -glycopeptides and the coreceptor ccr by stdd nmr and surface plasmon resonance (spr). spr experiments were carried out by immobilizing the (glyco)peptides on an spr chip by amide coupling. several concentrations of ccr overexpressing human osteosarcoma (hos-r ) cells were passed over the sensor surface. the role of individual amino acids in the binding process to ccr can be analyzed by using different v -peptides and -glycopeptides. additionally, the observation of interactions between the glycopeptide ligands and the receptor proteins embedded in liposomes is possible by using the saturation transfer double difference (stdd) nmr technology. stdd nmr allows removal of all unwanted signals resulting from native binding processes in cells. ( ) . we used ccr liposomes derived from hos-r cells. substraction of an std spectrum of ccr liposomes from an std spectrum of the same concentration of liposomes incubated with a ligand allows recording of clean stdd nmr spectra presenting only signals of protons of the ligand interacting with the transmembrane receptor.( ) k d values of the ligand with respect to ccr have been determined in series of experiments with varying ligand concentrations. we could show that the binding epitope between hiv and ccr is formed by the carbohydrate at asn as well as the peptide. this knowledge is important for the design of new inhibitors. temperature dependant methionine proximity assays highlights conformational variations occurring through the mechanism of peptidergic gpcr activation arsenault, jason; clément, martin; leduc, richard; guillemette, gaétan; lavigne, pierre; escher, emanuel university of sherbrooke, canada g protein coupled receptors are invaluable for cell signal transduction mediated by external stimulus. pharmacological efforts towards these targets are of primordial importance albeit few efforts have resulted in structural characterisation, although rational drug design necessitates such information. recent advances in crystallisation of the -adrenergic receptor have been highly insightful and such a structure corroborates our results on the human angiotensin ii type receptor (hat ) using the methionin proximity assay (mpa) in identifying ligand receptor contact points. unfortunately, physical methods such as crystallography are still far from routine procedures and are limited to a static picture of a given receptor. in the present contribution we propose a more accessible method that permits analysis conformational variations of different receptor states through an energy landscape, spanning from low energy conformations to conformations at physiological temperatures. photolabelling, mpa mutants constructed on the wt receptor, the constitutively active receptor (cam) and a corresponding non-activable mutant across the temperature range reveal activation-status dependent labelling patterns. as an example, position becomes signifi cantly less accessible in the cam receptor compared to the wt receptor. ligand accessibility can be classifi ed from easily accessible (low temperature photolabeling) to less accessible residues (higher temperature photolabeling). this labelling pattern can be associated to the activation status of the receptor and may allow quantifying and identifying structural changes occurring during receptor activation. such structural understanding is crucial for future endeavours in rational drug design. capillary electrophoresis for diffi cult characterization of hardly soluble polypeptides recently, we have designed and synthesized polypeptides able to stimulate the natural plant defenses. these homopeptides were obtained by ring opening polymerization of n-carboxyanhydride (nca) with several initiators. ratio nca/initiator is determinant for the length of the polymers. on the bases of elicitor activity, we identifi ed a leader, called lapp , which results from l-ala-nca polymerization initiated by alaninol. the mixture of poly(alanine) with different lengths is diffi cult to analyze considering important solubility problems. malditof confi rmed polymerization despite limitation due to molecular discrimination during ionization. nmr in deuterated tfa was possible but only afforded the number-average degree of polymerization (dp). to obtain more detailed characterizations, we developed separation by capillary electrophoresis in new solvents mixtures based on hexafl uoroisopropanol (hfip) and water. this technique allowed us to separate the oligomers with baseline resolution. different parameters infl uencing electrophoretic separation were investigated and optimized conditions were established. this technique enabled a full characterization of the polymer distribution, with determination of number-average dp, weight-average dp and polydispersity index. the pertinence and the reliability of capillary electrophoresis for characterization of non-water soluble polypeptides have been confi rmed, with analysis of short and long peptide chains. weakly polar interactions support polypeptide structures although the sequence of a polypeptide appears to determine the secondary and tertiary structure, weak inter-residue interactions such as between aromatic side chains and other aromatic side chains, aliphatic side chains and the peptide backbone may contribute signifi cantly to the stability of the fi nal folded structure. recent advances in structural bioiformatics and computational chemistry made it possible to study precise strurctural features and energetics of these interactions in model peptides and miniproteins such as tc b, app and c-vhp. the interaction energies of the weakly polar interactions are of the same order as of the hydrogen bonds which occur in biopolymers ( - kj/ mol). furthermore, these interactions not only stabilize local secondary structures but also entire tertiary folds of miniproteins. acknowledgements: this work was supported by the nih-inbre grant (p rr ) and the carpenter endowed chair in biochemistry, creighton university trusova, valeriya; gorbenko, galyna v.n. karazin kharkov national university, ukraine a number of so-called conformational diseases including neurological disorders (parkinson's, alzheimer's and huntington's diseases), type ii diabetes, spongiform encephalopathies, systemic amyloidosis, etc., are associated with the deposition in tissue of highly ordered aggregates of specifi c peptides and proteins. despite the main structural elements of amyolid fi brils (particularly, cross--structure) are well-characterized, the mechanisms of fi brillogenesis remains poorly understood. accumulating evidence substantiates the idea that formation of fi brillar structures can be initiated and modulated by peptide/protein-lipid interactions. membrane-related determinants of fi brillization are thought to involve conformational changes of the peptide/protein, increase of its local concentration at lipid-water interface, specifi c orientation of aggregating species, neutralization of the peptide/protein surface charges by anionic lipid headgroups, particular arrangement of the inserted and solvent exposed segments of the peptide/protein molecule, etc. the present study was undertaken to explore the formation of lysozyme (lz) amyloidlike fi brils in the model lipid-protein systems. lipid component of the model system was represented by liposomes prepared from zwitterionic phosphatidylcholine (pc) and anionic phosphatidylglycerol (pg) lipids in the molar ratio : . fluorescence microscopy studies performed with fl uorescein-labeled and rhodamine-labeled lz showed the presence of long lz fi bers (length > ìm). the to elucidate the nature of the events preceding lz self-assembly into amyloid-like structures, the protein adsorption onto pc:pg vesicles was examined by monitoring fl uorescence changes of fl uorescein-labeled lysozyme. the observed sigmoidal shape of the adsorption isotherm is strongly suggestive of oligomerization of membrane-bound protein. it seems highly probable that such oligomers serve as nuclei in the membrane-assisted lysozyme fi brillogenesis structure and dynamics of photosystem ii lightharvesting complex revealed by high-resolution fticr mass spectrometric proteome analysis structure and dynamics of membrane-bound light-harvesting pigmentprotein complexes (lhcs), that collect and transmit light energy for photosynthesis and thereby play an essential role in the regulation of photosynthesis and photoprotection, were identifi ed and characterized using high-resolution fticr mass spectrometry. lhcs from photosystem ii (lhcii) were isolated from the thylakoid membrane of arabidopsis thaliana leaves after light stress treatment using sucrose density gradient centrifugation, and separated by gel fi ltration into lhcii subcomplexes. tsekova, daniela university of chemical technology and metallurgy, bulgaria formation of fi brous supramolecular complexes from l-val derivatives and their arrangement in spherulites was studied applying spectrophotometric approach and electron-microscopic observations. experiments show that boiling one and the same compound with different initial concentrations in water to completely dissolving and posterior cooling to room temperature lead to formation of stable supramolecular complexes with different sizes and properties. they behave like polymer molecules which specifi c numbers of monomers depend on the initial concentration of the boiling solution, moreover at higher supersaturations they crystallize in sperulites which is typical for crystallization of polymer molecules. metal -organic gels based on the self-assembly of peptidomimetics and cu (ii) ions peptidomimetics constructed from l-val and nicotinic/isonicotinic acid are good low molecular weight gelators and their supersaturated solutions in a number of solvents turn into gels. they make complexes with some metal ions. mixing of pure solutions of the peptidomimetic and some cu (ii) salts lead to immediate gel formation in some solvents. this kind of compounds, named metal-organic frameworks (mofs), are new class of nanoporous materials and are very promising ones for applications in catalysis, pharmaceutical industry, etc. the stability and molecular structure of these complexes in some solvents is in the process of investigation. peptide metalloconstructs often possess particular conformations and hence display increased activities and metabolic stabilities. we investigated new rgd peptide analogs cyclized through oxorhenium / oxotechnetium coordination. the rgd sequence is known to bind specifi cally to of the known integrins, a family of integral proteins that plays an important role in tumor neoangiogenesis, development and proliferation. several cyclic rgd pentapeptides bearing an exocyclic tc- m oxotechnetium core have been proposed for molecular imaging, however few of them have displayed attractive selectivities and metabolic stabilities. structure, biological activity and metabolic stabilities of metallated peptides cannot be predicted. therefore, we preferred a combinatorial approach to generate a panel of tracers that may be evaluated by tumor imaging in mice. tracers were constructed from a rgd model of general formula : ns -x -x -x -rs where x , , are respectively arginine, glycine and aspartic acid analogs and r is a series of linkers that feature various lengths and geometries (ns is a n-bis(ethylthio) moiety). first attempts for synthesizing these peptides by the versatile ugi multicomponent reaction did not yield the peptides with suffi cient purities. a representative library of peptides was obtained by standard parallel peptide synthesis and purifi cation of all members. peptides were metallated either with rhenium or technetium using standard procedures. their resistance towards mice serum, glutathione and tumor extracts was evaluated and showed that compounds containing an aminoethanethiol linker displayed higher stabilities. infl uence of the chemical environment on isomers ratios of the metallated peptides was also investigated. finally, oxorhenium peptide coordinates were assayed as specifi c ligands of integrins. their oxotechnetium equivalents were evaluated for tumor imaging in mice. degradation products of desmopressin in phosphate/ citrate buffer it shows, that introduction of unsaturated residue to the peptide chain could be useful tool to design bioactive compounds with desirable structure [ ] [ ] . therefore full knowledge about relation between presence of dehydroamino acid and peptide's conformation is necessary to predict biological proper and to design newdrug. for that reason we have undertook conformational investigations of numerous peptides containing two dehydroamino acid residues ( z phe, e phe, ala) in peptide chain, in different position. the investigations were based on nmr measurements (standard d techniques and d experiments, typical for detection of hydrogen bonding) and theoretical calculations. conformational preferences of investigated systems were obtained on base of roesy and noesy experiments and calculations by use of x-plor and quantum chemical calculation is concentration overload or volume overload the best strategy for synthetic peptide purifi cation? as the complexity of synthetic peptides increases so the demands on the synthesis and purifi cation increase. whilst improvements in synthesis resins and techniques enables higher purity peptides to be produced, % purity is not achieved. for many applications post-synthesis purifi cation is still required. methods for the purifi cation at the mg level can be relatively straightforward but where there is a need to develop methods which may be used for larger scale production there are a number of additionly considerations. the method developed must be scaleable and the economics of the process must be compatible with the fi nal product costs. when looking to develop a purifi cation method the loading will be critical to the throughput and fi nal production costs. the selection of the purifi cation media and the purifi cation conditions are two of the major infl uences on loading as these determine capacity and resolution. however, it is also important to consider the the physical properties of the pepitide -especially its solubility. as part of the purifi cation method development a loading study must be performed -increasing the amount of peptide loaded onto the column. the most common way of doing this is to increase the concentration of the sample and keep the injection volume constant, concentration overload. but this does require that the peptide is readily soluble in a solvent compatible with the hplc purifi cation conditions. alternatively volume overload can be used where the concentration of the peptide solution is kept constant and the volume purifi ed increases. the most commonly used method is concentration overload. the work presented in this poster compares the two overload strategies, concentration overload and volume overload, for the purifi cation of synthetic peptides. the suitability of the two strategies for large scale manufacture will be explored. expansion of human stem cells by passive transmembrane transfer of homeoproteins . these results clarify the effect of hoxb in the early stages of human lymphopoiesis, emphasizing the contribution of this homeoprotein to the intrinsic lympho-myeloid differentiation potential of defi ned lymphoid progenitor subsets. finally, this supports the potential use of hoxb for hsc and hpc expansion in a therapeutic setting, by means of direct transmembrane protein transfer. tumor selective delivery by cell-penetrating peptides systemic delivery of therapeutic agents used for cancer chemotherapy today lead to undesired side effects and toxicity. increasing the selectivity of the anti-cancer agent will not only facilitate lower dosage for equal therapeutic effect, but also decrease the frequency of drug resistance. we have studied two different targeting approaches both based on cellpenetrating peptides. the fi rst approach is a chimera between cancer homing peptides and a cpp the other is a enzyme activated prodrug design. the cyclic peptide ccpgpegagc (pega) is a homing peptide that has previously been shown to accumulate in breast tumor tissue in mice. pega peptide does not cross the plasma membrane per se; however, when attached to the cell-penetrating peptide pvec, the conjugate is taken up by different breast cancer cells in vitro. additionally, the homing capacity of the pega-pvec is conserved in vivo, where the conjugate mainly accumulates in blood vessels in breast tumor tissue and, consequently is taken up. matrix metalloproteinases (mmps) are over-expressed in a variety of tumor tissues and cell lines, and their expression is highly correlated to tumor invasion and metastasis. to exploit these characteristics, we designed a tumor cell-selective prodrug, by constructing modifi ed version of the already shown to be functional mtx-yta conjugate. it is an inactive pro form of a cell-penetrating peptide (nope) conjugated to the cytostatic agent metothrexate (mtx), selectively cleavable and thereby activated by mmps. characterization of hantavirus envelope structure hepojoki, jussi; strandin, tomas; vaheri, antti; lankinen, hilkka university of helsinki / haartman institute, finland hantaviruses are zoonotic viruses carried by different rodent species and if transmitted to man cause two severe diseases, the hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome to which there is no specifi c therapy. the hantavirus envelope consists of spike structures formed by surface glycoproteins gn and gc which anchorage to the viral lipid bilayer. the quaternary complexes formed by glycoproteins have not been solved. to begin with, it is important to understand the structure of virus particle in order to treat infection for example by prevention of membrane fusion. using pepspot technique the interaction site between gn and gc proteins was mapped to peptide level. to interpret interaction mapping results three dimensional ( d) models of gc protein were created. the structure of hantavirus was also investigated in this study by cryo-electronmicroscopy (cryo-em). according to our results, hantavirus spike consists of either four or eight glycoprotein units and the spike would consist of equimolar associations of both glycoproteins. overall it seems likely that the glycoproteins of hantavirus form a heterodimeric base unit analogous to semliki forest virus e -e glycoprotein complex, where the interaction between e -e proteins hides the fusion peptide in e protein and thus prevents premature fusion. isolation of a novel kda protein from ginger rhizomes having anti-fungal and anti-proliferative activity gill medicinal properties of ginger have been known for long. ginger has been used in traditional indian and chinese medicine and is effective on a wide range of ailments including diarrhea, nausea, respiratory disorders, infl ammatory diseases, arthritis etc. a number of constituents and active ingredients are present in ginger. recent studies have shown the role of ginger extract in the modulation of biochemical pathways involved in chronic infl ammation and thus providing evidences for the anti-infl ammatory role of ginger. mainly the medicinal properties and anti-proliferative activity of the ginger is because of the presence of certain pungent vallinoids, viz. .-gingerol and .-paradol, as well as some other constituents like shogaols, zingerone etc. we have identifi ed and purifi ed a novel anti-fungal and anti-proliferative protein with a molecular mass of kda from the crude extract of ginger rhizobium (zingiber offi cinales), belonging to the zingiberaceae family. the isolation procedure involved ion exchange chromatography using deae-cellulose and affi nity chromatography using affi -gel blue gel. crude extract was loaded on the deae-cellulose column equilibrated with mm tris-hcl buffer (ph . ). the unadsorbed protein fraction from deae-cellulose was further loaded on affi -gel blue gel column equilibrated with mm tris-hcl buffer (ph . ), from which the elution of adsorbed protein was done with the same buffer. the purifi ed protein of kda exhibited a potent anti-fungal activity against the mycelial growth in different fungal species, for example aspergillus fumigatus. in addition, the antifungal activity is also seen against important fungi, viz, fusarium and candida species. further, the purifi ed protein also showed % inhibition of cell proliferation at m concentration. the anti-proliferative activity was checked on human oral cancer (kb cells). search for native interacting partners of fl uorinated amino acids using phage display the introduction of fl uorine has proven to be a successful concept for improving the biological and pharmaceutical properties of drug candidates. the unique properties of fl uorine as well as its absence from the pool of canonical amino acids make fl uorinated amino acids a promising tool in the development of peptide based drugs.( ) however, the application of fl uorinated amino acids for rational protein design requires a comprehensive knowledge of their properties within a native protein environment. our research focuses on the effects caused by single substitutions by fl uorinated amino acids within a polypeptide environment.( ) based on a parallel, heterodimeric -helical coiled coil peptide we applied phage display technology ( ) to screen for preferred interaction partners of fl uorinated building blocks within the pool of the twenty canonical amino acids. three fl uorinated amino acids were introduced either at an a-or a d-position into one of the coiled coil monomers. the second coiled coil monomer was randomized at the four positions that represent the direct interaction partners of the fl uorinated amino acid within the dimerization domain. coiled coil pairing selectivity was used to determine the best binding partner out of the library. using the acylation method, fatty acyl chains are covalently linked to the free amino residues forming a stable amide bond. in this study, a novel method for acylation of proteins was developed using in situ prepared fatty acyl chloride dispersion in aqueous acetonitrile solution, which allows protein modifi cation under very mild conditions. chicken cystatin, a reversible kda inhibitor of papain-like cysteine proteases, was selected as a model protein due to its high potential to inhibit intracellular cathepsins. the protein was modifi ed using fatty acyl chlorides with , , , , , , and carbon atoms. based on the cell culture assays, we examined the transport properties of fatty acylated cystatin, the effectiveness of its internalization and effi ciency to inhibit intracellular enzymes, which was measured indirectly by cathepsin b inhibition. the experiments showed that acylated cystatin quickly internalized into the cells and effectively inhibited cathepsin b. in contrast, non-acylated cystatin didn't cause inhibition as it was unable to enter the cell. the permeability enhancement effect was shown to depend on the length of the attached fatty acyl chain as the strongest inhibition was caused by cystatin acylated with carbon atoms long stearoyl chloride. additionally, chemical modifi cation did not infl uence the protein's immunogenicity. the results of our study provide clear evidence that fatty acylation greatly improves membrane permeabilization properties of proteins. daisogel wing takes your process separation to a higher level. custom made solution for your process peptide purifi cation problem can be easily delivered using the daisogel wing polymer grafted silica based platform. "silica or polymer?"-it used to be an evergreen debate when it came to choose the adequate stationary phase for process scale separation or purifi cation. silica based stationary phases feature much higher mechanical strength and stability, better controlled porosity and as a result higher separation effi ciency, while polymers boast wider ph range. the attempts to bring the good aspects of these opposite methods together failed so far, or failed to provide fl exible solutions. the new daisogel wing platform for silica surface polymer grafting preceding chemical modifi cation is presented. the revolutionary new technique offers high versatility: most variations of the base silica (different pore sizes or particle sizes of choice) can be combined with your choice of familiar chemical surface modifi cations to tailor make the perfect stationary phase for your given chromatography challenge. the polymer graft on the silica surface provides extreme shielding effect, the produced stationary phase displays outstanding ph stability and durability. any of your preferred and trusted regular silica surface modifi cations can be done on the top of the grafted polymer layer. you may enjoy the usual high performance separation, excellent effi ciency, high plate numbers, mechanical strength of silica based stationary phases with a dramatically extended ph range you expected so far only from polymer resins. you have the full range of choices of particle and pore sizes with the full choice of desired bonding chemistries. finally daisogel wing is here to deliver you the most versatile polymer grafted silica hybrid platform to provide the best solution for your demanding process scale peptide separations. przybylski, józef; rogala, piotr; siemaszko-przybylska, krystyna melanin, a natural compound formed in the tyrosine metabolic process. according to occurrence, melanin is divided into that present in true skin and in internal organs. the melanin synthesis and secretion process takes place in melanocytes under the control of two neurohormons: msh -melanocyte stimulating hormone and mch -melanocyte concentrating hormone. research has shown the signifi cant role of melanin in the process of breeding and storing, in regulating metabolism of fat tissue, and in carbohydrates regulation. melanin biopolymer fi nds application in it technology as neurotransmitters for non cellular intelligence. biologically active collagen constitutes the architectonics of all tissues and organs. due to its piezoelectric and dielectric properties it also provides storage and relay functions. for the needs of medical practice, transplants, pharmacy, cosmetology and information technology the key issue is to obtain a compound of melanin with biologically active collagen. this compound we named melanocollagen type i. melanocollagen type i is formed in result of protonating collagen amino acids with melanin, which yields coloured gel ranging: grey, graphite and black. /melanin (h+) + collagen melanocollagen type i/. the therapeutical capacity of biologically active collagen type i with melanin is an ideal formulation inhibiting aging and mitigating related neurovegetative and dementia symptoms, and in vivo transplantology. freeze dried melanocollagen type i retains the features of both collagen type i and melanin. it can be a formulation on its own or a supplement for in vivo and in vitro tissue engineering, biotechnology, information technology, pharmaceutical industry and cosmetology. a patent application for melanocollagen was placed with the polish patent offi ce on . . as p- , entitled: melanocollagen type i -method of obtaining. [ , ] . , -dhp lipid structure was calculated with ab initio quantum mechanics to obtain the charges for molecular dynamics with amber . force fi eld. dhp-lipid molecules were subjected to molecular dynamics from the initial structure of a periodic lipid bilayerwater box, with a small amount of excessive water on the lipid edges to ensure the mobility of lipid molecules. after ns of md simulation the lipid molecules with the fatty acid tails started to squeeze from one bilayer layer to another one. after ns few lipid molecules turned with their charged heads to the side of the lipid bilayer and after ns a profound tubular micelle structure began to form. the tubular micellae structure becomes more perfect during the course of simulation of ns. conclusion is that one of the gene transfection agent , -dhp lipid structures is a tubular micellae, and we could expect that such the micellaes are capable to form lipoplex for the dna transfection or peptide delivery. introduction a newly developed hydrophilic polymer-based ion exchange chromatography column for biomolecules. ion exchange chromatography (iec) is a widely used for analysis and purifi cation of biomolecules. we have newly developed polymer-based iec column, named ymc-biopro series, specially designed for separation of proteins, peptides and nucleic acids. the completely spherical and monodispersed porous / nonporous beads ( m), optimally packed with advanced technology, provide high theoretical plate number and symmetric peak shape. excellent resolution is achieved from the high column effi ciency coupled with the excellent selectivity of qa and sp chemistries. in this paper, we will show features and benefi ts of ymc-biopro series. james p. tam, school of biological science, nanyang technological university, nanyang drive, singapore . viral entry requires fusion of the viral and cellular membranes. in all class- envelope viruses including hiv- , fusion is mediated by envelope glycoprotein which has a homotrimeric quaternary structure which forms a hairpin-like assembly of six-helix bundle ( hb) during the fusion event. the hb employs a -on- locking mechanism in which hrc (heptad repeating-carboxyl region) chains crosslink hrn (heptad repeating-aminal region) chains to enable membrane fusion. this locking mechanism confers avidity due to multi-chain interactions and a high genetic barrier to mutations in the viral entry strategy despite the high mutation rate of their envelope protein. this mechanism also provides inspiration to our approach in designing mutation-resistant entry inhibitors of hiv- . t /enfuvirtide, a synthetic hrc-peptide monomer designed to interrupt membrane fusion, is the fi rst and only fda-approved entry inhibitor against hiv- . however, t becomes ineffective in % of aids patients due to acquired resistance to t- resistant during the treatment course. our inhibitor design is based on a novel quaternary protein mimetic approach. key elements include a covalent-link parallel-chain construct mimicking the quaternary structure of gp in its pre-fusion state and an inhibition mechanism mimicking the multimeric interactions found in the highly conserved hb formation. our hypothesis is that covalent-linked, -chain quaternary mimetics (called mimetics) of hrc peptides can confer multi-chain binding to the hrn region in a multi-chain locking mechanism that may lead to mutation-resistant hiv fusion inhibitors. we also extend our design to -chain hrc mimetics (called mimetics) which may bind to the hrn as a -on- locking mechanism. in contrast, t and other single-chain peptide inhibitors with a -on- binding mechanism lacks the advantages offered by the quaternary mimetics and is susceptible to gp mutations. this report will describe all three types of inhibitors as a model to further our understanding of gp mutations and viral fusion mechanism in developing mutation-resistant entry inhibitors. references: . lain et al. cancer cell costentin, beauvillain, vaudry, proc. natl. acad. sci pnas , , . . references: . a.a.zamyatnin. fragmentimics of proteins and natural oligopeptides. biofi zika the fi rst genome sequence of an elite grapevine cultivar (pinot noir vitis vinifera l.): coping with a highly heterozygous genome the grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla the erop-moscow oligopeptide database press references: . alcaro galactosylated peptides, their preparation and use in autoimmune disease diagnosis new coupling reagents: development and industrial aspects assessment of new -cl-hobt based coupling reagents for peptide synthesis. part : coupling effi ciency study fast conventional synthesis of chemokine sdf- ( - ) on the symphony® references: . pardridge, w. m. drug discov.today alberto ; rondina, maria ; rosato maura ; quintieri synthesis and biological activity of new linear and cyclic shp- n-sh -ligands the sequence specifi city for most sh domains is dictated by the amino acids surrounding the py-residue (position ) and the c-terminal positions relative to py ( ). in contrast, the sh domains of shp- in addition to py+ and py+ depend on position py- ( ). it was further demonstrated that beyond this minimal consensus sequence the positions py+ and py+ also signifi cantly affect the binding affi nity and specifi city of the shp- sh domains ( ). for the investigation of shp- mediated signaling pathways, inhibitors of this phosphatase are of great interest. therefore, we focused our research on linear and cyclic peptide ligands based on the previous studies [ - ]. the new ligands were c-terminally prolongated according to the recognition determinants for py+ and py+ . except, an additional motif designed to occupy a basic gap on the surface of the ptp-domain was introduced. the latter was predicted to impair the n-sh -ptp dissociation process design of a directed molecular network restructuring artifi cial peptide networks by external triggering the road to non-enzymatic molecular networks boolean logic functions of a synthetic peptide network systems chemistry: logic gates, arithmetic units and network motifs in small networks peptides: the wave of the future references: . d'andrea l.d.; iaccarino g.; fattorusso r.; sorriento d.; carannante c.; capasso d.; trimarco b.; pedone c proc. natl. acad. sci agnieszka ; gofman nir ; willumeit, regine gkss research centre, germany; hasylab/desy, germany urtti novel cationic amphiphilic , -dihydropyridine derivatives for dna delivery dioleoyl phosphatidylethanolamine and peg-lipid conjugates modify dna delivery mediated by , -dihydropyridine amphiphiles masako ; kuriyama, naohiro poster abstracts lacking his-pro residues and stabilized by introduction of hval resulted in an analog h- hval-tyr-ile-tic-oh (al- ) that was very selective for irap versus ap-n and at( ) . the targeting properties of cell penetrating peptides cell penetrating peptides (cpps) offer the ability to penetrate across the plasma membrane of mammalian cells and to carry cargoes such as proteins, oligonucleotides and liposomes. due to this, cpps have gained high attention to improve the cellular uptake of the delivery of 'biologicals'. however, most of the research that has been performed with cpps restricted to in vivo studies. preclinical investigations are rare and the clinical validation of potential of cpps is required. a series of cpps were synthesized by solid phase peptide synthesis (spps) on an abi a synthesizer. the stability of these peptides in human serum was determined. subsequently, the uptake was studied in the following cell lines: sw , pc- , mh wt, hno in summary, the in vitro uptake studies did not reveal great differences that might have revealed a tumor type specifi city. in vivo studies revealed neither a distinct tissue uptake or tumor specifi city. design and synthesis of a tripartate paclitaxel prodrug for melanoma therapy therapy of melanoma continues to be a challenge since, regardless of the treatment used, long-term survival is quite uncommon. in an attempt to improve the effectiveness and decrease the toxicity of anticancer chemotherapy, one useful approach might be to administer a prodrug that specifi cally releases the active cytotoxic drug at the tumor site. in this work, we describe the synthesis of a peptide conjugate of paclitaxel potentially useful in the treatment of human melanoma, and characterized by the simultaneous presence of three functional domains: a "targeting domain", an "activation sequence", and the antitumor drug paclitaxel. the "targeting domain" of the prodrug is represented by an rgd-containing cyclic peptide, able to bind selectively to alpha-v beta- integrin, which is known to be highly over-expressed by both metastatic human melanoma cells, and endothelial cells of tumor vessels. the "activation sequence", responsible for the selective release of the drug, is a short peptide which is cleaved specifi cally by cathepsin b, a protease highly up-regulated in malignant tumors. the results of nmr conformational studies, as well as those of biological experiments aimed at evaluating the plasma stability of the prodrug and its ability to inhibit alpha-v beta- -mediated tumor cell adhesion to vitronectin, will be also presented. angiogenesis is a remodeling process characterized by the sprouting of new blood vessels from pre-existing ones. it occurs during embryogenesis and to a limited extent in the adult. vegf is a homodimeric protein and has been characterized as a prime regulator of angiogenesis and vasculogenesis; when cells lose the ability to control the synthesis of vegf, angiogenic disease ensues ( ) . in vitro studies show that vegf is a potent and specifi c angiogenic factor involved in the development of the vascular system and in the differentiation of endothelial cells ( ) . vegf biological function is mediated through binding to two receptor tyrosine kinases: the kinase domain receptor (kdr) and the fms-like tyrosine kinase (flt- ), which are localized on the cell surface of various endothelial cell types. this binding activates signal transduction and can regulate both physiological and pathological angiogenesis ( ) . vegf and its receptors are overexpressed in pathological angiogenesis, making this system a potential target for therapeutic and diagnostic applications ( ) . the extracellular portion of vegf receptors is comprised of immunoglobulin-like domains; deletion studies have shown that the ligand binding function resides within the fi rst three domains of flt- and in domains and of kdr. actually, no structural data are known on the extracellular portion of these receptors except for the second domain of flt- .. so, our aim is the cloning and the expression of part of extracellular domains of both vegf receptors for structural characterization and to be used in interaction studies with peptide ligands or small organic molecule. we are interested in an activation mechanism of receptor tyrosine kinases (rtks), especially, how the transmembrane (tm) and the intracellular juxtamembrane (jm) region couple ligand binding to tyrosine phopsphorylation. one of the rtks that we are working on is neu. neu (erbb ) is one of the four members of erbb receptor family. this rtk with a mutation in the transmembrane region (v e) has been recognized as a potent transforming oncogene product. structure of the tm region dimer and its structural difference between the wild type and v e mutant have been reported by smith et.al. ( ) , providing structural constraints for modeling the tm dimer. recently, we have performed a structural study on a tm-jm region of egfr (erbb ) showing that the tm helix breaks at the membrane interface and the unfolded jm region binds electrostatically to the membrane. we also have shown that ca + complex with calmodulin (ca +/cam) binds to the positively charged jm region and pulls this portion off the membrane. these results are consistent with an electrostatic engine model, postulated by mclaughlin and coworkers ( ) , for the autoinhibition and activation of the egfr. in this research, we are revisiting the structure of tm-jm region of neu to see if we can apply the electrostatic engine model to neu and to see if there is a structural difference between the wild type and the mutant tm-jm sequence. here we describe structural studies on the tm-jm sequence, neu( - ), reconstituted into bilayer vesicles. a combination of solid state nmr, infrared and fl uorescence measurements are used to draw conclusions that the unfolded jm binds electrostatically to the membrane and binding of ca + -calmodulin to the positively charged jm sequence can, under some conditions, reverse its charge and release it from the membrane. @the collagen triple-helix consists of three polypeptide chains, each of which takes the poly-l-proline ii form in a left-handed helix and undergoes a transition to a single coil state as the temperature increases. this characteristic structure is ascribed to the unique amino acid sequence x-y-gly, where x and y are commonly pro or (r)-hydroxyproline (hyp r ). @so far, various nmr studies have been done to explain the mechanism of folding and the thermal stability of the triple helical structure by using model peptides rich in imino acids. however, the complexity of nmr spectra imposed severe limitations on detailed analysis. the spectrum is complicated because of a large number of conformational isomers due to cis/trans isomerization around each imino acid causing various chemical shifts with small differences in h-nmr spectra. it has been demonstrated that the site directed n enrichment allows real time nmr monitoring of the folding of residues at specifi c locations of collagen model peptides ( ) . @to approach this problem, we have applied f-nmr study on various collagen model peptides containing (r)-fl uoroproline (fpro r ). compared to h and c, f chemical-shifts are extremely sensitive to small environmental changes around the nuclei and show a wide dispersion of chemical shifts. as a result, we not only distinguished the signals from the triple helix and unfolded states, but also differentiated the signals at intermediates states in d f-nmr spectrum of (pro-fpro r -gly) . @here, in this study, a combination of d nmr experiments including exsy, hoesy, and tocsy has been used to investigate detailed equilibrium properties of collagen model peptides. especially, in the d f-exsy spectra, we successfully observed many exchange cross peaks at high temperature. the f-nmr method shown here provides a clue to analyze the conformational transition, dynamics and stability of collagen triple-helix. by contrast to well-folded natural peptides, oligomers consisting of nonnatural building blocks, so-called foldamers, are highly stable towards proteolytic degradation which is a key feature in the development of peptide-based drugs. aside from the fact that foldamers consisting ofand -amino acid building blocks are able to display different secondary structures, the combination of different homologous building blocks to hybrid peptides has recently shown some promising results. [ , ] the ab initio mo theoretical studies have shown that / -hybrid peptides composed of alternating -and amino acid building blocks might be very well suited to mimic helical conformations. ( ) here we report the fi rst examples of -helical coiled coil formation with synthetic foldamers. based on the computational studies, we synthesized heterogeneous peptides by automated solid phase peptide synthesis (spps). purifi cation was carried out by hplc, and products were confi rmed by esi-tof-ms. furthermore, temperature-dependent cd spectroscopy was applied to investigate the stability of hetero-oligomers formed between, either / -or / / -hybrid peptides with a naturalhelical coiled coil peptide. in addition, the interaction potential with their natural -helical counterparts in aqueous solution were investigated by cd, and förster resonance energy transfer (fret). using reversed phase high performance liquid chromatography and two-dimensional gel electrophoresis, the lhcii proteins, lhcb - and fi brillins were effi ciently separated, and identifi ed by fticr-ms proteome analysis. some of the lhcii subcomplexes were shown to migrate from photosystem ii to photosystem i as a result of short term adaptation to changes in light intensity. in the mobile lhcii subcomplexes, decreased levels of fi brillins and a modifi ed composition of lhcii protein isoforms were identifi ed compared to the tightlybound lhcii subcomplexes. in addition, fticr-mass spectrometric analysis revealed several oxidative modifi cations of lhcii proteins ( ) . a number of protein spots in d-gels were found to contain a mixture of proteins, illustrating the feasibility of high resolution mass spectrometry to identify proteins that remain unseparated in d-gels even upon extended ph gradients. phosphinic analogue of the homophenylalanyl-phenylalanine dipeptide was demonstrated to be a potent, competitive inhibitor of cytosolic leucine aminopeptidase (lap, e.c. . . . ), mimicking the transition state of the reaction catalysed by the enzyme. exhibiting ki value at low nanomolar range it was ranked among the most potent inactivators of lap reported so far ( ) . recently, the compound has been also successfully employed to inhibit leucyl aminopeptidase of p. falciparum, a potential target protease for the development of new antimalarials ( ) . thus, its structure represented an attractive lead for the design and construction of next generations of modifi ed analogues. they were targeted towards both lap as well as a related metalloprotease -microsomal leucine aminopeptidase (apm, e.c. . . . ). these achievements, including recent results of studies on synthesis and activity, will be summarized here. we recently described the synthesis of some cyclic tetrapeptides bearing imidazole side chains and we analyzed, in detail, their copper(ii) binding properties in aqueous solution. the copper(ii) species obtained are of interest in relation to copper-protein active-site biomimetics. in particular, a -membered ring cyclic tetrapeptide c(lys-dhis-ala-his) (dk ) was synthesized by the solid-phase peptide synthesis method and its copper(ii) coordination properties were studied. surprisingly, all collected data strongly support the presence, at alkaline ph, of a stable peptide/copper(iii) complex that is formed in solution by atmospheric dioxygen oxidation. in order to clarify the mechanism of the copper oxidation through the average cu-n bond distance and to get information on the local geometry around copper, depending on the peptide sequence, we have collected experimental xanes and exafs spectra. the investigated cyclopeptide/copper complex shows pre-edge peak energy position and integrated intensity consistent with those of cu + model compounds. also, edge energy is consistent with the presence of trivalent copper. then, calculations performed on the exafs measures should give information on the local geometry, confi rming the square planar geometry proposed by us for this dk /cu(iii) complex. improved expressed protein ligation method for consecutive coupling of polypeptide fragments in the post-genomic era, to support the structural and functional studies of proteins, there is a growing demand for novel methods with which a wider range of selective protein modifi cations achievable. expressed protein ligation (epl) can be the method of choice for the preparation of unique protein derivatives not available by recombinant methods when the protein fragments extend the size accessible by solid phase peptide synthesis, and when extra chemical information (i.e. a special covalent modifi cation) is introduced into a well-defi ned part of the sequence. however, this method is not generally applicable, especially in the case when the target protein derivative is assembled from three or more polypeptide fragments. we demonstrate here an improved epl method that facilitates the effective ligation of large fragments in a consecutive way. the method employs a novel protection-deprotection scheme. supported by otka f and jános bolyai fellowship (cs.t.). protein ligation using protein trans-splicing , in order to facilitate protein ligation using synthetic peptides. highly effi cient fl uorescent labeling of proteins has been demonstrated in a site-specifi c manner by protein ligation using the newly designed intein. this approach could be applied for sitespecifi c modifi cation of proteins in vivo because protein trans-splicing is an autocatalytic process requiring no additional cofactor. moreover, protein trans-splicing approach could be extended to dual site-specifi c modifi cation using an additional intein. la doppel (dpl) is a glycosylphosphatidylinositol-anchored protein that exhibits % sequence homology with prion protein but lacks the octarepeat region. the role of prion is not yet completely known but prpc seems to be involved in cu (ii) traffi cking from synaptic clefts and in preventing neuronal oxidative damage. doppel is expressed in heart and testis and has been shown a regulator of male fertility. therefore, despite a high sequence homology and a similar three-dimensional fold, it's possible to hypothesize that the function of the two protein is not correlated. in the literature is reported that both prpc and of dpl are able to bind cu (ii) but the characterization of the complex species is well defi ned only for prpc. several binding sites of dpl are involved in the complexation with cu (ii). [ , ] in the present work we have focused our attention to the third alpha-helix of the dpl which is the preferential binding site for the metal ion. we have synthesized two peptide fragments relative to the - sequence of the dpl. the fi rst peptide has the native sequence whereas in the second fragment the asp was replaced by a asn residue. this substitution was performed to understand the role played by the carboxylate group of the asp residue in the complexation of cu (ii). cd ad nmr spectra were carried out on the two peptides in absence and presence of cu (ii) to investigate conformational features upon cu (ii) binding. finally, the complex species formed were completely characterized by using spectroscopic (nmr, cd, uv-vis, epr) and thermodynamic measurements (potentiometric titrations). three-dimensional ( d) domain swapping of proteins is a phenomenon associated with formation of oligomers and fi brils of several amyloidogenic proteins ( ) . the propensity of a particular protein to undergo domain swapping depends on factors like changes in environment or presence of specifi c mutations, but also on some topological determinants. it was proposed that domain swapping-prone proteins have some common "hot spots" in their structures that facilitate the process. one of these motives are so called "hinge loops" -turns showing enhanced propensity for unfolding due to conformational constraints ( ). cystatin c (hcc), main inhibitor of cysteine proteases in human body was shown to dimerize ( ) and oligomerize ( ) through domain swapping. hcc contains highly conserved throughout the cystatin family loop l , connecting two beta strands which, together with the only helix in the protein fold create a structural unit that undergoes the d process. in order to confi rm important role of distortions in l loop, which are centered around valine residue in position in the dimerization/ oligomerization process of hcc, we designed point mutations which should inhibit or promote aforementioned processes. as it was expected, substitution of val with either asparagine or aspartic acid residues abolished dimerization process almost completely. in contrast, v p mutant shows enhanced dimerization propensity. above observations are in good agreement with theoretical studies, performed on entire hcc and its fragment encompassing the hairpin structure centered on loop l . the expansion of the cag trinucleotide that produces polyglutamine (polyq) segments in several proteins is responsible for at least eight neurodegenerative diseases. a possible therapeutic approach would be to inhibit the polyq self-assembly process using disrupting agents. although, screening studies have identifi ed several polyq aggregation inhibitors, their mechanism of action remains unknown. this is due to the poor aqueous solubility and fast aggregation of uncharged polyq peptides, which complicate their study. the most common strategy to resolve these problems is to produce polyq stretches fl anked with charged residues. however, this strategy was found to modify the aggregation behaviour of polyqs, their aggregate structure and their affi nity for disrupting molecules. to circumvent these problems, polyq peptides containing morpholine moieties, whose charge is ph-dependent, were produced using fmoc-based chemistry on spps. following an acid treatment, the designed peptides were soluble in acidic aqueous solutions and their aggregation rate could be controlled by ph variations and followed by dynamic light scattering. furthermore, aggregation initiated at physiological ph provided uncharged fi brils allowing the evaluation of the effects of charged disrupting agents. the actions of known polyq aggregation inhibitors such as polyq-binding peptide (qbp ), trehalose and congo red were studied. peptide-protein and protein-protein interactions play key roles in most biological processes, and therefore represent valuable targets for drug discovery. an approach in the search of new chemical entities able to interfere with these interactions is the use of small molecules able to mimic or to induce precise aspects of specifi c peptide secondary structures. among the secondary structure elements, reverse turns have been shown relevant in many biomolecular recognition events. thus, different efforts have been directed to the design of turn mimetics or inducers. in this sense, -substituted azetidine- -carboxylates, synthesized by our group through a versatile procedure from amino acids, possess the dihedral angle restricted to approximately º or - º, depending on the absolute confi guration at the asymmetric carbon. these values are similar to those reported for the central residues of main types of -and -turns, and in fact, recent studies have shown the ability of the , -disubstituted azetidines to induce -turns when incorporated at the i+ position of model peptides. to know if this conformational behavior is distinctive of these restricted amino acids, we now investigate the infl uence of the incorporation of these amino acids at the i+ position of model peptides. with this aim, we have synthesized a series of simplifi ed tetrapeptide models, rco-l-ala-azx-nhme, in which the rco and nhme groups are simplifi cations of the i and i+ residues of the turn, respectively. for comparative purposes, dipeptide derivatives incorporating azetidine- -carboxylate, pro and -mepro have also been prepared. the turn inducing capacities of all these model tetrapeptides have been analyzed by molecular modelling, ¹h rmn, ft-ir and x-ray methodologies. the results have shown the importance of the , -disubstitution at the heterocyclic amino acid for stabilizing reverse turns, and the infl uence of the ring size on the induced turn type infl uence of position of dehydroamino acid in peptide chain on conformation of dehydropeptides containing two dehydroamino acid residues it is known that presence of c -c double bound in dehydroamino acid infl uences on dramatic limitation of conformational space, not only side-chain but also main-chain [ ] [ ] . according to the peptide's length and neighboring amino acid residues, dehydroamino acid exerts s-shaped, -turn or helical conformation in peptides [ ] [ ] [ ] [ ] [ ] . angiotensin-i converting enzyme (ace) somatic form bears two catalytic domains with two zn metal ions, while the testis form bears remarkable similarity to the aminoacid sequence of ace c-catalytic domain and only one zn metal ion. however, both exhibit their hydrolytic activity to vasoactive peptides such as angiotensin i (angi) and bradykinin (bk), though with different effi cacy. in order to study experimentally the possible interaction and binding events between the ace active site(s) and known ace substrates or inhibitors, we constructed peptide-based catalytic site maquettes (csm). -residue peptides were synthesized through solid-phase peptide synthesis having the aminoacid sequence that correspond to the ace val -ala n-domain segment and to the val -ala c-domain segment and enzyme's active sites were reconstructed through addition of zinc metal ion in solution. the resulting complexes have the metal ion bound in a native-like mode, as manifested by previously reported nmr data. the reconstituted peptides were titrated separately by captopril and angiotensin-i with a molar ratio ace:captopril/angi : and : , respectively. titration was followed by h nmr spectroscopy and spectral changes (chemical shifts, line broadening, etc.) were recorded and analyzed. after the end of angi titration the solution of interacting peptides was titrated with captopril and the titration was followed by h nmr spectroscopy. at the end of each titration d homonuclear h- h tocsy and noesy spectra were recorded. analysis of high-resolution nmr data probes the conformational variation of ace csm and peptide substrates upon interaction, and in the presence of captopril in ace-angi solutions. contemporary development in nanotechnology is fabrication of nanostructured materials using the peptide-based self-assembly. importance of peptide as monomeric components is their capacity to be engineered to mediate spontaneous supramolecular self-assembly and their inherent chemical nature that facilitates chemical and biological recognition process. tubular self-assembled peptide nanostructures are of special interest since these can serve in various applications including 'bottom-up' fabrication of molecular scaffolds, nanoelectromechanical systems and nanomachines. an important discovery by gazit group revealed that the simple dipeptide molecule of phenylalanine which is core recognition motif of the alzheimer's -amyloid polypeptide effi ciently self-assembles into well-ordered nanotubular structure ( ) which could be controlled by relatively simple chemical modifi cations at its termini. based on these observations and our interest in the synthesis of softmaterials for nanotechnology, we have successfully synthesized novel monomeric units of di-peptides with ureido functional group at nterminal of various hydrophobic l-amino acids. these derivatives have been prepared by using solid phase peptide synthesis protocol with cost effective process retaining the chirality of molecule. these ureido dipeptide formed nanostructures with high yield under mild and controlled condition in aqueous media. to fully understand the molecular and supramolecular mechanism guiding the formation of nanostructures we have selected a multidisciplinary approach by combining spectroscopic studies with advanced electron microscopic techniques. the morphology of nanostructures can be controlled by using variety of l-amino acids. these novel ureido di-peptides nanotubes can be used in fabrication of biocompatible peptide-based nanostructures and they will undoubtedly have nanotechnological applications. biomolecules are excellent in hierarchically organizing a characteristic structure by self-assembly. the spontaneous assembly of biomolecules has advanced a bottom-up approach for fabrication of nanostructured materials. we have developed fabrication of controlled nanofi bers through self-assembly of simple and short de novo designedsheet peptides with unique sequences [ , ] . the single straight nanofi ber with high regularity constructed by -sheet fi peptide (sequence: pkfkiiefep) was functionalized with proteins by biotinylated peptides ( ) and using functional anchors that have binding and functional groups. conjugation of self-assembled peptide nanofi bers with biomolecules such as peptides and proteins will expand their potentialities of the nanofi bers for various applications. to identify peptides binding to the self-assembled fi peptide nanofi ber, a phage display combinatorial screening using a random peptide library was performed. after fi ve rounds of biopanning, phage pools with highly specifi c affi nities to fi nanofi bers were screened. as a result of dna sequencing after phage cloning, phage clones were identifi ed. binding affi nities of these clones were quantitatively investigated by an enzyme-linked immunosorbent assay. these clones showed specifi c affi nities to fi nanofi bers, as compared with ff and vi nanofi bers having slightly differences of amino acid sequences. affi nities and specifi cities were dependent on the sequence of each peptide, indicating that different amino acid sequences contributed to peptide interactions with nanofi bers. infrared spectroscopy studies by using chemically-synthesized peptides showed that the peptide binds specifi cally to the fi nanofi ber with structural transitions. owing to their diversity in size and shape, easy access, and biocompatibility, peptides represent versatile units for the construction of h-bonded tubular assemblies and other biomimetic materials with potentially useful applications. so-called peptide nanotubes (pnts) have been obtained through multiple and complementary approaches ( ). originally designed from -peptides made of d-and l-amino acids ( ) , fl at macrocyclic systems forming cylindricalsheet like assemblies have diversifi ed to include oligoamides made from higher amino acid homologs as well as peptide hybrids (e.g. ,peptides). tubular sheet-like assemblies however are not restricted to oligoamides. the urea group which shares a number of features with the amide linkage, i.e. rigidity, planarity, polarity, and hydrogen bonding capacity is an interesting surrogate. we and other have demonstrated that macrocyclic biotic and abiotic n,n'-linked oligoureas have a unique propensity to self-organize into polar h-bonded nanotubes ( ). partial peptide backbone n-methylation has been introduced as a general strategy to generate truncated stacks (i.e. h-bonded dimers) useful to gain access to the thermodynamics of nanotube formation ( ). herein, we describe biotic macrocyclic amide/urea hybrids with partially nalkylated backbones as new candidates for the formation of h-bonded dimers. a cysteine branched pga (polyglutamic acid) hydrogel is being investigated for therapeutic peptide and protein controlled delivery. entrapment of hgh as a model protein in this hydrogel has been carried out in order to check its later release in different aqueous buffered media and stability. different physico-chemical analysis (by maldi-tof, sec-mals-ri, h-nmr, cd, …) of the released hgh showed no effect on destabilization of the protein. controlled release systems of proteins involve important challenges such as maintaining the protein integrity. researchers need an exhaustive understanding of protein stability issue in drug delivery formulations. common degradative reactions in proteins involve: oxidation, deamidation, stability of disulfi de bonds and aggregation, which may affect secondary and tertiary structure of proteins and thus, their biological activity. the high water content which imbibes the polymer network of hydrogels enables a good accommodation for proteins. polyamino acids have been already studied in some biotechnological applications, however, in spite of their potential, they have barely been taken into account for the development of controlled release microparticles. we show that proteins could be easily loaded into cys cross-linked pga hydrogels. the protein hgh has been loaded and released preserving its physico-chemical integrity. thus, these hydrogels are promising for their use as therapeutic protein delivery systems. totally synthetic collagen-like gels by intermolecular folding of designed peptides our goal is to develop artifi cial collagens that can use as safer and functional biomaterials. here, we report the development of collagenlike gels by means of the intermolecular folding of chemically synthesized peptides. the peptides are disulfi de-linked trimers of collagenous gly-x-y triplet repeats with self-complementary shapes. the self-complementary peptides are able to form elongated triple-helix through spontaneous intermolecular folding. upon cooling of the peptide solutions, hydrogels of peptide supramolecules formed. the gel-sol transition was appeared to be reversible, and the transition temperatures were found to be tunable by the design of the peptides. our strategy for the totally synthetic collagen will offer possibilities for the development of innovative biomaterials. recent advance in biotechnology enables us to fi nd the peptides with affi nity for nonbiological materials and with function of mineralizing inorganic materials. the use of the functional peptides is attracting a growing interest for bottom-up fabrication approaches of nanoscale devise. zinc oxide (zno), a semiconductor with a wide direct band gap, possess unique optical, acoustic, and electronic properties, so that it is one of most widely studied metal oxides for solar cells, ultra violet nanolaser, blue light-emitting diode and so on. this wide variety of applications requires various fabrications of morphologically and functionally distinct zno nanostructures. here, the peptides which are selected from the phage-displayed peptide library with a -mer on the surface, can bind zno particle but not other metal oxides particle, and further, the zno-binding peptides play an important role on the crystal growth of zno in its synthesis. the anti-zno peptide can assist the synthesis of zno nanoparticles from a zn(oh) solution even at ºc, and further, the peptide leads to the self-assembly of synthesized zno particles to fl ower-type morphologies. we describe the biomimetic zno synthesis using the artifi cial peptide with affi nity for zno. the design of nanoparticles suitably functionalized for applications in biology or medicine is an ongoing challenge for both chemists and biologists. nanoparticles such as quantum dots, gold nanoparticles or carbon based-materials offer, in addition to their remarkable physical properties, the possibility to be functionalized by biomolecules, making them suitable for sensing, detection, diagnostic, and/or therapeutic applications. recently, quantum dots have been designed for biological imaging owing to their unique size-dependent fl uorescence properties. however, their plausible toxicity still remains a major concern for in vitro and in vivo applications. nanodiamonds, (nds) are also candidates for biomedical applications considering their intrinsic or induced fl uorescence and biocompatibility. the work will describe: i) the functionalisation and the characterization of nm non-fl uorescent cnds and ii) their use in toxicity and transport studies in living cells after the grafting of a fl uorescent model peptide. we chose to introduce a nonpermeant fl uorescent peptide for the tracking of the nanoparticles on or inside the cells. nanodiamonds (cnds, nm) coated by silanisation or with polyelectrolyte layers have been grafted with a fl uorescent thiolated peptide via a maleimido function, leading to aqueous colloidal suspensions stable for months. for each step of the preparation, the diameter of the nds measured by dynamic light scattering (dls), the zeta potential, the amount of amino groups grafted onto the surface, as well as the stability of the different nds suspensions no cytotoxicity was observed up to hours incubation with cho cells with any of the prepared ( g/ml). their capacity to enter mammalian cells, and their localisation inside cho cells, have been ascertained by confocal microscopy, refl ected light and fl uorescence. hepatitis b virus is one of the most common problem and potential danger of all over the world and also in our country. it is known that the use of proteins and peptide antigens as vaccines has several potential advantages over whole viral or bacterial preparations. to elicit the maximum immunogenic response from synthetic peptide antigens ,it is generally necessary to bind the peptide to carrier protein. moreover, to realize the full potential of synthetic peptide antigens protein-peptide conjugate will have to be used with an adjuvant. we have developed new approches for obtaining highly immunogenic peptide conjugatessynthetic polyelectrolytes (pe) were used for the conjugation with peptide molecules in which pe carry out the carrier and adjuvant roles simultaneously. this concept drive us to create new immunogenic bioconjugates of hepatitis b surface antigenic polypeptides (hbsag) with synthetic pe. in this study we used the region - of the s gene. the synthesis of peptides was performed by explorer pls ® automated microwave synthesis workstation (cem), by using f-moc chemistry. copolymers of acrylic acid with different monomers were used as a pe for the conjugation with polypeptides. pe-peptide conjugates was synthesized by microwave assisted method. composition and structure of bioconjugates werw characterized by hplc, spectrofl uorometry and different spectrophotometric methods. protein-protein and protein-ligand interactions are among the few essential cell processes whose understanding provide us insights into fundamental events of the life cycle. aside from in silico methods, natively folded proteins are an absolute prerequisite in all current biotechnological tools used for the study of these interactions. however, the recently developed ianus peptide array has a potential to evolve in to a protein-free method for detection of protein-protein interaction sites. using this assay, protein-protein interactions could be represented by and investigated as peptide-peptide interactions using peptide pairs immobilized on a solid support.( ) . two main obstacles had to be overcome for successful implementation of this array: (i) the library size and (ii) identifi cation of interacting peptide pairs. if, for example, interactions between two small proteins of only approx. amino acids each should be analyzed, a library of ca. peptide pairs would be needed to cover all possible combinations of overlapping peptides derived from these proteins. although libraries of several thousand peptides were already successfully synthesized, the library size could be reduced drastically if the binding pocket of one protein is already known or if the protein-ligand interactions are studied. up to date, two methods for the identifi cation of interacting peptides in a library have been developed in our group. both methods will be demonstrated in studies of protein-protein and protein-ligand interactions. the role of the non-helical tailpiece in myosin ii assembly the hebrew university of jerusalem, israel self-assembly of macromolecules into large complexes is often mediated by folding of disordered regions. we used peptides to investigate the role of the non-helical tailpiece from non-muscle myosin iic (nm-iic) in its self-assembly process. nm-iic is a motor protein composed of a globular motor domain in the n-terminal region and a coiled-coil rod domain in the c-terminal region, which terminates with a non-helical -residue tailpiece (residues - ) . nm-iic molecules undergo self-assembly into fi laments that are necessary for its contractile activity. the tailpiece has an unfolded character and participates in the assembly process of nm-iic. the n-terminal region of the tailpiece (residues - ) has a net positive charge of + while its c-terminal region (residues - ) has a net negative charge of - . we designed peptides that correspond to the entire tailpiece and to its negative and positive regions. these peptides were used to study interactions with the rod, their structures in the free and bound states, and structural changes they undergo upon binding to residues - of nm-iic rod. fluorescence anisotropy binding studies showed that a peptide corresponding to the positive region of the tailpiece (residues - ) bound the nm-iic rod (residues - ) with an affi nity of m. circular dichroism studies showed that the positively charged peptide became folded upon binding the rod, indicated by a shift of the spectral minimum from nm to nm upon binding. a peptide corresponding to the negative region of the tailpiece (residues - ) did not bind nm-iic (residues - ). based on our results, we suggest that the positive region of the tailpiece is required for ordering and aligning neighboring molecules by electrostatic attraction, leading to fi lament formation. our results provide molecular insight into the role of the structurally disordered tailpiece of nm-iic in the selfassembly process. stefanidakis, michael; karjalainen, katja; pasqualini, renata; arap, wadih; koivunen, erkki md anderson cancer center, united states acute myelogenous leukemias (aml) are characterized with non-random patterns of medullary and extramedullary invasion. we hypothesized that a supramolecular complex, the leukemia cell invadosome, which contains certain integrins, matrix metalloproteinases (mmps) and other as yet unidentifi ed proteins, is essential for tissue invasion and may be central to the phenotypic diversity observed in the clinic. here we show that the specifi c binding of mmp- to leukocyte surface ám integrin is required for pericellular proteolysis and migration of amlderived cells. an effi cient antileukemia effect was obtained by peptide inhibitors that prevented prommp- cell surface binding, transmigration through an endothelial cell layer, and extracellular matrix degradation. notably, the functional protein anchorage between ám integrin and prommp- described in this study does not involve the enzymatic active sites targeted by any of the existing mmp inhibitors. taken together, our results provide a biochemical working defi nition for the human leukemia invadosome. disruption of specifi c protein complexes within this supramolecular target complex may yield a new class of anti-aml drugs with anti-invasion (rather than cytotoxic) attributes. the molecular mechanisms of action of the polycationic lysine homoand heterodendrimers on functional activity of adenylyl cyclase signaling system (ac system) in the myocardium and the brain of rats were studied. the lysine homodendrimers of the third (i), the fourth (ii) and the fi fth (iii) generations, as well as the lysine heterodendrimers of the fi fth generation -[(nh ) (lys-glu) (lys-glu) (lys-glu) (lys-glu) (lys-glu) lys-ala-ala-lys(clac)-ala-nh ] (iv), [(nh ) (lys-ala) (lys-ala) (lys-ala) (lys-ala) (lys-ala) lys-ala-lys(clac)-ala-ala-nh ] (v) and [(nh ) (lys-gly-gly) (lys-gly-gly) (lys-gly-gly) (lys-gly-gly) (lys-gly-gly) lys-gly-gly-lys(clac)-ala-ala-nh ] (vi) stimulated by receptor-independent mechanism the activity of heterotrimeric g proteins, preferably of inhibitory type, and interacted with c-terminal regions of their -subunits. the homodendrimers ii and iii and heterodendrimer v were more effective g protein activators. the treatment of the membranes with pertussis toxin (inactivating g i protein), but not with cholera toxin, led to a decrease of g protein activation effect of the dendrimers. the lysine dendrimers disturbed the functional coupling of the receptors of biogenic amines and peptides hormones with g i proteins and, to a smaller extent, with g s proteins. it was illustrated by the decrease of regulatory effects of the hormones on ac activity and g protein gtp binding and by the decrease of receptor affi nity to agonists in the presence of the lysine dendrimers, as result of receptor-g protein complex dissociation. it was shown that the molecular mechanisms of the action and the g protein selectivity of the polylysine dendrimers are similar to those of mastoparan and melittin, natural toxins of insect venom, which also preferably activate g i/o proteins and inhibit g i/o -coupled signaling. acknowledgements: supported by rfbi (grant - - ) and «russian science support foundation». genetics, bielefeld university, germany dna-protein interactions are a key element in the regulation of cellular processes. transcription factors are able to recognize their cognate dna sequences and regulate the expression of proteins. as a model system, the specifi c dna binding of the transcription factor phob from e. coli is investigated in single molecule experiments. structurally, phob belongs to the family of winged helix-turn-helix proteins. it is composed of a transactivation domain (amino acids - ) and a dna binding domain (amino acids - ). after phosphorylation of the transactivation domain, the protein binds to specifi c dna sequences containing a tgtca consensus sequence. different c-terminally modifi ed protein epitopes representing parts of the dna binding domain of phob were chemically synthesized using microwave assisted solid phase peptide synthesis. the binding contributions of these molecules are compared to the complete dna binding domain ( - ). this protein was purifi ed using intein mediated protein splicing, an additional cysteine was ligated to the protein by intein mediated ligation. performing single molecule force spectroscopy experiments, kinetic off-rates were obtained, and sequence specifi c dna-binding of both peptide and protein was proven in competition experiments. alanine scans of strategic residues revealed the contributions of single amino acid residues for peptides and proteins. structural investigations of the peptides, the proteins and dna/protein complexes were performed using circular dichroism measurements. this method revealed structural differences of the peptides, proteins and dna upon complex formation. furthermore, the protein/dna or peptide/dna interaction was determined by surface plasmon resonance experiments and electrophoretic mobility shift assays. acknowledgment: this project was supported by dfg (sfb ). reversed-phase chromatography is important in the development of well-characterized peptide pharmaceuticals. several factors infl uence sensitivity, resolution, and retention for lc and lc-ms applications: bonding chemistry, pore size, particle size, column confi guration, and solvent composition. by decreasing the particle size of hplc packings, column effi ciency is increased. we demonstrate the use of short -mm length hplc columns packed with . m, Å c silica for ultrafast, one-minute peptide analysis with moderate backpressures (< psi) using a conventional hplc system. for more complex peptide samples, such as protein digests, on conventional hplc, a novel column confi guration packed with . m, Å c is described for -to- min. separations that traditionally take to minutes. we also discuss fast two-minute separations on an ultra-high pressure system using a variety of . m, small pore columns with unique selectivity. "unknown-genome" proteomics-based identifi cation of a new nadp-epimerase/dehydratase from desulf. phosphitoxidans by inverted-pcr, edman-sequencing and high resolution mass spectrometry protein identifi cation in proteomics is generally based on the availability of genomic data. using amenable databases, identifi cation in "bottom-up" proteomics is often straightforward but is highly complex in the absence of genome data, which typically requires "de novo"-identifi cation approaches. we present here a new approach for identifi cation of proteins from a bacterial strain, desulfotignum phosphitoxidans, with unknown genomic background, using a combination of (i), inverted pcr of degenerate primers derived from n-terminal edman sequencing, and (ii) high resolution maldi-fticr mass spectrometric peptide mass fi ngerprint-proteomics of expressed proteins. desulfotignum phosphitoxidans is an anaerobic sulfatereducing bacterium from marine sediment in which phosphite oxidation was found crucial for energy metabolism. culturing under different growth conditions provided specifi cally expressed proteins with molecular masses of ca. kda in the presence of phosphate, which were subjected to d-gel electrophoretical separation for soluble and membrane fractions using pdquest comparative analysis. n-terminal sequences of the proteins, determined by edman analysis were used for inverted pcr of degenerate primers, and provided a series of orf candidates, one of which coded for a putative nad-dependent dehydratase. in a complementary approach, protein spots from the d-gels were excised, digested with lys-c protease, and digestion mixtures analysed by maldi-fticr-ms. the accurate peptide masses unequivocally matched to identify a new nad-dependant epimerase/ dehydratase. the detailed functional characterization of the new protein, and further development and application of this approach to proteome analysis with unknown genomic background, are currently in progress. based on specially treated large pore silica and enhanced with a proprietary bonding process, vydac ms reversed-phase (rp) hplc columns offer superior performance for peptides and proteins. the deamidation of human growth hormone (hgh) has been monitored for many years by rp using vydac columns. the vydac ms c column provides the best overall performance characteristics (recovery, resolution, and peak symmetry) for the common important assay of hgh and desamido hgh. although hydrophobic membrane proteins are particularly diffi cult to separate, the vydac ms c column provides better separation and recovery (up to % higher vs. other leading columns) for a reptilian reovirus p protein and myristolyated form, a component of a potentially new vaccine delivery system. separation of the trypsin digest of fetuin, a glycoprotein, exhibits improved selectivity for peptide mapping on a vydac ms c column compared to other c columns, revealing some peaks otherwise not seen. the improved selectivity for peptides on the vydac ms columns results in better primary structure defi nition and easier identifi cation of degradation products and other protein characteristics. hemoglobin peptides in mammalian tissues: facts or artifacts?yatskin, oleg; karelin, andrei; ivanov, vadim shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, russian federation comparative rp-hplc and ms analysis of peptide composition of rat brain, heart, lung and spleen acidic extracts was performed. the extracts were prepared from snap-frozen organs both in the presence and in absence of protease inhibitors ( - m pepstatin a, - m pmsf, mm edta) which stabilize the peptide composition of tissue acidic homogenates. the absence of inhibitors led to appearance of novel peptides and did not affect the concentration of predominant peptide components detected in samples prepared with protease inhibitors. therefore, the latter were considered as present in the tissues prior to extraction, i.e. as endogenous. the majority of predominant peptide components were found common in the studied tissues. we believe that the presence of predominant (> pmol/g) peptide components in the protease-inactivated tissue extracts can be reliably extrapolated to their in vivo presence in the tissues. the endogeneity of minor (< pmol/g) components requires a separate consideration. generally, the results of peptidomic studies strongly depend on sample preparation procedures involved, making diffi cult the straightforward comparison of the results obtained by different research groups. urine is a promising source of endogenous peptide biomarkers, because the protein concentration is -fold lower than in plasma but the peptide concentration is equal. urine contains small metabolites that interfere with peptide analysis and urine fl ow varies between subjects and within a day. these variations pose a challenge to urine sample preparation, which, in case of serum, has been shown to be critical for reproducibility. we aim to optimize the enrichment method for urine peptides, to characterize these and to fi nd potential biomarkers. all methods resulted in good peptide recovery if the peptide concentration was normal and metabolite content low. slightly different peptides were obtained with different methods. the df preferentially recovered hydrophilic peptides, while sec-spe recovered high molecular weight peptides better than the df-method. sec-spe provided the best peptide enrichment especially for samples with high metabolite concentration. normalization was critical especially with very dilute samples. without normalization the number of peptides detected was highly dependent on urine concentration. normalization against specifi c gravity proved the most reproducible results. we have identifi ed several peptides found in all samples and some differences between cancer and control samples. validation of these differences is currently under way. jalili, pegah ; ball, haydn sigma-aldrich, united states; in order to improve the detection of phosphorylated peptides/proteins, we developed a novel protocol that involves the chemical derivatization of phosphate groups with a chemically engineered biotinylated-tag (biotintag), possessing three functional domains; a biotin group for binding to avidin, a base-labile -carboxy fl uorenyl methoxy-carbonyl ( -carboxy fmoc) group, and a nucleophilic sulfhydryl moiety on the side-chain of cysteine. using this approach, the derivatized, enzymatically digested peptides were selectively separated from unrelated sequences and impurities on immobilized avidin. unlike previously published phosphopeptide enrichment procedures, this approach upon treatment with mild base, liberates a covalently bound gly-cys analog of the peptide(s) of interest, exhibiting improved rp-hplc retention and ms ionization properties compared to the precursor phosphopeptide sequence. the results obtained for a model peptide akt- and ovalbumin protein digest, demonstrated that the method is highly specifi c and allows selective enrichment of phosphorylated peptides at low concentrations of femtomoles/ l. in search of physiological substrates of brain prolyl oligopeptidase. prolyl oligopeptidase (pop) is a serine protease, which cleaves small peptides at the carboxyl side of an internal proline residue. substance p, arginine-vasopressin, thyroliberin and gonadoliberin are proposed physiological substrates of this protease. pop has been implicated in a variety of brain processes including learning, memory, and mood regulation, as well as in pathologies such as neurodegeneration, hypertension and mood disorders. however, there is no defi nite information about the physiological substrates of pop in brain. we have determined previously that some pop inhibitors, when administrated orally or intraperitoneal, are able to cross the blood-brain barrier and inhibit pop ex vivo. in this work also inhibition duration according to the doses were determined. using this system we studied the natural occurring peptide profi le in brain tissues from inhibited animals and compare it with the profi le in control tissue. we were able to design a method in which the background peptide level was importantly decreased which allowed us to identify, with high degree of confi dence, the peptides which were changed in rat hypothalamus upon pop inhibitor treatment. conclusions about the physiological substrates of pop are discussed. [ , ] . in the present work, we further studied the dependence of peptide production by the cells on their functional state. using rat hepatocytes as a model, we compared peptide sets generated by: ( ) primary hepatocytes in a differentiated state; ( ) dedifferentiated primary hepatocytes with fi broblast-like phenotype; ( ) rat hepatoma cells. peptides were extracted from the cellular lysates and supernatants by solid-phase extraction and then separated by rp-hplc. peaks were analyzed with maldi-tof and/or maldi-tof/tof. acknowledgements: this work was supported by ras presidium grant "molecular and cellular biology"; russian president grant "scientifi c schools" # . . ; rfbr grant # - - -a. triostin a is a naturally occurring quinoxaline depsipeptide antibiotic originally isolated from streptomyces s- - . with its two quinoxaline moieties it bis-intercalates into dna in a gc selective manner. triostin a contains n-methylated l-valine and l-cystein. the unmethylated analog tandem binds at selectively due to a different hydrogen bonding pattern. substitution of the disulfi de bridge of tandem by an azobenzene moiety leads to photoswitchable analogs. their photoswitchability was investigated and quantifi ed using uv, cd and nmr spectroscopy as well as hplc. in order to determine the three-dimensional structure, conformational analysis by nmr spectroscopy in combination with molecular dynamics calculations was carried out. in this process, distance restraints were obtained from nmr spectra measured in dmso-d and applied in distance geometry/simulated annealing followed by molecular dynamics calculations. as it is possible to distinguish between cis-and transisomer in nmr spectra due to the difference in the chemical shifts, structures of both isomers can be investigated. dna binding studies were carried out using an optical tweezers setup with -dna. furthermore, uv melting curves were recorded for the tandem derivatives in complex with dna. cd spectroscopy was applied to observe changes in dna structure upon formation of the complex. in addition to structural investigations, the antibiotic activity of the tandem derivatives was investigated in minimal inhibition concentration assays against gram-positive bacillus subtilis. acknowledgment: this work was supported by the dfg (sfb ). k. gaus gratefully acknowledges the fi nancial support (phd grant) by the international nrw graduate school of bioinformatics and genome research. cyclic peptide nanotubes are formed by self-assembly of cyclic peptides with alternating l-and d-amino acid. however, peptide nanotube selfassembly phenomenon had not yet been made cleared. intermolecular electrostatic interaction is one of the important driving forces to assemble cyclic peptides to peptide nanotubes. here in, a series of cycilc hexapeptides, cyclo(-l-lys-gly-) , cyclo(-l-glu-gly-) , cyclo(-l-lys-d-ala-) , cyclo(-d-glu-l-ala-) , cyclo(-l-trp-d-lys-) , and cyclo(-l-trp-d-glu-) , were designed and synthesized by solid-phase peptide synthesis using fmoc strategy. turbidity study revealed that the abilities of self-assembly formations of the : mixtures of cyclo(-l-trp-d-lys-) and cyclo(-l-trp-d-glu-) in aqueous solution are extremely higher than the abilities of each individual positively or negatively charged cyclic peptides. transmission electron microscope (tem) experiments showed that mixture of cyclo(-lys-xxx-) and cyclo(-glu-xxx-) formed fi brous structure. tem images indicated that, the diameters of the nanotubes were approximately - nm. diameter of similar cyclic peptides was found to be nm approx. these results also supported that the bundle formation of peptide nanotube. conformations of monomer cyclic hexapeptides were calculated based on nmr observation. estimated formation of peptide nanotube is consisted with the bundle structure. koèevar, nina; obermajer, nataša; kreft, samo faculty of pharmacy, slovenia therapeutic proteins offer a promising potential as effective drug compounds. however, proteins are generally poorly transported across biological membranes due to hydrophilicity. fatty acylation with reactive fatty acid derivatives represents one of the basic methods for increasing the proteins' hydrophobicity and improving membrane permeability. key: cord- -w lczb authors: robson, b. title: bioinformatics studies on a function of the sars-cov- spike glycoprotein as the binding of host sialic acid glycans date: - - journal: comput biol med doi: . /j.compbiomed. . sha: doc_id: cord_uid: w lczb sars-cov and sars-cov- do not appear to have functions of a hemagglutinin and neuraminidase. this is a mystery, because sugar binding activities appear essential to many other viruses including influenza and even most other coronaviruses in order to bind to and escape from the glycans (sugars, oligosaccharides or polysaccharides) characteristic of cell surfaces and saliva and mucin. the s n terminal domains (s -ntd) of the spike protein, largely responsible for the bulk of the characteristic knobs at the end of the spikes of sars-cov and sars-cov- , are here predicted to be “hiding” sites for recognizing and binding glycans containing sialic acid. this may be important for infection and the ability of the virus to locate ace as its known main host cell surface receptor, and if so it becomes a pharmaceutical target. it might even open up the possibility of an alternative receptor to ace . the prediction method developed, which uses amino acid residue sequence alone to predict domains or proteins that bind to sialic acids, is naïve, and will be advanced in future work. nonetheless, it was surprising that such a very simple approach was so useful, and it can easily be reproduced in a very few lines of computer program to help make quick comparisons between sars-cov- sequences and to consider the effects of viral mutations. as far as is known to history, no coronavirus [ ] has been as disturbing to humanity as the human pandemic of the novel coronavirus [ ] now known as sars-cov- . in quick response to determination of the final version of the rna sequence of the wuhan seafood market isolate, the present author examined functional sites of sars-cov- that are highly conserved across the coronaviruses [ , ] , and which thus likely to exhibit escape mutation that can quickly undo the good work of the developers of vaccines and therapeutic agents. so far, the published papers have concerned the spike glycoprotein [ ] [ ] [ ] . exploration of known and newly found proteolytic cleavage sites in the spike glycoprotein of sars-cov- that are well conserved is a popular area of inquiry for sars researchers because such sites can interact with human host airway proteases that could be the target for protease inhibitors as potential drugs (e.g. ref [ ] ). the difficulty is that inhibiting the action of human proteins could have undesirable effects on the host [ ] , so parallel work on other kinds of functional sites in the coronavirus proteins is of great importance. the present paper explores another potential functional site in the spike protein, but this one is a different because the site is not a proteolytic cleavage site, and it is not well conserved, except, it is argued, for a characteristic composition of particular amino acid residues. expressed another way, there can exist certain subsequences of a protein sequence that are well conserved, but only in respect to some pattern or property that is less obvious than the order of amino acids. finding them (or as is more correctly stated, predicting them) may therefore require a more subtle and, in the present case, novel bioinformatics tool, compared with the standard bioinformatics tools which were essential in the preceding papers [ ] [ ] [ ] . comparisons with other proteins as described below suggest that the subsequence of interest in this paper could have a crucial function, and a high degree of conservation is, even by itself, also a clue as having a role important to the virus [ ] . hence such a site may represent a potential therapeutic target, perhaps as well as representing a synthetic vaccine target. however, until very recently, that crucial function did not even seem to be possessed by sars-cov and sars-cov- , and the details have yet to be elucidated. the particular virus function that is considered in the present paper is noncovalent binding to the sialic acid glycans, i.e. oligosaccharides or polysaccharides that contain sialic acid residues. they are sometimes called sialylated glycans. interest in this binding arose as follows. it seems unlikely (although of course possible) that functions important for many different kinds of virus are of little importance to others, especially if they have a common lifestyle such as infection of the respiratory system or alimentary tract, typically reflected by common symptoms. if such functions are absent, it begs the question of how the virus copes. though glycan binding of sars-cov and sars-cov- seems absent, diminished, or relatively neglected in the literature (see section . ), many coronaviruses such as human coronavirus oc and bovine coronavirus appear to recognize sialic acid as a receptor. however, most biology students are more familiar with the hemagglutinin and neuraminidase of influenza, the h and n in, for example h n (the numbers such as being based on immunological typing of these proteins), that bind to glycans, (sugar chains, oligosaccharides or polysaccharides) at cell surfaces notably those chemically bound to membrane proteins, hence called glycoproteins, of host cells. the surfaces of many animal and all by host enzymes and so would appear, again at first consideration, to be more specific. enveloped viruses such as sars-cov- also have their own bound sialic acid glycans (the spike protein is usually referred to as the spike glycoprotein), but these are of less direct interest here although they can clearly influence binding of a virus to various receptors. despite their diversity, and perhaps because of it (i.e. because that diversity implies more information content) sialic acid glycans of host cells are key molecular recognition features not only for entry of viruses such as influenza, but also in embryonic development, neurodevelopment, reprogramming, and oncogenesis. correctly speaking, even sialic acid itself is diverse. it is a generic term for a family of derivatives of the nine-carbon sugar neuraminic acid. the sialic acid family includes some derivatives of neuraminic acid, but these acids rarely appear free in nature. members include n-acetylneuraminic acid, -keto- -deoxy-d-glycero-d-galactononulosonic acid, , -diamino- , , , -tetra-deoxy-d-glycero-d-galacto-nonulosonic acid, and , -diamino- , , , -tetra-deoxy-l-glycero-l-manno-nonulosonic acid. if the term "sialic acid" is used unqualified, it usually refers to the representative member of this group, n-acetylneuraminic acid. the variability of glycans is not random but reflects their modes of synthesis. in eukaryotes generally, a typical n-linked glycan has an initial core that consists of residues ( glucose, mannose, and n-acetylglucosamine). this preassembled glycan is usually transferred by a glycosyltransferase oligosaccharyltransferase to a nascent peptide chain within the reticular lumen. this initial core -sugar unit is assembled in the cytoplasm and endoplasmic reticulum and other sugars may be added later. in contrast, o-linked glycans are assembled one sugar at a time at the outset on proteins in the golgi apparatus. there are some specific features of medical interest as relevant to the human host of viruses (but by no means unique to humans). the lung epithelial glycans are typical by having sialic acids as the distal residues, and it is these that the influenza neuraminidase cleaves away. most soluble secreted proteins are also similarly decorated with such glycans. that includes the proteins that make up saliva and mucus in the airway, and are in general important for viral infection. both n-and o-and glycosphingolipid-glycans are found in human lungs, and they include large and complex-type n-glycans with linear poly-n-acetyllactosamine [- galβ - glcnacβ -] n extensions, which are predominantly terminated in α , -linked sialic acid. in contrast, the smaller n-glycans lack poly-n-acetyllactosamine but are enriched in α , -linked sialic acids. there are also large glycosphingolipid glycans, which also consists of poly-n-acetyllactosamine, usually terminating in α , -linked sialic acid. while it is commonly maintained that viruses such as influenza virus bind to the sialylated glycans, and this is assumed in the present paper, some care is required, because there are also nonsialylated glycans in human lungs on which viral binding could occur. while it is binding of viruses to sialic acid glycan that is of interest here, it should be kept in mind that it is often associated with a catalytic activity in which the sialic acid glycan is the substrate. in influenza these two aspects are particularly distinct by being on separate surface proteins, the hemagglutinin and neuraminidase respectively, but that separation is not true of all viruses. not surprisingly, as noted above, most coronaviruses of the coronaviridae family also have capabilities to bind to sialic acid glycans, but they also have the ability to cleave the glycans, which is often described by authors as a "receptor destroying activity". like influenza c viruses, purified bovine coronavirus preparations have an esterase activity which inactivates o-acetylsialic acidcontaining receptors on erythrocytes; diisopropyl fluorophosphate completely inhibited this receptor-destroying activity suggesting that the viral enzyme is in this case a serine esterase [ ] . this is believed to facilitate the spread of virus infection by removing receptor determinants from the surface of infected cells (see discussion below) and prevent the formation of virus aggregates. another coronavirus, porcine transmissible gastroenteritis virus (tgev) recognizes n-glycolylneuraminic acid. nor does it depend on the sialic acid binding activity for infection of cultured cells, but interaction with sialoglycoconjugates appears to help the virus to pass through the sialic acid-rich mucus layer in the epithelium of the small intestine. hemagglutinin-esterases are a family of viral envelope glycoproteins that mediate reversible attachment to oacetylated sialic acids. these too are said to be receptor-destroying, but the enzymic activity reaction is in this case not a cleavage in the sense used concerning neuraminidase, but rather a change in molecular recognition by removal from of the acetyl group from the c position of the above acetylated neuraminic acid residues. the other and probably major reason for researchers thinking of these actions as "receptor destroying" is because the picture is a little more complex than just allowing entry and exit from the host cell. because many viruses attain host cell specificity by being selective for particular types of sialic acid, these may occur as decoys to the virus on off-target host cells and on free molecules in the extracellular environment. to prevent irreversible binding to these decoys, many viruses including many coronaviruses have receptor-destroying enzymes that are therefore interesting targets for antiviral intervention, exemplified by the influenza a virus neuraminidase [ ] . even though such glycan binding domains and enzymes as neuraminidases are found in many coronaviruses, there seems to be no such enzymes in sars-cov and sars-cov- . viruses of the lower respiratory tract, such as influenza virus, respiratory syncytial virus, and sars-related coronaviruses, are generally considered as having key differences that require different therapeutics [ ] even though relatively little is lost in considering already approved drugs for one of such viruses against the other (e.g. ref [ ] ). typically, the apparent absence of glycan binding and enzymic sites in sars-cov and sars-cov- has been dismissed as due to the fact that the virus enters on ace , i.e. angiotensin converting enzyme type (e.g. see ref [ ] for discussion), not on a glycoprotein. this does not, however, escape from the intuitively important need for preliminary binding, cell entry and exit through the glycan layer, and probably the decoy-related function discussed above. there appears to be growing evidence of significant lectin-binding capability. lectins are the carbohydrate-binding proteins that are highly specific for sugar groups of other molecules. activation of ctype lectin receptor and other similar receptors contributes to pro-inflammatory response to many coronavirus infections. there also are studies over several years that locate glycan binding and even related catalytic activities in the spike glycoprotein. it has been noted that e protein of bovine coronavirus is a receptor-destroying enzyme with acetylesterase activity [ ] , and the d structure of coronavirus hemagglutinin-esterase offered insight into coronavirus and influenza virus evolution, with implications for drug and antibody discovery [ ] . the location of any sialic acid glycan binding region of sars-cov- is, a priori unclear, although intuitively (a) it would likely be associated with the cap or knob at the outer end of the spike protein, or (b) at least not involve exactly the same domain as is required for other important functions. although throughout the coronaviruses various external proteins and domains can recognize either protein or sugar receptors or both, the majority of such studies like those above implicate the s region in their spike glycoproteins, but as discussed in the present paper, there are other potential sugar binding sites that are still within the spike protein. overall, the sars-cov- spike glycoprotein has amino acid residues and until early understanding of structure was heavily based on sars-cov spike glycoprotein ( amino acids) with - % amino acid residues similarity among non-sars coronaviruses. most of the spike protein appears to be involved in the specific stages of cell entry. the spike glycoprotein of sars-cov and sars-cov- is translated as a large polypeptide that is later cleaved to s and s sites. after binding to the main receptor that that is held to be primarily ace , the host proteases activate the virus by cleaving first at the s /s boundary (i.e. s /s site) and then within s , i.e. at the s ' site. the spike of similar coronavirus have long been considered as being in two main states (i) the pre-fusion form (the form of the mature virion) and (ii) post-fusion form, the form after membrane fusion has been completed). more detailed studies have split the latter into a prehairpin intermediate state, and post-fusion hairpin state. somewhat like in all virus class i fusion proteins, the s protein contains two heptad repeat regions (hrs) of which one (hr ) is located close to the transmembrane anchor. membrane fusion occurs when there is a conformational change in the hrs to form a fusion core. the hrs of the protein fold into a coiled-coil structure, known as the "fusogenic state". as virus and target cell membranes fuse, the coiled coil regions (called heptad repeats) become a trimer-of-hairpins structure. the s ' cleavage site appears particularly important by being well conserved [ ] [ ] [ ] and proteolysis by cathepsin appears sufficient to expose the fusion peptide of s and activate fusion within the host cell endosome. in general, s ' is now considered as the key viral fusion peptide which is unmasked following s cleavage. subsequently, s dissociates from s , allowing s to transition to the post-fusion structure. the following locations in the sequences of amino acid residues apply specifically to sars-cov- . these vary somewhat with author, and the following are used here. the signal peptide (sp) comprises residues - . on the inside of the lipid membrane, the carboxyl terminus (c-terminus) is comprised of the transmembrane region (tm) comprising residues - and the cytoplasmic tail (ct) residues - . the extracellular domain of the spike glycoprotein is comprised of n-terminal domain (s -ntd) comprising residues - , and is of particular interest here. the host cell receptor binding domain (rbd) comprising residues - . in summary the key regions are as follows. sp see text in regard to the significance of the other colors. fig. shows the external part - of the spike glycoprotein of sars-cov- in the closed state prior to ace binding, with s -ntd domain (the "ears", dark blue) of interest here, rbd (at tip, subdomains light blue, blue-green), s (subdomains, orange, green, yellow). the orange-white, green-white and yellow-white helical structures are the α-helices of the trimer that form the neck associated with s , and the red-white helical structures are the start of the transmembrane α-helices tm. in at least some coronaviruses, s -ntd is known to be involved in binding host proteins or glycans, but coronaviruses show great diversity in their binding which presumably underlies their ability to jump between very different host species. while the role of s -ntd compared with the current reasonably detailed knowledge of the remarkable mechanism of cell entry involving ace and changes to the spike protein on cleavage, the specific function of s -ntd of sars-cov- has not been elucidated (at least, not by the time of writing in april ). as noted above, s in sars-cov- is now well known to have a region which is the receptor binding domain to human ace but also, significantly for what follows in the text below, sars-like coronaviruses can bind clec m/dc-signr c-type lectin domains on host cells. see ref [ ] for review of the diverse receptor recognition mechanisms of coronaviruses up to , which represented the body of understanding until the cov- pandemic. bovine coronavirus is an example of a coronavirus for which it seems clear that s -ntd has an established glycan-binding function. although the structure of a sugar-bound bovine cov s -ntd was not available, some conclusions could be reached by researchers using structureguided mutagenesis and comparisons with different coronaviruses. as well as evidence in linking hemagglutinin-esterase to the s domain of at least some coronaviruses [ ] , zhang and yap [ ] had reported in a rational d model for s domain of sars-cov spike protein by fold recognition and molecular modeling techniques, and there they noted a suggestive structure similarity between s protein and influenza virus neuraminidase [ ] . this opened up the possibility for those authors that existing antiinfluenza virus inhibitors and anti-neuraminidase antibody could be used as a starting point for designing anti-sars drugs, vaccines and antibodies [ ] . based on such observations and discussion so far, it is therefore reasonable to propose that s -ntd could be important in the binding of certain alternative host cell surface receptors, or perhaps which aid in targeting the virus to ace , and so might provide a helpful therapeutic target (as well as candidate antigenic site for synthetic vaccine design). nonetheless, such functions if present in sars-cov- could, a priori, reside in other domains at the virus surface. the challenge for research here is that the substantial knowledge concerning such matters in well-studied coronaviruses is not readily transferable. variously throughout the coronaviruses, s -ntd, ctd and s regions can recognize either protein or sugar receptors or both in various cases, and very similar coronavirus spike protein domains within the same genus may recognize different host cell receptors, while many very different coronaviruses may recognize the same host cell receptor. the studies mentioned above also suggested that at least some coronavirus s -ntds are evolutionarily related to human galectins, the term typically used for the lectins as carbohydrate-binding proteins that are specifically involved in inflammation, immune responses, cell migration, autophagy and signaling; however the viral domains derived from them have diverged with specificities for different sugar receptors [ ] . further review is given throughout results section where appropriate. another challenge discussed in results section is that key regions for which an experimental d structure would help resolve the matter are disordered, and hence invisible, in current available spike protein structures. less familiar theoretical principles do arise in the knowledge gathering, inference, and prediction methods developed by the author and colleagues are used in the present cov- project as described in ref [ ] . they also include approaches to facilitate interaction with the standard bioinformatics web tools which are stated below in methods section . however, while tools of these various kinds do speed and facilitate a project of this nature, the usual methods of investigating literature and accessing bioinformatics data bases and tool are sufficient for reproducibility of the work described in this paper, at least by researchers reasonably familiar with bioinformatics and protein structure. an algorithm for predicting the domains and proteins involved in sialic acid glycan binding is developed in the course of the project described in results section , but this is primarily of a highly empirical nature. future work to advance this algorithm on a sounder theoretical basis is underway. see brief discussion on future work in discussion section . , where some of the above-mentioned theoretical approaches of the author and colleagues, also to be used in the development of the algorithm, are cited. the overall approach comprised the following steps. i. automated gathering of information from the world wide web regarding hemagglutinins, neuraminidases, and sugar binding proteins, particularly but not solely of viruses, using "autosurfing", natural language processing, and knowledge extraction techniques [ ] . note that it is in particular non-covalent sialic acid glycan binding sites that are being explored, not for example asparagine or serine or threonine sites to which glycans are covalently linked. this knowledge gathering approach, as described in ref [ ] , is not essential for reproducing the present work or for carrying out comparable studies, but it does greatly accelerate research and preparation of the scientific paper, allowing fast responses to a new epidemic [ ] [ ] [ ] . ii. attempted discovery of continuous short sequences of amino acid residues (potential "sequence motifs") with patterns and amino acid content common to sars-cov- spike protein amino acid sequences and hemagglutinins and neuraminidases, particularly those of influenza viruses. also, more generally, in preparative work, comparison of the spike protein sequence of the spike protein with sialic acid glycan binding proteins and other sugar binding proteins, or domains of them. protein sequences or parts of them used as input for any part of the study were obtained from genbank https://www.ncbi.nlm.nih.gov/genbank/. the standard method of bioinformatics used for detecting in large protein sequence databases any amino acid residue sequences similar to those of an input sequence was primarily blastp at https://blast.ncbi.nlm.nih.gov/ blast.cgi. the standard tool for a more formal and typically multi-sequence alignment was clustal omega at https://www.ebi.ac.uk/ tools/msa/clustalo/. these tools can be automatically accessed by the present author's methods [ ] but again that is not essential for reproducibility of the present work. iii. examination of patterns in potential or known short subsequences in small proteins or domains known to have a function involving non-covalent sialic acid binding and, in absence of any clear patterns, study of the amino acid content of the subsequences. this established a preliminary sialic acid glycan binding score (sabs) for the twenty naturally occurring amino acid residues. however, the short subsequences identified were to be considered as "signals" or "fingerprints" for sialic acid glycan binding domains as a whole. that is, direct contact with sialic acid was not necessarily at (or solely at) the specific short subsequence, for reasons discussed in results section . this, plus a sequence rather than three dimensional structure perspective, and a specific focus on binding sialic acid glycans rather than sugars in general, resulted in a substantial difference in scores from another major method of predicting sugar binding regions of proteins also discussed later below. iv. development of the above as an algorithm sabr-p for identifying potential small proteins or domains of proteins that non-covalently bind sialic acid glycans, by predictions on a test data set of protein sequences. as noted above the subsequences predicted are taken to indicate the glycan binding domain as a whole, not necessarily the sialic acid sites per se, but they may be. this approach involved noting true positive and negative predictions and false positives and negative predictions so as to optimize sensitivity and specificity. this was done specifically in regard to non-covalent binding of sialic acid glycans. in other words, it was done so as to distinguished sialic acid glycan binding domains not only from those domains known not to bind sugars but also from those that bind sugars and glycans that do not contain sialic acid. v. examination of the three dimensional structures of regions of the regions of the sars-cov- spike protein predicted as binding sialic acid glycans to propose and locate a sialic acid binding function of sars-cov- (possibly but not necessarily associated with some kind of enzymic activity). results and discussion in the present paper used the same amino acid codes as the above tools and data bank use, i.e. the iupac (international union of pure and applied chemistry) one letter amino acid codes, given in table below. for completeness, conservative replacements in column of table are given. they relate largely to substitutions that can usefully be made in the design of synthetic peptides [ , ] . this is an application which is not specifically discussed in the present paper but which could be a basis for design of synthetic vaccines and preventative or therapeutic agents [ , ] , in this case targeted at sialic acid glycan binding site of a virus. as discussed in the sequence of steps for the methodology above, the sialic acid glycan binding motifs are taken to be indicators of the sialic acid binding domain and not necessarily of the target sites per se, but they may be, and often are, potential target sites. the list of conservative replacements also remains useful for considering substitutions that are conservative in maintaining similar amino acid properties when detecting and comparing related sequences. since for both reasons they be useful in deeper consideration of many results in the present paper, some comment may be useful to researchers less familiar with bioinformatics. see ref [ ] for a further account. note that the work of considering what is a conservative replacement is done automatically by the standard bioinformatics tools used. the replacements in table are consistent with the conservative replacement rules implied by the tables of weights implemented automatically in blastp and clustal omega mentioned above, which are discussed at those sites. however, the original intent as an application to peptide design means that in table there is a degree of asymmetry based on the author's experience in peptide design [ ] because one is going from a natural protein state to less natural one without evolution making compensatory changes in the rest of the protein or system. for example, empirical studies show that serine (s) can be replaced by alanine (a) or threonine (t) but it is frequently important that a replacement to threonine should be isoleucine (i) in order to retain stability of a βpleated sheet in which they occur. strictly speaking, these are just fairly crude rules-of-thumb: the best replacements are dependent on more specific circumstances and detailed conformational and binding calculations. the assignment in table are not seen as controversial because apart from the asymmetry they relate to the "interchangability" or "alternative rule" of amino acid residues by many authors that are intended as universal, i.e. intended to apply to all proteins. this is because they relate to similarity of amino acid residues in terms of physicochemical, conformational, as well as biological properties of many sequences that are at least universal to, say, vertebrates. however, they are historically more directly empirically based on wellknown studies probabilities of amino acid differences found by comparing amino acid residue sequences amongst fairly related proteins from a wide range of sets of different proteins, such that one is comparing sequences of hemoglobins, or of lysozymes, or of cytochromes c, and so on. as is to some extent customary in the field, three letter codes (such as gly for glycine) are used for the amino acids in the molecular graphics figures; these codes are fairly self-evident at least in the direction of deducing the full name of the amino acid being represented. there was also use of data and the associated graphics tools in the protein data bank (pdb) at https://www.rcsb.org/ and in japan https://pdbj.org/ which was used for fig. . energy calculations by the author's own krunch and by a commercial sculpt protein modeling package were used in ref [ ] , but were not required for the present study, with the exception that some calculations by these tools were used to obtain earlier unpublished results on sugar binding to amino acid residue sidechains. this provided a check on the preliminary sialic acid binding capability shown in column of table , used initially in the present paper. krunch is a molecular mechanics modeling package that essentially functions like many standard molecular modeling packages. there is the arguable exception that it gives much more attention than usual to novel algorithms for navigating through multiple energy minima and discovering new conformers, but that capability did not appear to be too important in the present study. for the much greater part, however, these binding assessments were based on the amino acid residues observed by the author in sequences involved in sugar binding sites in proteins (found by visual examination of binding sites of entries in the pdb) and similar qualitative observations by other authors. that is, they also reflect rather general opinion of what amino acids are involved in sugar binding and in its most general formulation this intuitively comprises aromatic residues, and hydrogen bonding residues to interact with the sugar hydroxyl groups. at the outset, as a starting point only, column of table of these preliminary sialic acid binding amino acid scores (sabs) are really assignments that are qualitative, using for not often present in sialic acid glycan binding sites and for often present,. however, tryptophan was assigned a double score of reflecting its larger size and double ring. how reliable these assignments are in regard to sialic acid glycan binding is what is assessed on a more objective basis by the prediction method developed in this paper, including a degree of recalibration. the marginally modified parameters are also shown in the last column table for convenience of comparison. while as a methodological strategy it was tempting to start from an alternative more objective and established approach discussed in results section , or at least to use it as a starting point or as an important "gold standard" for comparison, it has substantially different aims. as a first step in an investigation making use of bioinformatics, a common strategy is the use of protein sequence alignments so that a functional part that is known in one protein might suggest an analogous functional part in another. as discussed here, this approach has proven somewhat less successful for sialic acid glycan binding sites than for other functions considered elsewhere (e.g. refs [ ] [ ] [ ] ), and necessitated development of alternative techniques of which discussion occupies the major part of this paper, but the results are consistent and to some degree suportive. as the basis of discussion and starting point for the present study, the following is a sars-cov- spike protein sequence in which the s -ntd is shown in italics. the short section of sequence underline emerges below as of particular, but by no means sole, interest. more subtle computational techniques based on protein conformation have in this project suggested relationships between sars-cov and hemagglutinins and neuraminidases of other viruses, but they essentially support prior work. recall that zhang and yap [ ] noted a suggestive structure similarity between sars-cov s and influenza virus neuraminidase. using various computational techniques they compared the d structure of sars-cov s protein data bank d structures iny (neuraminidase from influenza a virus complexed with a sialic acid phosphonate analogue inhibitor) and b t (neuraminidase from influenza b virus with novel aromatic inhibitors). this observation does not necessarily suggest by itself a common function, because many protein folding patterns are used by nature for diverse purposes, but armed with that information a more careful use of sequence alignment is helpful. the following part of a clustalw sequence alignment done in the present study also includes the hemagglutinin esterase sites in e of the bovine coronavirus [ ] (e -cov-q eb . below) and that studied by zeng et al. [ ] (hem-est-cov- cl below). substrate binding residues in deduced for sars-cov s by zhan and yap [ ] are shown by #. : :. . *: . ::.: *. ---qiifyegvnfnphhrf------kcffngsndvwifnkvrfyralysnmalfryltfv . : *:*:. .: : :.. * . :: the situation is of course not clear when the degree of amino acid residue match between sequences is poor because similarities can arise by chance, but aligning more sequences can be insightful, and in this case including influenza a and b sequences seemed particularly helpful. although still barely conserved compared with the motifs in the current project (published [ , ] , submitted, or under investigation) the most plausible match for further study is at sssgwtagaaayy of sarscov -s-mn . , because others would include at least one extensive deletion areas from one protein. including influenza a and b sequences interferes with much of the alignment but it does preserve the above match. this preserved match region also places the notable tryptophan (w) within short distance of alignment in the second subsequence corresponding to the zhan-yap binding site, but in procedures of this kind it is important to establish that such features are not simply artifacts of the particular sequences included. the essential features of the above alignments at the binding site are not perturbed by removing the influenza neuraminidases which provided a basis for the zhan-yap catalytic regions, as follows. : :. . *: . ::.: *. ---qiifyegvnfnphhrf------kcffngsndvwifnkvrfyralysnmalfryltfv . : *:*:. .: : :.. * . :: . . #### alignment studies like those above are helpful in forming initial hypotheses, but they are clearly not convincing by themselves when the degrees of match between aligned residues is weak; however, it is possible that the actual composition in terms of amino acids in the subsequence and potential motif of interest is more important than their actual order in that subsequence. based on many studies exemplified by the above, it was noted that tryptophan is a recurrent, albeit not absolutely essential, feature of sugar binding including sialic acids. references to that also recur throughout the biochemical literature. for example, see e.g. ref [ ] , and also fig. shows the tryptophan interaction with and sialic acid in the influenza virus b neuraminidase (pdb entry bat). as might be expected by their similar aromatic character, the alternative amino acid residues as sugar binders, and residues frequently supporting tryptophan in the binding site, tend to be aromatic sidechains, notably tyrosine (y), sometimes phenylalanine (f) and histidine (h). a preliminary survey of sequence motif patterns in sugar binding proteins suggests that invariant amino acid residues across a family of proteins tend be one or more of the above aromatic residues supported by negatively charged aspartate (d), asparagine (n), serine (s), threonine (t) glycine (g) and sometimes alanine (a) that provide the hydrogen bonding. however, particularly in regard to the non-aromatic residues, the binding of acidic and non-acidic sugars should probably be distinguished. as discussed later below, charged amino acid residues glutamate (e), arginine (r), and lysine (k) also frequently make intimate contact with sialic acids but that is in three dimensions, not together in a subsequence. a likely relevant observation was that the first set of amino acid residues (the set containing aspartate) and binding sialic acids tended to occur in a subsequence that adopted a local loop conformation, while the second set (that containing glutamate) were frequently associated with α-helices and particularly their termini. however, this was an empirical and qualitative observation regarding a tendency, and a more objective quantification of the importance of the aspartate set is the purpose of the prediction algorithm developed below. three dimensional considerations, however, give insight and sometimes explain why influences can be somewhat indirect. hydrogen bonding that occurs between the hydroxyl groups of carbohydrate ligands and polar amino acid residues at the binding site is typically supported by water-mediated hydrogen bonding networks in which serine and threonine are fairly commonly involved. nonetheless, the most outstanding feature of carbohydrate binding sites from a three dimensional perspective would appear to be the position and orientation of tryptophan (w), tyrosine (y), and/or phenylalanine (f), which usually provide a hydrophobic plate for close interaction with the planar face of sugar rings, an interaction resembling hydrophobic stacking interactions, as in fig. . the importance of these and to some extent of histidine (h) in a sequence motif seems reasonable. along with the occasional appearance of cysteine (c), sometimes as a serine (s) and particularly a threonine (t) substitution, the residues mentioned above can be used as the basis of a preliminary and essentially qualitative model for assessment of sugar binding as given column of table . often a valine (v) substitution was seen in potential glycan binding sites, although this was not significantly supported by the optimization of the predictive technique described below. however, glycans containing sialic acids appear to bind somewhat differently to other sugars. taking this as a hypothesis and focusing on these, protein sites for binding them may have a variety of affinities for different subtypes. for example, all influenza a virus strains critically depend on sialic acid to bind to host cells and the different forms of sialic acids all show different affinities that change with influenza a virus variety, important because it determines which species can be infected. there has also been very relevant work that can complicate the details of what can be meant by, for example, "sugar binding motif". indeed, zhang and yap [ ] themselves noted that tryptophan was frequently involved in strong protein fold interactions that stabilized the sugar binding domain fold, yet lacked any direct interactions with sugars. in the influenza neuraminidase they comprised three pairs of main chain-side chain interactions: tryptophan (w) (donor) and phenylalanine (f) (acceptor), alanine (a) (donor) and phenylalanine (f) (acceptor), leucine(l) (donor) and tryptophan (w) (acceptor). for example, tryptophan accepted one n-h⋯π bond from leucine (l) and donates one n-h⋯π bond to phenylalanine . it is possible that the aromatic residues could be induced to be more exposed in certain types of binding, but the above interactions appeared to stabilize the structure of s fold motif while reducing the active site cavity for ligand binding [ ] . features of sialic acid glycan binding regions represented by a run of amino acid residues have diverse sequence patterns, but evidently the simple presence of the above residues in a section of sequence is itself a strong signal feature; this seems particularly so for tryptophan. to examine this further, many sequence alignments were examined that relate to role of the aromatic amino acids and the contribution of tryptophan to known or suspected sialic acid glycan binding, and these were were investigated in a more quantitative way. for example, the following represent a summary of influenza subsequences that contain tryptophan. with it is associated the preliminary, essentially qualitative sialic acid binding residue score (sab-s) discussed above and based on observation of multiple sugar binding sites and literature survey, to each of the amino acids mentioned above. at this stage, there is no significant algorithm except that the sum of scores over the residues in the short sequence is divided by the number of residues (mostly or ) so that it expressed on an averaged, per residue, basis for that sequence. recall that it is qualitative that the amino acid residues are given a score of (and otherwise), except that tryptophan is given an extra weight of . in many cases the sidechain is involved, especially for the aromatic residues, but this not obligatory: it could be a backbone interaction (as is necessary for glycine (g) that lacks a sidechain). this is the "qualitative" model that was shown in table , which will be extended to the sabr-p method later below. there is in the following an attempt at local alignment to highlight similar features because there is often some indication that a motif is reused even within a protein, although that assertion is not required for present purposes. the sum of score is divided by the sequence length that excludes relative deletions '-'. many hemaglutanin and neuraminidase matches were found with sssgwtagaaayy using blastp, the top varying from % match and % identities that preserve the tryptophan, such as hemagglutinin influenza a virus, genbank axb . ssgw--gavn, and neuraminidase, influenza a virus afk . sgw----aay, and neuraminidase, influenza a virus anz . , ssawsasa. looking at the larger sequence context, % of these scored over . on the above system. however, nucleocapsid proteins of influenza a virus such as sequence qiq with sg-tagaa and aaz . as stsg-aagaa also appear in the top matches along with the above and with comparable scores, but with one obvious and significant difference, that the tryptophan (w) is missing. the possible importance of tryptophan in binding in sars-cov- is exemplified in the following sequence of the sars-cov- s -ntd section of the spike glycoprotein (including the signal peptide, in order to conserve standard numbering). the sequence is from the s -ntd of sars-cov- genbank entry mn . , along with a brief description of accessibility in pdb entry vvx, which is for the spike protein in the closed state. trtqlppaytnsftrgvyypdkvfrssvlhstqdlflpffsnvtwfhaihv - w exposed sgtngtkrfdnpvlpfndgvyfasteksniirgwifgttldsktqsllivnnatnvvikvcefqfcndpf - cleft lgvyyhknnkswmesefrvyssannctfeyvsqpflmdlegkqgnfknlrefvfknidgyfkiyskhtpi - disord. nlvrdlpqgfsaleplvdlpiginitrfqtllalhrsyltpgdsssgwtagaaayyvgylqprtfllkyn - disord. engtit here, "disord." means a disordered (flexible) loop: the tryptophan (w) and adjacent residues are not seen in the experimental d structure determination vvx. this is also true of pdb entry vsb the spike protein in the open state, vyb and other sars-cov- accessible to the author at the time of writing. see fig. . the fact that disordered suggest an open loop like structure that be may be capable of binding to, and perhaps adjusting to, disordered targets, and certainly does not prohibit functional significance. the following summary table has comments on the status of exposure of the tryptophan and surrounding residues, and on the involvement in binding based on alignments with the zhang-yap analysis. evidently the above is not perfect as a basis for a prediction method, and the next step in this study required more detailed considerations. it also required a more comprehensive analysis of predictive capability that allows for false positives and false negatives. the method developed here takes account of the above results in the light of several pieces of experimental and theoretical evidence, which is usefully briefly reviewed at this point in the narrative. several kinds of evidence were taken into account in development of sabr-p, the sialic acid binding region prediction. the emerging principles used, not all of which are immediately intuitive, are argued as follows so that they may help researchers develop improved versions. the method specifically concerns predicting regions that interact with glycans containing sialic acid residues. the development of the method started with the qualitative sialic acid binding region sabs score in table and used in preliminary studies above. other prediction methods such as that discussed below also address sugars or oligoscharrides in general. recall, however, that even the sialic acid components comprise a fairly diverse family of sugar types (see introduction section . ). also, while described as a sialic acid binding region prediction, and this is believed to be the naturally emerging emphasis, it is formally the binding to glycans that contain sialic acids that matters. (ii) the emphasis is also on binding, not catalysis. the method is not specifically concerned with catalytic amino acid residues involved in neurominidase action (nor any other activity of cleaving modifying the glycan to reduce virus binding, e.g. esterases, deacetylases). while evidence of neuraminidase activity in sars-cov- would be very important, the current evidence is that focus should be on sialic acid binding. this is because of lack of any strong evidence for such enzymic activities at the time of writing, and also because neuraminidase inhibitors approved as dugs, oseltamivir (tamiflu), and zanamivir (relenza) have been tested on sars-cov- in vitro and were not found effective [ ] . it is nonetheless the binding of the appropriate glycans containing sialic acid that may still be important for sars-cov and sars-cov- infection in vivo, as it is for other viruses [ ] . the fact that other viruses, including other coronaviruses, require that function, followed by any demonstration that sars-cov and sars-cov- still possess that function, would suggest that it could still be a target for pharmaceutical drug development, at least as a preventative. this argument is not unique in its general form. for example, fantani and colleagues believe that the sars-cov- also uses sialic acids linked to host cell surface gangliosides to somehow facilitate host cell entry and so could represent a therepeutic target [ ] . (iii) the method seeks to predict whole domains and proteins that bind sialic acid, not specific short sequence motifs. that is the case even though the predictions assign a sialic acid binding score to each residue in the domain or protein, which can of course still be inspected as of potential involvement in direct binding. the threshold and scale of the sabr-p prediction method is set such that any residue (in practice it is almost always a continuous run of residues) with a sialic acid binding score of more than signals that the whole domain or protein, whichever was provided as the whole sequence in input, is a binding module for glycans containing sialic acid. there are several reasons for using this as a marker of a domain or protein of interest. one is that the binding is to the whole glycan as an oligosaccharide or polysaccharide, not necessarily the sialic acid components per se, and therefore the ligand is a large structure that could involve several binding sites. recall that tryptophan was found to be frequently involved in strong protein fold interactions that stabilized the sugar binding domain fold, yet were lacking any direct interactions with sugars and even buried [ ] . there are at least somewhere between and amino acid residue sequence motifs known to be associated with sugar binding in general, and they fall into some fold motifs [ ] . also, as the function of sialic acid recognition may be to guide the virus to and across the host cell surface [ ] , sites on the virus that enable this motility and hunt out points for catalysis or simply the strong binding appear to be as important as key strong binding and catalytic sites themselves (e.g. ref [ ] ). in addition, predictions of localized regions of proteins as sugar binding sites are naturally not as good as those based on predicting that a whole domain or protein is involved in sugar binding, but such a prediction remains valuable and indeed well suited to the present study. in a particularly detailed investigation by taroni et al., analysis of the characteristic properties of sugar binding sites was performed on a set of sugar binding proteins [ ] . their prediction was optimized on a training set of non-homologous carbohydrate binding structures and tested on a test set of protein-carbohydrate complexes. the thoroughness of that study and the inclusion of many kinds of information would make it a reasonable choice of "gold standard" for comparison, except that the aims were substantially different, and the overall accuracy of prediction achieved was only %. it is true nonetheless that results were very good for carbohydrate-binding enzymes as opposed to lectins, with a rate of success of %. this emphasis on enzyme catalytic sites again argues for avoiding the use of this kind of approach in the present paper, as covered by point (i) above. in summary, it may be that a large domain or protein that has strong signals for sialic acid binding may be indicative of a sialic acid binding and/or catalytic function even if a specific short subsection of the sequence predicted as binding sialic acids is not in the position for which a strong binding or catalysis has been observed. subsequences. only the sequence is considered in the method, not residues brought together by the folding of the protein in space. most prediction methods for predicting sugar binding such as the example of taroni et al. [ ] discussed above are not based on sequence and the sugar-binding propensity of amino acids in short segments alone, but on regions on a protein surface that require account of d structural information, analogous to "discontinuous" epitopes or "discontinuous determinants" of a pathogen protein in the study of immune response and in synthetic vaccine design. this kind of approach was abandoned in the present study not just because it took away the emphasis on the binding functions of whole domains or proteins but most importantly because many of the sites of particular interest are in disordered regions, or otherwise invisible in the available experimental d structure, as discussed later below. it might even be that a degree of disorder is important for binding some sugars. also and not least, d structural information is not always available. (v) glycophilicty parameters for amino acid residues or sequence patterns as obtained by other workers were not used. this is particularly because, the aims, i.e. what things are wanted to be known, are usually very different in these methods. although taroni et al. showed that out of partially parameters partially dependent on d information the sugar binding propensities of certain amino acids was prominent in having discriminatory power, this was in regard to the tendency for being in putative a sugar binding patch compared with protein surfaces in general (requiring ordered conformation and experimental information about it). at the same time, it still concerns a specific region of a kind rather than predictions of domain or proteins as sugar binding as a whole. perhaps most importantly these studies were concerned with binding sugars in general (not specifically sialic acids). not surprisingly, therefore, the amino acid residue propensities have no significant overall correlation with the propensities in table used and developed in the present paper. notably, alanine (a), serine (s), threonine (t) and cysteine (c) have a propensity against sugar binding in their approach. there is nonetheless the significant exception that tryptophan (w) is the strongest in both that and the present study. (vi) a simple predictive model was developed based on optimized parameters. if a simpler method works as well as a more rigorous approach, it can sometimes provide insight and help build even better rigorous approaches. the method used in the present case was initially based on the gor method [ ] for protein secondary structure prediction in which sialic acid glycan binding state of residues replaced α-helix, β-sheet, and coil (or loop) states of residues. however, it was found that the results were essentially reproduced by a simpler model. this is primarily because the directional effects (in terms of n-terminal direction or c-terminal direction along the amino acid residue sequence) was found to be equivalent (i.e., symmetrical) and to persist for some residues in both directions, very like the influence of alanine on α-helix formation in the gor method [ ] . consequently the final method used in the present study consisted of just two changes to that used in previous results section . above, the second being described in point (vii) below. the qualitative scores as parameters to types of amino acid residue as shown in table were based on visual observations by the present author and a survey of sites in the literature, and this was improved by optimization on essentially the same set of proteins examined. the amino acids were initially assigned the qualitative parameters of table , then these parameters were optimized to optimize sialic acid glycan binding predictions as positive for sialic acid glycan binding proteins, and negative for proteins not considered as binding sugars, and proteins such as lectins that bind other sugars that do not contain sialic acids. this gave as before glycine (g), alanine (a) aspartate (d), asparagine (n), serine (s), threonine (t), cystine/cysteine (c) each with a score of , and the rest assigned , except for tyrosine (y phenylalanine (f) and histidine (h) that were now assigned larger parameter values of and tryptophan (w) that was assigned a larger parameter value of . (vii) parameters describing directional influences of amino acid residues were modeled in a simple way. in part this is a response to the above point that the directional effects (in terms of n-terminal direction or c-terminal direction along the amino acid residue sequence) are symmetrical, but more specifically the essential features of the interactions between neighboring residues can be deduced from parameters like those in table in much the same way as the shape of an equilateral triangle standing on its base can be deduced from its height. as well as using new parameters, the initial score for a particular residue was computed in the same way as that for the whole segment of residues in section . , but was specifically considered as associated with the central residue, and the sum was retained as that sum rather than the overall score being averaged by the number of residues in the segment. deletions to consider alignments of segments were not applied in the sabr-p method. the above sum associated with each central, i.e. th , residue, was then averaged over the corresponding sums from the residues up to away on the n-terminal direction up to residues in the c-terminal direction including the central residue being addressed. this was done for every residue in the input sequence. it formally models parameters describing the directional characteristics of different types of amino acid residue, albeit in a highly empirical way. however, it is simply analogous to a smoothing of the propensity plot for residues along the overall input sequence. (viii) plot scaling was applied to produce a convenient decision threshold. an appropriate height scale of the plot, in which a residue with a score of over would be considered as the whole input sequence indicative of a sialic acid residue binding domain or protein, was obtained by an empirical pseudo-normalization consisting of dividing each residue score by . whenever the final results by sabr-p are expressed as a percentage-like score by multiplying all residue scores on the plot by , this is of course equivalent to dividing by . on that percentage basis, there seems no benefit it describing scores more accurately than to the nearest integer. in summary, the essential features of the simple resulting algorithm are as follows. ( ) residue binding parameter assignment. examine every residue in the sequence and assign it the parameter , , , , or of the sabr-p prediction method refined parameter (last column of table ). ( ) basic motif score. for each residue number i, obtain a score by summing over the run of residues of which i is the central ( th ) residue and assign the resulting sum (the score) to each residue i (providing that a residue numbers i- , i- etc. up to i+ lies within the sequence). [ ] by repeating step ( ) with the resulting scores, but do not add the basic motif score for each residue i to itself. more specifically stated, examine the above basic motif score for each i th residue from i= to end of sequence in turn, and add it to the score of the residue at i- , i- , etc up to i- , and to the score of the residue i+ , i+ , etc. up to i+ (providing that the residue numbers i- etc. lie within the sequence). ( ) normalized score as sabr-p propensity. examine each smoothed score resulting from the above for each residue i, and divide by . this is the current value of the optimized normalization parameter that sets the threshold as above which sialic acid glycan binding is predicted. ( ) reporting. the normalized score (sabr-p propensity) is plotted as a function of residue number from to end of the sequence, i.e. as a "sabr-p propensity plot". the actual predictions are also written out separately as text and these report only the amino acid residue (as g, a, v, etc.) along with the score, for those residues for which the normalized scores exceed . particularly because of step ( ), these will tend to occur conveniently in runs of approximately - contiguous residues, but sometimes shorter. in the present study the normalized score was formalized as a simple measure by rounding to the nearest integer prior to reporting, though this made no significant difference in the present paper. this simple approach will be better developed as a gor-like method, but for present purposes it suffices to give an estimate of the likely sialic acid glycan binding domains on the spike protein and proceed with further investigations and results described below. at the above optimized threshold, this the example sabr-p propensity plots shown in fig. . the initial studies using a random selection (more correctly stated, an arbitrary selection) of proteins except that a predominance of sialic acid or sialic acid glycan binding proteins were sought, the method initially gave true positives out of proteins representing group a, i.e. those that are known to bind sialic acids or glycan molecules containing them, true negatives for prediction of the above same kind of sialic acid binding out of group b, i.e. proteins believed not to bind any kinds of sugars significantly, and true negatives for the proteins comprising group c, i.e. proteins that bind sugars but not those containing sialic acids, and so representing the biggest challenge. this initial result represented % accuracy, % sensitivity, and % specificity, a reasonable preliminary result for such a simple model. note that there were initially no false negatives for those proteins not believed to bind any kinds of sugars, so a specific search for at least one case of a false negative (discussed below), which is strictly speaking a bias, dropped the prediction quality to % accuracy, sensitivity %, and % specificity. a recent number of further studies exemplified in the discussion below were also, strictly speaking, a bias including comparisons between weakly homologous proteins, extended the sample to proteins and reproduced the original quality to within the nearest percentage: % accuracy, % sensitivity, and % specificity. fig. . examples of prediction of sialic acid binding sites by sabr-p. in each case, the abscissa (x axis) is the distance along the sequence (residue number) and the ordinate (y axis) is the predicted sialic acid glycan binding propensity. scores above a threshold of (red line) for any residues are taken as a prediction that the domain or protein binds sialic acid glycans. at this stage of the present project the predictive method was only required to give approximate results as guidelines as to the regions the spike protein that might be further examine for sialic acid glycan binding capability, but the method appears promising and justifies some further comments and some further exploratory investigation, as follows. in group a, proteins being tested are believed to be able to accommodate sialic acid, sialic acid glycan, and related compounds by non-covalent binding. predicted correctly, these would represent the true positives, but predicted incorrectly, they would represent false negatives. such proteins include various hemagglutinins such as that of influenza a at genbank caa . generally arbitrarily selected neuraminidases and hemagglutinins are true positives but the above kind of pattern, a segment of approximately - residues with a score exceeding and centered on tryptophan with the peak score, is not universal. sometimes it is another hydrophobic residue. in a "haemaglutinin repeat-containing protein" of a yet to be fully classified micororganism candidatus kentron a tryptophan (w) with a score of is slightly displaced from the central peak of associated with a run of three glycines (g). it is two glutamate residues (e), negatively charged that are in the vicinity in the sequence that appear to perturb the usual central role of tryptophan. similarly, a rat neuraminidase is of some interest in that the predicted sequence sldhghtw surrounds the glycine (g) with peak score and terminates at tryptophan (w) with a score of . the tryptophan is, however, immediately followed by a glutamate (e). the significance of the above comments is as follows. of the amino acids with charged sidechains, only aspartate (d) and histidine (h) appear to favor such binding in the present author's analysis, while glutamate (e) arginine (r), lysine (k) have zero valued parameters and so are contraindications of sialic acid or sialic acid glycan binding. however, the latter three charged amino acid residue can sometimes be found in scialic acid binding sites and in sites predicted as such by the present method. indeed, they are often dominant features of residues directly interacting with sialic acid. in the sialic acid binding site of the globular head region of the newcastle disease virus haemagluttinin a glutamate (e), three arginine residues (r) and a lysine (k) are intimate contact with sialic acid ligand. a difference is that these residues make intimate contact in space and are not together in a one or very few subsequences. that is, they do not necessarily constitute a sequence motif. the actual extent of any counter-predictive impact of the above charged residues other than aspartate (d) and histidine (h), when they together in a subsequence of amino acids examined by the present algorithm, can be seen in false negatives. the neuraminidase of the plant striga asiatica (asian witchweed), was one of the false negatives: the analogous region to many of the above, at least in the sense of surrounding the only tryptophan (w) and with the peak value, is egavdrwrgeanf where the tryptophan (w) has only a peak value of , and has an arginine (r), positively charged, on both sides. the other false negative in the original study was a bacterial (chryseobacterium) haemagluttinin with the peak value of at a tryptophan, but with two lysine residues (k), also positively charged sidechains, in the vicinity. later studies also noted that n-acetyl neuraminic acid synthetase neub of e. coli, which has score of any residue to more than . for example, in the case of the witchweed neuraminidase, changing rwr to sws raised the peak value at the tryptophan to , a significant increase but still not exceeding . in some cases a value exceeding can of course attained. a false negative also found in later studies investigating these issues was the ox neuraminidase, with the subsequence ddhgvswrygggvs containing the tryptophan (w) with peak value . changing to an serine (s) the arginine (r) adjacent to the tryptophan (w) did have an effect, albeit that the only change was having the tryptophan as the only residue predicted, with a marginal score of . two kinds of potential true negative or false negative were distinguished in the study. the group that appeared to do particularly well at predicting those proteins that do not binding sialic acid or sialic acid glycan was, perhaps not surprisingly, group b, i.e. those proteins not expected to bind any kind of sugars. out of the original sample of proteins not expected to bind any kinds of sugars significantly, and that also confirmed that expectation, i.e. true negatives as far as predicting sialic acid binding is concerned, two (i) hemoglobin and (ii) trypsin precursor for which the prediction plots for which are shown in fig. . the others not shown are mostly quite large proteins containing more than tryptophan residues (w) sites, for which the method still correctly predicted as non-sialic-acid binders, and so worthy of some comments. they included (iii) human ubiquitin c of residues and no tryptophans (w) and no residue score exceeding , in contrast to (iv) human progesterone receptor of residues of which were tryptophan but none of which exceeded (one had the highest score of ). the remaining true negative cases are (v) fatty acid oxidation complex subunit alpha fadb of acinetobacter calcoaceticus of a substantial residues, (vi) the mitochondrial nadh-ubiquinone oxidoreductase kda subunit of the camel, which comprised a substantial residues but did notably not exceed a score of for any residue, (vii) human cytochrome c with a maximum score of for the second tyrosine (y) in tgqapgysytataankn, and (viii) alcohol dehydrogenase (human, a) that did not exceed a score of for any residue despite the "concern" that ethanol having basic sugar-like features and so could, a priori, be marginal. perhaps unfairly included as rather small, (ix) proinsulin nonetheless does contain a tryptophan (w) which correctly did not exceed and indeed only had a score of in llallalwgpdpaaa; the phenylalanine (f) in gpdpaaafvnqhlcg had a highest score of in the sequence. in the initial study, the case most closely approaching a false positive in this group was (x) human prothrombin with a substantial number of residues, there was only one residue, glycine (g), that reached a score of , and a residue score should exceed to classify the whole domain or protein as sialic acid binding. it is possibly best declared as an example of a marginal case. although at the outset false positives were expected to appear in this non-sugar-binding group groups as the sample is increased, not least because of the preliminary nature of the method. human angiotensin converting enzyme type (ace ) was the first exception found and it is a significant exception in that it had two substantial regions - and - both exceeding scores of throughout and peaking at substantial scores of . this group is interesting in that persistently predicting these as false positives might incline a researcher to abandon specifically predicting sialic acid glycan binding and instead consider their method as better positioned to predict sugar binding in general, but this turned out not to be the case. for group c, i.e. those sugar-binding proteins that are believed to bind sugars but not to bind sialic acids or glycans containing them, the prediction plots for the true negatives are shown to the lower right in fig. . human α-amylase, not shown, was an interesting false positive with subsequence of residues all only marginally exceeding ysgwdfwgegw but containing three tryptophans (w) of which the first had the highest score of . the human lysozyme precursor was also a false positive, albeit only marginally so, with one sequence kwesgyntra exceeding and with the peak at tyrosine (y) with a score of . in later studies examining the effect of considering weakly homologous sequences, the significantly different hen lysozyme precursor is interesting as still being a false positive but representing and even more marginal case, having one short subsequence with residues exceeding , namely wv, with tryptophan (w) with final score followed by valine (v) with final score . while there are significant amounts of sialic acids in hen egg white, the author is not aware of any extract of hen egg white lysozyme that has these bound to the protein. evidently comparisons of homologous proteins might be helpful to improve prediction power. another false positive is the human atp-dependent translocase abcb isoform which has a ribose binding site has a considerable size of , residues yet with just one subsequence, syalafwygmmyfsyagcf, exceeding , and has the peak of at the tryptophan (w). for such reasons, one may suspect that a fairer set of criteria for assessing predictive performance would be an average per residue basis. one of the more recent studies included the sars-cov- polyprotein which contains proteins with known rna and ribose binding functions. out of residues, residues exceeded scores of . the proteins and domains there, however, include several of which all possible functions are not yet known. evidently, those regions with scores over , by virtue of being in proteins of sars-cov- , are certainly worthy of future further examination in this project. however, this was considered beyond present scope for the present paper, as the focus is on the spike glycoprotein. the more recent studies of sugar binding proteins that did not include suspected sialic acid or sialic acid glycan binding regions included sugar isomerases, such as the l-rhamnose isomerase of rubinisphaera brasiliensis, which with residues containing tryptophans (w) was a true negative. they also included a comparison between sugar transporter proteins, such as the udp-galactose transporter of drosophila melanogaster had residues including tryptophans (w), the pts fructose transporter subunit eiic of aeromonas hydrophila with residues containing tryptophans, and a facilitated glucose transporter member , which despite having residues containing tryptophans (w), all of which were true positives by not predicting any sialic acid or sialic acid glycan ability. however, some sugar transporters were false positives. for example, the arabinose transporter of e. coli had the subsequence fwlyta which exceeded with the tryptophan scoring . armed with the above predictions and insights, one may make better informed judgements as to whether a domain for non-covalently binding host sialic acid glcycans may exist in the sars-cov- spike protein. the above results suggesting the ability to distinguish between three classes of protein in relation to sialic acid glycan binding is perhaps surprising, not least because non-sialic sugars such as fucose and mannose can occur in cell surface glycans (including sialic acid glycans) as indicated in introduction section . . this is discussed in discussion section . . at this stage, only the ability to show propensities in different regions of the sars-cov- (genbank entry mn . ) spike glycoprotein is required. in this stage of the study the predicted regions of the sars-cov spike protein were examined in more detail. while as discussed above tryptophan can be involved in the fundamental structure of a sialic acid binding domain, an exposed tryptophan like that binding a sialic acid glycan in fig. would be particularly persuasive. the first predicted sialic acid binding motif is segment ffsnvtwfhaihv - with sabr-p score ranging from for valine (v) to for tryptophan (w) and for phenylalanine ( as shown in fig. , the tryptophan sidechain in ffsnvtwfhaihv as residues - of the sars-cov- spike glycoprotein is exposed in a site that has all the appearance of a sialic acid glycan binding site, comparable with the influenza virus b neuraminidase (pdb entry bat) tryptophan site known to have interaction with and sialic acid, that was shown in fig. . the tryptophan sidechain in ffsnvtwfhaihv - of spike glycoprotein is exposed in a site that has all the appearance of a sialic acid glycan binding site. it is useful to see how recurrent a potential more universal motif may be, to give insight, to avoid cross reactions of vaccine in human and veterinary patients as hosts, to detect an underlying common function that one might not wish to inhibit in the host with an anti-viral therapeutic, and so on. a blast search on non-viruses picks this subsequence up as ffsnvtniawihai of parasteatoda tepidariorum, the common house spider, and related sequences, a zinc figure domain because it contains fyve the zinc finger motif, but more abundantly it picks up sugar-binding proteins such as glycosyl transferases such as ffspvwartpnvtwfh-hv of actinobacteria, ribulosephosphate -epimerase of animals, a-amylase of the chitinophagia ("chitin eating") bacteria, c-type lectin db-like of drosophila hydei, and related sugar binding proteins, all varying around % cover and % match, % cover % match, % cover % match, and so on. the second predicted sialic acid binding subsequence sssgwtagaaa has been of interest in the preceding sections . - . , where it seemed a likely sugar binding site based on circumstantial evidence such as homologies to neurmaindiases and glycan esterases. unfortunately, it lies in the range that generally disordered in experimental d structure, e.g. residues - in vvx (spike closed state) and - in vyb (spike open state). it also has modest maximum scores of , but the above considerations do not prohibit its potential importance. again, as for the previous subsequence, it is useful to see how recurrent a potential more universal motif may be. blastp searches of non-viruses find subsequences in sugar binding proteins such as ssagwtagaa of microbacteria ( % cover % match), but compared with ffsnvtwfhaihv discussed above, searche generate a lot of closer matches (many % matches with the top at % cover % match or better) which, however, involve an even more diverse set of proteins. at first examination most appear less directly relevant to sugar, but many of these merit more examination. details of each case are beyond present scope, but for example a particularly recurrent match example is sssgwtaga or similar sequences of proteins that contain the twin-arginine translocation pathway signal of most bacteria and archaea. for example such as ruegeria marisrubri, of the rhodobacteraceae has the above matching subsequence. the twin-arginine translocation pathway transports folded proteins across the cytoplasmic membrane of these microorganisms. the proteins are targeted by signal peptides containing a conserved twin-arginine motif, and the literature does not always mention any n-glycosylation and indeed there may not in every case be any directly relevant evidence of such. however, there is certainly known involvement in the production of secretory and extracellular n-linked glycoproteins in bacteria such as escherichia coli. the third predicted subsequence is hwkwpwyiwl - with a peak score of relates to ikwpwyiwl in the original wuhan seafood market isolate (genbank mn . ) and lies at the boundary between the c-terminal end of s and the transmembrane part - . its d structure is not, to the present author's knowledge, available, as it lies in residues onward are generally excluded from experimental structure (e.g. vvx and vyb). again, matches with non-virus or host proteins may be of interest as biologically and medically important. blastp searches on non-viruses with the sequence as query inappropriately pick up a lot of coronaviruses by accidentally relating to the host name, but this is indicative of a strong recurrence of the motif across coronaviruses. otherwise, there is a diverse set of matches especially but not solely with bacterial proteins, and unfortunately most are described as hypothetical proteins for which the function is typically unclear. of those that are named, there are some indications of involvements with sugar binding in many cases. many are ribosome proteins or phosphatases relevant directly or indirectly to rna or ribose binding. the pap superfamily is characterized by a core consisting of a -helical bundle and includes functions involving glycerol phosphates but also sugars such as in the case of glucose- -phosphatase. this subsequence hwkwpwyiwl also matches sequences in proteins with an spfh domain which is implicated in regulating targeted protein turnover in stomatins and other membrane-associated proteins. hwkwpwyiwl has of course a notable tryptophan (w) repeat that hints a special role of its own, certainly worthy of analysis but outside present scope. in general, however, it does appear that across many proteins it has other functions than sugar binding (perhaps diverse or multiple functions), although sugar binding is not excluded. the significance and innovation of the present work is that it proposes a sialic acid glycan binding function for the sars-cov- spike protein that has been largely neglected by other workers, apparently on the rationale that ace- binding is the important first step in cell entry. sites involved in the characteristic cap or knob of the spike protein appear partially persuasive in the light of their role as binding to host cells. there is a further possible site towards the base of the external part of the spike protein, which seems less likely by virtue of its position and weaker prediction. interaction with sialic acid glycan with or without associated catalytic activity would be consistent with such functions observed in many respiratory and alimentary tract viruses, and not least in many or most other coronaviruses, and so such a function must be important to these viruses. on these grounds, it may be a target for therapeutic agents against sars-cov- , particularly perhaps preventatives as well as means of impeding spread from lung cell to lung cell, and an exposed target for antibodies raised by synthetic vaccines. although other authors have recently touched on such a glycan binding ability in sars (as discussed in this paper above and particularly below), it has not been to the present author's knowledge analyzed in comparable detail and do not appear to relate to the same site. nor do they propose a general prediction method for sialic acid glycan binding as described in the present paper. of course, in the present paper this is still a prediction and not an experimental result, but it will hopefully encourage experimental researchers to investigate the glycan binding properties of sars-cov- more extensively. a further innovative feature is that predictive method, which is expected to be worthy of investigation for the proteins of other viruses and even of other organisms. like many predictive methods in bioinformatics it is not perfect, i.e. there are false positives and false negatives in prediction, so it is actually conceivable that the method is useful even if it is not correct in the particular case of sars-cov- . in that sense, it may emerge as the more important contribution. work. the current sabr-p predictive algorithm is naïve and it is not expected that it will resemble closely the final refined form of the algorithm, which will based on more rigorously on principles closer to those of the gor method [ ] , the hyperbolic dirac net [ ] [ ] [ ] , the association q-uel language, and the bioningine implementation including its new algorithms [ ] [ ] [ ] [ ] . the impression of good performance for the current sabr-p method largely arises from the fact that it is only required to predict the sialic acid glycan binding properties of whole domains or proteins, not highly localized subsequences or surface patches. in essence, the method is really doing little more than capture and quantify in an algorithm the visual inspection of sugar binding domains and proteins and the observations of other workers as discussed above. however, the method was only required to help explore potential non-covalent sialic acid glycan binding sites in the spike glycoprotein, and in that regard it has proven adequate and valuable for present purposes. it also suggests a more refined approach may perform well because false positives and false negatives were mainly just over the boundary and just under it respectively. the current predictions also indicate a research direction in which to explore. the parameters for the general sugar binding capacity of amino acids residues are very different to those used by other workers and here the focus has been on sialic glycans versus other saccharide-based molecules. in this, perhaps the most surprising finding of all is the apparent ability of the method to distinguish between sialic acid containing glycans and other sugars in the case of lectins. this is because non-sialic sugars such as mannose and fucose can occur in in sialic acid glycans, and prediction results hint that there is likely to be some distinguishing feature for a majority of cases that makes a specific recognition. in this respect it would seem initially of concern for the sensibleness of the predictions that, for example, mannose-binding lectin binds to a range of sugars that also include n-acetyl-d-glucosamine, n-acetyl-mannosamine, fucose and glucose. it is therefore possible that research in this direction will not be so profitable because the above distinguishing behavior of the algorithm might be to some extent coincidental. be that as it may, the predictions are remarkably much better than expected, and should certainly be challenged by researchers in order to improve such methods. specifically, a larger sample may require a threshold adjustment or corresponding rescaling, perhaps resulting in a deterioration of performance particularly in regard to distinguishing lectins. nonetheless, it is noteworthy that ultimately human glycan binding proteins have to overcome the same problem as the above kind of prediction algorithm. while this broad range of sugar recognition by the mannose-binding lection permits that lectin to interact with a wide selection of pathogens (viruses, bacteria, yeasts, fungi and protozoa) decorated with such sugars, there must be some kind of distinguishing aspect such that is not decoyed by the sialic acid glycans of the human host. a more mundane problem in extending the study is that the correct state as sialic acid glycan binding, other sugar binding, or not binding any kind of sugar, may be uncertain or a matter of degree. further studies at time of writing suggested only about % for each of accuracy, sensitivity, and specificity, but this larger set is, as yet, of dubious quality for the purpose. some proteins were believed, rather than known, to bind sialic acid glycans, binding might be weak or less specific or of multiple types, or the domain or approximate location of the binding site can be unclear. related to that is a difficulty that the performance of any prediction method of this kind is defensible, and possibly unfairly defensible, in regard to false positives: it may be that experiment shows that a particular virus predicted to bind sialic acid glycans does not specifically do so, but perhaps it once did, in evolutionary terms. this is particularly relevant in regard to studying coronaviruses because, as discussed above, many coronaviruses certainly do bind sialic acid glycans. of course, the prediction method would then still be subject to the criticism that it insufficiently sophisticated to manage the impact of small changes. for purely theoretical methods, that may be an issue for some time: in the present author's experience even simulations of binding of sugars to proteins in atomic detail tend to be difficult in view of the complex role of water molecules. for example, water molecules commonly represent protein-to-sugar bridges as discussed in this paper. potential biological implications arguably support the above prediction for sars-cov- spike protein. that is, the story "makes sense". although the involvement of sars-cov and sars-cov- with sialic acid glycans has been rather neglected in the literature (but see below), such involvement represents a prominent and well known feature in the life history of influenza and other viruses, and appears no less important in the life of many other coronaviruses. admittedly, hiv and many other enveloped viruses do not encode hemagglutinin for sialic acid binding. instead, they interact using n-terminal sialic acid bound to envelope-associated proteins, like gp on hiv- . however, the mode of infection is different. the sars-cov- coronavirus cannot jump in a magical way from contaminated surfaces to the lung, and it is doubtful that infective small loads of virus rely on chance to travel from the infecting person to the lung cell ace receptor of the next human host. it is as yet unclear how many virus particles of sars-cov- are needed for infection, but the virus is clearly very contagious, and this may be because rather few particles are needed for infection. in any event, the virus has to survive, and ideally even benefit for its survival, stages in a complex journey in mucus of a sneeze or on hands, face, eye, nose, or mouth, and in the various stages of the airway. initial cell entry points are unlikely to be only the lung epithelium. sars-cov- entry factors are highly expressed in nasal epithelial cells together with innate immune genes [ ] . viral mechanisms relating to these various surfaces could be fairly sophisticated. in biology in general, flexibility in carbohydrate recognition contributes to the targeting efficiency of carbohydrate-active enzymes in environments where there is diverse range of saccharides [ ] . in a virus, more than one saccharide-binding site or multiple sugar binding sites in a protein could act to increase or decrease the overall affinity and increase or decrease virus mobility at different locations, while conformational changes could make available some sites and not others could regulate the extent of movement of the virus. some binding sites have evolved to distinguish not just the sugar residue components but several types of monosaccharide or glycosidic bond linkage. once having reached the vicinity of a cell with an ace receptor, the virus still needs to recognize the cell surface and raft across the cell surface to reach the ace receptor. fantani et. al [ ] argued that a new type of ganglioside-binding domain exists at the tip of the n-terminal domain of the sars-cov- s protein, and that the subsequence - , conserved among clinical isolates, may improve attachment of the virus to lipid rafts and facilitate contact with the ace- receptor. this study also showed that, in the presence of clq or its more active derivative, hydroxychloroquine, the spike protein is no longer able to bind gangliosides [ ] . the present study does not support (nor necessarily refute) their conclusions in terms of such specific details, but the general argument concerning guidance to the ace- receptor is compatible. very recently milanetti and colleagues have made available a preprint [ ] that is tune with such ideas, and specifically states that binding silvic acids provides a second means of entry, other than ace . rather like the approach of thornton et al. [ ] , this is based more on interactions between surfaces of molecules in three dimensional space. the results and conclusions of this study are speculative in the sense that they are applications of computers (using the techniques of bioinformatics and a new predictive method), and hence they are essentially theoretical. their role has been to highlight the likelihood that the sars-cov- spike has a biological function of binding host cell sialic acid glycans (and probably across cells surfaces by that means, as discussed below). in particular, a domain in the cap or knob of the sars-cov- spike, which has so far been somewhat neglected, is involved in the non-covalent binding of host sialic acid glycans. it is perhaps curious that subsequences found as conserved by use of bioinformatics tools such blastp and clustal omega (also used here as described in methods section . ), or detected as a known or new functional motif, often seem in the literature to be considered as having the status of experiment or observation, while consideration of more complex patterns with more sequence options tend to be treated as theory and prediction. this caution is justified in the present study because further study and confirmation is required along the lines discussed in discussion section above. above. to the extent that it is a prediction, it is a prediction for sars-cov- made in advance of experiment in order to provide an objective and fair test of the methodology and it is hoped that it will stimulate experimental study in this area whether the experiments confirm or refute that prediction. either result would likely be of ultimate medical importance. this is essentially typical of the more interesting roles of computers in biomedical research, although the general infrastructure and support that they provide for more routine tasks is of course of great importance. the present paper possibly still stands as the first reported attempt to establish means of making use of sequence motifs that could be recognized between strains, albeit that the order and to some extent precise nature of the amino acid residues of amino acid residues appears less important, or perhaps more subtle, than has been considered in previous papers in this series [ , ] , which is why it required the development of the predictive technique (sabr-p). this will be advanced in future work, but the present method already helps to make quick comparisons between sars-cov- sequences and to consider the effects of viral mutations. however, it was surprising that a very simple approach was so useful, and it can easily be reproduced in a very few lines of computer program. the important consequence of the present study, however, is that there are already a variety of inhibitors of sialic acid binding that may serve as anti-viral agents, and this will be examined elsewhere. • this paper extends the studies of the author's previous sars-cov- papers. • designing vaccine and drugs must seek to avoid escape mutations. • strangely, sars-cov and sars-cov- appear to lack sialic acid binding functions. • sequence motifs are found, but they require a simple prediction method. the molecular biology of coronaviruses genomic characterization and epidemiology of novel coronavirus: implications for virus origins and receptor binding, www.thelancet preliminary bioinformatics studies on the design of synthetic vaccines and preventative peptidomimetic antagonists against the wuhan seafood market coronavirus. possible importance of the krsfiedllfnkv motif preliminary bioinformatics studies on the design of a synthetic vaccine and a preventative peptidomimetic antagonist against the sars-cov- ( -ncov, covid- ) coronavirus covid- coronavirus spike protein analysis for synthetic vaccines, a peptidomimetic antagonist, and therapeutic drugs, and analysis of a proposed achilles' heel conserved region to minimize probability of escape mutations and drug resistance the spike glycoprotein of the 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antiviral drugs sialic acids as receptor determinants for coronaviruses structural and molecular modelling studies reveal a new mechanism of action of chloroquine and hydroxychloroquine against sars-cov- infection carbohydrate-binding modules analysis and prediction of carbohydrate binding sites gor method for predicting protein secondary structure from amino acid sequence hyperbolic dirac nets for medical decision support. theory, methods, and comparison with bayes nets split-complex numbers and dirac bra-kets bidirectional general graphs for inference. principles and implications for medicine suggestions for a web based universal exchange and inference language for medicine implementation of a web based universal exchange and inference language for medicine. sparse data, probabilities and inference in data mining of clinical data repositories studies in using a universal exchange and inference language for evidence based medicine. semi-automated learning and reasoning for pico methodology, systematic review, and environmental epidemiology studies in the extensively automatic construction of large odds-based inference networks from structured data. examples from medical, bioinformatics, and health insurance claims data extension of the quantum universal exchange language to precision medicine and drug lead discovery. preliminary example studies using the mitochondrial genome sars-cov- entry factors are highly expressed in nasal epithelial cells together with innate immune genes in-silico evidence for two receptors based strategy of sars-cov- barry was a pioneer in bioinformatics, protein modelling, and computer-aided drug design, interests that he actively continues today. he is the recipient of several honours including the asklepios award for outstanding vision in science and technology at the future of health technology congress at m.i.t. in . he has helped start up several other companies or divisions in the uk and usa. barry continues as ceo of the dirac foundation in the uk, and distinguished scientist prior to that, he was cso of gryphon sciences (later gryphon pharmaceuticals) in south san francisco, california, a bio-nanotechnology ultrastructural chemistry start-up largely held and then acquired by smithkline beecham. before moving to the us, barry was the scientific founder of proteus international plc in the uk, designing and leading the development of the prometheus expert system and its underlying global expert system, bioinformatics and simulation language for drug, vaccine, and diagnostic discovery. it sold for the equivalent of $ . million to the pharmaceutical industry in the mid- s. at proteus, he also led the team that used the above expert system to invent and patent several diagnostics and vaccines including the mad cow disease diagnostic subsequently marketed worldwide by abbott as a whitepaper to the president of the united states. he was also an advisor in relation to a major scientific computer-aided drug design collaboration and network for peter feinstein consultants between work between us scientists and the russian science city arzamas. for five years, barry was a nature "news and views" correspondent on biomolecules. he was visiting scholar stanford university school of medicine - , professorial lecturer mount sinai nyc during part of his period at ibm corporation, and held visiting positions and professorships in inra and u quantum universal exchange language and hyperbolic dirac nets for precision medicine and drug design. proposals with examples from mitochondrial studies studies in the use of data mining, prediction algorithms, and a universal exchange and inference language in the analysis of socioeconomic health data bidirectional general graphs for inference. principles and implications for medicine studies in the extensively automatic construction of large odds-based inference networks from structured data. examples from medical, bioinformatics, and health insurance claims data studies in using a universal exchange and inference language for evidence based medicine. semi-automated learning and reasoning for pico methodology, systematic review, and environmental epidemiology studies of the role of a smart web for precision medicine supported by biobanking data-mining to build a knowledge representation store for clinical decision support. studies on curation and validation based on machine performance in multiple choice medical licensing examinations interesting things for computer systems to do: keeping and data mining millions of patient records, guiding patients and physicians, and passing medical licensing exams implementation of a web based universal exchange and inference language for medicine. sparse data, probabilities and inference in data mining of clinical data repositories split-complex numbers and dirac bra-kets suggestions for a web based universal exchange and inference language for medicine. continuity of patient care with pcast disaggregation popper, a simple programming language for probabilistic semantic inference in medicine hyperbolic dirac nets for medical decision support. theory, methods, and comparison with bayes nets suggestions for a web based universal exchange and inference language for medicine the concept of novel compositions of matter. a theoretical analysis towards new tools for pharmacoepidemiology the role of information drug discovery using very large numbers of patents. general strategy with extensive use of match and edit operations considerations , for a universal exchange language for healthcare quantum mechanics in artificial intelligence and decision support. a review this project used some of the knowledge gathering methods described in preceding papers although the work is described entirely in terms of standard bioinformatics tools. these knowledge gathering methods are used, amongst many others in an integrated way, in the algorithms and internal architectural features of the bioingine.com, a distributed system developed by ingine inc. cleveland, ohio, for the mining of, and inference from, very big data for commercial purposes. key: cord- -q b ym authors: murakami, makoto title: lipoquality control by phospholipase a( ) enzymes date: - - journal: proc jpn acad ser b phys biol sci doi: . /pjab. . sha: doc_id: cord_uid: q b ym the phospholipase a( ) (pla( )) family comprises a group of lipolytic enzymes that typically hydrolyze the sn- position of glycerophospholipids to give rise to fatty acids and lysophospholipids. the mammalian genome encodes more than pla( )s or related enzymes, which are classified into several subfamilies on the basis of their structures and functions. from a general viewpoint, the pla( ) family has mainly been implicated in signal transduction, producing bioactive lipid mediators derived from fatty acids and lysophospholipids. recent evidence indicates that pla( )s also contribute to phospholipid remodeling for membrane homeostasis or energy production for fatty acid β-oxidation. accordingly, pla( ) enzymes can be regarded as one of the key regulators of the quality of lipids, which i herein refer to as lipoquality. disturbance of pla( )-regulated lipoquality hampers tissue and cellular homeostasis and can be linked to various diseases. here i overview the current state of understanding of the classification, enzymatic properties, and physiological functions of the pla( ) family. in terms of signal transduction, the phospholipase a (pla ) reaction, which hydrolyzes the sn- position of phospholipids to yield fatty acids and lysophospholipids, has been considered to be of particular importance, since arachidonic acid (aa, c : ), one of the polyunsaturated fatty acids (pufas) released from membrane phospholipids by pla , is metabolized by cyclooxygenases (coxs) and lipoxygenases (loxs) to lipid mediators including prostaglandins (pgs) and leukotrienes (lts), which are often referred to as eicosanoids (fig. ) . lysophospholipids or their metabolites, such as lysophosphatidic acid (lpa) and platelet-activating factor (paf), are categorized into another class of pla -driven lipid mediators ( fig. a, b) . more recently, a novel class of anti-inflammatory lipid mediators derived from b pufas, such as eicosapentaenoic acid (epa, c : ) and docosahexaenoic acid (dha, c : ), has also been attracting much attention (fig. c ). these lipid mediators exert numerous biological actions on target cells mainly by acting on their cognate g protein-coupled receptors. the pathophysiological roles of individual lipid mediators have been summarized in recent reviews. )- ) however, this principal concept appears to be insufficient to fully explain the biological aspects and physiological roles of the pla family. phospholipids comprise numerous molecular species that contain various combinations of fatty acids esterified at the sn- and sn- positions and several polar head groups at the sn- position. many, if not all, pla enzymes recognize such differences in the fatty acyl and/or head group moieties in their substrate phospholipids. moreover, several enzymes in the pla family also catalyze the phospholipase a (pla ), lysophospholipase, neutral lipid lipase, or even transacylase/ acyltransferase reaction rather than or in addition to the genuine pla reaction. therefore, the fatty acids and lysophospholipids released by different pla s are not always identical; rather, in many situations, specific fatty acids and lysophosholipids can be released by a particular pla in the presence of a given microenvironmental cue. in this context, pla enzymes act as one of the critical regulators of spatiotemporal lipid profiles, namely the quality of lipids (lipoquality). to comprehensively understand the lipoquality regulation by individual pla s in various pathophysiological contexts, their precise enzymatic, biochemical and cell biological properties, tissue and cellular distributions, and availability of phospholipid substrates in various pathophysiological settings should be taken into consideration. herein, i overview current understanding of the biological aspects of various pla enzymes in the context of lipoquality. obviously, the substrate specificity of individual pla s is the critical determinant of lipoquality. the in vitro enzymatic activity of pla s may be influenced by the assay conditions employed, such as the composition of the substrate phospholipids, concentrations of pla s and substrates, presence of detergents, and ph. hence, the enzymatic properties of individual pla s determined in different studies may not be entirely identical. since natural membranes contain numerous phospholipid molecular species, the results obtained using artificial phospho- lipid vesicles comprising only one or a few phospholipid species may not always reflect the true enzymatic properties of a given pla . addition of an excess amount of recombinant or purified pla to an enzyme assay often results in hydrolysis of bulk phospholipids, which makes precise evaluation of its substrate specificity difficult. the results obtained using a commercially available pla assay kit, in which a synthetic, chromophoric phospholipid is used as a substrate, should be interpreted carefully, since some pla s are unable to hydrolyze it efficiently. in this regard, mass spectrometric examination of the in vitro hydrolysis of natural membrane phospholipids extracted from the affected tissues or cells by pla , particularly at a low (physiologically relevant) concentration of the enzyme, could provide a valuable clue to the in vivo substrates and products of this enzyme. )- ) the overall tendency in this in vitro assay using natural membranes is recapitulated in several in vivo systems, often with even more selective patterns of hydrolysis that are relevant to the results of studies using pla knockout and/or transgenic mice (see below). importantly, the mobilization of distinct lipids by pla s in vivo relies not only on their intrinsic enzymatic properties, but also on tissue-or disease-specific contexts such as the lipid composition of target membranes, the spatiotemporal availability of downstream lipid-metabolizing enzymes, or the presence of cofactor(s) that can modulate the enzymatic function, which may account for why distinct pla enzymes even in the same subfamily exert specific functions with different lipid profiles in distinct settings. hereafter, i describe the current understanding of various pla s in the context of lipoquality. the classification, distributions, properties and functions of individual pla s, whose pathophysiological functions have currently been studied using their gene-manipulated mice, are summarized in table enzymes whose in vivo functions have been analyzed using knockout mice are summarized. the cpla family. the cytosolic pla (cpla ) family comprises isoforms (,- ), among which cpla o, /, c and map to the same chromosomal locus (fig. a) . ) cpla , (also known as group iva pla ) is undoubtedly the best known pla and its biological roles in association with lipoquality have been well documented. ) cpla , is the only pla that shows a striking substrate specificity for aa-containing phospholipids. strictly speaking, cpla , can also hydrolyze phospholipids containing epa, yet the low abundance of this b pufa relative to other fatty acids including b aa in cell membranes allows cpla , to release aa rather specifically in most situations. upon cell activation, cpla , translocates from the cytosol to the phosphatidylcholine (pc)-rich perinuclear, endoplasmic reticulum (er) and golgi membranes (particularly golgi) in response to an increase in the µm range of cytosolic ca d concentration, and is maximally activated by phosphorylation through mitogen-activated protein kinases (mapks) and other kinases. ), ) in addition, the phosphoinositide pip and ceramide- -phosphate modulate the subcellular localization and activation of cpla ,. ), ) the aa released by cpla , is converted by the sequential action of constitutive cox- or inducible cox- and terminal pg synthases to pgs or by the sequential action of -lox and terminal lt synthases to lts (fig. b ). mice deficient in cpla , display a number of phenotypes that can be explained by reductions of pgs and/or lts. under physiological conditions, cpla ,-deficient mice display a hemorrhagic tendency, impaired female reproduction, gastrointestinal ulcer, and renal malfunction, among others. )- ) under pathological conditions, cpla ,-deficient mice are protected against bronchial asthma, pulmonary fibrosis, cerebral infarction, alzheimer's disease, experimental autoimmune encephalomyelitis, collagen-induced arthritis, metabolic diseases, intestinal cancer and so on, whereas they suffer from more severe colitis and spinal cord injury. ), )- ) most of these phenotypes are recapitulated in mice lacking one or more of the biosynthetic enzymes or receptors for pgs and lts, lending strong support to the notion that cpla , lies upstream of eicosanoid biosynthesis in many situations. for instance, as is the case for cpla ,-deficient mice, mice lacking ltc synthase (ltc s), ltd receptor (cyslt ), ltb receptor (blt ), or pgd receptor (dp ) are protected from asthma, )- ) revealing the critical role of the cpla ,-ltb /ltc /pgd axis in this allergic disease. likewise, the decrease of pge in cpla ,-deficient mice can account largely, even if not solely, for the mitigation of arthritis, autoimmune encephalomyelitis, cancer and neurodegeneration as well as the exacerbation of colitis, since these phenotypes are mimicked by mice lacking pge synthase (mpges- ) or either of the four pge receptors (ep ). )- ) furthermore, cpla ,-triggered release of aa by platelets is coupled not only with biosynthesis of the pro-thrombotic eicosanoid thromboxane a (txa ), but also with o-oxidationmediated bioenergetics for blood clotting. ) importantly, inherited human cpla , mutations are associated with reduced eicosanoid biosynthesis, platelet dysfunction, and intestinal ulceration, ) , ) thus mimicking cpla , deletion in mice. on the other hand, the enzymatic activities and biological functions of cpla isoforms other than cpla , have remained largely unknown. reportedly, cpla o (group ivb pla ), which has a unique jimc domain in the n-terminal region, display pla , pla and lysophospholipase activities. ) cpla . (group ivc pla ), which uniquely lacks the c domain characteristic of the cpla family, is cterminally farnesylated and possesses lysophospholipase and transacylase activities in addition to pla activity. ) cpla / (group ivd pla ), whose expression is elevated in human psoriatic skin, ) shows pla activity in preference to pla activity. ) cpla c (group ive pla ) exhibits a unique transacylase activity that transfers sn- fatty acid of pc to an amino residue of phosphatidylethanolamine (pe) to form n-acyl-pe, a precursor of the endocannabinoid lipid mediator n-acylethanolamine. ) cpla (group ivf pla ) displays both pla and pla activities without fatty acid selectivity. ) however, these enzymatic properties of cpla o- vary according to the in vitro assays employed, implying that analyses using gene-manipulated mice for these enzymes will be necessary for clarifying their biological roles in the context of lipoquality. the ipla /pnpla family. the human genome encodes ca d -independent pla (ipla ) enzymes (fig. ) . these enzymes are now more generally referred to as patatin-like phospholipase domain-containing lipases (pnpla ), as all members in this family share a patatin domain, which was initially discovered in patatin (ipla ,), a potato protein. ), ) mammalian ipla /pnpla isoforms include lipid hydrolases or transacylases with specificities for diverse lipids such as phospholipids, neutral lipids, sphingolipids, and retinol esters. generally speaking, enzymes bearing a large and unique n-terminal region (pnpla ) act mainly on phospholipids (phospholipase type), whereas those lacking the n-terminal domain (pnpla ) act on neutral lipids (lipase type). analysis of mutant mouse models and clinical symptoms of patients with mutations for these enzymes have provided valuable insights into the physiological roles of the ipla / pnpla family in various forms of homeostatic lipid metabolism that are fundamental for life. among the ipla /pnpla family, pnpla (ipla o, also known as group via pla ) is the only isoform that acts primarily as a pla with poor fatty acid selectivity. ), ) although pnpla (ipla . or group vib pla ) displays pla activity, it acts as a pla toward phospholipids bearing sn- pufa. ), ) accordingly, hydrolysis of pufa-bearing phospholipids by pnpla /ipla . typically gives rise to -lysophospholipids (having a pufa at the sn- position) rather than -lysophospholipids (having a saturated or monounsaturated fatty acid at the sn- position). pnpla (ipla /) and its closest paralog pnpla (ipla ) have lysophospholipase activity that cleaves lysophosphatidylcholine to yield fatty acid and glycerophosphocholine. ), ) genetic mutations or deletions of these phospholipid-targeting pnplas cause various forms of metabolic dysfunction and neurodegeneration. )- ) in particular, pnpla /ipla o is also referred to as the parkinsonism-associated protein park , whose mutations impair ca d signaling in dopaminergic neurons. ) apart from the metabolic and neurodegenerative phenotypes, the lack of pnpla /ipla o leads to male infertility through an unknown mechanism. ) pnpla (ipla ), more generally known as adipose triglyceride lipase (atgl), is a major lipase that hydrolyzes triglycerides in lipid droplets to release fatty acids as a fuel for o-oxidation-coupled energy production, a process known as lipolysis. ) genetic deletion or mutation of pnpla leads to massive accumulation of triglycerides in multiple tissues leading to multi-organ failures, ) while protecting from cancer-associated cachexia by preventing fat loss. ) the activity of pnpla is regulated positively by abhd (see below) and negatively by perilipin and g s , which modulate the accessibility of pnpla to lipid droplets. ) the fatty acids released from lipid droplets by pnpla act as endogenous ligands for the nuclear receptor ppar, or ppar/, which accelerates energy consumption. ), ) the regulatory mechanisms and metabolic roles of pnpla have been detailed in other elegant reviews. ), ) mutations of pnpla (ipla c) are highly associated with non-alcoholic fatty liver disease. ) although the catalytic activity of pnpla is controversial, it may serve as a triglyceride lipase, since its loss-of function mutation increases cellular triglyceride levels. ) furthermore, recent evidence suggests that pnpla acts as a retinyl-palmitate lipase in hepatic stellate cells to fine-tune the plasma levels of retinoids. the expressions of pnpla and pnpla are nutritionally regulated in a reciprocal way; pnpla is upregulated, while pnpla is downregulated, upon starvation, and vice versa upon feeding. ) biochemical and cell biological studies have suggested that pnpla (ipla , which is absent in mice) might be involved in retinol ester metabolism ) and that pnpla might participate in triglyceride lipolysis coupled with autophagosome formation, ) although the in vivo relevance of these in vitro observations is unclear. unlike most pnpla isoforms that are ubiquitously expressed in many tissues, pnpla is localized predominantly in the upper layer of the epidermis. pnpla acts as a unique transacylase, catalyzing the transfer of linoleic acid (la; c : ) in triglyceride to the b-hydroxy residue of ultra-longchain fatty acid in ceramide to form b-o-acylceramide, a lipid component essential for skin barrier function. ), ) accordingly, genetic deletion or mutation of pnpla hampers epidermal b-o-acylceramide formation, thereby severely impairing skin barrier function and causing ichthyosis. the unique role of pnpla in the acylceramide-metabolic pathway in the epidermis is depicted in fig. . the pafah family. the paf-acetylhydrolase (pafah) family comprises one extracellular and three intracellular pla s that were originally found to have the capacity to deacetylate and thereby inactivate the lysophospholipid-derived lipid mediator paf. ), ) type-i pafah is a heterotrimer composed of two catalytic subunits, group xiiia and xiiib pla s, and a regulatory subunit lis- , the causative gene for a type of miller diecker syndrome. ) deficiency of type-i pafah leads to male infertility through an unknown mechanism. ) type-ii pafah (group viib pla ) preferentially hydrolyzes oxidized phospholipids (i.e., phospholipids having an oxygenated fatty acid at the sn- position) in cellular membranes, thereby protecting cells from oxidative damage. ) although plasma-type pafah (group viia pla ) is a secreted protein, it is described here as its structure is close to type-ii pafah. plasma-type pafah is now more generally called lipoprotein-associated pla (lp-pla ), existing as a low-density lipoprotein (ldl)-bound form in human plasma. ) a series of studies have revealed the correlation of lp-pla with atherosclerosis, likely because this enzyme liberates toxic oxidized fatty acids from modified ldl with pro-atherogenic potential. ), ) furthermore, deficiency of lp-pla decreases intestinal polyposis and colon tumorigenesis in apc min/d mice, ) suggesting an anti-tumorigenic role for paf in this setting. lysosomal pla . lysosomal pla (lpla ), also known as group xv pla , is homologous with lecithin cholesterol acyltransferase (lcat) and catalytically active under mildly acidic conditions. ) lpla hydrolyzes both sn- and sn- fatty acids in phospholipids and contributes to phospholipid degradation in lysosomes. genetic deletion of lpla results in unusual accumulation of non-degraded lung surfactant phospholipids in lysosomes of alveolar macrophages, leading to phospholipidosis, ) perturbed presentation of endogenous lysophospholipid antigens to cd d by invariant natural killer t (inkt) cells, ) and impairment of adaptive t cell immunity against mycobacterium. ) the plaat family. the pla-acyltransferase (plaat) family ( enzymes in humans and enzymes in mice) is structurally similar to lecithin retinol acyltransferase (lrat). members of this family, including group xvi pla (pla g ), display pla and pla activities, as well as acyltransferase activity that synthesizes n-acyl-pe, to various degrees. ) pla g is highly expressed in adipocytes, and pla g -deficient mice are resistant to diet-induced obesity. ) pla g and its paralogs in this family have also been implicated in tumor invasion and metastasis, ) vitamin a metabolism, ) peroxisome biogenesis, ) and cellular entry and clearance of picornaviruses. ) the abhd family. the ,/o hydrolase (abhd) family is a newly recognized group of lipolytic enzymes, comprising at least enzymes in humans. ) enzymes in this family typically possess both hydrolase and acyltransferase motifs. although the functions of many of the abhd isoforms still remain uncertain, some of them have been demonstrated to act on neutral lipids or phospholipids as lipid hydrolases. abhd selectively hydrolyzes phospholipids with medium-chain fatty acids. lipoquality control by phospholipase a enzymes no. ] abhd releases fatty acids from multiple classes of n-acyl-phospholipids to produce n-acyl-lysophospholipids. ) abhd acts as lysophospholipase or monoacylglycerol lipase, the latter being possibly related to the regulation of -arachidonoyl glycerol ( -ag) signaling. ), ) -ag is an endocannabinoid lipid mediator that plays a role in the retrograde neurotransmission and is considered to be produced mainly by diacylglycerol lipase ,. ) interestingly, in the brain, the aa released from -ag by monoacylglycerol lipase, rather than that released from phospholipids by cpla , (see above), is linked to the production of a pool of pge that promotes fever. ), ) abhd hydrolyzes lysophosphatidylserine (lysops), and is therefore referred to as lysops lipase. ) mutations in the human abhd gene result in accumulation of lysops in the brain and cause a disease called pharc, which is characterized by polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract. ) abhd a acts as a phosphatidylserine (ps)-selective pla (referred to as ps lipase), being located upstream of abhd in the ps-catabolic pathway. ) although abhd (also called cgi- ) does not have a catalytic activity because of the absence of a serine residue in the catalytic center, it greatly enhances pnpla directed hydrolysis of triglycerides in lipid droplets by acting as an essential lipolytic cofactor. ) . lipoquality control by secreted pla s general aspects. the secreted pla (spla ) family contains catalytically active isoforms and one inactive isoform in mammals. ), ) based on the structural and evolutional relationships, these enzymes are categorized into classical (ib, iia, iic, iid, iie, iif, v and x) and atypical (iii and xii) classes (fig. ) . the spla family strictly hydrolyzes the sn- position of phospholipids, a feature that differs from intracellular pla s that often display pla , lysophospholipase, lipase, or transacylase/acyltransferase activity (see above). individual spla s exhibit unique tissue and cellular distributions, suggesting their distinct biological roles. as spla s are secreted and require ca d in the mm range for their catalytic action, their principal targets are phospholipids in the extracellular space, such as microparticles, surfactant, lipoproteins, and foreign phospholipids in microbe membranes or dietary components. the biochemical properties and pathophysiological functions of spla s have been detailed in several recent reviews. ), ) here, i describe several key features of lipoquality regulation by the spla family. in terms of the lipoquality, spla s have long been considered to display no apparent selectivity for sn- fatty acid species in the substrate phospholipids. this view was based on the fact that spla -ib and -iia, two prototypic spla s that were initially identified through classical protein purification from the pancreas and sites of inflammation, respectively, ), ) as well as a number of snake venom pla s that belong to group i and ii spla s, are capable of releasing fatty acids non-selectively. however, recent lipidomics-based evaluation of the substrate specificity of spla s toward natural membranes (see above) has revealed that several spla s can distinguish sn- fatty acyl moieties in phospholipids under physiologically relevant conditions. in general terms, spla -ib, -iia and -iie do not discriminate fatty acid species, spla -v tends to prefer those with a lower degree of unsaturation such as oleic acid (oa; c : ), and spla -iid, -iif, -iii and -x tend to prefer pufas including aa and dha. several spla s can also distinguish differences in the polar head groups of phospholipids. for instance, spla -x is very active on pc, while spla -iia has much higher affinity for pe than for pc, and this substrate selectivity has been partly ascribed to their crystal structures. ), ) therefore, in order to comprehensively understand the specific biological roles of this enzyme family, it is important to consider when and where different spla s are expressed, which isoforms are involved in what types of pathophysiology, why they are needed, and how they exhibit their unique functions by driving specific types of lipid metabolism. classical spla s. spla -ib, also known as "pancreatic spla ", is synthesized as an inactive zymogen in the pancreas, and its n-terminal propeptide is cleaved by trypsin to yield an active enzyme in the duodenum. ) the main role of spla -ib is to digest dietary and biliary phospholipids in the intestinal lumen. perturbation of this process by gene disruption or pharmacological inhibition of spla -ib leads to resistance to diet-induced obesity, insulin resistance, and atherosclerosis due to decreased phospholipid digestion and absorption in the gastrointestinal tract. )- ) the human pla g b gene maps to an obesity-susceptible locus. ) spla -iia is often referred to as "inflammatory spla ", since its expression is induced by proinflammatory cytokines such as tnf, and il- o or by bacterial products such as lipopolysaccharide. ) in mice, however, spla -iia in mice is distributed only in intestinal paneth cells (in balb/c, c h, nzb and dba, etc.) or not expressed at all due to a natural frameshift mutation (in c bl/ , a/j, c / j, p/j, /sv and b .riii, etc.). ), ) the bestknown physiological function of spla -iia is the degradation of bacterial membranes, thereby providing the first line of antimicrobial defense in the host. ), ) consistent with this, spla -iia preferentially hydrolyzes pe and phosphatidylglycerol, which are enriched in bacterial membranes. under sterile conditions, spla -iia attacks phospholipids in microparticles, particularly those in extracellular mitochondria (an organelle that evolutionally originated from bacteria), which are released from activated platelets or leukocytes at inflamed sites. ) hydrolysis of microparticular phospholipids by spla -iia results in production of pro-inflammatory eicosanoids and lysophospholipids as well as in release of mitochondrial dna as a danger-associated molecular pattern (damp). thus, spla -iia is primarily involved in host defense by killing bacteria and triggering innate immunity, while over-amplification of the response leads to exacerbation of inflammation. spla -iia, -iic, -iid, -iie and -iif are often classified into the group ii subfamily (spla -iic is a pseudogene in human), since they share structural characteristics and map to the same chromosome locus. spla -iid is constitutively expressed in dendritic cells (dcs) in lymphoid organs. spla -iid is an "immunosuppressive spla " that attenuates dc-mediated adaptive immunity by hydrolyzing pe probably in microparticles to mobilize antiinflammatory b pufas and their metabolites such as resolvin d (rvd ). ) as such, spla -iid-null mice exhibit more severe contact hypersensitivity and psoriasis, whereas they are protected against infection and cancer because of enhanced anti-viral and anti-tumor immunity. ), ), ) unlike spla -iia, which is stimulus-inducible (see above), spla -iid is downregulated by pro-inflammatory stimuli, consistent with its anti-inflammatory role. in mice, spla -iie instead of spla -iia is upregulated in several tissues under inflammatory or other conditions. spla -iie is expressed in hair follicles in association with the growth phase of the hair cycle ) and induced in adipose tissue in association with obesity in mice. ) spla -iie hydrolyzes pe without apparent fatty acid selectivity in hair follicles and lipoproteins, and accordingly, spla -iie-deficient mice display subtle abnormalities in hair follicles ) and are modestly protected from diet-induced obesity and hyperlipidemia. ) spla -iif has a long c-terminal extension containing a free cysteine, which might contribute to formation of a homodimer, and is more hydrophobic than other spla s. ) physiologically, spla -iif is an "epidermal spla " that is expressed predominantly in the upper epidermis and induced by il- , a th cytokine, in psoriatic skin. ) spla -iif preferentially hydrolyzes pufa-containing plasmalogen-type pe in keratinocyte-secreted phospholipids to produce plasmalogen-type lysophosphatidylethanolamine (p-lpe; lysoplasmalogen), which in turn promotes epidermal hyperplasia (fig. a-c) . accordingly, spla -iif-null mice are protected against epidermal-hyperplasic diseases such as psor- iasis and skin cancer, while spla -iif-transgenic mice spontaneously develop psoriasis-like skin. ) although spla -v was previously thought to be a regulator of aa metabolism, ), ) it is now becoming obvious that this spla has a preference for phospholipids having fatty acids with a lower degree of unsaturation. spla -v is markedly induced in adipocytes during obesity as a "metabolic spla " and hydrolyzes pc in hyperlipidemic ldl to release oa and to a lesser extent la, which counteract adipose tissue inflammation and thereby ameliorates obesity-associated metabolic disorders. ) transgenic overexpression of spla -v, but not other spla s, results in neonatal death due to a respiratory defect, which is attributable to the ability of spla -v to potently hydrolyze pc with palmitic acid (pa, c : ), a major component of lung surfactant. ) this unique substrate preference of spla -v has also been supported by a recent lipidomics analysis of the spleen (a tissue where spla -v is abundantly expressed), in which the levels of fatty acids with a lower degree of unsaturation (e.g. pa, oa and la), rather than pufas (aa, epa and dha), are significantly reduced in spla -v-deficient mice relative to wild-type mice (fig. ) . this is in contrast to the spleen of spla -iid-deficient mice, in which b pufas and their metabolites are selectively diminished, ) revealing distinct lipoquality regulation . e+ . e+ . e+ . e+ . e+ , were significantly reduced in spla -v-deficient mice relative to control mice. accordingly, la metabolites, including -and -hydroxyoctadecadienoic acids (hodes) among others, were substantially decreased in mutant mice relative to control mice, whereas none of the aa, epa and dha metabolites differed significantly between the genotypes. these results are consistent with the view that spla -v has a propensity to preferentially hydrolyze phospholipids having sn- fatty acids with a lower degree of unsaturation, as illustrated at right bottom. by different spla s. another intriguing feature of spla -v is that it is the only "th -prone spla " induced in m macrophages by the th cytokines il- and il- and promotes th -driven pathology such as asthma. gene ablation of spla -v perturbs proper polarization and function of m macrophages in association with decreased th immunity, ) although the underlying lipid metabolism responsible for this event remains obscure. probably because of this alteration in the macrophage phenotype, spla -v-null macrophages have a reduced ability to phagocytose extracellular materials. accordingly, spla -v-null mice are more susceptible to fungal infection and arthritis due to defective clearance of hazardous fungi and immune complexes, respectively. ), ) likewise, spla -v-null mice suffer from more severe lung inflammation caused by bacterial or viral infection, ) which could also be explained by poor clearance of these microbes by alveolar macrophages. among the mammalian spla s, spla -x has the highest affinity for pc leading to release of fatty acids, with an apparent tendency for pufa preference. spla -x is activated by cleavage of the nterminal propeptide by furin-type convertases. ) spla -x is expressed abundantly in colorectal epithelial and goblet cells and has a protective role in colitis by mobilizing anti-inflammatory b pufas. ) consistently, spla -x-transgenic mice exhibit global anti-inflammatory phenotypes in association with elevation of systemic b pufa levels. ) in the process of reproduction, spla -x secreted from the acrosomes of activated spermatozoa hydrolyzes sperm membrane phospholipids to release dha and docosapentaenioc acid (dpa, c : ), the latter facilitating fertilization. ), ) additionally, spla -x-null mice are protected from asthma, accompanied by decreased levels of pulmonary b aa-derived eicosanoids. ) unlike the situation in spla -v-null mice (see above), however, the th response per se is not affected in the asthma model ) and the lung damage is milder following influenza infection ) in spla -x-null mice, illustrating the distinct actions of different spla s in the same tissue. atypical spla s. spla -iii is unusual in that it consists of three domains, in which the central spla domain similar to bee venom group iii spla is flanked by large and unique n-and cterminal domains. ) the enzyme is processed to the spla domain-only form that retains full enzymatic activity. ) although spla -iii does not discriminate the polar head groups, it tends to prefer sn- pufas in the substrate phospholipids. spla -iii is expressed in the epididymal epithelium and acts on immature sperm cells passing through the epididymal duct in a paracrine manner to allow sperm membrane phospholipid remodeling, a process that is prerequisite for sperm motility. ) spla -iii is also secreted from mast cells and acts on microenvironmental fibroblasts to produce pgd , which in turn promotes proper maturation of mast cells. ) accordingly, mice lacking spla -iii exhibit male hypofertility and reduced anaphylactic responses. spla -xiia is evolutionally far distant from other spla s. ) spla -xiia is expressed in many tissues at relatively high levels, yet its enzymatic activity is weaker than that of other spla s. the properties and physiological roles of spla -xiia are currently unclear and await future studies using spla -xiia-deficient mice. apart from lipoquality regulation, spla -xiib is a catalytically inactive protein due to substitution of the catalytic center histidine by leucine. ) spla -xiib deficiency impairs hepatic lipoprotein secretion, ) although the mechanism is unclear. beyond the lipoquality control by spla s, several spla s binds to spla receptor (pla r , also known as the c-type lectin clec c) with different affinities. ) in mice, pla r binds to spla -ib, -iia, -iie, -iif and -x with high affinity, spla -v with moderate affinity, and spla -iid, -iii and -xiia with low or no affinity. ) pla r is homologous to spla -inhibitory proteins present in snake plasma and exists as an integral membrane protein or as a soluble protein resulting from shedding or alternative splicing. pla r may act as a clearance receptor or endogenous inhibitor that inactivates spla s, as a signaling receptor that transduces spla -dependent signals in a catalytic activity-independent manner, or as a pleiotropic receptor that binds to non-spla ligands. in support of its clearance role, pla r !/! mice show more severe asthma, likely due to defective clearance of pro-asthmatic spla -x. ) in support of its signaling role, pla r , probably through binding to myocardial spla s or other ways, promotes the migration and growth of myofibroblasts and thereby protects against cardiac rupture in a model of myocardial infarction. ) pla r has recently attracted attention as a major autoantigen in membranous nephropathy, a severe autoimmune disease leading to podocyte injury and proteinuria, ) although it is not clear whether this role of pla r is spla dependent or -independent. by applying lipidomics approaches to knockout or transgenic mice for various pla s, it has become evident that individual enzymes regulate specific forms of lipid metabolism, perturbation of which can be eventually linked to distinct pathophysiological outcomes. knowledge of lipoquality control by individual pla s acquired from studies using animal models should be translated to humans. current knowledges on the relationship between pla gene mutations and human diseases are summarized in table . nonetheless, future development of more comprehensive and highly sensitive lipidomics techniques will contribute to the discovery of novel pla driven lipid pathways that could be biomarkers or druggable targets for particular diseases. nakanishi, h., ikeda, k., taguchi, r., kabashima deletion of cytosolic phospholipase a promotes striated muscle growth lipid signaling in cytosolic phospholipase a ,-cyclooxygenase- cascade mediates cerebellar longterm depression and motor learning acute lung injury by sepsis and acid aspiration: a key role for cytosolic phospholipase a cytosolic phospholipase a ,-deficient mice are resistant to collagen-induced arthritis an essential role of cytosolic phospholipase a , in prostaglandin e -mediated bone resorption associated with inflammation cytosolic phospholipase a ,-deficient mice are resistant to experimental autoimmune encephalomyelitis phospholipase a reduction ameliorates cognitive deficits in a mouse model of alzheimer's disease group x secreted phospholipase a releases b polyunsaturated fatty acids, suppresses colitis, and promotes sperm fertility cysteinyl leukotrienes regulate th celldependent pulmonary inflammation leukotriene b receptor blt mediates early effector t cell recruitment prostaglandin d as a mediator of allergic asthma dual roles of pge -ep prostaglandin e -ep signaling promotes immune inflammation through th cell differentiation and th cell expansion acceleration of intestinal polyposis through prostaglandin receptor ep in apc " knockout mice involvement of prostaglandin e in production of amyloid-o peptides both in vitro and in vivo the prostaglandin receptor ep suppresses colitis, mucosal damage and cd cell activation in the gut mapping the human platelet lipidome reveals cytosolic phospholipase a as a regulator of mitochondrial bioenergetics during activation the enteropathy of prostaglandin deficiency inherited human cpla , deficiency is associated with impaired eicosanoid biosynthesis, small intestinal ulceration, and platelet dysfunction interfacial kinetic and binding properties of mammalian group ivb phospholipase a (cpla o) and comparison with the other cpla isoforms a novel calcium-independent phospholipase a , cpla ., that is prenylated and contains homology to cpla cloning of a gene for a novel epithelium-specific cytosolic phospholipase a , cpla /, induced in psoriatic skin a calcium-dependent acyltransferase that produces n-acyl phosphatidylethanolamines function, activity, and membrane targeting of cytosolic phospholipase a in mouse lung fibroblasts mammalian patatin domain containing proteins: a family with diverse lipolytic activities involved in multiple biological functions recent progress in phospholipase a research: from cells to animals to humans a novel cytosolic calcium-independent phospholipase a contains eight ankyrin motifs multiple splice variants of the human calcium-independent phospholipase a and their effect on enzyme activity cyclooxygenase- mediated oxidation of -arachidonoyl-lysophospholipids identifies unknown lipid signaling pathways activation of mitochondrial calcium-independent phospholipase a . (ipla .) by divalent cations mediating arachidonate release and production of downstream eicosanoids identification of an insulin-regulated lysophospholipase with homology to neuropathy target esterase evidence that mouse brain neuropathy target esterase is a lysophospholipase genetic ablation of calcium-independent phospholipase a . prevents obesity and insulin resistance during high fat feeding by mitochondrial uncoupling and increased adipocyte fatty acid oxidation loss of function variants in human pnpla encoding calcium-independent phospholipase a . recapitulate the mitochondriopathy of the homologous null mouse loss-offunction mutations in pnpla encoding neuropathy target esterase underlie pubertal failure and neurological deficits in gordon holmes syndrome mutations in pnpla are linked to photoreceptor degeneration and various forms of childhood blindness impairment of park -dependent ca d signalling is a novel determinant of parkinson's disease male mice that do not express group via phospholipase a produce spermatozoa with impaired motility and have greatly reduced fertility fat mobilization in adipose tissue is promoted by adipose triglyceride lipase defective lipolysis and altered energy metabolism in mice lacking adipose triglyceride lipase adipose triglyceride lipase contributes to cancer-associated cachexia the g /g switch gene regulates adipose lipolysis through association with adipose triglyceride lipase atgl-mediated fat catabolism regulates cardiac mitochondrial function via ppar-, and pgc- lipolysis -a highly regulated multienzyme complex mediates the catabolism of cellular fat stores biochemistry and pathophysiology of intravascular and intracellular lipolysis genetic variation in pnpla confers susceptibility to nonalcoholic fatty liver disease chronic overexpression of pnpla i m in mouse liver causes hepatic steatosis a feedforward loop amplifies nutritional regulation of pnpla identification of a novel keratinocyte retinyl ester hydrolase as a transacylase and lipase neutral lipid stores and lipase pnpla contribute to autophagosome biogenesis has a crucial role in skin barrier function by directing acylceramide biosynthesis pnpla is a transacylase essential for the generation of the skin barrier lipid b-o-acylceramide intracellular pafacetylhydrolase type i intracellular plateletactivating factor acetylhydrolase, type ii: a unique cellular phospholipase a that hydrolyzes oxidatively modified phospholipids brain acetylhydrolase that inactivates plateletactivating factor is a g-protein-like trimer targeted disruption of intracellular type i platelet activating factor-acetylhydrolase catalytic subunits causes severe impairment in spermatogenesis protection against oxidative stress-induced hepatic injury by intracellular type ii platelet-activating factor acetylhydrolase by metabolism of oxidized phospholipids in vivo antiinflammatory properties of a platelet-activating factor acetylhydrolase inhibition of lipoprotein-associated phospholipase a reduces complex coronary atherosclerotic plaque development phospholipase a inhibitors in atherosclerosis: the race is on deficiency of phospholipase a group decreases intestinal polyposis and colon tumorigenesis in apc min/d mice lysosomal phospholipase a is selectively expressed in alveolar macrophages lysosomal phospholipase a and phospholipidosis role for lysosomal phospholipase a in inkt cell-mediated cd d recognition lysosomal phospholipase a : a novel player in host immunity to mycobacterium tuberculosis generation of n-acylphosphatidylethanolamine by members of the phospholipase a/acyltransferase adpla ablation increases lipolysis and prevents obesity induced by high-fat feeding or leptin deficiency pla g phospholipase mediates gain-of-function activities of mutant p lrat-specific domain facilitates vitamin a metabolism by domain swapping in hrasls interaction of phospholipase a/ acyltransferase- with pex p: a possible involvement in the down-regulation of peroxisomes represents a switch between entry and clearance of picornaviridae in vivo metabolite profiling as a means to identify uncharacterized lipase function: recent success stories within the alpha beta hydrolase domain (abhd) enzyme family metabolomics annotates abhd as a physiologic regulator of mediumchain phospholipids abhd regulates multiple classes of n-acyl phospholipids in the mammalian central nervous system the serine hydrolase abhd controls the accumulation and efficacy of -ag at cannabinoid receptors the serine hydrolase abhd is a critical regulator of the metabolic syndrome the endocannabinoid -arachidonoylglycerol produced by diacylglycerol lipase , mediates retrograde suppression of synaptic transmission fever is mediated by conversion of endocannabinoid -arachidonoylglycerol to prostaglandin e immunomodulatory lysophosphatidylserines are regulated by abhd a and abhd interplay abhd controls brain lysophosphatidylserine pathways that are deregulated in a murine model of the neurodegenerative disease pharc adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by cgi- and defective in chanarin-dorfman syndrome a new era of secreted phospholipase a the roles of the secreted phospholipase a gene family in immunology pancreatic phospholipase a : isolation of the human gene and cdnas from porcine pancreas and human lung cloning and recombinant expression of phospholipase a present in rheumatoid arthritic synovial fluid crystal structure of human group x secreted phospholipase a . electrostatically neutral interfacial surface targets zwitterionic membranes structures of free and inhibited human secretory phospholipase a from inflammatory exudate protection against diet-induced obesity and obesity-related insulin resistance in group b pla -deficient mice group b phospholipase a -mediated lysophospholipid absorption directly contributes to postprandial hyperglycemia the phospholipase a inhibitor methyl indoxam suppresses diet-induced obesity and glucose intolerance in mice group b phospholipase a inactivation suppresses atherosclerosis and metabolic diseases in ldl receptor-deficient mice linkage and potential association of obesity-related phenotypes with two genes on chromosome q in a female dizygous twin cohort phospholipase a -a mediator between proximal and distal effectors of inflammation a natural disruption of the secretory group ii phospholipase a gene in inbred mouse strains the secretory phospholipase a gene is a candidate for the mom locus, a major modifier of apc min -induced intestinal neoplasia mobilization of potent plasma bactericidal activity during systemic bacterial challenge. role of group iia phospholipase a staphylococcus aureus adenosine inhibits spla -iia-mediated host killing in the airways platelets release mitochondria serving as substrate for bactericidal group iia-secreted phospholipase a to promote inflammation dual roles of group iid phospholipase a in inflammation and cancer critical role of phospholipase a group iid in age-related susceptibility to severe acute respiratory syndrome-cov infection expression and function of group iie phospholipase a in mouse skin the adipocyte-inducible secreted phospholipases pla g and pla g e play distinct roles in obesity on the diversity of secreted phospholipases a . cloning, tissue distribution, and functional expression of two novel mouse group ii enzymes the functions of five distinct mammalian phospholipase a s in regulating arachidonic acid release. type iia and type v secretory phospholipase a s are functionally redundant and act in concert with cytosolic phospholipase a regulation of delayed prostaglandin production in activated p d macrophages by group iv cytosolic and group v secretory phospholipase a s transgenic expression of group v, but not group x, secreted phospholipase a in mice leads to neonatal lethality because of lung dysfunction group v secretory phospholipase a is involved in macrophage activation and is sufficient for macrophage effector functions in allergic pulmonary inflammation group v secretory phospholipase a modulates phagosome maturation and regulates the innate immune response against candida albicans a novel anti-inflammatory role for secretory phospholipase a in immune complexmediated arthritis group v phospholipase a in bone marrowderived myeloid cells and bronchial epithelial cells promotes bacterial clearance after escherichia coli pneumonia group x secreted phospholipase a proenzyme is matured by a furin-like proprotein convertase and releases arachidonic acid inside of human hek cells group x phospholipase a is released during sperm acrosome reaction and controls fertility outcome in mice importance of group x-secreted phospholipase a in allergen-induced airway inflammation and remodeling in a mouse asthma model key role of group v secreted phospholipase a in th cytokine and dendritic celldriven airway hyperresponsiveness and remodeling lack of group x secreted phospholipase a increases survival following pandemic h n influenza infection novel human secreted phospholipase a with homology to the group iii bee venom enzyme cellular arachidonate-releasing function of novel classes of secretory phospholipase a s (groups iii and xii) group iii secreted phospholipase a regulates epididymal sperm maturation and fertility in mice mast cell maturation is driven via a group cloning and recombinant expression of a structurally novel human secreted phospholipase a novel mammalian group xii secreted phospholipase a lacking enzymatic activity hepatocyte nuclear factor- , regulates liver triglyceride metabolism in part through secreted phospholipase a gxiib biochemistry and physiology of mammalian secreted phospholipases a deficiency of phospholipase a receptor exacerbates ovalbumin-induced lung inflammation circulating levels of secretory type ii phospholipase a predict coronary events in patients with coronary artery disease thrombospondin type- domain-containing a in idiopathic membranous nephropathy efficient phagocytosis requires triacylglycerol hydrolysis by adipose triglyceride lipase desnutrin/ atgl activates ppar/ to promote mitochondrial function for insulin secretion in islet o cells patatin-like phospholipase domain-containing protein is involved in hepatic fatty acid and triglyceride metabolism through x-box binding protein and modulation of endoplasmic reticulum stress in mice patatin-like phospholipase domain-containing / adiponutrin deficiency in mice is not associated with fatty liver disease brainspecific deletion of neuropathy target esterase/ swisscheese results in neurodegeneration group vib calcium-independent phospholipase a (ipla .) regulates platelet activation, hemostasis and thrombosis in mice mitochondrial dysfunction and reduced prostaglandin synthesis in skeletal muscle of group vib ca d -independent phospholipase a .-deficient mice mice deficient in group vib phospholipase a (ipla .) exhibit relative resistance to obesity and metabolic abnormalities induced by a western diet genetic ablation of calcium-independent phospholipase a . leads to alterations in hippocampal cardiolipin content and molecular species distribution, mitochondrial degeneration, autophagy, and cognitive dysfunction genetic ablation of calcium-independent phospholipase a . leads to alterations in mitochondrial lipid metabolism and function resulting in a deficient mitochondrial bioenergetic phenotype smooth muscle cell arachidonic acid release, migration, and proliferation are markedly attenuated in mice null for calcium-independent phospholipase a o age-related changes in bone morphology are accelerated in group via phospholipase a (ipla o)-null mice neuroaxonal dystrophy caused by group via phospholipase a deficiency in mice: a model of human neurodegenerative disease group via phospholipase a in both host and tumor cells is involved in ovarian cancer development platelet-activating factor and metastasis: calciumindependent phospholipase a o deficiency protects against breast cancer metastasis to the lung loss of pafah b reduces amyloid-o generation by promoting the degradation of amyloid precursor protein c-terminal fragments paf-ah catalytic subunits modulate the wnt pathway in developing gabaergic neurons increase of smooth muscle cell migration and of intimal hyperplasia in mice lacking the ,/o hydrolase domain containing gene age-related pulmonary emphysema in mice lacking ,/o hydrolase domain containing gene skin barrier development depends on cgi- protein expression during late-stage keratinocyte differentiation growth retardation, impaired triacylglycerol catabolism, hepatic steatosis, and lethal skin barrier defect in mice lacking comparative gene identification- (cgi- ) /o-hydrolase domain deletion induces adipose browning and prevents obesity and type diabetes platelet microparticles are internalized in neutrophils via the concerted activity of -lipoxygenase and secreted phospholipase a -iia secreted phospholipases a are intestinal stem cell niche factors with distinct roles in homeostasis, inflammation, and cancer secretory group v phospholipase a regulates acute lung injury and neutrophilic inflammation caused by lps in mice group v secretory phospholipase a promotes atherosclerosis: evidence from genetically altered mice group v secretory phospholipase a plays a pathogenic role in myocardial ischaemia-reperfusion injury group v secretory phospholipase a enhances the progression of angiotensin ii-induced abdominal aortic aneurysms but confers protection against angiotensin ii-induced cardiac fibrosis in apoe-deficient mice group x secretory phospholipase a regulates insulin secretion through a cyclooxygenase- -dependent mechanism group x secretory phospholipase a enhances tlr signaling in macrophages group x secreted phospholipase a limits the development of atherosclerosis in ldl receptor-null mice group x secretory phospholipase a augments angiotensin ii-induced inflammatory responses and abdominal aortic aneurysm formation in apoe-deficient mice group x secretory pla in neutrophils plays a pathogenic role in abdominal aortic aneurysms in mice mutations cause autosomal recessive congenital ichthyosis in golden retriever dogs and humans the gene encoding adipose triglyceride lipase (pnpla ) is mutated in neutral lipid storage disease with myopathy neuropathy target esterase gene mutations cause motor neuron disease genetic associations of nonsynonymous exonic variants with psychophysiological endophenotypes genome-wide association study identifies variants at p and q associated with development of cutaneous nevi lipoprotein-associated phospholipase a adds to risk prediction of incident coronary events by c-reactive protein in apparently healthy middle-aged men from the general population: results from the -year follow-up of a large cohort from southern germany tagging-snp haplotype analysis of the secretory pla iia gene pla g a shows strong association with serum levels of spla iia: results from the udacs study phospholipase a group iia expression in gastric adenocarcinoma is associated with prolonged survival and less frequent metastasis a novel polymorphism in secretory phospholipase a -iid is associated with body weight loss in chronic obstructive pulmonary disease phospholipase a group iii and group x have opposing associations with prognosis in colorectal cancer a gene involved in oxidative stress induced death, is associated with alzheimer's disease tagging snp haplotype analysis of the secretory pla -v gene, pla g , shows strong association with ldl and oxldl levels, suggesting functional distinction from spla -iia: results from the udacs study biallelic mutations in pla g , encoding group v phospholipase a , cause benign fleck retina key: cord- - ujiscdt authors: kitajima, ken; varki, nissi; sato, chihiro title: advanced technologies in sialic acid and sialoglycoconjugate analysis date: - - journal: sialoglyco chemistry and biology ii doi: . / _ _ sha: doc_id: cord_uid: ujiscdt although the structural diversity of sialic acid (sia) is rapidly expanding, understanding of its biological significance has lagged behind. advanced technologies to detect and probe diverse structures of sia are absolutely necessary not only to understand further biological significance but also to pursue medicinal and industrial applications. here we describe analytical methods for detection of sia that have recently been developed or improved, with a special focus on -o-acetylated n-acetylneuraminic acid (neu , ac), n-glycolylneuraminic acid (neu gc), deaminoneuraminic acid (kdn), o-sulfated sia (sias), and di-, oligo-, and polysialic acid (disia/oligosia/polysia) in glycoproteins and glycolipids. much more attention has been paid to these sia and sialoglycoconjugates during the last decade, in terms of regulation of the immune system, neural development and function, tumorigenesis, and aging. all cells are covered with glycoproteins and glycolipids, the biosynthesis of which takes place in the endoplasmic reticulum and golgi compartments, and involves a variety of enzymes. the expression of some of these enzymes has been shown to be important in embryogenesis, cancer, pathogen recognition, and inflammation. n-and o-glycans and glycosphingolipids are often terminated with sialic acid (sia), a family of nine carbon carboxylated monosaccharides, with high structural diversity. sia consists of n-acetylneuraminic acid (neu ac), n-glycolylneuraminic acid (neu gc), deaminoneuraminic acid (kdn), and their derivatives with modifications, such as acetylation, lactylation, methylation, and sulfation at the , , , and positions (fig. ) [ ] [ ] [ ] . these modifications of sia have been implicated in embryonic development, protection from microbes and viruses, and modulation of complement activation [ , ] . sia exists as either free or bound sugar linked to galactose, n-acetylgalactosamine, and other sugar residues in various positions. furthermore, sia links to sia itself to form dimeric, oligomeric, and polymeric structures (disia/oligosia/polysia). thus, sia shows extremely high structural diversity due to monosaccharide species as well as linkage modes. no other monosaccharide exhibits this structural diversity. although there are some reports showing the biological significance of neu gc and neu , ac [ , [ ] [ ] [ ] , for the majority of modified sia, biological roles have not been well elucidated. for this purpose, specific methods or probes to detect specifically modified sia are absolutely necessary. in this chapter we summarize current chemical and immunochemical methods to detect modified sia species, with a special focus on o-acetylated neu ac, neu gc, kdn, o-sulfated sia, as well as disia, oligosia, and polysia. because other chapters in this volume specifically focus on o-acetylated neu ac [ ] , neu gc [ ] , and polysia [ ] , we restrict ourselves in this chapter to a brief introduction of the different sia species. the o-acetylated sia (siaac) at the , , , and positions frequently occurs in glycoproteins and glycolipids. siaac is expressed in cell type-specific and developmental stage-specific manners, and is involved in various biological phenomena, such as immune reactions, virus-cell adhesion, malignancy, and others [ , ] . sialate:o-acetyl-transferases [ , ] and sialate:o-acetyl-esterases [ ] [ ] [ ] are important enzymes for acetylation and deacetylation of sia. the importance of -o-acetylated sia during early development was evidenced by the transgenic expression of influenza c hemagglutinin esterase in mice, leading to mice deficient in -o-acetylated sia [ ] . recent analyses on sialate:o-acetyl-esterase deficient mice showed impairment of b cell-related functions that could be related to the inhibitory activity of siglec- ( [ ] ; siglecs are discussed by schwardt et al. [ ] ). neu gc is broadly expressed in mammals. neu gc is synthesized by the cmp-neu ac hydroxylase (cmah), a converting enzyme of cmp-neu ac to cmp-neu gc [ , ] . due to a genetic defect in cmah, humans do not express a functional cmah and thus do not endogenously produce neu gc. however, neu gc can be metabolically incorporated from dietary sources (particularly red meat and milk) into glycoproteins and glycolipids. human fetuses, tumors, and some normal tissues were shown by mass spectrometry to incorporate neu gc. moreover, neu gc has been demonstrated to be metabolically incorporated and covalently expressed on cultured human cell surfaces. metabolically incorporated neu gc is immunogenic in humans [ ] , and the presence of human anti-neu gc antibodies has been associated with tumor progression [ ] and vascular inflammation [ ] . for additional details see shilova et al. [ ] and davies and varki [ ] . kdn contains a hydroxyl group at the c- position, instead of the acetamide-group in neu ac (fig. ) . kdn was discovered in glycoproteins from rainbow trout eggs [ , ] . in mammalian tissues, kdn occurs at a very low level and amounts to < % of total sia. however, expression of kdn increases in certain ovarian cancers, and the amount is proportional to the degree of malignancy [ ] . under hypoxic conditions free kdn increases in mammalian tumor cells. as hypoxia-resistance is a frequent property found in tumors, it is likely that kdn supports this state of mammalian cells [ ] . in rat liver, cytosolic kdn increases in an age-dependent manner. the level of free kdn was reported to be high in erythrocytes of the umbilical vein. these findings demonstrate that attention has to be paid to the physiological significance of kdn in malignancy and in aging [ ] . of interest in this context, kdn residues, in contrast to neu ac and neu gc, are resistant to known bacterial and mammalian sialidases [ , ] . in salmonid fish, kdn caps polysia chains in egg polysialoglycoproteins, thus stopping chain elongation and possibly protecting polysia from bacterial sialidase attacks [ , ] . biological roles of kdn in mammals remain to be elucidated, but increase of the kdn level in cells and tissues may be a biomarker for some cancers and in aging cells. sias are sometimes esterified with sulfate group to give o-sulfated sia (sias). methods for the detection of sias are not so well developed, and occurrence of sias has been only demonstrated in a limited number of animal species so far [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in sea urchin, -o-sulfated neu ac (neu ac s) and neu gc s are present in glycolipids [ ] [ ] [ ] , in glycoproteins from the vitelline layer polysialylated glycoprotein [ ] , and in the sperm flagellar glycoprotein, flagellasialin [ , ] . in mammals, neu ac s and neu gc s residues are found in gangliosides from bovine gastric mucosa [ , ] , and neu ac s was detected in tissues from human, rat, and mouse [ , ] . the glycoconjugates carrying sias in mammals have remained undetermined with the exception of the bovine gastric gangliosides [ , ] . similarly, the biological significance of the sias in animal tissues is not known. it is interesting, however, that chemically synthesized sias-containing compounds have been applied as artificial inhibitors for fertilization [ ] and bacterial infections [ ] , irrespective of the very limited knowledge on the natural occurrence of sias. these facts point to the potential importance of the sias residues in various biologic processes. in most cases, sias are present as α , or α , -linked monosialyl residues at the non-reducing terminal positions of glycan chains on glycoproteins and glycolipids. in two cases sia has been identified as an internal sugar between neutral sugars: fucα !o glycolyl neu gcα - neu acα - glc -in echinoderms [ ] and a polymer of ! neu acα ! glc/gal disaccharide in the capsular polysaccharides of neisseria meningitidis serogroups y and w- , respectively [ ] . moreover, sias can be linked to each other to form disia, oligosia, and polysia (by definition dp > is termed polysia). first identified in the glycocalyx of neuroinvasive bacteria, polysia has so far been demonstrated to be a posttranslational modification on six glycoproteins, the fish egg polysialoglycoprotein (psgp), the neural cell adhesion molecule (ncam), a voltage-gated sodium channel in eel, cd in human milk, nrp in human lymphocytes, and syncam- in mouse brain (for recent reviews see [ ] [ ] [ ] ). polysialylation occurs in some tumors (probably on ncam) and is involved in metastasis. by virtue of its net negative charge at physiological ph and exclusive volume, polysia serves as a mediator of ligand-receptor and cell-cell interactions via an anti-adhesive effect [ ] . in addition, polysia has been demonstrated to function as a reservoir molecule for bdnf, dopamine, and fgf- [ , , [ ] [ ] [ ] [ ] [ ] and to be involved in the regulation of ion transport via interactions with channels [ , [ ] [ ] [ ] . compared to what is known about polysia, the information on oligosia is limited [ , , ] . disia and oligosia are common glyco-epitopes between glycolipids and glycoproteins. the functions of disia on the glycolipids (gd and gt b) have been well studied, while far less knowledge about the functions of disia and oligosia on the glycoproteins has been reported [ ] [ ] [ ] . interestingly, oligosia and polysia with the degree of polymerization up to have been recently found in glycolipids of sea urchin sperm [ ] , although their functions remain to be elucidated. to quantitate the amount of sia at . - μg, colorimetric analyses are most common with simple methods, and include the thiobarbituric acid [ , ] and resorcinol methods [ ] . in the resorcinol method, neu ac and neu gc in either free or bound form can be equally detected, while kdn gives no color. in the thiobarbituric acid, neu ac and neu gc are detected only in free form, while kdn can be detected in both free and bound forms. to quantitate the amount of sia below . μg, a highly sensitive fluorometric high-performance liquid chromatography (hplc)-based method is usually used. here we focus on the detailed description of fluorometric hplc technology. fluorometric hplc analysis is currently the most sensitive and reliable method for quantitative detection of sia in the pmol range. in this analysis, a free form of various sia species is labeled with the fluorescent dye , -diamino- , -methylenedioxybenzene (dmb), and fluorometrically analyzed on hplc [ , ] . dmb is a reagent which specifically reacts with α-keto acids, and detects not only sia but also typical α-keto acids such as pyruvate and α-ketoglutarate. fluorometric hplc analysis consists of the following steps: step , acid hydrolysis of sialoglycoconjugates to release free sia; step , fluorescent labeling of released sia with dmb; step , separation and quantification of dmb-labeled sia on hplc. fluorometric hplc analysis involves two acidic conditions. one is a hydrolysis, . n trifluoroacetic acid at c for h to release free sia from sialoglycoconjugates at step . the second is the dmb labeling which takes place in . n trifluoroacetic acid at c for h (step ). attention must therefore be given to the acid lability of some sia substituents. no significant degradation of neu ac, neu gc, and kdn has been reported. for sias, the glycosidic bonds of sias were completely hydrolyzed under the hydrolysis conditions, while their sulfate esters are hydrolyzed by, at most, % [ ] . no apparent desulfation occurred under the labeling conditions. the o-acetyl groups on sia are very labile to basic conditions and more stable under acidic conditions. although no quantitative data are available for the lability of o-acetyl group during the dmb-derivatization procedures, quantitative analysis is basically possible as long as authentic o-acetylated sia compounds are used as a standard. it is of interest to note that the -o-acetyl ester is more labile than a -o-acetyl ester, and acetyl-groups tend to migrate to the c- position at the end, even under neutral conditions at room temperature [ ] . however, no such migration takes place with the -o-sulfate ester of sia. in step , dmb derivatives of neu ac, neu gc, and kdn are separately quantitated by hplc on an octadecylsilyl (ods) column using acetonitrile/methanol/water ( : : by volume) as the elution solution [ ] . the o-acetylated sia are eluted more slowly than the corresponding non-o-acetylated sia. to separate the various sias from each other, an acidic solution of acetonitrile/methanol/ . % trifluoroacetic acid ( : : by volume) [ ] is recommended. this solution will enable the separation of dmb derivatives of neu ac s, neu ac s, neu gc s, and neu gc s. for the separation of dmb derivatives of kdn s and kdn s, a solution with higher polarity, acetonitrile/methanol/ . % trifluoroacetic acid ( : : by volume), is appropriate [ ] . the described protocol enabled the separation of dmb derivatives of nine different sulfated and non-sulfated neu ac, neu gc, and kdn (fig. ). the relative peak area represents the proportion of the peak area of each dmb-sia to that of dmb-neu ac, and can be used as an index for relative detection efficiency. typical relative peak areas to that of dmb-neu ac are shown in table . when samples containing disia, oligosia, and polysia structures at - μg are analyzed, conventional methods including methylation analysis [ ] , nmr (nuclear magnetic resonance) [ ] , and mild acid hydrolysis-tlc (thin layer chromatography) [ ] can be applied. however, the amount of these types of glycoproteins is often too small to be analyzed by conventional methods. the fact that the di-, oligo-, and polysia-modification of glycoproteins has not often been reported suggests that these species in an organism is rare. as highly sensitive chemical methods to analyze minute amounts of di-, oligo-, and polysia were developed using highly sensitive fluorescent reagents [ , [ ] [ ] [ ] [ ] , the number of studies identifying di-, oligo-, and polysia increased gradually [ ] . therefore, in this chapter we are focusing on the chemical and immunochemical detection of small amounts (picogram to nanogram amounts) of sialic acid which may serve as powerful tools to reveal the importance of di-, oligo-, and polysia in nature. the polysia glycotope exhibits structural diversity in the sialic acid components (neu ac, neu gc, and kdn) in the inter-sialyl linkages (α ! o glycolyl , α ! , α ! , and α ! / ) and in the degree of polymerization (dp) [ , ] . with a highly specific reagent for α-keto acid [ ] , a specific labeling of di-, oligo-, and polysia became possible and products could be detected by anion exchange chromatography. this broadly used method was first developed by us [ ] and was further improved in later studies [ ] . with this method, di-, oligo-, and polysia on glycoproteins were frequently demonstrated [ , , ] . additionally, this method can be applied to determine the glycosidic linkage (α ! , α ! , and α ! ) of disia units (neu ac!neu ac, neu ac!neu gc, neu gc!neu ac, neu gc!neu gc, kdn!kdn, and so on) and their component sia species [ ] according to their elution time. if the sample is pure sialyloligo or -polymer, it can be detected on a uv detector or a pulse amperometric detector (pad) [ ] , instead of the dmb-derivatization/fluorometric detection method. to increase the sensitivity, especially in higher dp, because the labeling site is one per chain, it is recommended to separate the polysia on an anion exchange column and to label each fraction with dmb labeling after hydrolysis of the sample with . n tfa at c for - h [ ] . the sensitivity increases depending on their dp. the periodate treatment of sialic acid residing in non-reducing terminal ends is a well-known method to modify the terminal sialic acid [ ] . to observe and quantify the internal sialic acids of α ! -linked di-, oligo-, and polysia-containing glycoconjugates, sensitive chemical methods were developed with highly sensitive fluorescent labeling reagents (dmb) as described above [ ] . to perform the fluorometric c /c analysis, several reagents are prepared. the dmb-labeled sialic acids are applied to the hplc equipped with a tsk-gel ods- t column (  . mm i.d., tosoh), and a fluorescence detector (fp- , jasco). the column is equilibrated using acetonitrile/methanol/water ( : : by volume) at c. then - μl of the supernatants are applied to hplc analysis for an isocratically flow at . ml/min and the dmb-labeled sialic acid is detected with a fluorescence detector at an excitation of nm and an emission at nm (fig. ) . the estimate of the ratios of the quantity of internal sialic acid residues (c (sia)) to that of total sialic acid residues (c (sia) + c (sia)) can then be obtained. the following limitations should be noted with respect to this method. first, this method is applicable to only α ! -linked oligo/polymer of n-acylneuramininic acid, and cannot be used for dp analyses of α ! , α ! /α ! -mixed linkage polymers, or α ! o glycolyl -linkage. second, the c -derivatives formed do not always arise from α ! -linkages, because -o-substituted neu acyl residues may also give the same c -derivatives. therefore, mild alkali treatment of samples is usually carried out prior to periodate oxidation. third, the molar proportion of c -derivatives to c -derivatives does not directly represent the dp unless it is a linear polysia chain. thus, the method does not yield the dp for multiply sialylated chains present in the same sample. in general, glycoproteins have more than one glycan chain and even one glycan chain bears two to four non-reducing terminal residues that may be terminated by monosia or oligosia residues. immunochemical analyses of sialic acid and sialoglycoconjugates sialic acids kdn residues are resistant to various bacterial sialidases that are often used as reagents. however, a bacterial sialidase from sphingobacterium multivorum, named kdn'ase sm, has a unique property in that it cannot cleave the glycosidic linkages of neu ac and neu gc in any free glycans and glycoconjugates but can specifically cleave the kdn glycosidic linkages in free glycans, glycolipids and glycoproteins [ ] . similar kdn'ases are also known in hepatopancreas of oyster. these enzymes strictly discriminate kdn from neu ac or neu gc, and thus provide evidence for kdn-specific recognition phenomena in these organisms. two monoclonal antibodies recognizing kdn-containing epitopes have been developed. kdn kdn, a mouse igm, recognizes α , -linked oligo/polykdn with more than two residues [ ] . mab.kdn g, a mouse igg, recognizes the kdnα , galstructure [ ] . in immunohistochemistry, enzyme-linked immunosorbent assay (elisa), and western blotting using these antibodies, the results would be more reliable, if the loss of antigenicity by digestion with kdn'ase sm, a bacterial kdn-specific sialidase (see below), was confirmed. of the known lectins that recognize neu ac, sambucus sieboldiana lectin (ssa) can recognize the kdnα , gal-linkage better than neu acα , gal-linkage [ ] . in a combination of ssa and kdn'ase sm, kdnα , gal-structure can be detected. kdn can be detected in free glycans, glycolipids, and glycoproteins in various cells and tissues from animals that express neu ac. the expression level is very low in mammals. two monoclonal antibodies, mab. g and mab. c , specifically recognizing sias, have been reported. the mab. g is a mouse igm, and is highly specific for neu ac s. the antibody was generated using sea urchin sperm as an immunogen k. kitajima et al. [ ] . the mab. c is also a mouse igm and recognizes neu ac s and neu gc s, which can compensate for the binding specificity of mab. g . this antibody was prepared using sea urchin egg low density detergent-insoluble membrane as an immunogen [ ] . detection limits of mab. g ( μg/ml) and mab. c (culture supernatant) for neu ac sα ! neu acα ! glc-cer in elisa were . and . pg/well, respectively [ ] . as in the case with kdn'ase sm in immunodetection of kdn, an enzyme specifically destroying the sias epitopes would be a powerful tool for reliable detection. commercially available arylsulfatases, which can remove sulfate group from sulfatide containing -o-sulfated gal residue, have no activities on sulfate group on sia. there are no known sulfatases that can act on the sulfate ester on sias. therefore, immunochemical detection should be confirmed at least by chemical evidence to obtain reliable conclusions of the localization of sias. western blotting of sperm lysate of hemicentrotus pulcherrimus using mab. g detects unique smear at - kda, which corresponds to flagellasialin containing neu ac s-capped α , -neu ac-linked polymers [ , ] . acids by immunoblotting and elisa antibodies are powerful tools for studying the structure and function of glycotopes. however, it is very important that the immunospecificities toward linkage, dp, and component are determined in detail before use. for α ! -linked polysia glycotopes, several antibodies have been developed and used for the past decades. among the "so-called anti-polysia antibodies," the horse polyclonal antibodies h. [ ] and mouse mab [ ] had been the only two whose immunospecificities were specifically determined by inhibition assays [ , ] . the immunospecificity of the majority of other "anti-polysia antibodies" remained unknown until a comprehensive examination of the immunospecificity of these "anti-polysia" antibodies was carried our using an elisa-based method with phosphatidylethanolamine-conjugated oligo-and polysia chains as antigens [ ] . it was demonstrated that the "anti-polysia antibodies" discriminate in terms of the species of sia residues and chain length. our group has also developed a new anti-disia antibody using copolymers of α ! -linked n-acetylneuraminyl p-vinylbenzylamide and acrylamide as an immunogen [ ] . thus, a large number of antibodies recognizing di-, oligo-, and/or polysia structures with defined specificities now exist and are summarized in table . interestingly, the anti-oligo/polysia antibodies can be classified into three groups, based on the immunospecificity for chain length and the involvement of the non-reducing terminus in the antibody recognition site. group i consists of the "anti-polysia antibodies" that recognize chains of α ! -linked sia with a dp ! , including fully extended polysia chains with a dp~ - . these antibodies recognize the helical conformation formed by sia residues within the internal region of the polysia chains, but not the non-reducing terminal residues. group ii antibodies, designated as "anti-oligo + polysia antibodies," recognize both oligosia with dp - and polysia chains. these antibodies recognize the distal portion of oligo/polysia chains, including the non-reducing termini. group iii antibodies, designated as "anti-oligosia antibodies," recognize specific conformations of di-and oligosia with a dp - , but do not bind polysia. group iii antibodies can be further classified into two subgroups. one group is composed of "anti-disia antibodies" that recognize the dineu ac structure (s - , e ), and the other includes the "anti-oligosia antibodies" (ac ). group ii and iii antibodies would be useful for detecting and determining di-and oligosia structures, in combination with treatment with exo-and endo-sialidases, as described below ( table ). the antibodies can be applied to western blotting (fig. a) , immunocytochemistry (fig. b) , and facs analysis (fig. c) [ ] . table reactivity of di-, oligo-, and polysia chains toward biochemical probes biochemical probes disia oligosia polysia dp ¼ d p ¼ - dp ! group i antibody À À + group ii antibody À + + group iii antibody + + or À À endo-sialidase (endon) À À or + a + endosialidase b À + + α , -sialidase/α , -sialidase À À À α , -, α , , α , -sialidase + + + + reactive or sensitive, À unreactive or insensitive a + in case of dp ¼ , b refer to [ ] advanced technologies in sialic acid and sialoglycoconjugate analysis surprisingly, anti-disia antibodies obtained after immunization with gangliosides, developed, and used as anti-ganglioside antibodies can recognize cytosolic proteins such as carbonic anhydrase ii [ ] although they recognize disia epitope on glycoproteins. these phenomena might explain the cytosolic immunostaining with those anti-ganglioside antibodies in part. the protein mimicry should be considered and confirm the epitope with those antibodies. endo-sialidase (for a detailed description see jakobsson et al. [ ] ) can serve as a specific molecular probe to detect and selectively modify α ! -linked polysia chains. the soluble enzyme derived from bacteriophage k f, designated endon, catalyzes the depolymerization of polysia chains as follows: (! neu acylα !) n Àx (n ! ) ! (! neu acylα !) À + (! neu acylα !) Àx. two other types of endo-sialidases whose substrate specificities are different from the endon of bacteriophage k f [ ] have been isolated: endone [ ] and an endosialidase [ ] from a bacteriophage. the minimum chain length required for cleavage is dp ! and dp ! , respectively. exo-sialidases have been isolated that cleave specific linkages, for example, α , -sialidase (nanase i), α , -, α , -sialidase (nanase ii), α , -, α , -, α , -sialidase (nanase iii), and α , -, α , -, α , -, and α , sialidase. as the endon is insensitive toward di-and oligosia structures and they should be cleavable by these exosialidases, it is possible to confirm the length of given di-, oligo-, and polysia chains in conjunction with endo-and exo-sialidase treatments before and after immunostaining with anti-disia, oligosia, and polysia antibodies (see table ) [ , , ] . finne et al. established a specific probe for detection of polysia from endone that inactivates its enzymatic activity but not its ability to bind to polysia [ ] . they succeeded in the detection of polysia-ncam with this probe. when tissues are removed from the body during surgery, or during an autopsy, the onset of autolysis occurs promptly. this can thwart efforts to isolate nucleotides or certain enzymes and proteins, or to perform high quality histology. thus tissue ideally has to be flash-frozen for extracts, or frozen in cryoprotective agents, or fixed, using different procedure-dependent fixatives, for analysis using histopathology. immunohistochemistry is the process whereby tissue sections are probed with k. kitajima et al. antibodies to determine which cells contain epitopes of interest. the bound antibody is then detected using secondary and/or tertiary reagents, using fluorescent tags, or enzyme labels. if using enzyme labels, detection is facilitated using a number of different substrates, which produce precipitates, which are visible under the microscope. the nuclei are counterstained so that the morphology of the tissue can then be recognized during the analysis of the slides. in all immunohistochemistry experiments, to determine specificity, it is important to use specific blocking or inhibitory reagents. processing of tissues into paraffin destroys many antigenic epitopes and additional unmasking methods often have to be used to detect antigens that are left after the processing. however, some changes are irreversible, such as the extraction of glycolipids during the paraffin embedding process. immunohistochemistry remains an exact science, and appropriate use of controls and blocking agents are critical while analyzing results. lectins are carbohydrate-binding proteins, usually derived from plants, and have been used as tools to detect changes in glycan branching presentation on different cells. mice that have been genetically altered to be deficient for specific sialyltransferases are then useful as important controls to demonstrate specificity of some lectin binding patterns. this was demonstrated nicely in a report [ ] describing mice deficient in the sialyltransferases st gal-i (transfers α - -linked sia to galβ - glcnac units, mostly on n-glycans), or st gal-i (transfers α - linked sia to galβ - galnac units, mostly on o-glycans). sambucus nigra agglutinin (sna) staining was lost in essentially all tissues of adult st gal-i null mice, indicating that this is the only enzyme generating the siaα - galβ - glcnac sequence. lectin histochemistry with the α - -sia-specific maackia amurensis hemagglutinin (mah or mal-ii) showed loss of binding in almost all tissues of st gal-i null mice. however, use of the other isolectins of the maackia amurensis seeds, the mal-i lectin, or the maa lectin, showed continued binding to many tissues from the st gal-i null animals (fig. ). different isolectins have been isolated from maackia amurensis seeds [ , ] and have been used as probes for sialic acids that are α - -linked to the penultimate galactose residues. the two most commonly used isolectins are the leukoagglutinin (mal) and the hemagglutinin (mah). some commercial vendors use the term maa to denote probes for sialic acids that are α - -linked, but in fact maa is a mixture of mal and mah [ , ] . in both mal and mah the glycine and asparagines involved in sugar binding were substituted by lysine and aspartic acid, respectively, [ , ] and shown to be important in sialic acid binding. investigators, using biochemical methods, have described the carbohydrate binding specificity of these lectins, and some have also described the lectin histochemistry profile. however, many of the histochemistry studies use mal or maa, and only rarely has mah been used to ascribe the binding being used to identify α - -linked sialic acids. most recently, it was shown that effective glycoanalysis with maackia amurensis lectins requires a clear understanding of their binding specificities [ ] . in mammalian tissues, -o-acetylation is often associated with gut mucins, neural gangliosides, and erythrocytes. direct demonstration of the presence of -oacetylated sias in mammalian tissues was indirect (modifications of the pas technique) until the development of a chimeric dual-functional probe derived from influenza-c hemagglutinin esterase (infche). this probe was constructed using a soluble and versatile form of the infche that retains both the hemagglutinin and esterase activity and also has the binding properties of igg fc. the esterase activity is very similar to that of the native protein, and ph conditions can be adjusted to remove either -o-acetyl groups alone or both -and -o-acetyl groups completely (by causing migration of the latter to the -carbon position). dfp (di-isopropyl fluorophosphates) inactivation stabilizes and "unmasks" the hemagglutinin activity, resulting in a probe that specifically detects -o-acetylated sialic acids at ambient temperatures. since che-fcd and che-fc differ only by a single di-isopropyl group, each can be used as control for the other. thus, when the che-fc is used to remove -o-acetyl esters, the che-fcd can be used as an inactive control. conversely, when che-fcd is used to probe for -o-acetylated sias, the che-fc can be used as a control for nonspecific binding. the two forms can even be used sequentially; i.e., treatment with che-fc can be used to remove the -o-acetyl esters prior to probing with che-fcd, leaving only nonspecific staining, if any. it should be noted that the che-fc does have a masked hemagglutinin activity, which can give a weak positive response at low temperatures, and even at ambient temperatures if the density of o-acetylated sias is very high [ , [ ] [ ] [ ] [ ] [ ] [ ] . tissue expression of -o-acetylated sias was best analyzed using frozen sections, using the probe che-fcd pre-complexed to the secondary antibody. there were differences in expression of -o-acetylated sias in tissues isolated from rats and those from mice. as shown fig. , there was abundant expression in rat liver, rat lung, in the glomeruli of rat kidney, and gray matter of rat brain. however, although mouse red blood cells and blood vessels showed expression of -o-acetylated sias with the che-fcd probe, expression was not robust in mouse liver and mouse lung, but was instead present in the t-cell areas of lymphoid follicles in spleen, in the mature lymphoid medulla of the thymus, gray matter of the brain, and adrenal medulla. since the submaxillary gland mucins of many species have been reported to have high concentrations of -o-acetylated sias, it was surprising to find no significant staining in the parenchyma of the rat or mouse submaxillary gland. lipid overlays and protein blots confirmed that this is not due to inaccessibility of molecules on the tissue section. it is possible that the sialic acids of rat submaxillary mucins are -o-rather than -o-acetylated [ ] . the hemagglutinin esterases of influenza c viruses and certain nidoviruses, including group coronaviruses, recognize -o-acetylated sia-containing glycoconjugates on the surface of host cells, and some can remove the -o-acetyl moieties [ , , ] . recombinant soluble forms of the bovine coronavirus he with and without inactivation of the esterase active site have been reported, and these molecules may be more stable than the infche reagents. however, fig. differences in binding seen with the probe for -o-acetylated sialic acids, che-fcd, to mouse and to rat sections. as shown, -o-acetylated sialic acids are abundantly expressed in rat liver but not in mouse liver, although it is present on mouse red blood cells (right). it is also expressed on rat kidney glomeruli but not in the mouse kidney where it is present only on some k. kitajima et al. localization to tissue sections using immunohistochemistry has not yet been reported with the bovine coronaviruses reagents. humans are genetically defective in synthesizing the common mammalian sialic acid neu gc. it has been shown that neu gc can be metabolically incorporated and covalently expressed on cultured human cell surfaces. thus humans can also metabolically incorporate neu gc into glycoproteins and glycolipids of human tumors, fetuses, and some normal tissues, likely from dietary sources (particularly red meats). mass spectrometry confirmed the presence of small amounts of neu gc in these human tissues. as birds also do not synthesize neu gc [ ] , it is possible to raise good anti-neu gc antibodies in chickens, a polyclonal chicken anti-neu gc was purified using a novel affinity method, utilizing sequential columns of immobilized human and chimpanzee serum sialo-glycoproteins, followed by specific elution from the latter column by free neu gc, this purified anti-neu gc antibody was used to characterize the expression of neu gc in normal human and malignant tissues ( table ) . examination of frozen tissue sections showed expression in blood vessels, some epithelia, and in certain epithelial carcinomas [ , , ] . specificity of binding is confirmed by using a control igy, and by inhibition using neu gc rich chimpanzee serum. about % of breast, ovarian, and prostate carcinomas from humans showed expression of neu gc (fig. ) . other than expression within blood vessels within the malignant tissue, no expression was observed in melanomas and lymphomas using this three-step immunohistochemistry method. paraffin sections showed a marked loss of expression, as did pre-incubation of frozen sections with methanol, indicating that the neu gc epitopes are likely to be present mostly on glycolipids. ä fig. (continued) medullary vessels. there is a different distribution in spleen from rat and mouse, and is present in rat lungs but not in mouse lungs; and diffusely in rat brain (left) and only in gray matter of mouse brain (right). scale bar ¼ μm structural diversity of sia is not so large in each animal compared with that which we know in nature. in humans, neu ac is a dominant sia species, with neugc and kdn contributing to only small amounts. -o-acetylated neu ac is a major modified sia, but is still a minor form of total neu ac in human. in rainbow trout, on the other hand, neu ac, neu gc, and kdn appear to be equally expressed, although in a tissue-specific manner. therefore, which sia species each animal expresses for particular biological roles depends on the animal species. evolutional pressures for fig. examples of results from immunohistochemistry on human malignant tissues, using a purified polyclonal chicken anti-neu gc antibody. as shown, neu gc is expressed on blood vessels and on malignant cells (left side) and the binding is blocked when using neu gc rich chimpanzee serum. scale bar ¼ μm the selection of sia species may come from critical interactions for survival of life, such as pathogen-host interactions, and species-specific sperm-egg interactions in fertilization. thus, in some cases, a particular sia species is specifically functional; in other cases, no specific sia is necessary. the presence of kdn-specific sialidase in bacteria, the preferential recognition of neu gc by mouse siglec- , and an inhibitory effect of polysia, but not di-and oligosia, on homophilic binding of ncam may exemplify the significance of the structural diversity of sia species. importantly, modifications of sia are often changed dynamically, including the o-acetylation/ de-o-acetylation cycle and transient degradation of polysia to oligosia. however, currently there is very little knowledge available about the enzymes involved in these modification/re-modification reactions. future studies will be directed toward this identification of sia-modification enzymes and their genes. chemical diversity in the sialic acid and related α-keto acids: an evolutionary perspective chemistry, biochemistry and biology of sialic acid, glycoproteins ii sialic acids as ligands in recognition phenomena developmental abnormalities in transgenic mice expressing a sialic acid-specific -o-acetylesterase b cell antigen receptor signal strength and peripheral b cell development are regulated by a -o-acetyl sialic acid esterase o-acetylated n-acetylneuraminic acid as a novel target for therapy in human pre-b acute lymphoblastic leukemia functions and biosynthesis of o-acetylated sialic acids why is n-glycolylneuraminic acid rare in the vertebrate brain? polysialic acid in brain development and synaptic plasticity differentially regulated expression of -o-acetyl gd (cd b) and -o-acetyl-gd (cd c) during differentiation and maturation of human t and b lymphocytes properties and partial purification of sialate o-acetyltransferase from bovine submandibular glands biochemical and genetic evidence for distinct membranebound and cytosolic sialic acid o-acetyl-esterases: serine-active-site enzymes o-acetylation and de-o-acetylation of sialic acids in human colorectal carcinoma advanced technologies in sialic acid and sialoglycoconjugate analysis isolation and properties of two sialate-o-acetylesterases from horse liver with -and -o-acetyl specificities siglec antagonists a mutation in human cmp-sialic acid hydroxylase occurred after the homo-pan divergence the molecular basis for the absence of n-glycolylneuraminic acid in humans novel mechanism for the generation of human xenoautoantibodies against the nonhuman sialic acid n-glycolylneuraminic acid evidence for a human-specific mechanism for diet and antibody-mediated inflammation in carcinoma progression evidence for a novel human-specific xeno-auto-antibody response against vascular endothelium natural antibodies against sialoglycans its unique occurrence at the nonreducing ends of oligosialyl chains in polysialoglycoprotein of rainbow trout eggs kdn (deaminated neuraminic acid): dreamful past and exciting future of the newest member of the sialic acid family identification of -keto- -deoxy-d-glycero-d-galactonononic acid (kdn, deaminoneuraminic acid) residues in mammalian tissues and human lung carcinoma cells. chemical evidence of the occurrence of kdn glycoconjugates in mammals hypoxia-enhanced expression of free deaminoneuraminic acid in human cancer cells unusual gangliosides of eggs and embryos of the sea urchin strongylocentrotus intermedius. structure and density-dependence of surface localization isolation and structural studies of a sulfated sialosphingolipid from the sea urchin echinocardium cordatum gangliosides from the eggs of the sea urchin, anthocidaris crassispina isolation and identification of novel sulfated and nonsulfated oligosialyl glycosphingolipids from sea urchin sperm the occurrence of novel -o-sulfated n-glycolylneuraminic acid-capped α ! -o glycolyl -linked oligo/polyneu gc chains in sea urchin egg cell surface glycoprotein. identification of a new chain termination signal for polysialyltransferase a major flagellum sialoglycoprotein in sea urchin sperm contains a novel polysialic acid, an α acetylneuraminic acid chain, capped by an -o-sulfated sialic acid residue flagellasialin: a novel sulfated α , -linked polysialic acid glycoprotein of sea urchin sperm flagella characterization of the sulfated monosialosyltriglycosylceramide from bovine gastric mucosa structure of a novel sulfated sialoglycosphingolipid from bovine gastric mucosa specific distribution of sialic acids in animal tissues as examined by lc-esi-ms after derivatization with , -diamino- , -methylenedioxybenzene diversity of the human erythrocyte membrane sialic acids in relation with blood groups inhibition of in vitro fertilization of mouse gametes by sulfated sialic acid polymers antiviral activity of nmso against respiratory syncytial virus infection in vitro and in vivo determination of the absolute configuration of sialic acids in gangliosides from the sea cucumber cucumaria echinata structural determination of the polysaccharide antigens of neisseria meningitidis serogroups y, w- , and bo new functions of polysialic acid and its relationship to schizophrenia polysialic acid: versatile modification of ncam, syncam and neuropilin- polysialic acid polysialic acid in the plasticity of the developing and adult vertebrate nervous system complex formation of a brain-derived neurotrophic factor and glycosaminoglycans direct binding of polysialic acid to a brainderived neurotrophic factor depends on the degree of polymerization measurement of glycan-based interactions by frontal affinity chromatography and surface plasmon resonance structural and functional impairments of polysialic acid by a mutated polysialyltransferase found in schizophrenia novel regulation of fgf -mediated cell growth by polysialic acid neural cell adhesion molecule-associated polysialic acid potentiates α-amino- -hydroxy- -methylisoxazole- -propionic acid receptor currents neural cell adhesion molecule-associated polysialic acid inhibits nr b-containing n-methyl-d-aspartate receptors and prevents glutamate-induced cell death mechanism regulating ca + -dependent mechanosensory behaviour in sea urchin spermatozoa glycobiology of di-and oligosialyl glycotopes glycobiology of di-and oligosialyl glycotopes occurrence of oligosialic acids on integrin alpha- subunit and their involvement in cell adhesion to fibronectin neuronal differentiation-dependent expression of the disialic acid epitope on cd and its involvement in neurite formation in neuro a cells identification of disialic acidcontaining glycoproteins in mouse serum: a novel modification of immunoglobulin light chains, vitronectin, and plasminogen co-expression of two distinct polysialic acids, α , -and α , -linked polymers of n-acetylneuraminic acid, in distinct glycoproteins and glycolipids in sea urchin sperm methods for the quantitative estimation of n-acetylneuraminic acid and their application to hydrolysates of sialomucoids distribution of neuraminidase in arthrobacter and its purification by affinity chromatography quantitative estimation of sialic acids. ii. a colorimetric resorcinolhydrochloric acid method fluorometric highperformance liquid chromatography of n-acetyl-and n-glycolylneuraminic acids and its application to their microdetermination in human and animal sera, glycoproteins, and glycolipids determination of mono-o-acetylated n-acetylneuraminic acids in human and rat sera by fluorometric high-performance liquid chromatography development of sensitive chemical and immunochemical methods for detecting sulfated sialic acids and their application to glycoconjugates from sea urchin sperm and eggs migration of o-acetyl groups in n, o-acetylneuraminic acids occurrence of disialosyl groups in glycoproteins conformational differences between linear α , -linked homosialooligosaccharides and the epitope of the group b meningococcal polysaccharide structural diversity in the α - -linked polysialic acid chains in salmonid fish egg glycoproteins. occurrence of poly (neu ac), poly(neu gc), poly(neu ac, neu gc), poly(kdn), and their partially acetylated forms development of a highly sensitive chemical method for detecting α , -linked oligo/polysialic acid residues in glycoproteins blotted on the membrane fluorescent-assisted detection of oligosialyl units in glycoconjugates acid-catalyzed lactonization of α , -linked oligo/polysialic acids studied by high performance anion-exchange chromatography degree of polymerization (dp) of polysialic acid (polysia) on neural cell adhesion molecules (n-cams): development and application of a new strategy to accurately determine the dp of polysia chains on n-cams an ultrasensitive chemical method for polysialic acid analysis structure and biosynthesis of surface polymers containing polysialic acid in escherichia coli kdnase," which specifically hydrolyzes deaminoneuraminyl ( -deoxy-dglycero-d-galacto- -nonulosonic acid) but not n-acylneuraminyl linkages monoclonal antibody specific for α , -linked oligo deaminated neuraminic acid (kdn) sequences in glycoproteins. preparation and characterization of a monoclonal antibody and its application in immunohistochemistry monoclonal antibody specific to α , -linked deaminated neuraminyl beta-galactosyl sequence synthesis of neoglycoconjugates containing deaminated neuraminic acid (kdn) using rat liver α , -sialyltransferase isolation and characterization of low density detergent-insoluble membrane (ld-dim) fraction from sea urchin sperm epidemiology of escherichia coli k in healthy and diseased newborns bitter-suremann d ( ) nzb mouse system for production of monoclonal antibodies to weak bacterial antigens: isolation of an igg antibody to the polysaccharide capsules of escherichia coli k and group b meningococci determinant specificities of the groups b and c polysaccharides of neisseria meningitidis interaction of meningococcal group b monoclonal antibody and its fab fragment with α - -linked sialic acid polymers: requirement of a long oligosaccharide segment for binding characterization of the antigenic specificity of four different anti-(α , -linked polysialic acid) antibodies using lipid-conjugated oligo/polysialic acids frequent occurrence of pre-existing α ! -linked disialic and oligosialic acids with chain lengths up to sia residues in mammalian brain glycoproteins. prevalence revealed by highly sensitive chemical methods and anti-di-, oligo-, and poly-sia antibodies specific for defined chain lengths developmental stage-dependent expression of an α , -trisialic acid unit on glycoproteins in mouse brain identification of an inflammation-inducible serum protein recognized by anti-disialic acid antibodies as carbonic anhydrase ii endosialidases -versatile tools for the study ofpolysialic acid purification and properties of a bacteriophage-induced endo-n-acetylneuraminidase specific for poly-α- , -sialosyl carbohydrate units common cleavage pattern of polysialic acid by bacteriophage endosialidases of different properties and origins screening of bacteriophages producing endo-n-acetylneuraminidase mutant bacteriophage with non-catalytic endosialidase binds to both bacterial and eukaryotic polysialic acid and can be used as probe for its detection genetically altered mice with different sialyltransferase deficiencies show tissue-specific alterations in sialylation and sialic acid -o-acetylation sialic acid-binding motif of maackia amurensis lectins a sialic-acid binding lectin from the legume maackia fauriei: comparison with lectisn from m. amurensis effects of sialic acid substitutions on recognition by sambucus nigra agglutinin and maackia amurensis hemagglutinin sialic acid receptor detection in the human respiratory tract: evidence for widespread distribution of potential binding sites for human and avian influenza viruses an unusual carbohydrate binding site revealed by the structures of two maackia amurensis lectins complexed with sialic acid-containing oligosaccharides effective glycoanalysis with maackia amurensis lectins requires a clear understanding of their binding specificities the receptordestroying enzyme of influenza c virus is neuraminate o-acetylesterase the influenza c virus glycoprotein (he) exhibits receptor-binding (hemagglutinin) and receptor-destroying (esterase) activities use of influenza c virus for detection of -oacetylated sialic acids on immobilized glycoconjugates by esterase activity -o-acetylated sialic acids have widespread but selective expression: analysis using a chimeric dual-function probe derived from influenza c hemagglutinin-esterase sialic acid -o-acetylation on murine erythroleukemia cells affects complement activation, binding to i-type lectins, and tissue homing selective inactivation of influenza c esterase: a probe for detecting -o-acetylated sialic acids induction of protein and glycoprotein synthesis in rat submandibular glands by isoproterenol sialic acids as receptor determinants for coronaviruses attachment of mouse hepatitis virus to o-acetylated sialic acid is mediated by hemaglutinin-esterase and not by the spike protein sialic acids: chemistry, metabolism and function human uptake and incorporation of an immunogenic nonhuman dietary sialic acid sensitive and specific detection of the non-human sialic acid n-glycolyneuraminic acid in human tissues and biotherapeutic products advanced technologies in sialic acid and sialoglycoconjugate analysis key: cord- - tbc x authors: rustmeier, nils h.; strebl, michael; stehle, thilo title: the symmetry of viral sialic acid binding sites—implications for antiviral strategies date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: tbc x virus infections are initiated by the attachment of the viral particle to protein or carbohydrate receptors on the host cell. sialic acid-bearing glycan structures are prominently displayed at the cell surface, and, consequently, these structures can function as receptors for a large number of diverse viruses. structural biology research has helped to establish the molecular bases for many virus–sialic acid interactions. due to the icosahedral point group symmetry that underlies many viral capsids, the receptor binding sites are frequently arranged in a highly symmetric fashion and linked by five-fold, three-fold, or two-fold rotation axes. for the inhibition of viral attachment, one emerging strategy is based on developing multivalent sialic acid-based inhibitors that can simultaneously engage several of these binding sites, thus binding viral capsids with high avidity. in this review, we will evaluate the structures of non-enveloped virus capsid proteins bound to sialylated glycan receptors and discuss the potential of these structures for the development of potent antiviral attachment inhibitors. the cell membranes of eukaryotes are decorated with a large number of chemically and structurally diverse carbohydrates. these so-called glycans form a protective layer at the interface between a cell and its environment. components of this layer are synthesized and assembled by a large set of enzymes that differ among species, thus helping to provide host-specific glycan structures. despite some differences, the carbohydrate building blocks of the glycan layer (monosaccharides) are largely identical in all cases. commonly found monosaccharides in glycan structures are glucose (glc), galactose (gal), their n-acetylated forms n-acetylglucosamine (glcnac) and n-acetylgalactosamine (galnac), and mannose (man). these building blocks constitute the bulk of most glycans. however, two particularly important sugar classes are missing here: fucoses and sialic acids. while glc(-nac) and gal(-nac) are usually components of the scaffold, fucose (fuc) and sialic acids are often added as head groups to a glycan. while fucose is, for example, an important determinant in histo-blood group antigens (hbga), sialic acids are major components of the glycan portions of gangliosides and the glycan structures of many membranous proteins in eukaryotes. sialic acids are based on a nine-carbon acidic α-keto sugar framework ( figure ). due to their anionic nature, sialic acids contribute to the negative net charge of the cell surface. sialic acids are abundant in the animal kingdom and are found in different forms. their most common form is n-acetylneuraminic acid (neu ac), which also constitutes the chemical basis for other sialic acids. the hydroxylation of the n-acetyl group of neu ac gives rise to n-glycolylneuraminic acid (neu gc), another commonly found sialic acid in animals. while neu gc cannot be synthesized in humans due to a gene defect, humans glycans terminating in sialic acid are prominently expressed at the cell surface. they are, therefore, often easily accessible and serve as the initial contact points for many viruses in different families [ , ] . while some of these glycans function as attachment receptors to simply tether a virus to the target cell membrane, others act as entry receptors and mediate binding of the virus, as well as the delivery of the viral genome into the cytoplasm. in the latter case, the attachment step is often followed by the recruitment of secondary (co-)receptors and endocytosis factors, eventually leading to cell entry of the virus particle or its components and infection of the cell. among the enveloped viruses that recognize sialic acid-containing receptors are members of the families coronaviridae, paramyxoviridae and orthomyxoviridae [ ] [ ] [ ] [ ] [ ] . in non-enveloped viruses, sialic acid-containing glycans serve as attachment receptors for members of the parvoviridae, picornaviridae, caliciviridae, polyomaviridae, reoviridiae, and adenoviridae [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . structural biology has provided precise views of how these pathogens interact with sialylated glycans, and although the binding modes differ among the viruses listed above, several common principles have emerged. (i) the viral binding sites for sialylated glycans are typically surface-exposed and feature a small number of contacts. the affinities of the interactions are, therefore, quite low (in the millimolar range) [ ] [ ] [ ] [ ] . firm adhesion of the virus to the cell surface is achieved through the engagement of multiple receptors via identical binding sites, which is known as avidity. (ii) in all cases investigated to date, the sialic acid itself mediates the majority of contacts with the viral capsid, with a smaller number of additional contacts formed to neighboring monosaccharides. (iii) most viruses are highly specific in the context in which sialic acid is presented; that is, they only recognize sialylated glycans featuring, for example, α- , -linked sialic acid but do not engage sialylated glycans carrying α- , -linked or α- , -linked sialic acid. (iv) although the database remains small, some viruses can discriminate between the many different modifications of sialic acids, and, as some of these modifications, are species-specific, this phenomenon can contribute to the ability of a virus to only infect species that express a particular sialic acid modification. the available structural information on virus-receptor interactions is crucial to enable the rational design of therapeutic compounds. due to the surface-exposed binding mode and the weak individual interactions between sialic acids and their cognate virus proteins, modifying sialic acid to achieve high-affinity binding is challenging. however, viruses possess many identical binding sites that are often linked by symmetry operators, and thus multivalent and symmetric ligands that target several binding sites could result in high-affinity interactions. the strategy of employing a carbohydrate-based, multivalent, and symmetric inhibitor that matches the symmetry of the binding sites in a multimeric target protein was first applied in the context of the bacterial shiga-like toxin (slt). slt consists of an enzymatic domain a and a pentameric, cell-binding domain b [ ] . the crystal structures revealed that the b domain pentamer recognizes the p k trisaccharide portion (αgal - βgal - βglc) of its physiologic ganglioside receptor, globotriaosylceramide (gb ) [ , ] . in order to achieve high affinity binding, kitov et al. [ ] designed the starfish compound, a quasi-symmetric, pentavalent molecule with a central glucose motive carrying five linkers that terminate in dimeric p k trisaccharides ( figure ). x-ray crystallography of the toxin-inhibitor complex revealed a sandwich-like arrangement of two slt b-pentamers intercepted by one starfish molecule. all five b-pentamer binding sites were simultaneously occupied by the inhibitor. in line with this, affinity measurements showed an increase in the inhibition potency from a millimolar affinity for the monovalent receptor (p k trisaccharide) to a subnanomolar affinity for the starfish compound. this concept of targeting multiple, symmetric receptor binding sites by multivalent inhibitors is also applicable for many viruses, since viral capsids are often icosahedral and, therefore, highly symmetric structures. figure . an example of a tailored multivalent inhibitor. the globotriaosylceramide-binding b-subunit of shiga-like toxin (slt) forms pentamers and serves as target for the pentavalent inhibitory compound starfish, which has been functionalized with the p k trisaccharide. the starfish compound exploits the symmetric structure of its target and binds to slt with a subnanomolar affinity [ ] . the slt pentamer is shown as a protein surface with single protomers colored in grey, yellow, pink, green, and light blue, respectively. the starfish compound is shown in stick representation with carbon, nitrogen and oxygen atoms colored in orange, dark blue and red, respectively. missing parts of the scaffold structure are schematically indicated as orange lines (pdb id qnu). all protein representations in the figures of this review were generated using pymol (schrödinger inc.). in this chapter, we will introduce some universal concepts of virus capsid geometry and architecture, focusing in particular on non-enveloped viruses that bind sialic acid-based receptors. we will highlight the local symmetries that relate the sialic acid binding sites in different viral attachment proteins to each other. these local symmetries can serve as a useful framework for the rational design of multivalent virus-targeting inhibitors, similar to the approach used to develop the starfish compound. this strategy has been successfully applied to several viruses, as we will show in chapter . however, in order to evaluate this approach, we first need to introduce the symmetry elements that guide viral capsid assembly. typically, small viral genomes can only encode a low number of structural capsid proteins. these capsid proteins often form multimers (capsomers), which, again, assemble into a stable virus capsid that can house the viral genome and associated components (e.g., nucleoproteins or enzymes). thus, the assembled virus particles comprise many copies of capsid proteins and display a high (quasi-) symmetry that is often based on an icosahedron [ ] . in icosahedral capsids, the particle architecture can be expressed in terms of the triangulation number. an icosahedron is a polyhedron consisting of identical triangular faces that intersect at twelve vertices with five-fold rotational symmetry. three-fold rotational axes are located in the center of each triangular face. the edges between the faces are intersected by two-fold rotational axes. the combination of these symmetry operations gives rise to the point group of an icosahedron ( figure , lower right). in the context of an icosahedral virus particle, the triangulation number (t = h + hk + k ) can be interpreted as a measure of capsid size. it is calculated by the numbers of inter-capsomeric steps (iterated in h and k) that one has to traverse via (quasi-) six-fold symmetric capsomers, from one five-fold vertex to another. five-fold vertices and the associated capsid proteins are highlighted in blue. a schematic view of an icosahedron in the same orientation is shown on the lower right, with two-fold, three-fold and five-fold axes indicated as ellipse, triangle and pentagon, respectively. pdb ids b t (adenovirus), cse (orthoreovirus), kz (rotavirus), sid (polyomavirus), and q w (coxsackievirus). the smallest and simplest virus capsids have an architecture corresponding to a triangulation number of t= . in a t= capsid, each of the twelve five-fold symmetric vertices are occupied by a single capsid protein pentamer. neighboring capsomers are pentamers that are also associated with the five-fold icosahedral vertices. no additional capsomers are present, which gives rise to a total number of × = capsid proteins in the particle. to our knowledge, the only example of a t= sialic acid binding capsid is found in the adeno-associated virus (aav) of the parvoviridae family. sialic acid binding in aav was reported to occur at the interface between two single capsid protein monomer chains, resulting in a total number of binding sites in the capsid. the symmetry of the local binding site is defined by the shortest distance of a single binding site towards the rotational axis. the aav capsid proteins that are responsible for sialic acid binding form pentamers. however, the binding sites themselves locate closer to the icosahedral three-fold symmetry axis than the five-fold capsomer/vertex axis. this implies that the sialic acid binding sites of the aav capsid are arranged in twenty local three-fold rather than twelve local five-fold symmetries [ ] . virus particles comprising more than twelve capsomers must, by definition, have a higher triangulation number than t= . picornaviridae family members (such as coxsackieviruses, rhinoviruses, polioviruses, or enteroviruses) are viruses possessing three surface-exposed capsid proteins. the icosahedral five-fold penton positions are occupied by vp pentamers, which are bridged by one pseudo-hexon capsomer (vp -vp heterohexamer), giving rise to pseudo-t= geometry ( figure ). in the sialic acid-binding human coxsackievirus, a variant (cva v), the binding site is located at the interface of two vp protomers, close to the five-fold icosahedral axis. vp and vp do not bind sialic acid, which results in a total of binding sites, arranged in twelve local five-fold symmetries (figure a ) [ ] . the distance between an individual sialic acid binding site and the local five-fold rotation axis of the vp pentamer amounts to ca. . nm. in the members of both papillomaviridae and polyomaviridae, single capsid proteins named l and vp , respectively, constitute the outer capsid. both proteins exclusively form pentamers, which can occupy pentavalent and hexavalent positions in the mature particle, thus deviating from the quasi-equivalence principle proposed by caspar and klug in [ ] . here, a total number of pentameric capsomers are arranged in a t= d fashion, with a diameter of ca. nm in the mature particles ( figure ) [ ] [ ] [ ] . the formation of smaller, non-viable lower-symmetry t= virus-like particles (vlps) has also been described for both papillomaviruses and polyomaviruses [ ] [ ] [ ] . many members of the polyomavirus family bind sialic acid-based glycans using their vp proteins, so the binding sites on individual pentamers are always linked by local five-fold symmetry (figure a , tspyv). in all structures of polyomavirus-sialyl-oligosaccharide complexes, the majority of contacts between the protein and receptor can be attributed to sialic acid, with a small number of augmenting contacts to other saccharides providing specificity for a given glycan structure [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in contrast, papillomaviruses do not bind sialylated receptors, but instead interact with glycosaminoglycans to adhere to cells [ ] [ ] [ ] . reoviridae are a large family including, among others, the genera of orthoreoviruses and rotaviruses. members of the reoviridae family are double-shelled particles, in which the inner core layer consists of two core proteins arranged in a t= * order and the outer capsid possesses t= geometry [ ] [ ] [ ] . the diameter of mature virions is about - nm (figure ). in the case of mammalian reoviruses, the icosahedral vertex positions are occupied by the trimeric attachment protein sigma , which markedly protrudes as a thin fiber from the virion surface. the sigma protein is about nm long and has a head-and-tail morphology, with a globular head domain and an elongated tail that has flexible regions and partially inserts into the virion [ ] . the location of sialic acid binding is type dependent. type reoviruses bind sialylated receptors in the protruding head domain while type reoviruses use a binding site in a region near the mid-point of the tail (figure b , reov t d and t l) [ , ] . both sites primarily engage sialic acid with a small number of contacts, and in both cases, three-fold rotational symmetry is found between the binding sites within a single sigma trimer. thus, sigma trimers give rise to sialic acid binding sites in orthoreoviruses. rotaviruses also have protruding domains that recognize carbohydrate receptors, which are also dimers of the virus protein (vp ) [ ] . while some of the more pathogenic human viruses bind hbgas, animal rotaviruses primarily engage sialic acid-based receptors [ ] [ ] [ ] . each rotavirus vp subunit carries a single sialic acid binding site, which is related to the two-fold rotational symmetry in the dimer (figure a , rrv) [ ] . in further contrast to orthoreoviruses, these fibers do not coincide with the five-fold vertices but rather associate at the margins of the five capsomers around the five-fold vertices, resulting in a total number of vp dimers and sialic acid binding sites [ ] . adenoviridae members are large viruses with a diameter of about nm and a t= icosahedral capsid ( figure ) [ , ] . they display trimeric fibrous attachment proteins, known as the fiber, at their icosahedral vertices. similar to the reovirus sigma , the adenovirus fiber has a head-and-tail morphology and features a globular head domain (the knob) that projects from the virus surface, and a tail (the shaft) that inserts into the virus particle [ ] . the sialic acid binding sites of the different structurally characterized adenovirus types are located in the fiber knob domain [ , ] . while these sites differ in location in different adenoviruses, they are all linked by three-fold symmetry and lie in close proximity to each other (figure a, hadv and hadv ) . although this review focuses on non-enveloped viruses, it should still be mentioned that some of the concepts described above also apply to enveloped viruses, such as coronaviruses, paramyxoviruses, and orthomyxoviruses. while global symmetry measures for these viruses are elusive (since their attachment proteins are membrane-bound, somewhat mobile, and do not follow easily-appreciated assembly rules), the existing local binding site symmetries within multimeric attachment proteins can still be exploited for rational multivalent inhibitor design. a prominent example is the influenza a virus hemagglutinin, which is a homotrimeric protein bearing three individual sialic acid binding sites [ ] . these binding sites are related by local three-fold rotational symmetry, with a distance of . nm from the three-fold axis (figure a, iav) . the sialic acid moieties of glycosylated proteins and/or glycolipids are required for the attachment, entry, and productive infection of many viruses. in the majority of cases, the viral binding sites are surface-exposed and engage terminal sialic acid residues with a range of hydrophilic and hydrophobic contacts. the available structural data for receptor-ligand interactions at an atomic resolution can inform the synthesis of high affinity inhibitors via the chemical modifications of sialic acids. a prominent example of this approach is the inhibition of influenza virus neuraminidases. these enzymes recognize and hydrolyze terminal sialic acids of cell surface glycans and are vital for the release of viral progeny [ , ] . an early non-selective prototype inhibitor of neuraminidases was the sialic acid derivate -deoxy- , -didehydro-n-acetylneuraminic acid (neu ac en, dana) [ , ] . dana can engage and block the binding sites of neuraminidase of both ortho-and paramyxoviruses, thereby suppressing the activity of these enzymes. in the early s, mark von itzstein and colleagues made substantial advances towards selective and highly affine influenza a virus inhibitors. they discovered that the derivatization of the -o-hydroxyl group of dana with a guanidine moiety drastically increases affinity to the influenza a virus neuraminidase, which resulted in the anti-influenza drug zanamivir [ , ] . subsequently, the structures of the planar sialic acid transition state during the neuraminidase reaction became available. the analogous compounds of sialic acid's transition state based on benzoic and shikimic acids, and subsequently the drug oseltamivir [ ] [ ] [ ] [ ] , benefit from the increased affinity to neuraminidase compared to the native neu ac structure [ , ] . unfortunately, the family of compounds related to zanamivir or oseltamivir do not act against viruses that do not possess neuraminidase activity but instead engage undistorted sialic acids via a hemagglutinin (e.g., many non-enveloped viruses discussed in chapter of this review). in cases where affinities between sialylated glycans and hemagglutinins have been measured, the interactions were shown to have dissociation constants in the millimolar range [ ] [ ] [ ] [ ] . the firm cell attachment of these viruses is usually driven by high avidity, relying on a large number of identical binding sites that can engage receptors. in terms of the inhibition of attachment, monovalent sialic acid analogues are generally poor choices, as they are not able to engage a single binding site in a viral attachment protein with high affinity. therefore, polyvalent sialic acid-based compounds can sometimes be more effective. in parallel to the development of monovalent sialic acid transition state analogues against the influenza virus neuraminidases, polymeric or nanoparticle based sialic acid conjugates were developed to also prohibit influenza virus infection by extensive binding to the viral hemagglutinin, thereby shielding the virus particle. the influenza virus hemagglutinin can be blocked by multivalent sialosides that vary in chemical composition, size, branching complexity, and ligand density [ ] [ ] [ ] [ ] [ ] . particularly the effects of the nature of the scaffold (or platform) and spatial sialic acid distribution in polyvalent viral attachment inhibitors are still being investigated [ ] . recently, excellent reviews covering the composition and biophysical properties of multivalent sialosides were published by bhatia et al. [ ] and lu et al. [ ] , so we will not discuss this topic further here. next to the design of monovalent inhibitors or highly complex polyvalent macromolecular structures, a third and rather minimalistic design strategy makes use of the internal symmetry of the addressed targets. as elucidated earlier in this review, many viral attachment proteins employ a local symmetry of their receptor binding sites that represent a convenient framework for the rational design of small, tailored inhibitors. in contrast to monovalent inhibitors, oligovalent compounds can benefit from cooperative binding or avidity. nevertheless, they are small enough for nephric clearance and display a higher bioavailability than most macromolecular polyvalent structures [ ] . the design of oligovalent inhibitors is usually based on available crystal structures, taking the symmetry and topology of the target into account. the accessibility of the individual ligand binding sites and their distances from an eventual symmetry axis should be considered. for binding sites in close proximity to the local symmetry axis, it can be feasible to start from a central molecule, which can be derivatized by ligand terminating linkers, resulting in an inhibitor with a radial structure. for binding sites that are far from the local symmetry axis or in a recessed part of the target protein, it may be viable to directly link ligand molecules to each other instead of using a central scaffold. direct linking of ligand molecules may eventually result in the design of a circular oligovalent inhibitor. so far, structural information on attachment proteins bound by oligovalent inhibitors is scarce. x-ray crystallography analysis is usually hampered by the high flexibility of spacer groups and scaffolds. these non-binding parts of the inhibitors typically do not assume defined conformations, resulting in weak and uninterpretable electron density. this was the case in the study of the shiga-like toxin inhibitor starfish, described in the introduction of this review, where interpretable electron density was only present for the bound ligand moieties of the compound. still, the structural characterization of virus-inhibitor interactions in order to design or optimize anti-viral compounds has been the subject of extensive research. recently, a research paper by lu et al. [ ] reported on the trivalent design of sialic acid bearing inhibitors against influenza a virus hemagglutinin, showing a greater than -fold increase in affinity compared to the monovalent ligand. however, structural data of the interactions between the inhibitor and hemagglutinin were not reported in that study. one study of a central group-based anti-viral inhibitor, which contains structural data, can be found for adenoviruses. adenoviridae members carry trimeric fibers terminating in the knob domain, which engages sialic acid-based receptors in some adenoviruses. human adenovirus (hadv ), which causes epidemic keratoconjunctivitis (ekc), carries three individual binding sites for sialic acids in its fiber knob, which are located at a distance of Å from the local three-fold symmetric axis (figure a ) [ ] . the physiologic receptor of hadv is the glycan portion of ganglioside gd a. this glycan is a branched hexasaccharide with two terminal sialic acid moieties, both of which were shown to simultaneously occupy two of the three available sialic acid binding sites in the same fiber knob [ ] . based on this observation, spjut et al. ( ) [ ] designed and synthesized a symmetric, tridentate sialylated inhibitor, which is capable of occupying all three binding sites of the fiber knob at once. the first generation of these inhibitors was designed around a central tris( -aminoethyl) amine group. it utilized flexible spacers between the central group and the sialic acid ligand, in order to minimize the chance of steric hindrance of the inhibitor docking. the resulting compounds demonstrated a potency increase of four orders of magnitude compared to monomeric sialic acid [ ] . recently, in the second-generation inhibitors, the binding affinity could be improved even further by using a shorter, triazole-based linker structure, which was based on the results of the first-generation complex structures. the more compact and rigid design resulted in an additional -fold increase in potency [ ] . the crystal structures of the second-generation inhibitor molecules in a complex with adenovirus fiber knob proteins verify the trivalent binding mode by also displaying the electron density of the linkers (figure a,b, left) . an example of directly-linked ligand moieties was shown in a study of potential polyomavirus inhibitors, in which baier et al. [ ] synthesized so-called divalent sialylated glycooligopeptides. they solved the structures of two glycooligopeptide compounds in a complex with the major capsid protein (vp ) pentamer of the trichodysplasia spinulosa-associated polyomavirus (tspyv), which is associated with abnormal skin growth in immunocompromised patients. the vp pentamer carries five individual sialic acid binding sites at a distance of Å between neighboring sites [ , ] . the glycooligopeptide-vp complex structures displayed a similar ligand binding mode that was reported for sialic acid in an earlier study [ ] and showed, for the compounds, that the linker between the ligand and the scaffold occupies the space that is usually targeted by the natural glycan receptor moieties (figure a,b, right) . however, the interconnectivity of functional receptors by the scaffold remained undetermined (probably due to their flexibility) and are, therefore, the average of several potential bridging modes on top of the pentamers [ ] . many viral lectins or attachment proteins rely on the recognition of sialic acids. due to the high symmetry of viral particles and the occurrence of local symmetry within commonly multimeric viral proteins, sialic acid binding often occurs in a symmetrical context, too. this symmetry is a convenient framework for the design of tailor-made inhibitory ligands competing with the high avidity of virus-cell interactions. structural biology techniques, such as x-ray crystallography and single-particle electron cryo-microscopy (cryo-em), can now tackle the visualization of viral attachment and carbohydrate interactions with unprecedented scope and detail. the resulting structural data can be used for the optimization of anti-viral compounds, which could be developed further into high-affinity drug candidates. however, challenges in compound design remain. for example, the higher rigidity of a multivalent ligand does not necessarily translate into improved binding. in the case of had , a rigid compound bound -fold less well to the fiber knob than a related compound that had higher flexibility [ ] . this suggests that the perfect positioning of all sialic acids in the binding site, especially for larger inhibitor molecules, is difficult to achieve, and a certain degree of flexibility might help with the high-affinity binding of the inhibitor. another limiting factor for oligovalent inhibitors is the positioning of the binding pockets. in the case of the trivalent adenovirus inhibitor, the binding sites are on the very top of the knob domain, so there is enough space for linkers and a central core (figure a ). in contrast, the sialic acid binding sites of the reovirus sigma fibers are located on the side of the protein (figure b) , which makes it challenging to design an appropriate oligovalent inhibitor. additional challenges are the long-term stability, convenient synthesis, and, for later application, reasonable bioavailability of multivalent compounds. n-glycolylneuraminic acid deficiency in humans chemical diversity in the sialic acids and related α-keto acids: an evolutionary perspective diversity in cell surface sialic acid presentations: implications for biology and disease essentials of glycobiology sialic acid tissue distribution and influenza virus tropism structural features of glycan recognition among viral pathogens glycan engagement by viruses: receptor switches and specificity human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza c viruses sialic acids as receptor determinants for coronaviruses role of sialic acid-containing molecules in paramyxovirus entry into the host cell: a minireview structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid sialic acid species as a determinant of the host range of influenza a viruses binding of adeno-associated virus type to , -linked sialic acid is required for gene transfer adeno-associated virus serotype (aav ) and aav both require sialic acid binding for hemagglutination and efficient transduction but differ in sialic acid linkage specificity sialic acid functions in enterovirus binding and infection coxsackievirus a variant uses sialic acid-containing o-linked glycoconjugates as cellular receptors on human ocular cells sialic acid-dependent cell entry of human enterovirus d tulane virus recognizes sialic acids as cellular receptors infection of glial cells by the human polyomavirus jc is mediated by an n-linked glycoprotein containing terminal α( - )-linked sialic acids gangliosides are receptors for murine polyoma virus and sv identification of gangliosides gd b and gt b as receptors for bk virus ganglioside gt b is a putative host cell receptor for the merkel cell polyomavirus the α-anomeric form of sialic acid is the minimal receptor determinant recognized by reovirus initial interaction of rotavirus strains with n-acetylneuraminic (sialic) acid residues on the cell surface correlates with vp genotype, not species of origin adenovirus type uses sialic acid as a cellular receptor human adenovirus uses sialic acid-containing glycoproteins and the coxsackie and adenovirus receptor for binding to target cells structural basis of gm ganglioside recognition by simian virus structural and functional analysis of murine polyomavirus capsid proteins establish the determinants of ligand recognition and pathogenicity triazole linker-based trivalent sialic acid inhibitors of adenovirus type infection of human corneal epithelial cells hemagglutinins from two influenza virus variants bind to sialic acid derivatives with millimolar dissociation constants: a -mhz proton nuclear magnetic resonance study globotriosyl ceramide is specifically recognized by the escherichia coli verocytotoxin structure of the shiga-like toxin i b-pentamer complexed with an analogue of its receptor gb shiga-like toxins are neutralized by tailored multivalent carbohydrate ligands physical principles in the construction of regular viruses characterization of the adeno-associated virus and sialic acid binding site a sialic acid binding site in a human picornavirus atomic model of the papillomavirus capsid polyoma virus capsid structure at . . a resolution structure of simian virus at . -Å resolution structure of small virus-like particles assembled from the l protein of human papillomavirus polymorphism in the assembly of polyomavirus capsid protein vp structure and assembly of a t = virus-like particle in bk polyomavirus high-resolution structure of a polyomavirus vp -oligosaccharide complex: implications for assembly and receptor binding structure-function analysis of the human jc polyomavirus establishes the lstc pentasaccharide as a functional receptor motif structures of merkel cell polyomavirus vp complexes define a sialic acid binding site required for infection a structure-guided mutation in the major capsid protein retargets bk polyomavirus structures of b-lymphotropic polyomavirus vp in complex with oligosaccharide ligands crystallographic and glycan microarray analysis of human polyomavirus vp identifies n-glycolyl neuraminic acid as a receptor candidate trichodysplasia spinulosa-associated polyomavirus uses a displaced binding site on vp to engage sialylated glycolipids the l major capsid protein of human papillomavirus type recombinant virus-like particles interacts with heparin and cell-surface glycosaminoglycans on human keratinocytes human papillomavirus infection requires cell surface heparan sulfate human papillomavirus types , , and share similar endocytic requirements for entry the three-dimensional structure of reovirus obtained by cryo-electron microscopy structure of the reovirus core at . ? Å resolution x-ray crystal structure of the rotavirus inner capsid particle at . a resolution sigma protein of mammalian reoviruses extends from the surfaces of viral particles the gm glycan serves as a functional coreceptor for serotype reovirus crystal structure of reovirus attachment protein sigma in complex with sialylated oligosaccharides three-dimensional visualization of the rotavirus hemagglutinin structure comparison of human, simian, and bovine rotaviruses for requirement of sialic acid in hemagglutination and cell adsorption role of sialic acids in rotavirus infection sialic acid dependence in rotavirus host cell invasion the rhesus rotavirus vp sialic acid binding domain has a galectin fold with a novel carbohydrate binding site atomic model of an infectious rotavirus particle the structure of the adenovirus capsid image reconstruction reveals the complex molecular organization of adenovirus crystal structure of species d adenovirus fiber knobs and their sialic acid binding sites structural basis of preexisting immunity to the h n pandemic influenza virus functional significance of sialidase during influenza virus multiplication functional significance of sialidase during influenza virus multiplication: an electron microscope study inhibition of neuraminidase activity by derivatives of -deoxy- , -dehydro-n-acetylneuraminic acid inhibition of influenza and parainfluenza virus replication in tissue culture by -deoxy- , -dehydro-n-trifluoroacetylneuraminic acid (fana) rational design of potent sialidase-based inhibitors of influenza virus replication -guanidino- , -dideoxy- , -dehydro-n-acetylneuraminic acid is a highly effective inhibitor both of the sialidase (neuraminidase) and of growth of a wide range of influenza a and b viruses in vitro structure-based inhibitors of influenza virus sialidase. a benzoic acid lead with novel interaction synthesis and influenza neuraminidase inhibitory activity of aromatic analogs of sialic-acid influenza neuraminidase inhibitors possessing a novel hydrophobic interaction in the enzyme active site: design, synthesis, and structural analysis of carbocyclic sialic acid analogues with potent anti-influenza activity synthesis of a carbocyclic sialic acid analogue for the inhibition of influenza virus neuraminidase evidence for a sialosyl cation transition-state complex in the reaction of sialidase from influenza virus a study of the active site of influenza virus sialidase: an approach to the rational design of novel anti-influenza drugs polyacrylamides bearing pendant α-sialoside groups strongly inhibit agglutination of erythrocytes by influenza-virus effective inhibitors of hemagglutination by influenza virus synthesized from polymers having active ester groups. insight into mechanism of inhibition generation and in situ evaluation of libraries of poly(acrylic acid) presenting sialosides as side chains as polyvalent inhibitors of influenza-mediated hemagglutination inhibition of viral adhesion and infection by sialic-acid-conjugated dendritic polymers polymeric inhibitor of influenza virus attachment protects mice from experimental influenza infection linear polysialoside outperforms dendritic analogs for inhibition of influenza virus infection in vitro and in vivo pathogen inhibition by multivalent ligand architectures carbohydrate-protein interactions and multivalency: implications for the inhibition of influenza a virus infections properties of the glomerular barrier and mechanisms of proteinuria enhanced inhibition of influenza a virus adhesion by di-and trivalent hemagglutinin inhibitors the gd a glycan is a cellular receptor for adenoviruses causing epidemic keratoconjunctivitis a potent trivalent sialic acid inhibitor of adenovirus type infection of human corneal cells divalent sialylated precision glycooligomers binding to polyomaviruses and the effect of different linkers this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank the swiss institute of bioinformatics for the provision of the expasy viralzone and the scripps institute for the provision of the viper data bank-two very helpful resources of virus knowledge. we apologize to our many colleagues whose work could not be discussed and cited here due to space considerations. the authors declare no conflict of interest. key: cord- - i q c u authors: djakpo, odilon; yao, weirong title: rhus chinensis and galla chinensis – folklore to modern evidence: review date: - - journal: phytother res doi: . /ptr. sha: doc_id: cord_uid: i q c u the species rhus chinensis mill. (anacardiaceae) is an important representative of the genus rhus, which contains over individual species found in temperate and tropical regions worldwide. rhus chinensis has long been used by folk medicine practitioners in asia. leaves, roots, stem, bark, fruit and particularly the galls on rhus chinensis leaves, galla chinensis, are recognized to have preventative and therapeutic effects on different ailments (such as diarrhea, dysentery, rectal and intestinal cancer, diabetes mellitus, sepsis, oral diseases and inflammation). however, it is critical to separate evidence from anecdote. fortunately, recent scientific research has revealed that rhus chinensis compounds possess strong antiviral, antibacterial, anticancer, hepatoprotective, antidiarrheal and antioxidant activities. moreover, compounds isolated from the stem of rhus chinensis significantly suppressed hiv‐ activity in vitro. compounds from this plant were also found to inhibit enamel demineralization in vitro and enhance remineralization of dental enamel with fluoride. this review highlights claims from traditional and tribal medicinal lore and makes a contemporary summary of phytochemical, biological and pharmacological findings on this plant material. it aims to show that the pharmaceutical potential of this plant deserves closer attention. copyright © john wiley & sons, ltd. rhus chinensis belongs to the genus rhus and the family anacardiaceae (miller et al., ) . commonly called sumac, rhus consists of approximately individual species of fl owering plants, with six species found (four endemics) in china. like most sumacs, rhus chinensis is a dioecious shrub that can reach m in height. it bears odd pinnately compound leaves and creamywhite fl owers. the fruits (drupes) are orange or red in color at maturity and contain one seed (barkley, ; miller et al., ; tianlu and barfod, ) . the species grows in areas with marginal agricultural capacity, and is widely distributed in temperate, subtropical, and tropical regions, including china, japan, malaysia, taiwan and india (rayne and mazza, ; ren et al., ) . the species rhus chinensis has two distinct varieties, rhus chinensis var. chinensis (syn. rhus (tianlu and barfod, ; grin; tropicos) . galla chinensis or galla rhois is the term used to describe the gall caused by the chinese aphid, schlech-tendalia chinensis (bell), on the leaves of rhus chinensis (lee et al., ) . this gall is widely used as a separate drug. other species in this genus also produce galls that are considered to have an inferior quality. a plethora of traditional medicine references claim curative power for rhus chinensis, despite its widespread use, many of these claims of effi cacy were not supported by scientifi c evidence, whether for traditional use validation or for drug development endeavors. fortunately, recent scientifi c research on rhus chinensis has revealed promising health benefi ts, including anticancer, antiviral, antimicrobial, antidiarrheal and antiinfl ammatory properties (yang et al., ; gu et al., ; ahn et al., ; chen et al., ; kim et al., ) . in recent years, the chinese herbal medicine galla chinensis has been discussed widely as a new alternative for carious disease (chu et al., ) . so far, no comprehensive review has been compiled from the literature encompassing the effi cacy of this plant. widespread claims of the medicinal effectiveness of various rhus chinensis tree preparations motivated us to bridge the information gap in this area. among rhus species, rhus chinensis and its gall, galla chinensis, have a long history of use by indigenous peoples for medicinal care and others. numerous curative properties are ascribed to different parts of this tree, namely root, bark, stem, leaf, fruit, fl owers, seed and gall ( table ). the leaves and the root are used as depuratives, stimulating blood circulation. its decoction is used in the treatment of hemoptysis, infl ammations, laryngitis, snakebite, stomachache and traumatic fractures (duke and ayensu, ; kao, ; ouyang et al., ) . the ripe fruits of this plant have long been used in asia to treat dysentery and diarrhea, as well as other gastrointestinal disorders (kala, ; pradhan and badola, ; bose et al., ) . the fruit produces a sour juice when boiled with water. this juice, when diluted with water or/and mixed with raw eggs, treats diarrhea and dysentery (pradhan and badola, ) . it is used for the treatment of colic (chopra et al., ) and also as a food preservative (pradhan and badola, ) . the seed is used in the treatment of cough, dysentery, fever, jaundice, malaria and rheumatism (duke and ayensu, ; abbasi et al., ) . the gall of rhus chinensis has long been considered to possess natural medicinal properties with numerous benefi ts . galla chinensis is used internally for its astringent properties to treat disease such as diarrhea and hemorrhage (duke and ayensu, ) . it is a frequent ingredient in polyherbal prescriptions for diabetes mellitus (duke and ayensu, ) . it has hemostatic effects, often used to promote clotting following traumatic injuries and to treat burns (yeung, ) . it is also used to treat rectal and intestinal cancer, prolapse of the rectum, seminal enuresis and hemorrhoids (yeung, ; gao et al., ) . in addition to its antiphlogistic and antiseptic uses for treating diseases such as persistent cough, galla chinensis also has antiinfl ammatory properties (tian et al., ) . it is also used to counteract ulcers in the mouth and to treat fever and malaria (duke and ayensu, ; gao et al., ) . phytochemical studies on rhus species have been reported earlier and resulted in the characterization of several compound groups such as fl avonoids (taniguchi et al., ; lee et al., ; lin et al., ) , triterpenoids (kuo, ; parveen, ; lee et al., ) , phenolics (parveen and khan, ; lee et al., ; ouyang et al., ) , tannins (takechi et al., ) and aromatic alkanes (kuo et al., ; lee et al., ; ouyang et al., ) . the galls on rhus chinensis leaves are rich in gallotannin ( - %), a type of hydrolysable tannin (kee and walter, ; xiao et al., ) . gallotannins from galla chinensis consist of a central glucose core, which is surrounded by several gallic acid units, and further gallic acid units can be attached through depside bonding of additional galloyl residues. structures containing to galloyl residues result from such processes, yielding tri-, tetra-, penta-, hepta-and nonagalloylglucose, and others (xiang et al., ; tian et al., b) . pentagalloylglucose [ ], -galloyl-gallic acid and -galloyl-gallic acid isomers isolated from galla chinensis are reported to be the primary bioactive gallotannins, possessing numerous medicinal activities and health benefi ts sakai et al., ; bhimani et al., ; feldman et al., ; . rhus chinensis is rich in well known phenolic compounds, gallic acid [ ] and methyl gallate [ ] (ahn et al., (ahn et al., , bae et al., ; choi et al., ). according to buziashvili et al. ( ) galla chinensis is composed of nearly % gallic acid and % methyl gallate. a new benzofuranone, -hydroxy- -(propan- -ylidene)- -( , , , -tetramethylhexade-ca- , , , -tetraenyl)- ( h)-benzofuranone [ ], together with known bioactive compounds, including -hydroxy- -( , , , -tetramethylhexadeca- , , , -tetraenyl)- ( h)-benzofuranone [ ], -oxo- β-hydroxyolean- en- -oic acid [ ], -oxo- β-hydroxyolean- -en- -oic acid [ ] moronic acid [ ], betulonic acid [ ], gallicin [ ], dihydroxytoluene [ ] and dimethylcaffi c acid [ ] , have been isolated from the root stem of rhus chinensis (gu et al., ; wang et al., ) . phenol glycosides and lariciresinol-based ligan glycosides compounds have been shown to be present in the butanol extract of rhus chinensis root (ouyang et al., (ouyang et al., , . -pentadecylsalicylic acid, an antithrombotic compound [ ] (kuo, ) and fi setin ( , , -, -tetrahydroxyfl avone) [ ] an antiinfl ammatory, have also been isolated from the stem of rhus chinensis. the leaves of this plant are rich in essential oils, with palmitic acid, phytol and n-heptacosane as the major components (zhu et al., ) . the high level of gallotannins along with phenolic compounds, gallic acid and methyl gallate, known antimicrobial agents make galla chinensis very useful in bacterial control (wu-yuan et al., ; ahn et al., ; kang et al., ; tian et al., a tian et al., , b . extracts from galla chinensis inhibited several bacteria such as bacillus subtilis, b. cereus, escherichia coli, enterobacter cloacae, helicobacter pylori, klebsiella oxytoca, lactobacillus casei, l. acidophilus, l. salivarius, salmonella derby, s. minesota, s. typhimurium, s. enteritidis, shigella dysenteriae, staphylococcus aureus, streptococcus mutans, s. sobrinus, ureaplasma urealyticum, with the minimal inhibitory concentration (mic) in the range . - mg/ ml (wu-yuan et al., , bae et al., choi ii et al., ; kang et al., ; zhu et al., ; choi et al., ; tian et al., a) . tian et al. ( b) reported that different gallotannins from galla chinensis separated according to the number of galloylglucose had signifi cant antibacterial activities on bacillus cereus and salmonella typhimurium. structure activity relationship studies indicated that antibacterial activity was positively correlated with the numbers of galloyl groups and generally, gallotannins with higher molecular weights had strong antibacterial activities (tian et al., a (tian et al., , b . a methanol extract of galla chinensis was shown to have signifi cant growth-inhibitory activity towards harmful intestinal bacteria (ahn et al., (ahn et al., , . activity-directed fractionation of the methanol extract of galla chinensis has led to the isolation of gallic acid and its derivative methyl gallate as the major components involved in the observed antimicrobial activity. it was also reported that methyl gallate and gallic acid from galla chinensis had inhibitory effects on periodontopathic bacteria (mic = mg/ml) and signifi cantly reduced the in vitro biofi lm formation of s. mutans (methyl gallate, mg/ml gallic acid, mg/ml, p < . ) (kang et al., ) . anti-hiv activity. in a recent study, different fractions of rhus chinensis showed potent anti-hiv- activity (wang et al., ) . subsequent anti-hiv guided fractionation of rhus chinensis led to the isolation of compounds with potent anti-hiv- activity (gu et al., ; wang et al., ) . among those compounds, a new class of benzofuranone-type compounds -hydroxy- -(propan- -ylidene)- -( , , , -tetramethylhexadeca- , , , -tetraenyl)- ( h)-benzofuranone [ ] and -hydroxy- -( , , , -tetramethylhexadeca- , , , -tetraenyl)- ( h)-benzofuranone [ ] were found signifi cantly to suppress hiv- replication (gu et al., ) . compound [ ] possessed signifi cant anti-hiv- activity with a therapeutic index (ti) of . , whereas compound [ ] showed moderate anti-hiv- activity with a ti of . (gu et al., ) . furthermore, the action mechanisms of the two benzofuranonetype compounds were investigated by wang et al. ( ) . these authors found that both compounds [ ] and [ ] inhibited hiv- replication in chronically infected h cells and may target late-stages of the hiv- life cycle. betulonic acid [ ] an analogue of betulinic acid, a well known anti-hiv- agent (kashiwada et al., ; soler et al., ) exhibited moderate anti-hiv- activity with a ti value of . - . μm (gu et al., ; wang et al., ) . -oxo- β-hydroxyolean- -en- -oic acid [ ], -oxo- β-hydroxyolean- -en- -oic acid [ ] and moronic acid [ ] are oleanolic acid-related triterpenes previously reported to have potential anti-hiv- activity (pengsuparp et al., ; soler et al., ; kashiwada et al., ) . these compounds showed weak anti-hiv activity with ti values of . , . and . , respectively (gu et al., ; wang et al., ) . mengoni et al. ( ) described the anti-hiv and the mechanism of action for oleanolic acid, both of which suggested that oleanolic acid inhibits hiv- protease activity in vitro. gallicins [ ], gallic acid derivate-type compounds, have been reported to inhibit hiv- integrase . the work of wang et al. ( ) confi rmed the result in cell lines with a therapeutic index of . . dihydroxytoluene [ ] had the same extent of anti-hiv- activity with a ti of . . wang and coworkers also showed that dimethylcaffi c acid [ ] , caffeic acid phenylethyl ester derivate, has potent anti-hiv- activity with a ti value of . . these values are relatively low compared with the control azt (ti > ) but the resistance and the adverse side effects to available conventional anti-hiv drugs beg the need of identifi cation and development of additional small-molecule inhibitors that can be used in combination with currently available antiviral agents. in vivo studies performed in mice have shown that the hot-water extract of rhus chinensis had prophylactic and therapeutic efficacy against herpes simplex virus (hsv) type (hsv- ) (kurokawa et al., (kurokawa et al., , a (kurokawa et al., , b (kurokawa et al., , . this extract was also effective against acyclovir-resistant hsv- and hsv type (hsv- ) infections in mice (kurokawa et al., b) and improved the therapeutic effi cacy of acyclovir in mice infected with hsv- (kurokawa et al., a) . subsequently, nakano et al. ( ) also investigated the effi cacy of rhus chinensis extract in vivo, using a guinea-pig primarily infected intravaginally with hsv- . prophylactic oral administration of rhus chinensis at the dose corresponding to human use signifi cantly reduced the incidence and severity of spontaneous skin lesions compared with latently infected guinea-pigs administered water. when recurrent hsv- infection was induced by ultraviolet irradiation months after primary infection, prophylaxis with rhus chinensis was also signifi cantly effective in reducing the severity of ultraviolet-induced skin lesions. two terpene compounds, moronic acid [ ] and betulonic acid [ ], were separated from rhus chinensis and their subsequent anti-hsv activities were assessed in vitro and in vivo . the effective concentrations of moronic acid and betulonic acid for % plaque reduction for hsv- were consecutively, . and . μg/ml. the therapeutic index of moronic acid ( . - . ) was larger than that of betulonic acid o. djakpo and w. yao ( . ). oral administration of moronic acid thrice a day to mice infected cutaneously with hsv- , signifi cantly retarded the development of skin lesions and/or prolonged the mean survival of infected mice without toxicity compared with the control. moronic acid exerted stronger anti-hsv- activity in the brain of hsv- -infected mice than in the skin, similar to the hotwater extract of rhus chinensis (kurokawa et al., a . anti-hcv and anti-sars-cov activities. screening a library of traditional medicines, duan et al. ( ) found that the etoac extract fraction from galla chinensis was effi cient in inhibiting the ns protease activity of hepatitis carcinoma virus (hcv). , , -tri-o-galloyl-βd-glucose, , , , -tetra-o-galloyl-β-d-glucose and pentagalloylglucose [ ] were identifi ed as the active compounds. tri-, tetra-and pentagalloylglucose inhibited hcv ns protease with ic of . , . and . μm, respectively (duan et al., ) . likewise, tetra-o-galloyl-β-d-glucose isolated from galla chinensis exhibited prominent inhibition against severe acute respiratory syndrome coronavirus (sars-cov) with a % effective concentration of . μm (yi et al., ) . liu et al. ( ) found that crude aqueous extract of galla chinensis has the ability to inhibit enamel demineralization in vitro. in another study, chu et al. ( ) evaluated the effects of compounds from galla chinensis on the remineralization of initial enamel carious lesions using an in vitro ph cycling model. the group demonstrated the potential of three different fractions of galla chinensis to affect net rehardening of artifi cial carious lesions under dynamic ph-cyclic conditions. furthermore, zou et al. ( ) , using the same protocol, demonstrated the potential of galla chinensis extract to inhibit the demineralization of initial enamel carious lesions. the chemical compounds of galla chinensis showed effects and combined effects with fl uoride on enhancing remineralization of dental enamel (cheng et al., ) . at this point, the active compound of galla chinensis involved in remineralization or demineralization is still unknown. chu et al. ( ) isolated gallic acid [ ] and methyl gallate [ ], both of which showed poor activity compared with the crude extract. this result was confi rmed by cheng et al. ( ) testing the combined effects of galla chinensis extract or gallic acid with fl uoride on remineralization of artifi cial early enamel caries. they found that both the crude extract of galla chinensis and gallic acid had synergistic effects with fl uoride on remineralization, but with apparent differing mechanisms. thus, it seemed that gallic acid was not the only possible active constituent of galla chinensis to enhance remineralization. zou et al. ( ) similarly attempted to determine which of the constituent chemical fractions of galla chinensis conferred a potential anticaries benefi t by comparing the effects of four different fractions of galla chinensis on demineralization of a bovine enamel model. the crude extract was the most active one, prone to some losses of other active compounds during the separation process. cai et al. ( ) screened chinese medicinal plants for antioxidant activity; the results showed that the aqueous extract of galla chinensis contained the highest antioxidant concentration of μmol teac/ g. more recently, two similar studies have investigated the antioxidant activity of gallotannins in four different systems, namely , -diphenyl- -picrylhydrazyl (dpph) radical scavenging, ferric reducing antioxidant power, β-carotene linoleic acid system and hydroxyl radical scavenging assays. tian et al. ( a) tested the antioxidant activity of gallotannins with different polarities and found that all of the consecutive extracts of galla chinensis possessed remarkable antioxidant activity. for example, dpph radical scavenging activity, ec were in the sequence ethyl acetate ( . μg/ml) > ether ( . μg/ml) > ethanol ( . μg/ml) > water ( . μg/ml). generally, all fractions showed better capacity to scavenge free radicals than the controls, bht and trolox. the same results trend was observed with ferric reducing activity. antioxidant activity increased when the polarity of extracts decreased, suggesting that extracts with weaker polarities contained higher molecular weight tannins, and thus had stronger antioxidant effects. aware of this fi nding, tian et al. ( b) isolated different gallotannins, containing - galloylglucoses (gg), from galla chinensis and investigated their antioxidant activities in the above systems. generally, gallotannins of high degrees of galloylation ( - ggs) had stronger antioxidant activities than those of low degrees of galloylation ( - ggs).the same conclusion was drawn in earlier work by yokozawa et al. ( ) . similarly, methyl gallate and gallic acid have been shown through in vivo and in vitro studies to have antioxidant and radical scavenging activity (chen and zhang, ; whang et al., ; madsen and bertelsen, ; peyrat-maillard et al., ) . it has been demonstrated that pentagalloylglucose possesses antioxidant activity and protects rat neuronal cells from oxidative damage feldman et al., ; oh et al., ; pan et al., ) . piao et al. ( ) showed that pentagalloylglucose exerts antiapoptotic activity through antioxidant properties. yang et al. ( ) were the fi rst to report the anticancer activity of rhus chinensis extract on carcinogenic cdc phosphatases. several molecules found in rhus chinensis such as pentagalloylglucose and gallic acid have been shown to have anticancer activity (bhimani et al., ; madsen and bertelsen, ; chung et al., ; hu et al., ; kuo et al., ) . pentagalloylglucose has been shown to exhibit in vivo anticancer effects against prostate cancer (hu et al., ; kuo et al., ) , lung cancer (huh et al., ) and sarcoma (miyamoto et al., ) , and in vitro inhibitory effects on the growth and/ or invasion of breast cancer, leukemia, melanoma and liver cancer . pentagalloylglucose can exert anticancer activity via the inhibition of angiogenesis (lee et al., ; huh et al., ) and invasion of melanoma cells in metastasis (ho et al., ) . in vitro studies showed that pentagalloylglucose signifi cantly inhibited the proliferation and tube formation of bfgf- phytother. res. : - ( ) treated human umbilical vein endothelial cells (huvec) with an ic of μm (huh et al., ) . the result is similar to the in vitro antiangiogenic activity of pentagalloylglucose in vegf-treated huvecs (lee et al., ) . daily injection of and mg/kg of pentagalloylglucose signifi cantly inhibited the growth of the highly angiogenesis-dependent lewis lung cancer allograft by % and %, respectively (huh et al., ) . similarly, pentagalloylglucose inhibited the invasion of highly metastatic mouse melanoma b f cells in vitro in a dose-and time-dependent manner, with ic of μm (ho et al., ) . some other investigations have also demonstrated that derivatives of galloylglucose inhibit not only cancer cell growth (pan et al., ; hu et al., ) but also the invasion of ht human fi brosarcoma cells (ata et al., ) . several studies of natural hepatoprotective agents have revealed that the extract of galla chinensis showed promising hepatoprotective activity tian et al., ) . based on an activity-guided separation scheme an et al. ( ) purifi ed pentagalloylglucose [ ] and an equilibrium mixture of -galloyl-gallic acid and -galloyl-gallic acid isomer from the methanol extract of galla chinensis and validated their hepatoprotective activity. pentagalloylglucose [ ] and the mixture compounds were found to have marked protective effects on tacrine-induced cytotoxicity in human liver-derived hep g cells with ec values of . ± . and . ± . μm, respectively, and also inhibited nitrofurantoininduced cytotoxicity in hep g cells at . ± . and . ± . μm respectively. furthermore, pentagalloylglucose treatment was able to reduce both hepatocyte necrosis induced by tertbutyl hydroperoxide ( and μm) and apoptosis induced by glycochenodeoxycholic acid ( . to μm) in primary rat hepatocytes (park et al., ) . diabetes mellitus is a chronic metabolic disorder characterized by high blood glucose level due to agents such as α-glucosidase enzyme, which boosts the digestion of carbohydrate to monosaccharides in the process of intestinal absorption. therefore, shim et al. ( ) tested the inhibitory effect of an aqueous extract from the gall of rhus chinensis on α-glucosidase activity in in vitro and in vivo models. galla chinensis inhibited bacillus α-glucosidase activity with an ic of . μg/ml. its inhibition on α-glucosidase was determined to be noncompetitive and reversible when the enzyme-substrate mixture was simultaneously treated with galla chinensis. galla chinensis signifi cantly suppressed the increase of blood glucose level in rats after oral administration of sucrose. these results suggest that galla chinensis might exert antidiabetic effects by suppressing carbohydrate absorption from the intestine and thereby reducing the postprandial increase in the blood glucose. likewise, tannic acid, a mixture of gallotannins containing pentagalloylglucose, was found to have a hypo-glycemic effect in patients with type diabetes (gin et al., ) . aware of this result, li and coworkers hypothesized that pentagalloylglucose could have antidiabetic activity. using synthetic pentagalloylglucose in vitro and in an animal assay, it was demonstrated that pentagalloylglucose effectively reduced blood glucose and insulin levels in vitro and in animal models (li et al., ) . unlike most antidiabetic drugs, pentagalloylglucose may reduce blood glucose without increasing adiposity. the methanol extract of the dried ripe fruit of rhus chinensis was tested in experimental models of castor oil-induced diarrhea in swiss albino mice (tangpu and yadav, ; bose et al., ) . at graded doses, the extract showed remarkable antidiarrheal activity evidenced by an . % reduction in the rate of defecation of control animals at a dose of mg/kg body weight. the extract also reduced intestinal fl uid secretion induced by mgso and gastrointestinal motility after charcoal meal administration in albino mice (tangpu and yadav, ) . in the same way, galla chinensis extracts were found to be effective against enterotoxigenic escherichia coliinduced diarrhea that produces a heat-labile enterotoxin (lt), which binds to the ganglioside g m on the surface of intestinal epithelial cells (holmgren and svennerholm, ) leading to a massive loss of fl uids and ions from cells (chen et al., ) . using the patent mouse gut assay in vivo study, chen et al. ( ) found that galla chinensis extract exhibited an anti-lt-induced diarrheal effect, with an ic value of . ± . mg/ml. competitive gm -elisa assay showed that galla chinensis suppressed (ic = . ± . mg/ml) ltinduced fl uid accumulation by blocking the binding of ltb to g m . thin layer chromatography suggests that the most active fraction that inhibited the binding of ltb to gm was composed of mainly phenolics, especially gallic acid which signifi cantly blocked the binding of ltb to g m , with an ic value of . ± . mm, and suppressed the lt-induced fl uid accumulation in a dose-dependent manner, with an ic value of . ± . mm. the work of kim et al. ( ) showed that galla chinensis had antiinfl ammatory activity in in vivo and in vitro models. galla chinensis could control all of the infl ammatory mediators, such as histamine, heparin, lipidderived mediators and various cytokines in the model of immediate-type allergic reaction in a dose-dependent manner through different mechanisms. latter activityguided fractionation and purifi cation of the etoac fractions of the galla chinensis indicated that the main antiallergic component in galla chinensis was gallic acid. fisetin a fl avonoid found in the root of rhus chinensis (lin et al., ) was also found to down-regulate infl ammatory reactions in stimulated human mast cells . similarly, pentagalloylglucose has been shown through in vivo and in vitro studies to exercise a strong antiinfl ammatory effect (oh et al., ; lee et al., lee et al., , kang et al., ) . -pentadecylsalicylic acid has been isolated from airdried stems of rhus chinensis by bioassay-directed fractionation of the n-hexane extract of the stem (kuo, ) . this compound showed antithrombotic activity at μg/ml using the amidolytic method (kuo, ) . it also prolonged clotting time in a dose-dependent manner in the clotting assay of thrombin-fi brinogen interaction. rhus chinensis species have long been recognized by folk medicine practitioners as having value and have been revealed to have great medicinal potential, much of which was completely unknown to western scientists. over the past few decades, the research efforts on rhus chinensis extracts indicate that the extracts have promising potential as antiviral, anticarie, antidiarrheal, anticancer, antidiabetic and hepatoprotective agents, among others. although the work reviewed here substantiated most of the traditional claims on its health effectiveness, more research is required for validation of the uses of this plant. the available information on the different bioactive contents in samples of various parts of this medicinal plant is very limited, both qualitatively and quantitatively. the gall on rhus chinensis leaves has received much scientifi c attention because of its high gallotannin content and subsequent health potential. however, other parts, such as fruits, leaves, and seeds, can also be investigated based on traditional uses and the fi ndings in other rhus species. , -hydroxy- -(propan- -ylidene)- -( , , , -tetramethylhexade-ca- , , , -tetraenyl)- ( h)-benzofuranone [ ], -hydroxy- -( , , , -tetramethylhexadeca- , , , -tetraenyl)- ( h)-benzofuranone [ ], -oxo- β-hydroxyolean- -en- -oic acid [ ], -oxo- β-hydroxyolean- -en- -oic acid [ so far, galla chinensis is the only medicine proven to remineralize a hard tissue like enamel. this is a unique potential for this plant, but the active constituent is still unknown. the mechanistic activity of rhus chinensis material as prophylactic, therapeutic, anti-hsv, anti-hiv and antidiarrheal medicine needs to be further examined. on the other hand, efforts should also be made to survey other sumac species to determine if these properties are generalized across the rhus genus. the safety of rhus chinensis still needs to be rigorously established, since cases of toxicity from intake of gallotannins found in rhus chinensis have been reported in the literature. different gallotannins such as tri-, tetra-, hexa-, hepta-, octa-, nona-and decagalloylglucose can reduce blood pressure and blood urea nitrogen, as reported in animal studies in the literature (feldman et al., ; hofmann et al., ; nishizawa et al., ) . nonetheless, tannins diminish protein digestibility when present in high levels in diets with low protein content and also inhibit human salivary α-amylase, thereby causing potential negative effects on starch digestion and food taste. this needs to be taken in account when testing the effi cacy of rhus chinensis compounds in human beings by clinical trials for drug use validation. medicinal plants used for the treatment of jaundice and hepatitis 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the authors have declared that there is no confl ict of interest. key: cord- -a ni s e authors: jackson, ronald s. title: wine, food, and health date: - - journal: wine science doi: . /b - - - - . -x sha: doc_id: cord_uid: a ni s e wine has historically been associated with religious rights, used as a salubrious beverage, employed as a medication as well as a medicinal solvent, and consumed as a food accompaniment. it is the last use that is most intimately associated in the minds of most modern consumers. despite this, there is little flavor commonality on which pairing could be based. the first section of the chapter examines this feature and wine's primary role as a palate cleanser and food condiment. the synergistic role of food and wine in suppressing each other's least pleasant attributes is also explained. the final section deals with the latest evidence relating to the many beneficial health effects of moderate wine consumption, shortfalls in the data, headache induction, dental erosion, and conditions under which wine intake is contraindicated. wine and its association with food have generated an incredible cornucopia of literature. amazingly, few of the views and assumptions have been subjected to scientific scrutiny. only a few research papers deal with the topic. this may relate to their subject disciplines (enology and food science) being in separate departments. also, the funding is different. that for enology and viticulture comes largely from government or industry grants, often based on levies on vineyards and wineries. funding in the food sciences comes largely from commercial firms, much of it done internally by food companies, and such research remains proprietary. funding from disparate industries provides little incentive for collaboration on interdisciplinary projects, such as food and wine combination. much of what is written is by those with an arts rather than a science background. sommeliers, for whom the combination of wine and food is their field, are more practitioners than researcher/academicians. thus, the topic has experienced languor, devoid of scientific rigor. only recently have scientists ventured into a subject normally the prerogative of the epicurean. scientists, being inherently doubters, demand experimental verification. the latter is difficult as designing adequate controls is next to impossible. in addition, the laboratory conditions, required for experimentation, make the relevance of finding to "real life" situations dubious. with food and wine combinations, the social context is often more important to perception than actual sensory clues. finally, social pressures often lead to acceptance of supposed norms. what follows is a brief discussion of the issue of food and wine combination and what can be said with a degree of confidence. in addition, it is hoped that it will provide "food for thought" to those seriously interested in wine. the traditional view is that wine is in some fundamental way designed to be consumed with food. however, when one searches for evidence, it seems to be a mythda view so oft repeated as to be taken as gospeld an example of social contagion of collective memory. wine appears to have been the standard food beverage in the greek or roman worlds. in the near east and egypt, wine was almost the exclusive preserve of the secular and religious oligarchy. in contrast, beer was the beverage of the laboring classes. wine is mentioned in the bible but recommended primarily in relation to religious ceremonies and weddings. it is only in regions where wild grapes grew indigenously that vines came to provide the drink for the masses. its acidity, alcohol, and phenolic contents enhanced its antimicrobial property, making it considerably safer to drink than water. wine's mildly antiseptic properties became increasingly important as population numbers grew, hygienic conditions deteriorated, and water supplies became more polluted. although production was seasonal (unlike beer based on dry grains), wine's comparative resistance to spoilage (isolated from air) permitted wine to become the preferred beverage. in addition, wine's higher alcohol content could temporarily numb the afflictions of a peasant life. although safe and nutritious (calorie rich), there is nothing in its attributes that inherently makes wine ideal as a food beverage. it is actually better suited for one of its ancient secondary roles, a solvent in preparing extracts from medical herbs. the sharp acidity of many ancient wines, often extended with aged seawater and vinegar (especially for slaves), can hardly be considered an ideal food accompaniment, at least from a modern perspective. the resinous flavor donated by storage in standard amphoras (made watertight with an inner coating of pitch) would not have facilitated detecting any subtle fragrances they might have had. the quality of the ordinary wine available to the average roman is probably best left to the imagination. finer wines were available for the patrician classes. these were probably stored in amphoras with a vitreous inner surface, without the need for a pitch lining. the most famous seem to have been or became highly concentrated with age, being almost syrup-like, usually diluted before being consumed. several roman poets eulogized the wonders of particular vintages. even modern wines, with their predominant acidic, bitterish, and astringent character have little to suggest food compatibility. these characteristics are simply the natural consequence of grape chemistry, not conscious intent. admittedly, consuming wine with food does mollify its less pleasant aspects, unless one develops an appreciation for sour beverages with a bitter/astringent aspect. some humans seem adept at coming to appreciate, even crave, perceptions initially repulsive to painful. black coffee, capsicum peppers, durian, and limburger cheese are classic examples. in some instances, the iron content of the wine can induce a metallic sensation, by catalyzing lipid oxidation. this may be masked in combination with food. however, this appears to be unrelated to the fishy aftertaste associated with some white wines taken with seafood (tamura et al., ) . in most "compatible" combinations, both the cheese and wine appear betterdnot by directly enhancing their respective qualities but by mutually suppressing their less pleasing attributes (nygren et al., (nygren et al., , a (nygren et al., , b . other studies have extended these findings, lending support to the view that red wines pair better with cheeses, due to wine tannins appearing more silky (bastian et al., ) . the fatty acid content of the cheese may also contribute to reduced bitterness (homma et al., ) , possibly by coating taste receptors. lipoproteins, typically found in foods, can react with taste receptors, suppressing the perception of bitterness (katsuragi et al., ) . however, the bitter-suppressing aspect of cheeses may also relate to its salt content (frijters and schifferstein, ; breslin and beauchamp, ; keast et al., ) or the presence of glutamate and adenosine monophosphate (keast and breslin, ) . the fruity aspect of wine may also be enhanced when sampled with cheese (galmarini et al., ) . although most cheese/wine associations seem mutually beneficial, some are not, for example, sweet wines and cheese (bastian et al., ) . that compatibility originates from the negation of unpleasant sensations may not be what wine pundits and sommeliers profess, but it seems closer to the truth. this phenomenon may also apply to many supposedly food and wine "marriages." salt's suppression of bitterness (nakamura et al., ) might explain the seemingly ancient habit of adding seawater to wine, e.g., oinos thalassikos (younger, , p. ) . pliny (historia naturalis, . ) also records that salt enhanced the perceived smoothness of wines. columella (de res rustica . ) recommends the addition of salt, apparently to avoid moldy tastes. he also notes salt addition in a recipe for preparing "greek" wine. salt water was even used until recently in preparing new barrels to receive wine (nègre and françot, ) . salt is well known as a flavor enhancer. this may involve disrupting weak, nonvolatile complexes between matrix and aromatic compounds, promoting their liberation and retronasal detection (linscott and lim, ) . in addition, sodium ion hydration may decrease "free water," changing solution polarity. although salt increases aromatic volatility, saltiness is by itself appreciated (bolhuis et al., ) . when one searches for affinities among the attributes of food and wine, one comes up empty-handed. in contrast, there is extensive incongruity. table wines possess gustatory attributes predominantly characterized by sourness, bitterness, astringency, and burning sensations. although pronounced sour tastes are inherently unpleasant, wine's acidity is of value when used as a marinadedpromoting acid-induced hydrolysis of food proteins. wine phenolics can also act as antioxidants, reducing the toxicity of heterocyclic amines (viegas et al., ) and acrylamide (qi et al., ) generated during frying (viegas et al., ) . phenolics are also antimicrobial (nisiotou et al., ) . by comparison, sourness is a rare attribute in most world cuisines (see moskowitz et al., for a marked exception). acids typically are added only as a component in some condiments or flavorants, notably vinegar, lemon juice, or tamarind. they can enhance the flavor of otherwise bland foods. the bitterness and astringency of most red wines also find no equivalent in meat and fish. the protein content of food reacts with both wine acids and phenolics, limiting their activation of taste and touch receptors. in comparison with wines, solid foods are characterized salty, savory (glutamate), sweet, and sebaceous (fatty acids) sensations. sour, bitter, astringent, and hot spicy attributes are (or have been) less common in western cooking and then usually in condiments. the inherent, aversive reactions to such sapid sensations probably arose as a protective response to avoid or limit the consumption of potentially toxic (or rotten) foods. conversely, bitter/astringent/toxic compounds were probably selected during plant evolution to discourage their consumption, with the principal exception of ripe fruit. during domestication, crop variants with reduced aversive and enhanced pleasant-tasting constituents have been propagated. thus, lettuce and other vegetables became less bitter; apples, cherries, and other fruits sweeter and less sour or astringent, citrus fruit less acidic, and legumes less flatulent. food preparation, notably cooking, further facilitated the inactivation or removal of potential food toxins and antimetabolites. examples include fungal toxins, potato alkaloids, and casava cyanogenic glycosides. cooking meat also facilitates digestion (promoting collagen and protein fiber breakdown) and enhances flavor. disappointingly, some cooking processes generate their own toxins, notably roasting and searing. examples are acrylamide (a maillard by-product) and a variety of toxic, pyrolytic, smoke by-products. fermentation is another ancient technique that helped destroy antimetabolites. an example is the action of rhizopus oligosporus degrading soybean flatulence compounds during tempeh production. lactobacillus can also destroy soy saponins. fermentation also has the potential to break down difficult-to-digest oligosaccharides as well as help preserve perishable foods. the aromatic aspects of food and wine equally show little similarity, on which supposed compatibility could be based. wine aromas are most frequently described in terms of fresh fruit, jam, or flowers. none of these is characteristic of the main components of a meal and would be considered odd if present. the hints of "apple" in chardonnay wine may be compatible with chicken, the "pepper" of a shiraz pair with pepper steak, and the "walnut" of some sherries combine with nutcontaining salads (without vinaigrette). however, does the box wood or cat urine of sauvignon blanc, the rose of riesling, and the black currant of cabernet sauvignon really match with any main course? in addition, does the vanilla/coconut of oak or the leather aspect of aged red wines have any inherent compatibility with food? the supposed spiciness of gewü rztraminer wines is given as a reason for its combination with spicy asian foods. however, the wine has more litchi aroma than spiciness, making the stated logic dubious. it is only its intense aroma that may permit its presence to remain noticeable. personally, the best aspect of wine and food association is not their complementary natures but their contrasts. each cleanses the palate between alternate samplings, allowing fresh perceptions of each. thus, wine permits swift shifts in savory sensitivity. without comparable tastes or flavors, where is the supposed inherent rapport between food and wine? is its only justification the reduction of undesirable aspects of the partnership and as a palate cleanser? certainly, wine's ability to partially rinse the palate between food samples is important. it offers both gustatory, olfactory, and trigeminal receptors time to reestablish their native receptive state, countering adaptation and loss of sensory appeal. thus, food appreciation is enhanced by being sampled afresh. wine can, unromantically, be viewed as a savory mouthwash. in addition, volatility of food and wine flavorants is influenced by the dynamically changing concentrations of ethanol, phenols, carbohydrates, etc. supplied with the wine. conversely, food dilutes, masks, and eliminates most wine flavors. the result being that the next sample can be appreciated to its full, adaptation having been avoided. the other aspect of potential compatibility arises from their dissimilar attributes. they act in concert, in a manner similar to condiments, providing flavor accents to enhance and maintain flavor interest throughout the meal. the result can be the creation of a stimulating holistic experience. the typical starchy elements in a meal (rice, potatoes, pasta, bread) supplement the synergy, helping to cleanse the palate and generate a feeling of satiation. however, statements like "wine cuts through fat" are unsupported. if this view has any validity, the partial fat solubility of ethanol may reduce the oily mouth-feel of fats as well as limit the activation of fatty acid taste receptors (running et al., ) , but this is no more than speculation. the acids in wine might also have a similar effect. in addition, wine tannins may denature the g protein receptors responsible for detecting fatty acids in the oral epithelium, giving the impression of less fattiness. conversely, fatty acids can reduce the perception of sourness, astringency (peyrot des gachons et al., ) , and bitterness (mattes, ) , possibly improving mouth-feel. wine phenolics can either reduce (jung et al., ) or enhance (mitropoulou et al., ; villamor et al., ) volatility, depending on the phenolic and aromatic involved. carbohydrates also have the ability to bind aromatics, reducing volatility and significantly modifying wine flavor and its retronasal attributes (voilley and lubbers, ; villamor et al., ) . thus, both synergies of flavor as well as suppression could be involved in "ideal" pairings. saliva-induced changes in flavor chemistry is another compounding issue little investigated. whether such potential reactions are sufficiently marked and rapid to have significance, within the time frame of food consumption, is currently terra incognita. in all the standard discussions of wine and food combinations, the obvious is rarely if ever mentioned. it limits the rate and maximum amount of alcohol reaching in the blood ( fig. . ) . correspondingly, it reduces breath-alcohol content (sadler and fox, ) and diminishes alcohol's performance impairment (millar et al., ) . in addition, by reducing alcohol uptake, its tendency to promote overeating is represseddby limiting activation of hypothalamic agrp neuron activity (cains et al., ) . many aspects of food preference appear to develop in utero, based on what the mother ate during pregnancy (mennella et al., ) and infancy. thus, early exposure can play a significant role in developing personal preferences. subsequently, peer pressure and cultural influences may combine to modify these early dispositions. habituation may not be imbued with the effusive and social appeal associated with the image of predestined food and wine marriages but far better fits the facts than any supposedly heaven-inspired pairings. thus, there is little wonder why there has been scant inclination to investigate lovingly held shibboleths about food and wine associations. however, a dose of reality does not have to destroy long-held views. knowing that michelangelo's sistine chapel consists of no more than brush strokes of pigment on plaster, perceived by photoreceptors in the eye, converted to synaptic impulses reconstructed piecemeal into a perception at the back of the brain, need not ruin art appreciation. ideally, it should enhance the wonder it instills. scientific understanding augments adds new layers of appreciation. if supplemental knowledge were not considered to augment appreciation, why would connoisseurs be so concerned with vintage dates, wine geography, cultivars, or details of vineyard sites and wine producers? with knowledge, it really is the more the merrier. admittedly, scientific realism injected at the wrong time may be a despoiler. for special events, psychological appeal can be more significant than sensory reality. as in other aspects of life, science can occasionally be set aside to permit spontaneity and anticipated pleasure presides. the intrigue of magic is being fooled so effectively. under most circumstances, though, some basic rules of pairing should be kept in mind, to avoid glaring mistakes. in most fine cuisine, flavor balance, combined with suitable complexity, is central. nonetheless, the vagueness of this concept is evidenced by the almost infinite variety of combinations seen in cookbooks, food magazines, and culinary shows. accepted norms also vary markedly among cultures (rozin, (rozin, , . thus, it should not be surprising that almost any wine can pair with almost any meal. there are limits, however, for example a dry gewü rztraminer with dessert or a riesling auslese with bouillabaisse. to almost everyone, these would not be considered "marriages made in heaven." cabernet sauvignon and dark chocolate is another clash but seeming appreciated by some connoisseurs. except where there are clear flavor or intensity disparities, notably sweet/ acid or sweet/bitter-astringent, almost any combination will be found pleasing to some and acceptable to most. of the generalities oft quoted, the "white with white, red with red" dictum bears logic, within the context of balanced flavor intensity. nonetheless, it is often the food preparation mode (e.g., poached, fried, baked, broiled, barbequed) or the condiments added (e.g., chilies, curry, relish, olive oil, tomato sauce, garlic, herbs) that often have the greatest influence on flavor intensity, the basic character of the meal, and correspondingly a compatible wine. in most instances, premium-quality table wines are best sampled prior to the meal and premium-quality dessert wines after the meal. because of their aromatic complexity, detection of these attributes is compromised by combination with food or dessert. alternatively, the food or dessert should be designed to be a foil for the wine and be mild in character. in contrast, the more markedly acidic or bitter/astringent the wine, the more effectively these features will be mollified by association with food. might not this be the most justifiable reason for the pairing most wines with food (other than reduced alcohol uptake)? in addition, a lack in the wine's aromatic interest can be camouflaged by flavors from the food. where little attention is likely to be paid to the wine, inexpensive, nondescript red or white wines are both financial and logical choices. if some food and wine pairings are ill-conceived, are there seraphic duets? clearly some do pair better than others, but transcendental experiences? published accounts of such paradisaical experiences may be no more than figments of a fertile imagination, created to sell wine, newspapers, or magazines. admittedly there is an incredible range in human sensory sensitivityd those having higher than average acuity tending to prefer milder flavored foods, and those with below average acuity tending to prefer more intensely flavored foods. but these are no more than tendencies, with experience and social pressures capable of inducing significant shifts in preference, if not perception. psychological influences and a desire to be influenced can distort perception, creating impressions that are "real," only because of the conditions under which the experience occurred. the more unexpected and astounding the sensation, the stronger the memory trace created. the more the mind is studied, the more we come to realize how the brain can distort perception, based on past experiences. they generate mental models of reality, against which sensations are judged, interpreted, and potentially modified. thus, it is wise to doubt perceptions and attempt to separate experience-based memory patterns from actuality, unless it is a selective choice to allow the mind to potentially deceive us. in addition to the sensory pleasure wine can supply when taken with a meal, it also has health benefits. among other direct benefits noted below, dining with wine can reduce the absorption of oxidized lipids and thereby limit their cardiovascular damage (natella et al., ) . the contrasting social and antisocial effects of alcohol consumption must have become evident shortly after the discovery of winemakingdthe negatives being noted early in the old testament (genesis . ) and vividly described by pliny (historia naturalis, . ). time has only expanded our understanding of this dr. jekyllemr. hyde relationship. it is clear that excessive alcohol consumption, both acute and chronic, can have devastating effects on physical and mental well-being. abusive ethanol consumption can cause cirrhosis of the liver, increase the likelihood of hypertension and stroke, favor the development of breast and digestive tract cancers, induce fetal alcohol syndrome, among others. many of these effects seem to arise from excessive alcohol intake activating of free-radical release and associated immune perturbations (meagher et al., ) . other, more severe, negative consequences may arise indirectly, via the accumulation of acetaldehyde, a major breakdown product of ethanol metabolism (lachenmeier et al., ) . because the problems associated with alcoholism (abrams et al., ; schmitz and gray, ) and its eventual, irreversible, chemical modifications in the brain (nestler and malenka, ; heinz, ) have been extensively reported, they need not be elaborated here. on the other hand, it is becoming equally clear that moderate wine consumption (approximately ml/day) can potentially have health benefits. this is considerably less than the two bottles per day that brillat-savarin ( ) considered a healthy man could consume and live long; or the amounts considered appropriate in times past (younger, , p. ) . mark twain, in characteristic style, crystalized moderation in his view of the temperance movement: "temperate temperance is best" (mark twain's notebook, ) multiple epidemiological studies suggest that daily, moderate, alcohol consumption (thun et al., ; doll et al., ) , and notably wine (grønbaek et al., ; renaud et al., ) , is associated with a reduction in allcause mortality. this is expressed in the now famous jshaped (hormesis) curve ( fig. . ), with earlier mortality being associated with both excess alcohol intake and abstinence. this is particularly evident in the reduced incidence of cardiovascular disease in moderate alcohol consumers. in addition, it reduces the likelihood of type diabetes, combats hypertension, and is correlated with reduced frequency of certain cancers (boffetta and garfinkel, ) . although encouraging to those who enjoy wine, the sharp rise in death risk at much above moderate consumption is of concern. presumably, this relationship would apply equally to morbidity figures, were such data available. however, morbidity unlike mortality is qualitative rather than quantitative and thus its measurement fraught with difficulty. additional dangers associated with anything more than moderate consumption, especially alone, comes from alcohol's progressive enhancing of the memory circuitry involving addictive cravings. these epidemiological correlations are supported by in vivo studies that provide potential molecular explanations for these associations. the principal elements missing, in confirming a causal relationship, involves detailed information on the dynamics of absorption, metabolism, and elimination of the proposed active ingredients. faced with a chemical and beverage that can be not only salubrious but also addictive, the fluctuations in society's attitude toward alcohol are not surprising (musto, ; pittman, ; vallee, ) . thankfully for those in the wine industry, wine drinkers appear less likely to become heavy drinkers (jensen et al., ) or to illustrate those alcohol-related problems that have given alcohol a bad reputation (smart and walsh, ) . in addition, wine has a more positive social image than other alcohol-containing beverages (klein and pittman, ; unwin, ) . the major caveat is the derogatory epithet, wino, ascribed to some unfortunate members of society. the use of wine as a medicine or carrier for herbal extracts has an extensive history. it goes back at least to pharaonic egypt (lucia, ; soleas et al., ) . ancient greek and roman society used wine extensively as a solvent for medicinal infusions. this practice continued largely unabated until the beginning of the twentieth century. the excessive abuse of distilled alcoholic beverages, combined with religious and political conservatism, created a backlash against all beverages containing alcohol, notably in north america. alcohol was viewed as an agent of corruption to be annihilated. following the failure of prohibition, humans themselves, not alcohol, came to be viewed as the source of iniquity. alcoholism is now viewed as a developmental, multistage, chronic dependence, possessing a complex etiology (nurnberger and bierut, ) , with both genetic and environmental aspects. in this regard, it is similar to other addictions (ersche et al., ) . thus, the social climate is changing and the relationship between wine (as opposed to alcohol) and health is again being reassessed and investigated seriously. it is unlikely that doctors will soon be prescribing wine for its health benefits. too often, people have difficulty recognizing the limits of rational use and differ markedly in their metabolism (gross et al., ) . in addition, detrimental influences rapidly counter any benefits at more than light to moderate consumption (often viewed as< mg ethanol/day) (rehm et al., ) . erring on the side of restraint seems judicious, without excessively assuaging pleasure, especially if combined with food. even dietary flavonoid supplements (one of the benefits of wine consumption) can be detrimental, if taken in excess (skibola and smith, ) . wine can be wonderful in moderation but is no panacea. alcohol is the primary by-product of fermentation in many organisms. ethanol is also an energy source for an even larger number of species. thus, it is not surprising that enzymes involved in ethanol metabolism are found in most life forms. in humans, ethanol enters the bloodstream either via the consumption of beverages containing alcohol, and/or from ethanol synthesized by members of the intestinal flora. when the concentration of alcohol is low, most of it is metabolized in the liver before it enters the systemic blood supply. most of the blood coming from the digestive tract passes through the liver before being dispersed to the rest of the body. the liver metabolizes about % of the blood alcohol content, at about ml/h. the rest tends to be lost via the breath or secreted in the urine and other bodily fluids. the rate of alcohol loss is relatively constant over time, with w % reduction within h ( fig. . ). the liver possesses two ethanol metabolizing pathways. the primary, constitutive mechanism involves the oxidation of ethanol to acetaldehyde, via cytoplasmic alcohol dehydrogenases (adhs). of the seven known adh genes (crabb et al., ) , three function in the liver. the others act in the gastric epithelium and other tissues. subsequent metabolism converts acetaldehyde to acetic acid. this occurs principally under the action of mitochondrial acetaldehyde dehydrogenase (aldh ). the cytoplasmic acetaldehyde dehydrogenase (aldh ) is less active. acetic acid is subsequently secreted into the blood or directly converted to acetyl coa. from this point, metabolism may flow along any standard biochemical pathway (see fig. . ). alcohol metabolizing enzymes frequently occur in allelic forms (isozymes). their relative occurrence also tends to vary among ethnic groups. some isozymes possess distinct physiological attributes. for example, adh b* codes for a subunit that oxidizes ethanol slowly, whereas adh b* encodes a highly active subunit of the dimeric enzyme (about times more efficient) (thomasson et al., ) . correspondingly, those individuals who are homo-or heterozygous for the adh b* subunit, eliminate alcohol from the blood more rapidly. rapid alcohol oxidation may donate a degree of protection against alcoholism, by quickly converting ethanol to acetaldehyde. however, if combined with slow-acting alleles for acetaldehyde dehydrogenase (aldh ) (crabb et al., ) , the accumulation of toxic acetaldehyde is enhanced, predisposing the bearer to cancers of the oropharynx and esophagus. those also possessing malfunctional glyoxalase and methylglyoxalase repair enzymes are those most susceptible to such damage (see dingler and patel, ) . a second, ethanol-degradation hepatic pathway becomes activated when blood-alcohol levels become elevated. it involves a microsomal cytochrome, p e (cyp e ). it oxidizes ethanol to acetaldehyde, using molecular oxygen rather than nad þ . regrettably, the microsomal pathway generates free oxygen radicals (meagher et al., ) dmolecules (or ions) with one or more unpaired electrons. these highly reactive oxidants, or reactive oxygen species (ros), can be generated long after alcohol intake ceases. another variant of cyp e occurs in the gastric mucosa, seemingly being more active in men. although most ros are inactivated by glutathione, superoxide dismutase, and catalase, long-term exposure to their presence may induce the slow, progressive accumulation of irreparable cellular damage. metabolizing ethanol to acetate (acetic acid) has the advantage that tissue cells can regulate its transport. this is not the situation with ethanol, which can diffuse freely across cell membranes. transport control is a central tenet in proper cellular function. the ability of ethanol to displace water and its unregulated passage across cell membranes explains much of alcohol's toxicity. in addition, its oxidation to acetaldehyde tends to be more rapid than acetaldehyde's oxidation to acetate. thus, acetaldehyde may accumulate in the blood and other bodily fluids. this is often viewed as an important contributor to the toxicity associated with excessive alcohol consumption (lachenmeier et al., ) . differentiating between these direct and indirect toxic effects of excessive ethanol intake has proven difficult. one of the first physiologic effects of alcohol consumption is a suppression of cognitive brain function. this is most noticeable in enhanced sociabilitydby blocking social inhibitions regulated by higher brain functions. for others, it quickly induces drowsiness (stone, ) . this probably explains why taking a small amount ( e ml) of wine before sleeping often helps those suffering from insomnia (kastenbaum, ) . this amount often provides the benefits of sleep induction, without causing subsequent agitation and sleep apnead often associated with greater alcohol consumption. the effect on sleep may arise from alcohol's modulating the action of inhibitory g-aminobutyric acid (gaba) receptors, while suppressing the action of excitatory glutamate receptors. gaba and glutamate are estimated to be involved in about % of the neurocircuitry of the brain. the level of melatonin in wine is well below those prescribed as an insomnia medication (rodriguez-naranjo et al., ) . another effect on brain function results from a reduction in the secretion of vasopressin. as a consequence, urine production increases, producing the frequently reported diuretic effect associated with alcohol consumption. less well known is how alcohol acts as a crucial regulator of the hypothalamicepituitaryeadrenal axis, modulating the release of hormones such as adrenocorticotropic hormone and corticosterone (haddad, ) . although alcohol has a general depressive action on brain function, the levels of some brain modulators show transitory increases. examples are serotonin and histamine. the latter may activate a cascade of reactions leading to headache production. another of the multiple influences of alcohol is the conversion of hepatic glycogen to sugar. this results in a short-lived increase in plasma glucose content. this, in turn, can cause glucose loss in the urine as well as an increase in insulin release by the pancreas. both result in a drop in blood sugar content. if sufficiently marked, hypoglycemia results. this apparently causes the temporary weakness occasionally associated with alcohol consumption, especially excess intake. in addition to direct effects, the accumulation of acetaldehyde, as a by-product of ethanol metabolism, may have several undesirable consequences. at low rates of alcohol intake, acetaldehyde metabolism is sufficiently rapid to limit its accumulation and liberation from the liver. at higher concentrations, acetaldehyde production rapidly consumes the liver's glutathione reservesda central cellular antioxidant. this coincides with activation of the microsomal ethanol oxidation pathway that generates toxic free-oxygen radicals. in the absence of sufficient glutathione, free-oxygen radicals can accumulate, disrupting mitochondrial function. elsewhere in the body, acetaldehyde can bind with proteins and cellular constituents, forming stable complexes (niemela and parkkila, ) . these can lead to the production of immunogenic determinants, which can stimulate antibody production against acetaldehyde adducts (romanazzi et al., ) . this may induce some of the chronic tissue damage associated with alcohol abuse (niemela and israel, ) . the binding of acetaldehyde to the plasma membrane of red blood cells is known to increase rigidity. by limiting their ability to pass through the narrowest capillaries, oxygen supply to tissue cells may be restricted. this could participate in suppressed brain function. it is estimated that the brain consumes up to % of the blood's oxygen supply but constitutes only about . % of body mass. in addition, acetaldehyde can disrupt dna repair mechanisms. fortified wines (notably sherries) can be a significant source of acetaldehyde (lachenmeier and sohnius, ) . although ethanol and acetaldehyde can produce severe, progressive, and long-term damage to various organs, and incite alcohol dependence, these consequences are minimal to undetectable when alcohol consumption is moderate and taken with meals (serianni et al., ) . as the sections below demonstrate, moderate, daily, wine consumption can have health benefits for the majority of people. wine's major nutritional value comes from its rapidly metabolized, ethanolic, caloric content. alcohol does not need to be digested, prior to being absorbed through the intestinal wall. in rural viticultural areas, wine historically provided a significant source of metabolic energy for the adult population. the caloric value of ethanol ( . kcal/g) is nearly twice that of carbohydrates ( . kcal/g). thus, it constituted a valuable caloric source. it is estimated that alcohol may supply about % of the energy in the average american diet (halsted, ) .wine was also a potable beverage and helped disinfect water to which it was addeddsome bacterial inactivation occurs within seconds (vaz et al., ) . wine contains small quantities of several vitamins, notably several b vitamins, such as b (thiamine), b (riboflavin), and b (cobalamin). however, wine is virtually devoid of vitamins a, c, d, and k. in excess, ethanol can impair vitamin uptake. wine contains various minerals in readily available forms, especially potassium and iron (in the ferrous state). nevertheless, excessive alcohol consumption can disturb the uptake of calcium, magnesium, selenium, and zinc and increase the excretion of zinc via the kidneys. the low sodium/high potassium content of wine makes it one of the more effective sources of potassium for individuals on diuretics. although wine contains soluble dietary fiber, especially red wines (díaz-rubio and saura-calixto, ) , it is insufficient to contribute significantly to the daily recommended fiber content in the human diet. wine has several direct and indirect effects on food digestion. its phenolic (hyde and pangborn, ) and alcohol (martin and pangborn, ) contents activate the release of saliva. in addition, wine promotes the release of gastrin as well as gastric juices. the principal constituent activating the release of gastric juices in red wines is apparently succinic acid, whereas in white wines it is malic acid (liszt et al., ) . they do not activate gastrin release, however. the substance(s) involved in stimulating gastrin secretion are unknown. wine also significantly delays gastric emptying, both on an empty stomach (franke et al., ) or when consumed with food (benini et al., ) . the latter favors digestion by extending the duration of acid hydrolysis. delayed gastric emptying may be a consequence of wine phenolics activating stanniocalcin- cells in the stomach. these possess the same tas r system as bitter-sensitive receptors in the mouth (see finger and kinnamon, ). on stimulus, they release cholecystokinin, a peptide hormone that reduces gut mobility. in addition, wine slows plasma glucose uptake, independent of any insulin response (benini et al., ) . furthermore, at the levels found in most table wines, ethanol activates bile release. wine acids and aromatics also have the same effects. in contrast, the high alcohol contents (e.g., distilled spirits) can suppress digestive juice flow, the release of bile, and induce stomach spasms. wine also aids digestion indirectly by inactivating gastrointestinal pathogens. despite the general beneficial effects of moderate amounts of alcohol on digestion, the phenolic content of red wine may counter some of these influences. for example, tannins and phenolic acids can interfere with the action of certain digestive enzymes, notably a-amylase, lipase and trypsin (rohn et al., ; gu et al., ) . digestion may be further slowed by phenolics polymerizing with food proteins. these effects may be mollified by the presence of ionic carbohydrates found in food (gonçalves et al., ) as well as by salivary proteins. both monomers and proanthocyanidins bind with basic, proline-rich, and histatin proteins in the saliva. their bonding is reversible, depending on equilibrium conditions. they can become irreversible, though, in the presence of metal ions, upon oxidation, or with ph changes (luck et al., ) . normally, these insoluble saliva/tannin complexes remain stable in the stomach and upper alimentary tract (lu and bennick, ) . thus, the inactivation of digestive enzymes or the disruption of mineral uptake by tannins may be limited. degradation of tannin-protein polymers and their moieties subsequently occurs in the colon. in contrast, some pepsin-activated protein breakdown is activated by monomeric phenolics, notably quercetin, resveratrol, catechin, and epigallocatechin gallate (tagliazucchi et al., ) . clearly the action of wine phenolics is complex and much more needs to be known. not only are the effects potentially different in the stomach from that in the small and large intestines, but also the chemical composition of wine phenolics changes during passage through the digestive tract. the wine's phenolic content can also decrease iron and copper absorption in the intestinal tract (cook et al., ) . although nutritionally undesirable, limiting iron bioavailability may reduce the formation of toxic lipid hydroperoxides during digestion. the antioxidant effect of polyphenolics also applies to peroxide generation in the stomach kanner and lapidot ( ); fig. . . the activation of gastric juice release not only aids food digestion but also inactivates enzymes involved in ulceration. even more significant may be the antibiotic action of wine constituents against helicobacter pylori (fugelsang and muller, ) . h. pylori is often considered the primary causal agent of stomach ulceration. thus, moderate wine consumption may have a prophylactic effect in limiting ulcer initiation (brenner et al., ) . the bacterium has also been implicated in gastritis, vitamin b malabsorption, and gastric adenocarcinoma. however, chronic secretion of gastric juice can produce irritation and may provoke ulceration, heartburn, and favor the development of adenocarcinomas in the lower esophagus. wine may further aid human sustenance by increasing nutrient uptake. congeners in wine combine with metallic ions, vitamins, and fatty acids, facilitating their transport across the intestinal wall. consuming wine with food slows the rate of alcohol uptake in the blood ( fig. . ) . in the absence of food, w % of the alcohol is absorbed through the intestinal wall. although the absolute proportion absorbed via the intestines increases when wine is jointly consumed with food, uptake is dispersed over a much longer period. this results primarily by food retarding gastric emptying. consequently, alcohol transfer into the intestines is delayed. this gives the liver more time to metabolize the alcohol, lowering the maximal blood-alcohol level reached. however, taking sparkling wine on an empty stomach can increase short-term alcohol uptake by about % (ridout et al., ) . because the same wine, with its carbon dioxide removed, did not have the same influence, it is suspected that carbon dioxide was the active ingredient (ridout et al., ) . it has occasionally been proposed that carbon dioxide relaxes the pyloric sphincter, allowing earlier transfer of fluids from the stomach into the duodenum and thereby its absorption into the blood. the rate of alcohol metabolism differs considerably among individuals, with rates commonly varying between and mg/kg/h. a person's hormonal and nutritional state also affects their ethanol metabolic rate. gender is also an influencing factor (kaltenback et al., ) . the tendency of women to have a higher body fat content (into which ethanol does not infiltrate), results in their being more rapidly influenced by similar amounts of alcohol (kalant, ) . additional benefits that may accrue from wine consumption are derived from metabolic by-products of proanthocyanin degradation in the colon. they assist protecting the colonic mucosa from the toxic effects of p-cresol production (generated from l-tyrosine) (wong et al., ) . furthermore, wine phenolics may prevent or delay intestinal diseases associated with inflammation and oxidative stress (e.g., inflammatory bowel disease) (biasi et al., ) . finally, wine can have a beneficial cultural/psychologic effect on food intake and digestion. the association of wine with refined eating promotes slower food consumption, potentially permitting biofeedback mechanisms to regulate food intake. in addition, wine consumption can promote a more relaxed lifestyle, something increasingly valuable in our overly compulsive society. whether this explains the reported improved appetite of many elderly and anorectic patients, when wine is taken with the meal, is unknown. wine taken with a meal can enhance the pleasures derived from both but not necessarily those suffering gastroesophageal reflux (acid reflux). this is apparently correlated more with the consumption of white than red wines (pehl et al., ) . most investigations on the health benefits of moderate wine consumption have involved population (epidemiologic) and tissue-culture studies. however, to be confident in their interpretation, intermediate stages needs to be known. this involves details on the uptake and degradation in the intestinal tract, metabolism in the liver, transport, binding, and modification in the blood and lymph, elimination by the kidneys, and cellular uptake and metabolism. although absorption via the intestinal tract is a priori requirement for most activity, it alone does not imply bioavailability at the cellular level. however, this does not apply to influences in the oral cavity and digestive tract. in the mouth and upper intestinal tract, a wine's phenolic constituents remain largely unmodified, except for binding with proteins. in contrast, considerable degradation occurs in the colon. here, large phenolics tend to be metabolized, depending on an individual's colonic flora. for example, hydrolyzable tannins are converted to more easily absorbed, antiinflammatory/anticarcinogenic urolithins (tomás-barberán et al., ) . if absorbed, most phenolics are quickly metabolized in the liver, conjugated with various moieties (e.g., methyl or sulfur groups) by plasma enzymes, and/or eliminated via the kidneys. amounts found in the plasma are often % of that consumed, although this may increase with repeated daily exposure. the proportion of wine-derived flavonoids is estimated at about mg/day/person in the united states (chun et al., ) . this compares with about mg/day/person from all sources. this value would increase to about mg flavan- -ols and mg procyanidin dimers, based on ml of red wine per day (forester and waterhouse, ). that volume is near the upper limit of what is typically considered moderate wine consumption. in the mouth, mid-sized flavonoid polymers often bind to salivary proteins, forming stable complexes (de freitas and mateus, ; pizarro and lissi, ) , slightly delaying their transport to the stomach. passage through the stomach does not modify the majority of wine phenolics. among wine flavonoids, anthocyanins appear to be those that most quickly traverse the stomach and pass into the blood (passamonti et al., ) . flavonoid glucoside uptake is facilitated by gastric glucose transporters (oliveira et al., ) , whereas aglycone uptake occurs by passive diffusion. flavonoids are also effectively translocated across the small intestine lining (talavéra et al., ) . phenolic acids, such as caffeic acid (simonetti et al., ) and resveratrol (soleas et al., ) also readily pass into the plasma via the intestinal tract. in contrast, flavonoid polymers tend to remain in the intestine, until degraded to phenolic acids and aldehydes by the colonic flora (aura, , fig. . ) . also metabolized in the colon are any anthocyanins or catechins monomers that have not already been absorbed and/or microbially degraded. these may enhance the growth of beneficial bacteria (e.g., bifidobacterium and lactobacillus spp.) or their attachment to the intestinal wall (bustos et al., ) . depending on the compound, variable amounts may be absorbed into the blood (ward et al., ) . studies on the bioavailability of phenolics, once in the bloodstream, are still preliminary (williamson and manach, ) . although many simple flavonoids are quickly absorbed into the plasma, most appear to be rapidly conjugated (methylated or sulfated), bound to proteins, transformed to glucuronides, or otherwise modified (williams et al., ; forester and waterhouse, ; xiao and högger, ) . hydroxycinnamic acids are also rapidly absorbed and metabolized into glucuronide and sulfate conjugates (nardini et al., ). this both reduces their toxicity (potential carcinogenicity) as well as facilitating their excretion by the kidneys. however, the latter reduces their potential beneficial effects. in contrast, anthocyanin seems to be efficiently absorbed via the stomach wall, and their metabolites appear to remain in the plasma for several days (kalt et al., ) . short-term studies primarily detect the uptake of phenolic metabolites, whereas long-term studies detect more parental constituents (sandoval-ramírez et al., ) . small amounts of cinnamic acid-tartrate esters are also found in the plasma. these transformations could significantly affect their antioxidant and other attributes as well as their ability to move into tissue cells and their surrounding fluids. most phenolic metabolites retain one or more hydroxyl groups and thus may still possess antioxidant properties. nevertheless, there is growing evidence that phenolic metabolites act primarily as signaling molecules, notably in oxygen-stress-related pathways (williams et al., ) . consequently, smaller amounts are needed than for direct antioxidant reactions. this might explain the discrepancy between the low levels of free phenolics in the plasma and their apparent effects. an example may be the increased activity and the presence of phenolics in the plasma permits their likely uptake into most body tissues. this generality does not necessarily apply to the brain. except where there are specific transport proteins, most compounds above a molecular weight of da are excluded from the brain by the bloodebrain barrier. the barrier consists of tight connections between the endothelial lining of cerebral capillaries. it prevents the diffusion of most molecules from the blood into the cerebrospinal fluid. however, with anthocyanins (passamonti et al., ) and simple flavonols (youdim et al., ) , access to the brain apparently can occur within minutes of consumption. initial animal studies suggest uptake levels in the brain occur at about % that of other tissues (wu et al., ) . although fascinating, consumers are more interested (if at all) in what wines provide the maximal health benefits and under what conditions. regrettably, data on these vital concerns are lacking. grape cultivar, maturity, wine production, maturation and aging conditions all influence the amounts and types of phenolics present and thus their activity. the prophylactic action of wine against gastrointestinal diseases has been known for millennia, long before their microbial origins were ever suspected. in spite of this, the mechanism(s) by which this occurs remain poorly understood. the antimicrobial effect of alcohol was discovered in the late s. nevertheless, alcohol is not particularly antimicrobial, certainly at the concentrations found in wine (its sterilant action is optimal at about %). the antimicrobial action of wine is closely related to that of crushed grapes (Ö ncü l and karabiyikli, ). thus, the antibiotic action of wine likely relates more to its phenolic and acidic (vaz et al., ) contents, although wine's alcohol content undoubtedly augments their effectiveness. anthocyanins, which are weakly toxic to viruses, protozoans, and bacteria, become more so as a consequence of fermentation. other phenolic compounds in wine are bacteriostatic and fungistatic. for example, p-coumaric acid is particularly active against grampositive bacteria (e.g., staphylococcus and streptococcus), whereas compounds, such as quercetin, inhibit pathogenic gram-negative bacteria (e.g., escherichia, shigella, proteus, and vibrio) (vaquero et al., ) . phenolics may also be inhibitory to intestinal pathogens such as clostridium difficile, c. perfringens, and bacteroides (lee et al., ) . despite wine being more effective than mildly antimicrobial agents, such as bismuth salicylate (weisse et al., ) , full action may take several hours (møretrø and daeschel, ; dolara et al., ) . although most studies have involved bacteria grown on culture plates, wine has also been shown to be antimicrobial under simulated gastrointestinal conditions (vaz et al., ) . an indirect effect, limiting intestinal problems, is illustrated by the action of the colon flora on anthocyanin structure. it favors the growth of bifidobacterium and lactobacillus-enterococcus spp. (hidalgo et al., ) . these have been associated with a healthy gut microflora (hord, ) . there is also considerable variation in the effects of different flavanols and procyanidins, both promoting and inhibiting the adhesion of probiotic lactobacilli to the intestinal wall, depending on their metabolic modification during passage through the intestinal tract (bustos et al., ) . in addition, viniferin (a resveratrol derivative) can inhibit biofilm formation by pathogenic pseudomonas aeruginosa and escherichia coli (cho et al., ) . red wine can suppress biofilm formation by oral pathogens (muñ oz- . in most instances, the mechanism by which phenolics have their action is unknown. however, in the case of quercetin, the effect may be partially attributed to its inhibition of dna gyrase, whereas with epigallocatechin, disruption of cell membrane function appears central to its antibiotic action. alternative methods of action may involve suppression of cell adherence and colony formation on the gut lining (selma et al., ; truchado et al., ) . adherence is often a prerequisite for the cascade of events leading to disease development. low ph and the presence of various organic acids appear to accentuate the antimicrobial action of both wine phenolics and ethanol. organic acids may themselves be antimicrobial, as is the case with bacillus cereus (vaz et al., ) . wine is also active against several viruses including the herpes simplex virus, poliovirus, hepatitis a virus as well as rhinoviruses and coronaviruses. the effect on the latter two groups appears reflected in the reduced incidence of the common cold in moderate alcohol consumers (cohen et al., ) , particularly those drinking red wines (takkouche et al., ) . if you have to gargle, port is certainly one of the more pleasant. the antioxidant action of wine phenolics not only appears to play an important role in limiting low-density lipoprotein (ldl) peroxidation (maxwell et al., ; rice-evans et al., ) but also the action of lipoxygenases and enzymes generating ros. phenolics can also directly scavenge (quench) these radicals (e.g., superoxide and hydroxyl radicals), contributing to the action of cellular antioxidants. the oxidized flavonoid byproducts are much more stable (nonreactive) and tend to be quickly metabolized or eliminated by the kidneys. a flavonoid's quenching ability is largely dependent on the location and number of its oh groups as well as its glycosylation, sulfation, methylation, and acylation status (plaza et al., ) . in addition, phenolics can chelate iron and copper, limiting their involvement in radical formation (morel et al., ; rice-evans et al., ) or access to bacterial pathogens. phenolic can also limit the influx of calcium ions associated with oxidative stress (ishige et al., ). an antioxidant relatively unique to wine is resveratrol. it is a stilbene phenolic produced in response to plant stresses. it has greater antioxidant activity than common dietary antioxidants, such as vitamin e and ascorbic acid (frankel et al., ) . there is also direct evidence that resveratrol can enter the blood system at levels sufficient to suppress cyclooxygenase and lipoxygenase pathways. these are involved in the synthesis of proinflammatory mediators (bertelli, ) . in addition, resveratrol can activate proteins involved in nerve cell differentiation, synaptic plasticity, and neuronal survival (tredici et al., ) . supplemental protection may result from ethanol activating the cellular biosynthesis of hydroxytyrosol (a dopamine metabolite) (de la torre et al., ) . hydroxytyrosol is an important antioxidant and antiinflammatory agent. the most clearly established benefit of moderate alcohol consumption, notably wine, relates to a nearly %e % reduction in death rate due to cardiovascular disease (klatsky et al., (klatsky et al., , renaud and de lorgeril, , fig. . ) . alcohol consumption is also correlated with a decrease in the likelihood of intermittent claudication (pain or cramping in the calf of the leg). claudication is a common indicator of peripheral arterial disease. recent studies have confirmed that incidental factors, such as gender, race, lifestyle, educational level, etc. do not affect these results (see mukamal et al., ) . studies have also demonstrated that daily consumption of alcohol significantly reduces the incidence of other cardiovascular diseases, such as hypertension (keil et al., ) , heart attack (gaziano et al., ) , stroke (truelsen et al., ; hillbom, ) , and peripheral arterial disease (camargo et al., ) . those who consume wine moderately live, on average, . e . years longer than teetotalers and considerably longer than heavy drinkers. the prime area of contention is the degree to which these benefits accrue from the effects of ethanol vs. phenolic and/or other constituents (rimm et al., ) . atherosclerosis is the principal cause of most cardiovascular disease (libby, ) . it apparently results from chronic injury to the arteries ( fig. . ) . although associated with several independent factors, most damage is correlated with lipid oxidationda subgroup of cholesterol-apoproteins complexes (ldls). because of the hydrophobic nature of cholesterol and triglycerides, their transport in the plasma requires a special structure. as illustrated in fig. . , lipoprotein complexes consist of an outer membrane of phospholipids, within which apoproteins and free cholesterol occur. they enclose a hydrophobic core possessing numerous triglycerides and cholesteryl esters. metabolism of the enclosed lipids is regulated by the apoproteins in the outer membrane. normally, ldls supply cholesterol for cellular membrane repair and steroid synthesis. however, in high concentrations, they may accumulate in the artery wall. if they remain there for an extended period, their lipid content tends to become oxidized. in an oxidized state, lipids are cytotoxic and indirectly irritate the artery wall. as a consequence, special adhesion proteins attach to the artery wall. monocytes and helper t-cells of the immune system bond to these proteins. in addition, affected endothelial cells may secrete compounds, such as endothelin- . endothelin- activates monocyte and t-cell migration into the artery wall. procyanidins, principally found in red wines, are particularly effective in suppressing the production of endothelin- (corder et al., ) . anthocyanin metabolites are also effective modulators of endothelial function (edwards et al., ) . in the layer just underneath the endothelial lining (intima), accumulated monocytes mature into macrophages. both macrophages and t-cells may release a range of cytokines that further activate the immune system, involving localized inflammation. activated macrophages tend to engulf oxidized ldls. however, as the ldls are not degraded, their progressive accumulation gives the macrophage the appearance of being full of bubbles (termed foam cells). they are the first clear evidence of localized arterial swelling (plaques). occasionally plaques bulge into the vessel. more frequently, they initially enlarge outward into the surrounding tissue. action of immune cells in the plaque also induces migration of smooth muscle cells from the artery wall into the intima. here they proliferate and produce collagen, forming a fibrous cap over the plaque. additional ldls slowly collect, provoking further rounds of inflammation and plaque enlargement. these accretions may develop their own vasculature, becoming fibrous and inelastic. as the plaques enlarge, they may produce irregular protrusions into and block the artery lumen. even without restricting blood flow, plaques set the stage for platelet aggregation, clot formation (thrombus) and the blockage that can precipitate a heart attack or stroke. in the later phases of plaque formation, unknown factors enhance inflammatory changes in the plaque. these disrupt the integrity of the cap. for example, collagenases secreted by macrophages inhibit collagen synthesis by smooth muscle cells. sudden rupture of a plaque permits blood infiltration into the plaque. because plaques contain potent blood clotting factors, thrombus development is almost instantaneous. it is currently thought that plaque rupture is the principal factor inducting thrombus formation and precipitating a heart attack, stroke, or other cardiovascular trauma. atherosclerosis appears to be at least partially reversible, if risk factors such as smoking, high blood pressure, high dietary sources of cholesterol, and possibly infection by pathogens such as chlamydia pneumoniae and cytomegalovirus are eliminated. part of the reversal process involves the action of high-density lipoproteins (hdls). of the two principal forms, ethanol augments the presence of hdl , whereas exercise increases the level of hdl . the effect of ethanol on hdl concentration appears independent of beverage type (van der gaag et al., ) . either form of hdl favors the removal of cholesterol from the arteries, transferring it to the liver for metabolism. hdls also appear to interfere with ldl oxidation. because the hdl/ldl ratio affects the degree and rate of cholesterol turnover, the slower the rate, the greater the likelihood of oxidation (walzem et al., ) and eventual plaque formation. the beneficial effect of moderate alcohol consumption on the hdl/ldl ratio is now relatively clearly established. less well understood is its effect in lowering the concentration of c-reactive protein (crp) (levitan et al., ) . crp is an indicator of inflammation. its level usually rises in correlation with the risk of atherosclerosis. moderate alcohol consumption also reduces the incidence of another risk factor for cardiovascular diseased type diabetes. chronically high values of circulatory glucose, associated with type diabetes, appear to generate high plasma triglyceride and ldl levels. however, the benefits of wine's alcohol content on glucose and insulin metabolism appear not to occur if intake is not coincident with meal consumption (augustin et al., ) . phytoestrogens, such as resveratrol, have a similar effect in reducing triglyceride and ldl contents in the circulatory system (see bisson et al., ) . another of alcohol's beneficial influences involves disruption of events leading to clot formation. platelets are less "sticky" in the presence of alcohol and thus less likely to aggregate, limiting clot formation. alcohol also increases the level of prostacyclin (interferes with clotting) and raises the level of plasminogen activator (a clot-dissolving enzyme). clots, adhering or becoming stuck to the roughened surfaces of narrowed atherosclerotic vessels, may block blood flow. the oxygen deficiency and cell death that result are central to the damage caused by a heart attack or stroke. thus, it is not surprising that inhibitors of platelet aggregation reduce the frequency of these cardiovascular crises and their sequelae. it is the rationale for recommending the daily consumption of acetylsalicylic acid (an inhibitor of platelet aggregation). ethanol (renaud and ruf, ) as well as wine phenolics, such as resveratrol and anthocyanins, have similar effects (fig. . ). an additional example of the beneficial effects of limited alcohol intake is the relation between alcohol dehydrogenase (adh) genotype and the incidence of myocardial infarction. individuals homozygous for adh c* (slow metabolizers of ethanol) are significantly less likely to have a heart attack than heterozygous individuals and even less likely than homozygous individuals for adh c* (fast metabolizers of ethanol) (hines et al., ) . individually, many phenolics, such as resveratrol, catechin, epicatechin, and quercetin appear to have inhibitory effects on platelet aggregation (keli et al., ) . in an in vitro study, though, monomeric or low-molecular-weight flavonoids and hydroxycinnamic acids enhanced platelet aggregation and ldl oxidation, with only large polymers being inhibitory (shanmuganayagam et al., ) . in another investigation, the combined effect of several phenolics was superior to single compounds (wallerath et al., ) . the action partially results from the enhanced synthesis and release of nitric oxide by endothelial cells. this has been found to occur at resveratrol concentrations associated with moderate wine consumption (gresele et al., ) . chlorogenic acid also appears to activate nitric oxide production (mubarak et al., ) . nitric oxide induces vasodilation (by relaxing vascular smooth muscle), reduces blood pressure, and limits platelet adhesion to blood vessel endothelia. indicative of the complexities of such interactions is the observation that flavonoids may also inactivate nitric oxide (verhagen et al., ) . in addition, nitric oxide, notably as peroxynitrite, oxidizes ldls. clearly, much more still needs to be known before a clear picture emerges. in addition to affecting platelet aggregation, wine phenolics can bind directly with ldls (limiting their oxidation), indirectly reduce macrophage-mediated oxidation and preserve the action of paraoxonase (further protecting ldls from oxidation) (aviram and fuhrman, ) . furthermore, red wine phenolics directly or indirectly limit the migration of smooth muscle cells into the intima of artery walls. these influences probably explain some of the added benefits of wine vs. other alcoholic beverages in reducing the incidence and severity of cardiovascular disease. although flavonoids tend to suppress inflammation, conflicting observations put the clinical significance of their antiinflammatory action to atherosclerosis in question. whether this might also apply to the antiinflammatory effects of wine phytoprostanes (degradation products of linolenic acid) is unknown. red wines usually have been credited with superior health-related benefits than white wines, especially relative to cardiovascular disease. this presumably results from their higher flavonoid content (tian et al., ) . this view is supported by studies where white wine has shown the same effects as red wine, when supplemented with grape polyphenolics (fuhrman et al., ) . nevertheless, prolonged skin contact, or choice of particular cultivars, can enhance the presence of phenolic acids in white wine. common phenolics in white wine, such as caffeic and coumaric acids as well as flavonols such as quercetin, are well-known potent antioxidants. the low sodium content of wine is an incidental benefit. it may permit wine consumption by those on a low-sodium diet, for example those with high blood pressure or heart attack victims. the high potassium to sodium ratio of wine ( : ) is also advantageous. as noted, many of the beneficial influences of alcohol and wine consumption show a j-shaped curve ( fig. . ). this also applies to its effect on age-related macular degeneration (obisesan, ; fraser-bell et al., ) . the disease expresses itself as a progressive degeneration of the central region of the retina (macula), leading to blurred or distorted vision. it results as a consequence of local atherosclerosis that deprives the retina of oxygen and nutrients. it is the leading cause of blindness in adults over the age of . a similar relationship has been found for cataract development. in both conditions wine antioxidants are suspected to be the active protective agent. in this regard, quercetin appears to be more protective (against light-induced lipid peroxidation) than either anthocyanin-or phenolic acid-rich constituents (liu et al., ) . however, wines high in ethanol content may undo these benefits, by promoting pro-oxidant action. alzheimer's, a devastating neurodegenerative disease, afflicts more than million people. not surprisingly, researchers have investigated whether wine consumption affects the incidence of this and other neurodegenerative diseases (barnham et al., ) . flavonoids not only activate key respiratory enzymes in mitochondria (schmitt-schillig et al., ) , but also decrease the production of reactive oxygen species, by stimulating the production of catalase, superoxide dismutase, glutathione reductase, and glutathione peroxidase . a pattern appears to apply here, as with most health-related benefits of wine and alcohol consumptiondmoderate intake bing beneficial, whereas high consumption or abstinence is deleterious. alzheimer's disease has been correlated with the accumulation of extracellular amyloid b-peptide (plaque) and the formation of intracellular neurofibrillary tangles containing tau-protein. many in vitro studies have shown that antioxidant compounds, such as vitamin e, protect neurons from b-amyloid accumulation. tannins also inhibit the formation of and destabilize preexisting b-amyloid fibrils (ono et al., ; guéroux et al., ) , whereas resveratrol promotes the degradation of amyloid b-peptides (marambaud et al., ) . wine consumption is also linked to a reduction in the incidence of alzheimer's disease (truelsen et al., ; letenneur, ; luchsinger et al., ) . even mild cognitive impairment and the progression of idiopathic dementia may be reduced with moderate alcohol consumption (solfrizzi et al., ) . grape juice has also been found to be effective in this regard (krikorian et al., ) . like other health benefits, these finding may not, in and by themselves, justify wine consumption, but they are encouraging to those who choose wine as part of their preferred lifestyle. age-related bone mass loss affects both sexes but is more frequent in postmenopausal women. many risk factors including dietary influences and hormonal supplements, can affect its progress and severity. of these factors, moderate alcohol consumption has been found to favor bone retention (ganry et al., ; ilich et al., ) . tucker et al. ( ) found data consistent with higher benefits from wine than other alcoholcontaining beverages. the source of these benefits may be a combination of enhanced calcium uptake, associated with alcohol consumption (ilich et al., ) , the phytoestrogen effects of phenolics, such as resveratrol and kaempferol, or other unsuspected influences. a number of drugs used in treating arthritis tend to irritate the stomach lining. this side-effect may be counteracted by the mildly acidic, dilute alcohol content of table wines. other beneficial effects connected with moderate wine consumption may accrue from its mildly diuretic and muscle relaxant properties. the diuretic action of wine can help reduce water retention and minimize joint swelling. wine can also directly reduce muscle spasms and the stiffness associated with . wine, food, and health arthritis. the antiinflammatory influences of wine phenolics, notably resveratrol (yang et al., ) , may also play a role in diminishing the suffering associated with arthritis and other diseases associated with chronic inflammation (schueller et al., ) . wine consumption has been shown to attenuate insulin-resistance in type diabetes (dixon et al., ; napoli et al., ) . this may result from wine phenolics quenching oxygen radicals, thought to be pivotal in the damage associated with the disease. type diabetes appears to result when body cells fail to respond properly to the presence of insulin. the incidence of metabolic syndrome is also lower in wine drinkers (rosell et al., ) . these effects may be due to one or more of the following: the influences of alcohol on metabolism; the antidiabetic properties of the element vanadium (for which wine is a significant source) (brichard and henquin, ; teissèdre et al., ) ; the hypoglycemic and hypolipidemic effects of phenolics such as resveratrol (su et al., ) ; and/or through some effect on endothelial nitric oxidase synthase (leighton et al., ). it appears there may be considerable specificity. for example, in a comparison between malvidin-and delphinidin- -o-glucosides, only the predominant anthocyanin in grapes (malvidin) seems to have a significant hypoglycemic effect (lida et al., ) . relative to diabetes mellitus (type diabetes), moderate consumption of dry wine was found to present no adverse effects on sugar control (gin et al., ; bell, ) . although wine does contain residual sugars, the most common, fructose, is poorly transported across the gastrointestinal tract. most of what is absorbed is rapidly removed from the blood by the liver, where it is metabolized into glycerol and often stored as fat. it does not stimulate pancreatic insulin release. red wine (and a diet rich in antioxidants) appear to slow the progression of kidney damage (necropathy), occasionally associated with diabetes (zhu et al., ) . in an epidemiological study, knudsen et al. ( ) found a strong link between alcohol consumption and a reduced prevalence of goiter and solitary thyroid nodules. the origin of this apparent protective effect is unknown. drinking water has long been associated with reducing the development of kidney stones. increased urine production is thought to limit calcium oxalate crystallization. what is new is the observation that wine consumption further reduces the production of these painful and dangerous inclusions (curhan, ) . moderate wine consumption has been correlated with reduced incidence in some cancers (see bianchini and vainio, ) (e.g., the kidney) but increased risk for others (notably the throat and gastrointestinal tract) (ebeler and weber, ; parry et al., ) . this potential varies markedly, primarily based on the amounts habitually consumed. for other cancers, moderate consumption appears to be neither protective nor a risk factor (e.g., prostate cancer) (chao et al., ) . conversely, excessive consumption of alcoholic beverages increases the risk of a range of cancers, notably those of the oropharynx, larynx, esophagus, liver, colon, rectum, and breast (connor, ) . because ethanol is not directly carcinogenic, negative associations with alcohol consumption presumably relate to carcinogens potentially present in wines (e.g., ethyl carbamate). thankfully, its carcinogenicity is reduced at the alcohol concentration typical of table wines. more significant, though, maybe acetaldehyde, a common denominator in gastrointestinal cancers (salaspuro, ) . depending on the type of wine consumed, short-term but high concentrations of acetaldehyde may occur in the saliva and gastric juice (lachenmeier and sohnius, ) . acetaldehyde may also accumulate due to the action of alcohol dehydrogenase in the digestive tract, microbial alcohol metabolism (notably the colon), and malfunction of cellular acetaldehyde dehydrogenase. certain wine phenolics can be protective, whereas others mutagenic, especially at high concentrations. for example, quercetin can induce mutations in laboratory tissue cultures but is a potent anticarcinogen in whole-animal studies (fazal et al., ). this apparent anomaly may result from differences in the concentrations of quercetin used, and/or the low levels of metal ions and free oxygen found in the body (vs. tissue culture). in addition, quercetin, along with several other phenolics that are potential carcinogens, lose this attribute when present as a glycoside. most phenolics in the plasma occur in some conjugated state, not as free phenolics. in addition, phenolics may detoxify the small quantities of nitrites commonly found in food. however, in the presence of high nitrite concentrations (a preservative found in smoked and pickled foods), nitrites are converted into diazophenols (weisburger, ) . these appear to favor the development of oral and stomach cancers. potential health issues several phenolics can limit or prevent cancer development through a diversity of effects, such as dna repair, carcinogen detoxification, enhanced apoptosis (programmed cell death), disrupted cell division (hou, ; aggarwal et al., ) , or enhanced immunostimulation (tong et al., ) . for example, resveratrol induces the redistribution of the fas receptor. it is a cellular attachment site for tnf (tumor necrosis factor). its action is part of a sequence that can lead to cancer cell apoptosis (delmas et al., ) . resveratrol is an inhibitor of angiogenesisdthe production of new vasculature essential for most tumor growth. other effects of resveratrol include inhibition of cyclooxygenase- (subbaramaiah et al., ) and cytochrome p a (chun et al., ) . cyclooxygenase- is thought to be involved in carcinogenesis, whereas p a is an important hydroxylase. it can convert several environmental toxicants and procarcinogens into active carcinogens. flavones and flavonols strongly restrict the action of common dietary carcinogens, notably heterocyclic amines (kanazawa et al., ) . it is estimated that these amines, produced during cooking, are consumed at a rate of approximately . e mg per day (wakabayashi et al., ) . antitumor activity is also associated with acutissimin, a flavono-ellagitannin found in oakmatured red wine. the antiallergic and antiinflammatory properties of flavonoid phenolics probably also contribute to the anticancer aspects of these flavonoids (see middleton, ) . the major exception to the general benefit of moderate wine consumption may be breast cancer (viel et al., ) . the connection is more evident in those with the adh c* (fast metabolizers of ethanol to acetaldehyde) (terry et al., ) . however, findings from the long-duration framingham study indicate no relationship between moderate alcohol consumption and the incidence of breast cancer (zhang et al., ) . ethanol, although not itself a carcinogen, can enhance the transforming effect of some carcinogens. another example of a negative effect of wine consumption, at least in excess of moderate intake, is to increase the incidence of mouth and throat cancers (barra et al., ) . alcoholic beverages may also induce a diversity of allergic and allergy-like reactions. in sensitive individuals, these may express as rhinitis, itching, facial swelling, headache, cough, or asthma. the primary culprit inducing bronchial constriction, at least in some asthmatics, is sulfur dioxide (dahl et al., ) . wine containing an abnormally high sulfur dioxide concentration ( ppm sulfite) induce a rapid drop in forced expiratory volume (vally and thompson, ) , recovery taking about e min. the same individuals did not respond to wine containing , , or ppm sulfite. fig. . illustrates the range of sulfur dioxide contents potentially found in californian wine. thus, the sulfite levels normally found in wine seem not to be a major factor in wine-induced asthmatic responses (vally et al., ) . why sensitive asthmatics episodically react to wines with low so content may be related to changes in their asthma control. surprisingly, red wines appear to provoke more asthma problems than white wines, even though red wines typically have lower sulfur dioxide contents than white wines. californian wines (mg/l). reprinted from peterson, g.f., kirrane, m., hill, n., agapito, a., . a comprehensive survey of the total sulfur dioxide concentrations of american wines. am. j. enol. vitic. , e , permission conveyed through copyright clearance center. . wine, food, and health the rapidity of the reaction to sulfite suggests some malfunction in the amount of glutathione in lung tissue or the activity of glutathione s-transferase in reducing sulfite to glutathione s-sulfonate. normally, sulfite is rapidly converted to sulfate by sulfite oxidase in the blood. however, low levels of this enzyme could permit sulfite to persist, provoking a heightened response in hypersensitive individuals. at greater risk are individuals afflicted with a rare, autosomal, genetic disease, caused by a deficiency in sulfite oxidase (shih et al., ; crawhall, ) . affected individuals must live on a very restricted diet, low in sulfur-containing proteins. it is estimated that the synthesis of sulfite, associated with normal food metabolism, generates approximately . g sulfite/day. the sulfites in wine contribute only marginally to this amount but may be temporally significant. because of the gravity of sulfite oxidase deficiency, most affected individuals do not reach adulthood. another allergy-like reaction provokes rapid facial and neck flushing (cutaneous erythema). it develops shortly after alcohol consumption. other symptoms often include peripheral vasodilation, elevated heart rate, nausea, abdominal discomfort, and bronchoconstriction. the syndrome is associated with a malfunctional form of mitochondrial acetaldehyde dehydrogenase (aldh * ) (enomoto et al., ; eriksson et al., ) and is particularly pronounced in the homozygous state. aldh is the principal enzyme oxidizing acetaldehyde to acetic acid. it is estimated that up to % of eastern asians possess at least one malfunctional aldh allele and express some degree of allergy-like reaction to alcohol consumption. it has been suggested that the aldh mutant, frequently found in eastern asians, may reflect an evolutionary adaptation to the endemic occurrence of hepatitis b (lin and cheng, ) . the mutation could have induced alcohol aversion, thereby avoiding synergism between alcohol and hepatitis b-induced liver damage. elevated levels of acetaldehyde appears to activate the release of histamine from mast cells. it subsequently induces vasodilation and an associated influx of blood (flushing). the connection between acetaldehyde and histamine is supported by the action of antihistamines in reducing the reaction, if taken in advance of an alcohol challenge (miller et al., ). an alternative proposal is that this flushing reaction results from a direct, cutaneous, alcohol-induced vasodilation. the phenomenon tends to be suppressed by acetylsalicylic acid (aspirin), if taken in advance (truitt et al., ) . the unpleasant side-effects of acetaldehyde accumulation is used in treating alcoholism. it involves taking disulfiram (a potent inhibitor of aldh) prior to alcohol consumption. a facial flushing, concomitant with alcohol consumption but devoid of other symptoms, is occasionally experienced by caucasians. whether this is related to an aldh malfunction is unclear. the histamine content of wine has frequently been thought to contribute to several allergy-like reactions. however, wine is typically low in histamine content. fig. . illustrates the range found in some wines. thus, it seems unlikely that a wine's histamine content is a major inducer of allergy-like reactions. this is supported by a double-blind study of people, selfreportedly wine intolerant. reactions to two pinot noir wines, differing in histamine content ( . vs. . mg/ l) were not significantly different (kanny et al., ) . other common foods are considerably higher in histamine content, for example cheeses. this does not necessarily exonerate biogenic amines from being somehow involved. people vary in diamine oxidase activity (a histamine inactivator) (wantke et al., ) and how alcohol (via acetaldehyde) suppresses its action . alcohol also can enhance the permeability of the intestinal lining to histamine. in addition, acetaldehyde accumulation can activate histamine release (harada et al., ) . this may explain the benefit antihistamines have in diminishing the rhinitis occasionally associated with wine consumption (andersson et al., ) . in addition, antihistamines counteract the broncho constriction in individuals showing histamine intolerance. idiopathic allergic and other immune hypersensitive responses to wine are difficult to predict or diagnose. reactions may include the induction of headaches, nausea, vomiting, general malaise, or a combination of these. in a few instances, ige-related anaphylaxis reactions have been reported to grape pr proteins (endochitinase and thaumatin) (pastorello et al., ) . the effects may involve urticaria/angioedema (red patches or wheals on the skin/swelling) and occasionally shock. residual amounts of fining agents, such as egg whites, have also been implicated in some allergic reactions (marinkovich, ) . in a double-blind, placebocontrolled trial, wines fined with egg white, isinglass, or nongrape derived tannins presented "an extremely low risk of anaphylaxis" to egg-, fish-, or peanutallergic consumers (rolland et al., ) . in an elisa analysis, only egg white and lysozyme could be detected in wine samples (weber et al., ) . nevertheless, with more than compounds potentially occurring in wine, it is not surprising that some individuals may occasionally show some form of adverse reaction to some wines. in addition to physiological reactions to wine constituents, there is a wide range of equally important psychological responses (rozin and tuorila, ) , both positive and negative. traumatic memories, associated with the first exposure to, or excessive consumption of, a particular beverage can create an association that lasts a lifetime. other people have come to associate certain products with social groups, lifestyles, or behaviors. such attitudes can make the beverage either unacceptable or desirable as the case may be. in the s, there were many reports linking gout with wine consumption, notably port. gout is caused by the localized accumulation of uric acid crystals in the synovium of joints. their presence stimulates the synthesis and release of humoral and cellular inflammatory mediators (choi et al., ) . gout is also associated with reduced excretion of uric acid in the kidneys. mutations in the gene that encodes urease, the enzyme that metabolizes uric acid to allantoin (a soluble by-product), is often implicated in gout. dietary predisposing factors for gout include red meat, seafood, and beer. this is presumably because purines, the principal source of uric acid, are found in higher concentrations in these products than many other foods or beverages. alcohol consumption may occasionally aggravate gout by increasing lactic acid synthesis. it, in turn, favors uric acid reabsorption by the kidneys. despite this, wine consumption appears not be associated with an increased risk for gout. in contrast, it seems to favor reduced serum urate levels (choi and curhan, ) . medical historians suspect the nineteenth century gouteport association was connected with leadinduced kidney damage (yu, ; emsley, emsley, / . samples of port from the nineteenth century show high lead contents. lead contamination probably came from the stills used in preparing the wine spirits added in port production. in addition, the former use of pewter and lead-glazed drinking cups and prolonged storage of port in lead crystal decanters or stemware could have further augmented lead content (falcone, ; guadagnino et al., ) . people occasionally avoid wine because it induces headaches. regrettably, the wine/headache connection is still poorly understood. however, effective differentiation between wine-induced headaches may be pivotal to discovering their causes and possible solutions. one of the most severe headache syndromes, potentially associated with wine consumption, is the migraine. migraines appear to be induced, but inconsistently, by a wide range of environmental stimuli, often in tandem. an association between it and red wine consumption has been noted since roman times. the dilation of cerebral blood vessels, partially associated with histamine release, appears to be a common element in many headache syndromes. migraines may be one of them, although current thought suggests a neurological rather than a vascular origin. in addition, a double-blind study seemingly has exonerated histamine in most redwine-induced migraine headaches (masyczek and ough, ) . in addition, migraine attacks are more often associated with consuming spirits and sparkling wines, both lower in histamine content that table wines or beer (nicolodi and sicuteri, ) . however, the former are often taken alone, leading to more rapid and higher spikes in blood alcohol content. alcohol may be directly involved in migraine induction through vasodilationdby activating meningeal vessel-associated trigeminal neurons (nicoletti et al., ) . alcohol's potential to reduce cerebral glucose metabolism (volkow et al., ) could also be a contributing factor. other potential disruptive aspects on brain function may relate to the slow rate of alcohol metabolism by cerebral adh. thus, more alcohol may be metabolized by cytochrome p e, a process that generates acetaldehyde as well as ros. the more frequent association of red wines with several headache sequelae may be due to their higher phenolic content. on average, red wines contain about mg/l phenolics, vs. mg/l for white wines. some phenolics can suppress the action of platelet phenol sulfotransferase (pst) (jones et al., ; yeh and yen, ) , several isozymic forms (m and p) of which detoxify biogenic amines and phenolics via sulfation. low levels of platelet-bound pst-p have been correlated with migraine susceptibility (alam et al., ) . the accumulation of small phenolics (those readily absorbed) in the blood could prolong the action of potent hormones and nerve transmitters (e.g., histamine, serotonin, dopamine, adrenalin, and noradrenaline). small phenolics can also promote platelet aggregation and blood vessel dilation. the associated increase in intercranial pressure may participate in a migraine attack (pattichis et al., ) . abnormal and cyclical patterns in platelet sensitivity to -ht release in migraine-prone individuals (jones et al., ; peatfield et al., ) may explain the inconsistent association of wine consumption with migraine induction. in the treatment of cluster-headaches, small doses of lithium have been suggested as preventive (steiner et al., ) . because some red wines have a higher than average lithium content, the possibility exists that they might limit the development of, rather than induce, this headache syndrome. another recognized headache syndrome is called the red wine headache (kaufman, ) . it may develop within minutes of consuming red wine, often being dose-related. the headache reaches its peak within w h, tends to fade but returns roughly h later. the headache seems related to the release of type e prostaglandins, important chemicals involved in dilating blood vessels. their release can be activated by phenolics (padilla et al., ) as well as alcohol (parantainen, ) . prostaglandins may also activate pain receptors around blood vessels (wienecke et al., ). this association may explain why prostaglandin synthesis inhibitors (e.g., acetylsalicylic acid, acetaminophen, or ibuprofen) may limit the development of some winerelated headaches (if taken about h before consumption) (kaufman, ) . a separate wine-related headache has been dubbed the red head (goldberg, ) . it develops within an hour of waking, after drinking no more than two glasses of red wine the previous evening. the headache, associated with nausea, is particularly severe when reclining. although the headache is somewhat relieved by standing, it itself exacerbates the nausea. the headache usually lasts a few hours before dissipating. a similar phenomenon has been reported with some white wines, or mixtures of white wine, taken alone or with coffee or chocolates. its chemical cause is unknown (kaufman, ) . because tannins are poorly absorbed in the upper digestive tract, in contrast to monomeric phenolics, the latter are likely the primary phenolic headache activants. this may explain why aged red wines (in which most phenolics occur as large polymers) tend to be less associated with headaches than their younger counterparts. large tannin polymers remain largely unmodified until reaching the colon, where bacteria degrade them (déprez et al., ) . because this can take up to days, they presumably are not (or not recognized to be) involved in wine-induced headaches. phenolic absorbed into the blood are primarily detoxified by being methylated or sulfated but may also become more "toxic" (to o-quinones). the latter can retard the breakdown of dopamine and restrict access to m-opioid (painkilling) receptors, exacerbating the pain associated with cerebral blood vessel dilation. nonetheless, some phenolics (e.g., resveratrol) limit, rather than augment, headache development. it inhibits the expression of cyclooxygenases, involved in the synthesis of prostaglandins (jang and pezzuto, ) . although red wines are generally associated with headache production, white wines are occasionally associated with their production (relja et al., ) . their characteristics and etiology are even less well understood than those evoked by red wines. in some individuals, this situation may be associated with a sensitivity to sulfites but atypically. one of the most recognized alcohol-related headache phenomena is associated with binge drinkingdthe hangover (veisalgia) (wiese et al., ) . although not consistently associated with a headache, it is frequently part of the sequelae. hangovers are characterized by tremulousness, palpitations, tachycardia, sweating, loss of appetite, anxiety, nausea, and possibly vomiting and amnesia . when accompanied with a headache, it possesses symptoms resembling a migraine. the headache may be global but frequently concentrated anteriorly, associated with heavy, pulsesynchronous throbbing. it usually starts a few (w h) after the cessation of drinking, when blood alcohol level is declining and other hangover symptoms have already developed (sjaastad and bakketeig, ; verster et al., ) . duration is seldom more than h. despite its all-too-frequent occurrence, the causal mechanism(s) remains unclear. most data suggest that alcohol-induced cerebral inflammation is the primary cause . this may operate directly via tissue dehydration and electrolyte imbalance (due to vasopressin enhanced urination) or indirectly via the toxic effects of acetaldehyde (quertemont et al., ) . ethanol can also promote hepatic glycogen breakdown, glucose release, loss via the kidneys, and induction of hypoglycemia. in addition, activation of the liver's microsomal ethanol oxidation pathway releases ros, causing cellular damage and multiple metabolic disruptions. that the severity of a hangover may be reduced by prostaglandin synthesis inhibitors suggests that they may also play a role in hangover sequelae (kaivola et al., ) . as the old spanish proverb noted: wine hath drowned more men than the sea because glutathione inactivates free radicals, taking an amino acid supplement (n-acetyl-cysteine) has been suggested as a partial remedy. it is rich in cysteine, an amino acid that forms the core of glutathione. in addition, glutathione facilitates the conversion of acetaldehyde to acetic acid. disruption of membrane function and cerebral neurotransmitter action by acetaldehyde is presumably the rationale for commercial products, such as hangover helper and rebound. they are designed to counter the effects of acetaldehyde. congeners (such as fusel alcohols and methanol) could exacerbate the effects of ethanol and acetaldehyde. however, because their content in wine is low, they are unlikely to be involved in wine-induced hangovers. some purported remedies, such as artichoke extract, have not stood up to rigorous clinical testing (pittler et al., ) , but others, such as an opuntia fiscus-indica (prickly pear) extract, apparently reduced the severity of some hangover symptoms (wiese et al., ) . that hangovers have been associated with deregulation of cytokine pathways (kim et al., ) , may explain the reported value of pyritinol (a vitamin b derivative) as a treatment (khan et al., ) . mineral deficiencies have also been correlated with hangovers (min et al., ) . combined with restraint, taking wine with a meal is probably the best means by which to avoid a hangover. food delays the movement of alcohol into the intestinal tract, thereby slowing alcohol uptake ( fig. . ) and correlating uptake closer to the liver's ability to metabolize ethanol. in addition, delayed transfer to the intestinal tract slows phenolic uptake (and other potential provocateurs). wine tasting is not normally considered a hazardous occupation. however, recent studies show that dental erosion is an occupational hazard (mok et al., ; mandel, ; mulic et al., ) . damage results from the frequent and extended exposure to wine acids, correspondingly, white wines are generally more corrosive than reds (willershausen et al., ) . saliva is diluted and washed away, resulting in the oral ph falling to that of the wine (obreque-slier et al., ) . this causes calcium to dissolve out of tooth enamel, softening and making it susceptible to erosion by masticatory forces and tooth brushing. exposure times as short as min can be harmful (lupi-pegurier et al., ) . demineralization commences at about ph . . dental erosion is unlikely to be a significant problem for the typical consumer who takes wine with meals. food and salivary secretion limit, if not prevent, tooth enamel demineralization. after many years, professional wine tasters may experience tooth disfiguration, affecting both tooth shape and size. cupping, a depression in the enamel, exposing dentine at the tip of molar cusps, is a frequent clinical sign. erosion can also contribute to severe root abrasion at the gum line. the good news is that not all tasters are equally at risk (mulic et al., ) . protection is partially achieved by rinsing the mouth with an alkaline mouthwash after tasting, application of a fluoride gel (such as apf) and refraining from tooth brushing for at least h after tasting. the delay permits minerals in the saliva to rebind with enamel. for more protective protocols see ranjitkar et al. ( ) . the use of remineralizing agents, such as tooth mousse, also helps prevent dental erosion (piekarz et al., ) . in contrast to this risk factor, consuming red wine may have some direct oral benefits. proanthocyanidins can limit the adherence and biofilm-forming activity of caries-inducing streptococcus mutans (daglia et al., ) . gibbons ( ) provides a fascinating insight into the association of this bacterium with changes in human diet which resulted from a switch from a hunter-gather to an agriculture lifestyle. mark twain also made pronouncements about dental health, which might be equally applied to wine: "i always take it (scotch whiskey) at night as a preventive of toothache. i have never had the toothache; and what is more, i never intend to have it". from europe and elsewhere. fetal alcohol syndrome refers to a set of phenomena including suppressed growth, mild mental retardation, and subtle facial abnormalities (wattendorf and muenke, ) . it was first described in and appeared most markedly in the children of alcoholic mothers. they tended also to be heavy smokers, users of illicit drugs, consumers of large amounts of coffee, had poor nutrition, or a combination of these (scholten, ; whitten, ) . although associated with alcohol uptake, the accumulation of acetaldehyde may be the principal toxicant. even more subtle effects have now been associated with alcohol consumption, generating the fetal alcohol spectrum disorders. because the consequences may be lifelong, it is generally recommended that pregnant women, or those desirous of becoming pregnant, refrain from alcohol consumption. although abstinence may be unnecessary (kesmodel et al., ) , erring on the side of caution can supply desired peace-of-mind. this also applies to breast feedingd alcohol in breast milk could be detrimental to infant development. the presence of toxins in wine is seldom mentioned, outside academic circles, presumably because of their minimal presence. the only mycotoxin for which there may be regular analysis is ochratoxin a (o'brien and dietrich, ; varga and kozakiewicz, ) , produced by several black aspergilli (notably aspergillus carbonarius) (somma et al., ) . preliminary data suggests that most ochratoxin a is eliminated (destroyed/precipitated) during and after fermentation/maturation (fernandes et al., ) . other potential mycotoxins that could occur in wine include isofumigaclavine, festuclavine, and roquefortine, all produced by penicillium spp. (moller et al., ) , aflatoxins (el khoury et al., ) from aspergillus flavus, fumonisins from aspergillus niger (mogensen et al., ) , and trichothecenes by trichothecium roseum (schwenk et al., ) . because these fungi are secondary saprophytes, they typically occur only on rotted grapes (thankfully, unlikely on noble-rotted grapes). although the exclusion of all diseased grapes is essentially impossible, their inclusion is limited as much as feasibly possible. pesticide residues are other potential toxins. their levels are usually below those known to be toxic, partially due to regulations limiting their use, precipitation or metabolism during winemaking and degradation during maturation. in addition, most importing countries possess regulations on permissible levels and systems to check for compliance. achieving a zero concentration is probably impossible, if only because of our increasing technical ability to detect their presence at increasingly infinitesimal levels. methanol is present but in amounts insufficient to have any known negative consequences. the same also appears to be true for diacetyl and other potentially toxic compounds. ethyl carbamate, a carcinogen, is no longer likely to occur, since its origin during wine production can be effectively avoided. it may initially be disconcerting to think of trace amounts of toxins in wine, but this situation applies to all food, water, and air. xenobiotics are an inescapable aspect of life, both modern and ancient. their universal presence in the natural environment presumably provided the selective pressures that favored the evolution of organs of detoxification (e.g., the liver and kidneys) and the presence of multiple detoxifying enzyme systems. thankfully, our bodies inactivate most xenobiotics rapidly and effectively, without our conscious knowledge. in addition, governmental agencies set regulations and assess compliance to limit most toxicants to well below known safe limits. as long as exposure to toxicants is kept to a bare minimum, consumers can basically forget they exist. the most important wine contraindication relates to those with a past history of alcohol abuse. for the majority of adults (except pregnant women), moderate wine consumption appears to have significant health benefits. nevertheless, there are several situations in which wine consumption, even in moderate amounts, can complicate or diminish the effectiveness of disease treatment. the acidic nature of wine can aggravate inflammation and slow the healing of ulcers in the mouth, throat, stomach, and intestinal tract. other constituents in wine may also be detrimental in this regard. thus, all beverages containing alcohol are usually contraindicated in cases of gastritis, gastric cancer, and bleeding in the upper digestive tract. nevertheless, the prophylactic action of red wine against helicobacter pylori and the suppression of histamine production by the gastric mucosa (masquelier, ) may require a reconsideration of the old prohibition in mild cases. in the presence of pancreatitis, alcohol is absolutely contraindicated. wine, along with other alcoholic beverages, may provoke gastroesophageal (acid) reflux in individuals prone to this syndrome. with liver disease, the consumption of wine is normally contraindicated. the presence of alcohol puts additional stress on an already weakened vital organ. chronic alcohol abuse can lead to cirrhosis of the liver. in acute kidney infection, wine should be avoided. the consumption of alcohol increases the burden on an organ essential to eliminating toxic metabolic wastes. with prostatitis or genitourinary infections, the consumption of alcohol can complicate matters. the diuretic action of wine may increase the frequency of urination, or conversely it may induce highly painful urinary retention. in epilepsy, the consumption of even moderate amounts of wine may increase the frequency of seizures. consumption should be strictly limited in most situations of hypertension, hemorrhagic stroke, or atrial fibrillation. patients, about to undergo surgery, are advised to avoid all alcoholic beverages well before surgery. this avoids increasing any tendency to enhance intra-and postoperative bleeding (wolfort et al., ) , due to alcohol's reduction of platelet clotting. the consumption of alcohol is also ill advised when eating certain mushrooms. the most well-known example is the antabuse reaction associated if alcohol is consumed with coprinus atramentarius (inky cap). another mushroom generating the same response is boletus luridus (budmiger and kocher, ) . the antabuse reaction derives its name from the trade name of disulfiram, a medication used in the treatment of alcoholism. it functions as an inhibitor of acetaldehyde dehydrogenase. even small amounts of alcohol consumed while taking disulfiram can generate very unnerving reactions (e.g., flushing, sweating, weakness, vertigo, blurred vision, difficulty breathing, nausea, chest pain, palpitation, and tachycardia). in severe cases, the reaction can provoke acute congestive heart failure, convulsion, and death. similar symptoms may develop in sensitive individuals when alcohol beverages are consumed while taking certain medications (e.g., cephalosporins, griseofulvin, chloramphenicol, sulfonylurea, metronidazole). in addition to the reactions noted above, consumption of alcohol while taking certain medications can generate dangerous conditions. most of the literature comes from studies on alcoholics or binge drinkers. this limits the potential applicability of the data to conditions of moderate consumption and when taken with food. nevertheless, even small amounts of alcohol may cause loss of muscle control in people taking tricyclic antidepressants. in addition, red wines can reduce the effectiveness of mao (monoamine oxidase) inhibitors, used in controlling hypertension. long-term acetaminophen use can enhance alcohol-induced kidney damage. other contraindications involve the intensification of the effects of barbiturates and narcotics. in combination with certain antidiabetic agents, such as tolbutamide and chlorpropamide, alcohol can cause dizziness, hot flushes, and nausea. mild reactions may occur with a wide range of other medications, such as sulfanilamide, isoniazid, and aminopyrine. additional details may be found in adams ( ) , fraser ( ) , and weathermon and crabb ( ) . in conclusion, mark twain crystalizes what so often seems to be the 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key: cord- - gh tg authors: uversky, vladimir n title: the alphabet of intrinsic disorder: ii. various roles of glutamic acid in ordered and intrinsically disordered proteins date: - - journal: intrinsically disord proteins doi: . /idp. sha: doc_id: cord_uid: gh tg the ability of a protein to fold into unique functional state or to stay intrinsically disordered is encoded in its amino acid sequence. both ordered and intrinsically disordered proteins (idps) are natural polypeptides that use the same arsenal of proteinogenic amino acid residues as their major building blocks. the exceptional structural plasticity of idps, their capability to exist as heterogeneous structural ensembles and their wide array of important disorder-based biological functions that complements functional repertoire of ordered proteins are all rooted within the peculiar differential usage of these building blocks by ordered proteins and idps. in fact, some residues (so-called disorder-promoting residues) are noticeably more common in idps than in sequences of ordered proteins, which, in their turn, are enriched in several order-promoting residues. furthermore, residues can be arranged according to their “disorder promoting potencies,” which are evaluated based on the relative abundances of various amino acids in ordered and disordered proteins. this review continues a series of publications on the roles of different amino acids in defining the phenomenon of protein intrinsic disorder and concerns glutamic acid, which is the second most disorder-promoting residue. intrinsically disordered proteins (idps) and intrinsically disordered protein regions (idprs) are new exciting members of the protein kingdom. , they are highly abundant in nature, [ ] [ ] [ ] [ ] [ ] possess numerous intriguing properties, are intimately involved in various cellular processes [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and are commonly found to be related to the pathogenesis of various diseases. , [ ] [ ] [ ] [ ] [ ] [ ] the common theme of protein disorder-based functionality is recognition, and idps/idprs are frequently involved in complex protein-protein, protein-nucleic acid and protein-small molecule interactions. some of these interactions can induce a disorder-order transition in the entire idp or in its part. , [ ] [ ] [ ] [ ] , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] furthermore, intrinsic disorder opens a unique capability for one protein to be involved in interaction with several unrelated binding partners and to gain different bound structures. , some idps can form highly stable complexes; others are involved in signaling interactions where they undergo constant "bound-unbound" transitions, thus acting as dynamic and sensitive "on-off" switches. these proteins typically return to their intrinsically disordered state after the completion of a particular function. many of the idps/idprs can gain different conformations depending on the environmental peculiarities. , all this constitutes an important arsenal of the unique physiological properties of idps/idprs that determines their ability to exert different functions in different cellular contests according to a specific conformational state. the folding-at-binding principle is believed to help idps or idprs to obtain maximal specificity in a protein-protein interaction without very high affinity. this combination of high specificity with low affinity defines the broad utilization of intrinsic disorder in regulatory interactions where turning a signal off is as important as turning it on. although some partial folding during the idp/ idpr-based interactions is a widespread phenomenon, with significant fraction (~ / ) of the interacting residues in idps/idprs adopting α-helix, β-strand and irregular structures, , there are still many other idps/idprs that are involved in the formation of "fuzzy complexes," where an idp/idpr keeps a certain amount of disorder in its bound conformation. , [ ] [ ] [ ] often, the interacting regions in idps are observed as loosely structured fragments in their unbound forms. these disorderbased binding sites are known as molecular recognition elements or features (mores or morfs), , preformed structural elements or pre-structured motifs (presmos). although the existence of such loosely structured regions suggests that idps can adopt their bound structure(s) at a free-energy cost that is not too high, it is important to remember that increasing the stability of the bound conformation does not necessarily enhance the binding affinity. another important feature of the disorder-based interactions is their increased speed due to the greater capture radius and the ability to spatially search through interaction space (the so-called "fly-casting" mechanism) and to the fact that fewer encounter events are required for the binding because of lack of orientational restrains. linking all these form at phs greater than its pk a . (and thus glu is negatively charged at the physiological ph ranging from . - . ) . therefore, glutamic acid is one of two acidic amino acids found in proteins that play important roles as general acids in enzyme active centers, as well as in maintaining the solubility and ionic character of proteins. in fact, glutamic acid residue has a nonpolar surface of Å , and the estimated hydrophobic effect associated with the burial of this residue is . kcal/mol. in ordered proteins, glutamic acids are predominantly located on protein surface so that they have access to the solvent. in fact, % of glutamic acids in known structures of folded proteins are classified as exposed since they have solvent exposed areas of > Å , and only % of glutamic acids in folded proteins possess solvent exposed areas of < Å and therefore are buried. the carboxylate anions and salts of glutamic acid are known as glutamates. glutamic acid is one of the most common natural amino acids and the most abundant amino acid in the diet. besides being an important component of proteins and polypeptides (see below), being a substrate for the production of the krebscycle-related α-ketoglutarate intermediate, glutamine and proline, and being the precursor for the synthesis of the inhibitory γ-aminobutyric acid (gaba) in gaba-ergic neurons, glutamate is the principal excitatory neurotransmitter within the vertebrate nervous system. in fact, glutamate is known to act on several different types of receptors and has excitatory effects at ionotropic receptors [such as n-methyl-d-aspartate (nmda), α-amino- -hydroxy- -methyl- -isoxazolepropionic acid (ampa), and kainite, which all incorporate ion channels that are permeable to cations] and modulatory effects at metabotropic receptors [which are g protein-coupled glutamate receptors (mglur) that modify neuronal and glial excitability through g protein subunits acting on membrane ion channels and second messengers such as diacylglycerol and camp]. at chemical synapses of the glutamatergic neurons, glutamate is stored in vesicles and is released from the pre-synaptic cell by nerve impulses. in the opposing post-synaptic cell, binding of glutamate lead to activation of specific glutamate receptors such as nmda or ampa. glutamate plays an important role in synaptic plasticity in the brain and is involved in various cognitive functions, such as learning and memory. in fact, long-term potentiation (one of the plasticity forms) takes place at glutamatergic synapses in the neocortex, hippocampus and other parts of the brain. another important role of glutamate is its ability to generate volume transmission, where extrasynaptic signaling is created via the summation of glutamate released from a neighboring synapse. in addition to glutamate receptors, neuronal and glial membranes contain glutamate transporters that are responsible for rapid remove of glutamate from extracellular space. under stress conditions (such as brain injury or disease), glutamate transporters work in reverse leading to the accumulation of considerations with the recent report showing that idp affinities are tuned mostly by association rates suggests that the degree of pre-adoption of binding conformations in idps has to be limited, but not unfavorable. all the functional and structural peculiarities of idps/idprs are encoded in their amino acid sequences. it was recognized long ago that there are significant differences between ordered proteins/domains and idps/idprs at the level of their amino acid sequences. , , in fact, in comparison with ordered proteins, idps/idprs are characterized by noticeable biases in their amino acid compositions, , , , [ ] [ ] [ ] containing less of so-called "order-promoting" residues (cysteine, tryptophan, isoleucine, tyrosine, phenylalanine, leucine, histidine, valine, asparagines and methionine, which are mostly hydrophobic residues which are commonly found within the hydrophobic cores of foldable proteins) and more of "disorder-promoting" residues (lysine, glutamine, serine, glutamic acid and proline, which are mostly polar and charged residues, which are typically located at the surface of foldable proteins) (fig. a) . glutamic acid is second of the most common disorder-promoting residues. figure b and table represent the result of a statistical analysis of the amino acid compositions of proteins in four standard data sets (disprot, uniprot, pdb select and surface residues ) and shows that the glutamic acid content in these data sets is . ± . %, . ± . %, . ± . % and . ± . %, respectively (cprofiler.org/help. html). in other words, idps/idprs contain . -and . times more glutamic acid residues than the average natural proteins from uniprot or ordered proteins from pdb, respectively. furthermore, the glutamic acid content in idps/idprs is . times higher than that on the surfaces of ordered proteins. this article continues a series of publications on the intrinsic disorder alphabet dedicated to the exploration of the amino acid determinants of protein intrinsic disorder. i overview below some functions of glutamic acid in idps/idprs (as well as in ordered proteins and domains) and show that there is a variety of glutamic acid-specific functions in disordered proteins and regions. chemical structure of glutamic acid. glutamic acid (glutamate, glu, e, see fig. a ) is one of the proteinogenic amino acids encoded by the standard genetic code and its codons are gaa and gag. glutamic acid is a dibasic nonessential amino acid that has a molecular mass of . da (molecular mass of glu residue is . da), surface of Å , volume of . Å , pk a of side chain of . and pi . at °c. intriguingly, free glutamic acid is not very soluble, possessing solubility of . g/ g at °c, which is significantly lower than the solubility of free prolines ( . g/ g at °c), and the solubility of the vast majority of free amino acids (www.fli-leibniz.de/ image_aa.html). the side chain of glutamic acid contains two methylene group and the carboxylic acid functional group (see fig. a ) that exists in a negatively charged deprotonated carboxylate with stroke, autism, amyotrophic lateral sclerosis, lathyrism, some forms of mental retardation and alzheimer's disease. the decreased glutamate release is associated with phenylketonuria leading to the developmental disruption of glutamate receptor expression. , the excess glutamate in the extracellular space and promoting entrance of calcium to the cell via the nmda receptor channels. this process is known as excitotoxicity, and it results in neuronal damage and eventual cell death. the excitotoxicity might occur as part of the ischemic cascade that is associated figure . amino acid determinants defining structural and functional differences between the ordered and intrinsically disordered proteins. (a) fractional difference in the amino acid composition (compositional profile) between the typical idps from the disprot database and a set of completely ordered proteins calculated for each amino acid residue. the fractional difference was evaluated as (c disprot − c pdb )/c pdb , where c disprot is the content of a given amino acid in a disprot databse, and c pdb is the corresponding content in the data set of fully ordered proteins. positive bars correspond to residues found more abundantly in idps, whereas negative bars show residues, in which idps are depleted. amino acid types were ranked according to their decreasing disorder-promoting potential. (b) amino acid compositions of several data sets discussed in the text (disprot, uniprot, pdb select and surface residues ). linkages, or ion pairs. an electrostatic interaction is a non-covalent bond that is based on the attraction of two oppositely charged groups. it can easily be broken and reformed and is characterized by the optimal distance of . Å between the interacting groups. the strength of these interactions depends on the distance of the two charges and the properties of the medium between them. in proteins, electrostatic interactions typically occur between coo − in the side chain of glutamic and aspartic acids and nh + in the side chains of lysines and arginines. hydrogen bond (h-bond) is another non-covalent bond. this interaction depends on the sharing of one hydrogen atom (h-atom) between two other atoms, where the h-atom has a covalent bond to one of them (which therefore serves as the h-bond donor), and where the other atom, to which the h-atom has a weaker bond, serves as the acceptor, a. hydrogen bond is weaker than a covalent bond but stronger than a van der waals bond. similar to electrostatic interactions, h-bonds can easily be broken and reformed. among established geometrical criteria for h-bond are a set of optimal distances between the non-h atom of donor and acceptor (dono-acceptor < . Å) and between the h atom of donor and acceptor (h-acceptor < . Å). being negatively charged at physiological ph, glutamic acid can serve as a hydrogen bond acceptor, whereas at acidic ph, it also can be a hydrogen bond donor. glutamic acid in the ramachandran plot. the structure of a protein can be described using torsion angles-φ and ψ-of its backbone that provides a simple view of the conformation of a protein. in sequence order, φ is the n i- -c i -cα i -n i torsion angle, and ψ is the c i -cα i -n i -c i+ torsion angle. since most combinations of φ and ψ are sterically forbidden, the d plot of the torsion angles of the protein backbone, known as the ramachandran plot, provides a simple view of the conformation of a protein, since the φ-ψ angles cluster into distinct regions in the ramachandran plot, where each region corresponds to a particular secondary structure. in the generic ramachandran plot (see fig. b ) that refers to the non-glycine and non-proline amino acids, there are four distinct regions of density (the α (right-handed α-helix region), α l (mirror image of α), β s (region largely involved in β-sheet formation) and β p (region associated with extended polyproline-like helices but also observed in β-sheet). the shape of the generic ramachandran plot is determined mainly by the presence of specific steric clashes and backbone dipole-dipole interactions. [ ] [ ] [ ] glutamic acid in electrostatic interactions and hydrogen bonds. glutamic acid participates in electrostatic interactions, which are also known as ionic bonds, or salt bridges, or salt gcn leucine zipper dimer revealed that the free energy of helix stabilization associated with the hydrogen-bonding and hydrophobic interactions in this capping structure is − . kcal/ mol, illustrating that helix capping might play a significant role in protein folding. based on the analysis of α-helices the normalized frequencies for finding particular residues at the c cap position, the average fraction of buried surface area and the hydrogen bonding patterns of the c cap residue side-chain were calculated. this analysis revealed that the residue found in the c cap position is on average % buried and that there is a noticeable correlation between the relative burial of this residue and its hydrophobicity. furthermore, c cap residues with polar sidechains were shown to be involved in hydrogen bonding, where the longer side-chains of glutamic acid, glutamin, arginine, lysine and histidine form hydrogen bonds with residues located more than four residues apart, whereas the shorter side-chains glutamic acid and protein secondary structure. although protein secondary structure is determined by hydrogen bonds between donor and acceptor groups in the protein backbone, different amino acids are known to favor the formation of different secondary structure elements, such as α-helices, β-pleated sheets or loops. the α-helix-formers include alanine, cysteine, leucine, methionine, glutamic acid, glutamine, histidine and lysine, whereas valine, isoleucine, phenylalanine, tyrosine, tryptophan and threonine favor β-structure formation, and serine, glycine, uncharged aspartic acid, asparagine and proline are found most often in β-turns. it was pointed out that there is no apparent relationship between the chemical nature of the amino acid side chain and its secondary structure preferences. for example, although glutamic and aspartic acids are closely related chemically, glutamic acid is more likely to be found in helices and aspartic acid is predominantly located in β-turns. in fact, the helical propensity of glutamic acid is . , whereas aspartic acid has an helical propensity of . , the third largest value after proline and glycine. note that the helical propensity is defined as the difference in free energy Δ(Δg) estimated in kcal/mol per residue in an α-helical configuration relative to alanine, which has been set to zero because it is usually the amino acid with the most favorable helix propensity. here, the higher helical propensity values correspond to more positive free energies and therefore are related to residues which are less favored in α-helix. glutamic acid in α-helix caps. since α-helices in peptides and proteins have an overall dipole moments caused by the cumulative effects of all the individual dipoles from the carbonyl groups of the peptide bond pointing along the helix axis, the overall helical structure is destabilized due to the noticeable entropic effects. the effect of this helical dipole moment can be approximated by placing . - . positive unit charge near the n-terminus and . - . negative unit charge near the c-terminus of the helix. , one of the nature's strategies to neutralize this helix dipole is the specific capping of the n-terminal ends of α-helices by negatively charged residues, such as glutamic acids. , furthermore, careful analysis of α-helices revealed that their first and last four residues differ from the remaining residues by being unable to make intrα-helical hydrogen bonds. instead, these first four (> n-h) groups and last four (> c = o) groups in an α-helix are often capped by alternative hydrogen bond partners. [ ] [ ] [ ] physico-chemical and statistical analysis suggested that certain residues are more preferable at the c-and n-termini of an α-helix (the helical c-and n-caps). for example, based on the analysis of series of mutations in the two n-caps of barnase, it was concluded that a single n-cap can stabilize the protein by up to ~ . kcal/mol. importantly, the presence of a negative charge of the n-cap was shown to add ~ . kcal/mol of stabilization energy mostly due to the compensation effects for the macroscopic electrostatic dipole of the helix. from a global survey among proteins of known structure, seven distinct capping motifs are identified-three at the helix n-terminus and four at the c-terminus. one of these motifs is the helix-capping motif ser-x-x-glu, a sequence that occurs frequently at the n-termini of α-helices in proteins. [ ] [ ] [ ] thermodynamic analysis of this ser-x-x-glu motif from the ramachandran plots for backbone conformations of the non-glycine and non-proline amino acids. marked regions of density correspond to the right-handed α-helix region (α), mirror image of α (α l ), region largely involved in β-sheet formation (β s ), and region associated with extended polyproline-like helices, but also observed in β-sheet (β p ). by the four glutamic acid residues located at homologous positions within each of the four pore-forming segments and which form a single or multiple ca + -binding site(s) that entrap calcium ions, thus giving them a possibility to be electrostatically repulsed through the intracellular opening of the pore. in the bacterial kcsa and inwardly rectifying k + (kir) channels, glutamic acid is also involved in the action of the selectivity filter. here, the network of residues stabilizing the pore of kcsa involves a glu -asp carboxyl-carboxylate interaction behind the selectivity filter, whereas the structure of the pore in kir channels is stabilized by a glu-arg salt bridge. therefore, although glu is quite conserved among both types of channels, the network of interactions is not translatable from one channel to the other. this clearly shows that different potassium channels are characterized by diverse gating patterns. the presence of a highly conserved glutamic acid residue in the middle of a transmembrane domain is a characteristic feature of a family of transmembrane glycoproteins with two immunoglobulin-like domains, such as basigin (bsg, also known as cd or emmprin), embigin and neuroplastin. finally, a critical glutamic acid residue was recently identified in clc proteins, which constitute a large structurally defined family of cl − ion channels and h + /cl − antiporters which are found in prokaryotes and eukaryotes, and which perform their functions in the plasma membrane or in various intracellular organelles such as vesicles of the endosomal/lysosomal pathway or in synaptic vesicles. mutations in human clc channels are known to cause a set of very diverse diseases such as myotonia (muscle stiffness), bartter syndrome (renal salt loss) with or without deafness, dent's disease (proteinuria and kidney stones), osteopetrosis and neurodegeneration, and possibly epilepsy. the side chain of the aforementioned critical glutamic acid occupies a third cl − ion binding site in the closed state of the channel and moves away to allow cl − binding. glutamic acid valve. glutamic acid is known to play a unique role in regulation of the cytochrome-c oxidase (cco) activity. cco is the last enzyme of the respiratory electron transport chain in mitochondria (or bacteria) located in the inner mitochondrial (or bacterial) membrane, and it is responsible for reducing ~ % of the oxygen taken up in aerobic life. this protein powers the production of atp by generating an electrochemical proton gradient across the membrane via the catalysis of the oxygen reduction to water that takes place in the binuclear center (bnc) of the enzyme. cco uses four electrons taken up from the cytochrome c located at the positively charged p-side (outside) of the membrane and four "chemical" protons taken from the negatively charged n-side (inside) to reduce the dioxygen to two water molecules. in addition to this oxygen reduction reaction, four "pump" protons are translocated from the n-side to the p-side across the membrane against the opposing membrane potential, doubling the total amount of charge separated by the enzyme. [ ] [ ] [ ] [ ] therefore, the main role of cco is to serve as a proton pump and a generator of the electrochemical proton gradient or charge separation across the membrane, which is achieved via two separate processes. first, the reduction of oxygen to water by electrons and protons taken up from opposite sides of the membrane leads of aspartic acid, asparagine, serine and threonine form hydrogen bonds with residues located close in sequence. finally, based on the analysis of α-helical propensity of a series of dodecapeptides containing alanine, asparagine, aspartate, glutamine, glutamate and serine at the n-terminus and arginine, lysine and alanine at the c-terminus, it was concluded that the α-helix-stabilizing abilities of these residues can be ranged as follows: aspartate > asparagine > serine > glutamate > glutamine > alanine at the n-terminus and arginine > lysine > alanine at the c-terminus. glutamic acid and protein solubility. based on the analysis of solubility-changing substitutions in proteins it has been pointed out that together with two other hydrophilic residues (aspartic acid and serine) glutamic acid contributes significantly more favorably to protein solubility than other hydrophilic residues (asparagine, glutamine, threonine, lysine and arginine). based on this observation, an important strategy for solubility enhancement was proposed, were the hydrophilic residues that do not contribute favorably to protein solubility can be replaced with the hydrophilic residues that contribute more favorably. glutamic acids inside the pores of ion channels. being negatively charged at physiological ph, glutamic acid is perfectly suited for binding metal ions. this property is used in specific regulation of a variety of ion channels. for example, in cyclic nucleotide-gated (cng) channels (which are found in vertebrate photoreceptors and olfactory epithelium, elsewhere in the nervous system [ ] [ ] [ ] and in a variety of other cell types including kidney, testis and heart, and whose activation represents the final step in the transduction pathways in both vision and olfaction [ ] [ ] [ ] ), a single glutamic acid strategically located in the pore represents the binding site for multiple monovalent cations, the blocking site for external divalent cations and the site for the effect of protons on permeation. this is not too surprising since the pore region of the channel controls both the singlechannel conductance and the pore diameter of the channel. importantly, cng channels are permeable to ca + , which is an important element in the activation of intracellular targets, and which in addition to permeating cng channels can profoundly block the current flow carried by monovalent cations through the cng channels. this capability of ca + to block the monovalent cation flow is determined by the high-affinity binding of ca + to a single acidic amino acid residue located in the pore of the channel, which is glu for the rod cng channel and glu for the catfish olfactory cng channel. this same glutamic acid residue is also responsible for the external rapid proton block of cng channels, another characteristic that the cng channels share with ca + channels. glutamic acid also plays an important regulatory role in the voltage-dependent calcium channels that are located in the plasma membrane and form a highly selective conduit by which ca + ions enter all excitable cells and some nonexcitable cells. for these channels to operate, ca + ions must enter selectively through the pore, bypassing competition with other extracellular ions. the high selectivity of a unique ca + filter is determined pathway for protons utilized in the catalytic no reduction; the carboxylate group of glu , which is located at the backside of glu , contributes to the electro-negative environment of the binuclear center of cnor, and to the low redox potential of heme b iron; finally glu and glu are positioned in the loop connecting the transmembrane helices iii and iv, with glu serving as one of the ca + ligands (which is crucial for maintaining the configuration of heme b and b ) and assisting in the water-mediated proton transfer through interactions with a number of water molecules, and with glu serving as a key residue for maintaining the unique conformation of the long loop through interactions with the residues in transmembrane helix ii, which would stabilize the coordination of glu to ca + . mono-adp-ribosyltransferase, which is responsible for the mono-adp-ribosylation of proteins, possesses a critical glutamic acid at the catalytic cleft which functions to position nad for nucleophilic attack at the n-glycosidic linkage for either adpribose transfer or nad hydrolysis. the pronounced na + /k + selectivity of na,k-atpase relies on the strategic positioning of glutamic acid residues. here, intramembrane glu in transmembrane segment m , glu in m , asp and asp in m are essential for tight binding of k + and na + , whereas asn and glu in m , together with thr , asn and glu in the -ytltsnipeitp motif of m contribute to the na + / k + selectivity. in the family of thiamin diphosphate enzymes, a highly conserved glutamate is known to promote the c -h ionization and the thiamin diphosphate activation. the direct catalytic role of glutamic acid can be seen in matrix metalloproteinases, which are ubiquitous endopeptidases characterized by an active site where a zn + atom, coordinated by three histidines, plays the catalytic role, assisted by a glutamic acid that acts as a general base. for example, one of the wellknown zinc-binding metalloproteases that uses a glutamic acid residue as the fourth ligand to coordinate the zinc ion is thermolysin. in thermolysin, glutamic acid is amino acids downstream from the second histidine in the first motif and present in a small conserved motif (nexxsd). in the zincin and pdf groups of metalloproteases, the catalytic zinc-binding site contains the hexxhxxg motif. also, a glutamic acid residue may be catalytically active in the substrate-binding cleft of plant lysozymes. each enzyme in the α-amylase family of multidomain hydrolases and transferases has one glutamic acid and two aspartic acid residues necessary for activity. the irreversible dealkylation reaction catalyzed by the o -alkylguanine-dna alkyltransferase (agt) that directly repairs alkylation damage at the o -position of guanine is accomplished by an active-site cysteine that participates in a hydrogen bond network with invariant histidine and glutamic acid residues, reminiscent of the serine protease catalytic triad. the spore germination protease (gpr) that degrades small, acid soluble proteins (sasp) protecting spore's dna against damage, is a structurally and functionally unique protease that utilizes glutamic acid residue to catalyze sasp degradation. in the hydrolytic aldehyde dehydrogenases (aldhs), catalytic but flexible glutamic acid residues located within the active site serve as the general base that activates the hydrolytic water molecule in the deacylation step. to the net translocation of one electrical charge across the membrane per electron consumed. second, an additional proton is translocated vectorially across the membrane for each electron consumed, resulting in a net transport of two electrical charges per electron. the protons for the chemical reaction are extracted from the n-side of the membrane via two proton pathways, the d-and k-channels. the d-channel starts at a highly conserved residue, asp (bovine numbering; subunit i) near the n side, and continues to another highly conserved residue glu that donates protons to the bnc, whereas the key residue in the k-channel is a highly conserved lysine (k ). the d-channel is responsible for the delivery of four "pump" protons that are first transferred from glu to a "loading" site above the bnc and then delivered to the p side via a proton-exit channel. the mystery of this mechanism is in the ability of glu located at the end of the d-channel to somehow sort "pump" protons from "chemical" protons. to explain this behavior, the glutamate valve model has been proposed according to which the side chain of glu shuttles between a state protonically connected to the d channel, and a state connected to the bnc and the pump site. in this proton valve model, the glu motion depends on its protonation state, where the unprotonated residue remains predominantly in a "down" conformation, pointing toward the n side, and therefore facilitating the uptake of a proton, whereas protonation shifts the glu to the "up" conformation, where the side chain of this important residue is swung toward the p side by ~ Å. glutamic acid in the active sites of enzymes. in addition to serve multiple structural roles and being involved in regulation of various channels, glutamic acid residues, being positioned within or in the close proximity to the active sites, might have roles in the catalytic activities of various enzymes. one of the illustrative examples of the functional roles of glutamic acid can be found in bacterial nitric oxide reductase (nor), which is a membraneintegrated enzyme that catalyzes the reduction of nitric oxide no to nitrous oxide n o using a type of anaerobic respiration where cytotoxic no is immediately decomposed after its production from nitrite no − via the nitrite reductase-catalyzed reaction. [ ] [ ] [ ] three different nor types are found in bacteria, with the cytochrome c dependent nor (cnor) that consists of two subunits, norb and norc, being the most extensively studied enzyme. precise description of the complex catalytic mechanism of this important enzyme is outside the scopes of this review, and therefore only a small piece of the entire picture, where the roles of glutamic acid are emphasized, is briefly described below. the characteristic feature of cnors is the presence of five conserved glutamic acid residues (glu , glu , glu , glu and glu in p. aeruginosa cnor) within the norb subunit consisting of trans-membrane helices and containing the heme b and the binuclear center (heme b /fe b ) buried in the hydrophobic interior of its trans-membrane region. here, glu is involved in the coordination of fe b and its carboxylate functions as the shuttle for catalytic protons from glu to the bound-no; glu , which interacts with glu but is not involved in direct interaction with fe b , is an important player of the thr -ser -glu -glu network that acts as a delivery matrix communication) and their ligands, it has been concluded that divalent cations are critical for integrin interactions with almost all ligands. importantly, although divalent cations are bound to integrins, their coordination sphere is not completed and the interactions between integrin and its ligands typically involve completing the metal ion coordination with an acidic ligand residue. for example, complexes between the human intercellular adhesion molecule- (icam- ) and the i domain of its integrin receptor αlβ are stabilized by a critical glutamate residue that completes the magnesium coordination in integrin. similarly, in the crystal structure of a complex between the i domain of a b integrin and a triple-helical collagen peptide containing a critical gfoger motif, glutamate residue from the collagen peptide completes the coordination sphere of the i domain metal ion. based on these observations it has been concluded that a metalglutamate handshake represents a basic mechanism of integrin i domain interaction with its binding partners. furthermore, it is believed now that the general mechanism by which integrins, these αβ-heterodimeric cell-surface receptors that are vital to the survival and function of nucleated cells, recognize their structurally diverse ligands relies on specific glutamic-acid-or aspartic-acid-based sequence motifs that function in a divalent cation-dependent and conformationally sensitive manner. the levels of intracellular zinc in living cells are crucial for managing various cellular processes, such as growth, development and differentiation. zinc is involved in protein, nucleic acid, carbohydrate and lipid metabolism and also plays a role in the control of gene transcription and the coordination of other biological processes controlled by proteins containing dna-binding zinc finger motifs, ring fingers and lim domains. the physiologically relevant intracellular levels of zinc are controlled by specific zinc transporters which mostly transport zinc into cells from outside. members of one of the subfamilies of these transporters, liv- subfamily of zip zinc transporters (lzt), being similar to other zip transporters in secondary structure and ability to transport metal ions across the plasma membrane or intracellular membranes, possess a unique hexphexgd motif containing conserved proline and glutamic acid residues, that fits the consensus sequence for the catalytic zinc-biding site of matrix metalloproteinases (hexxhxxgxxh), and which is unprecedented in other zinc transporters. in addition to this set of specific examples, one should keep in mind that all structures of the ca + -binding domains have in common a high negative surface potential usually associated with asp or glu residues. therefore, important glutamic acid residues responsible for calcium coordination can be found in various members of the major ca + -binding proteins, such as ef-hand domains, egf-like domains, γ-carboxyl glutamic acid (gla)-rich domains, cadherin domains, ca + -dependent (c)-type lectin-like domains and ca + -binding pockets of family c g-protein-coupled receptors. a particularly intriguing role was described for the n-terminal glutamic acid residues in the canonical ca + -protein, α-lactabumin, which is frequently used as a model protein in folding studies and in studies on the effect of calcium binding on protein structure, stability and folding. for example, in nudix hydrolases (which is a family of mg + -requiring enzymes that catalyze the hydrolysis of nucleoside diphosphates linked to other moieties) there is a specific motif, nudix box (gx ex reuxeexgu, where u is a bulky hydrophobic residue), that forms a loop-α helix-loop structural motif that functions as a common mg + -binding and catalytic site. it was emphasized that the overall catalytic powers of nudix hydrolases consists in accelerating the reaction rate by to times. the reactions are accelerated - -times by general base catalysis by a glutamate residue within, or beyond the nudix box, or by a histidine beyond the nudix box. the additional - -fold rate acceleration is due to the lewis acid catalysis provided by one, two, or three divalent cations. one divalent cation is coordinated by two or three conserved residues of the nudix box, the initial glycine and one or two glutamate residues, together with a remote glutamate or glutamine ligand located outside the nudix box. glutamic acids at various binding sites. hemopexin is an important multifunctional plasma protein involved in the sequestering of heme released into the plasma from hemoglobin and myoglobin as the result of intravascular or extravascular hemolysis and due to skeletal muscle trauma or neuromuscular disease. it also possesses hyaluronidase activity, serine protease activity, pro-inflammatory and anti-inflammatory activity and is involved in the suppression of lymphocyte necrosis, inhibition of cellular adhesion, and binding of divalent metal ions. finally, hemopexin possesses two highly exposed arg-gly-glu sequences that may promote interaction with cell surfaces. glutamic acid plays an important role in defining the retinal binding site geometry of rhodopsin, which is the photoreceptor in vertebrate rod cells responsible for vision at low light intensities. -cis-retinal is the photoreactive chromophore located in the interior of the protein where it is covalently attached to a lysine side chain through a protonated schiff base (psb) linkage. based on the c-nmr chemical shift data, it was concluded that glu of rhodopsin is involved in charge interactions with the retinal psb, which are crucial for maintaining rhodopsin in the inactive state in the dark and whose breaking leads to the protein activation. a centrally located glutamic acid residue in position of transmembrane segment vii of the main ligand-binding crevice of the chemokine tm receptors (gluvii: ) is crucial for recognition and binding of small molecule non-peptide ligands that contain one or two centrally located, positively charged nitrogen atoms and are characterized by relatively similar elongated overall structure with terminal aromatic moieties. furthermore, since this gluvii: is crucial for the binding and hence the function of a number of non-peptide ligands in several chemokine receptors, such as the ccr , ccr and ccr receptors, it serves as a selective anchor point for the centrally located, positively charged nitrogen of the small molecule ligands. glutamic acid and metal binding. the role of glutamic acid residues in coordination of various metal ions was already emphasized in sections discussing ion channels. a few other illustrative examples are listed below. based on the analysis of the complexes formed between integrins (which are central molecules in the adhesion processes that mediate cell-cell and cell-extracellular group giving rise to the pyrrolidone carboxylic acid (pyro-glu). however, it was emphasized that pyro-glu is exclusively found at the n-terminal end of the thermal polymers when glutamic acid is a predominant amino acid in a mixture of amino acids subjected to thermal polymerization. another important glutamic acid-based ptm is gammacarboxylation catalyzed by the vitamin k-dependent carboxylase that transforms specific glutamate residues in proteins to gammacarboxy glutamic acid (gla) in the presence of reduced vitamin k, molecular oxygen and carbon dioxide. this modification is widely distributed in the animal kingdom and has a wide range of physiological implications, such as hemostasis, bone calcification and signal transduction. in addition to be a target for various ptms, glutamic acid itself can be used as an important protein modifier, giving raise to polyglutamylation, which is a specific ptm where polyglutamate chains of variable lengths are added to the modified protein. polyglutamylation is evolutionarily conserved and is commonly found in the microtubule (mt) building block, tubulin. this ptm, being primarily found within the tubulin c-terminal tail that participates in binding of many structural and motor mt-associated proteins, is believed to be crucial for the functional adaptation of mts. polyglutamylation is catalyzed by a family of specific enzymes and in addition to tubulin can be found in some other proteins. high content of charged residues is one of the tricks used by nature to make stable proteins in thermophilic and hyperthermophilic organisms. in fact, based on the correspondence analysis of the completely sequenced genomes available from the three domains of life (seven eukaryotes, archaeal and bacterial species) it has been concluded and the amino acid composition permits discrimination between the three known lifestyles (mesophily, thermophily or hyperthermophily). the most specific amino acid compositional biases that represent specific signatures of thermophilic and hyperthermophilic proteomes are a relative abundance in glutamic acid, concomitantly with a depletion in glutamine and a significant correlation between the relative abundance in glutamic acid (negative charge) and the increase in the lumped "pool" lysine + arginine (positive charges). being absent in mesophiles, these correlations could represent a physico-chemical basis of protein thermostability. curiously, the distribution of the remaining charged amino acid, i.e., aspartic acid, appears to be quite homogeneous throughout all the species suggesting that this residue does not participate significantly in the aforementioned compensatory negative/positive (charged) correlation in thermophiles and hyperthermophiles. on average, thermophilic and hyperthermophilic proteomes were shown to contain . %, . %, . % and . % of glutamine, glutamic acid, aspartic acid and lysine + arginine residues, respectively. importantly, some of these numbers are rather different from those found in idps/idprs, as shown in table . α-lactabumin was shown to possess significantly different thermal and structural stability in its calcium-bound and calciumfree apo-forms, with the apo-protein possessing molten globule-like properties at slightly elevated temperatures. , this strong dependence of the α-lactabumin structural properties on metal-binding is determined by the simple fact that in the apo-form, many acidic side chains have unfavorable chargecharge interactions, with residues (glu , glu , glu , asp , asp , asp , asp , asp , asp , asp and asp ) possessing significantly unfavorable charge-charge repultion. although calcium binding has the most pronounced effect on residues directly involved in cation coordination (asp , asp and asp ) and strongly affects the other two residues in the ca + -binding loop, asp and asp , ca + binding has relatively minor effects on residues more distant from the ca + -binding site (glu , glu , glu , asp and asp ), which mostly preserve unfavorable electrostatic interactions seen in the apo-form. it was also shown that the mutation-induced neutralization of unfavorable charge-charge interactions in the n-terminus (residues - of which are characterized by a high proportion of negatively charged residues that cluster on the surface of the native protein) results in stabilization of both the apo-and ca +bound protein. unexpectedly, the Δglu mutant, where the glu residue was removed, leaving an n-terminal methionine in its place, possessed almost one order of magnitude higher affinity for calcium and higher thermostability (both in the absence and presence of calcium) than the native protein isolated from milk. this unique tuning of the α-lactabumin structure and calcium binding suggested that the n-terminal region of this protein might have a direct effect on the calcium-binding loop (and perhaps other regions of the structure). the side chains of glutamic acid residues are subjected to several ptms. some cytoplasmic and nuclear proteins are known to be methylated, i.e., enzymatically modified by the addition of methyl groups from s-adenosylmethionine. methylation reactions typically occur on carboxyl groups (such as the side chain of glutamic acid) and modulate the activity of the target protein. glutamate methyl ester formation plays a major role in chemotactic signal transduction in prokaryotes. for example, methyl-accepting chemotaxis proteins are a family of chemotactic-signal transducers that respond to changes in the concentration of attractants and repellents in the environment, transduce a signal from the outside to the inside of the cell, and facilitate sensory adaptation through the variation of the level of methylation. , in some proteins and peptides, glutamic acids can be amidated. also, some glutamine residues in proteins undergo spontaneous (nonenzymatic) deamidation to glutamate with rates that depend upon the sequence and higher-order structure of the protein. functional groups within the protein can catalyze this reaction, acting as general acids, bases, or stabilizers of the transition state. in rare cases, glutamate residues can be modified by cyclization via condensation of the α-amino group with the side-chain carboxyl neutral ph was shown to be accompanied by the instantaneous formation of a gel-like precipitate with intermolecular antiparallel β-structure. in bacteria, pga may be composed of only d-, only l-or both d-and l-glutamate enantiomers, and pga filaments may be poly-γ-l-glutamate filaments (plga), pdga filaments or poly-γ-l-d-glutamate (pldga) filaments. the production and maintenance of sufficient d-glutamate pool levels required for the normal bacterial growth is controlled by the glutamate racemase, which is a member of the cofactor-independent, twothiol-based family of amino acid racemases. this enzyme is conserved and essential for growth across the bacterial kingdom and has a conserved overall topology and active site architecture. therefore, it represents an attractive target for the development of specific inhibitors that could act as possible therapeutic agents. in gram-negative bacteria, the complex responsible for the polyglutamate synthesis is encoded in specific loci. if the pga is associated with the bacterial surface and forms a capsule, then the corresponding genes are named cap (for "capsule"); however, if the pga is released, then the corresponding genes are named pgs (for polyglutamate synthase). the minimal gene sets contain four genes termed cap or pgs b, c, a and e, with all cap genes and the four pgs genes (pgsb, pgsc, pgsaa, pgse) being organized into operons. since pga is an idp, whose biochemical and biophysical properties are environment-dependent, and since pga can be found in an anchored to the bacterial surface form or in a released form, this biopolymer can play different roles in different organisms and in different environments. for example, when anchored to the bacterial surface, pga forms a capsule and act as a virulence factor. , in fact, the virulence of bacillus anthracis (a gram-positive sporulating bacterium, which is the causal agent of anthrax) was found to be determined by its capsule composed solely of pga. similarly, the virulence of staphylococcus epidermidis (another gram-positive bacterium that causes severe infection after penetrating the protective epidermal barriers of the human body) is dependent on the pga-based capsule. furthermore, pga in capsules of these bacteria consists of either a mixture of l-and d-enantiomers (s. epidermidis) or solely d-enantiomer (b. anthracis), which makes them particularly non-immunogenic. the released form of pga is used by the producing organism for rather different purposes, starting from the sequestration of toxic metal ions that increases the resistance of some soil bacteria to harsh conditions, to serving as a source of glutamate for bacteria in a starvation state during late stationary phase, to playing a role in decrease of the high local salt concentrations that helps extremophilic bacteria and archaea to survive in a hostile environment, , and in hydra, to control explosion of the special stringing cells, nematocysts, that are used to capture prey, for locomotion and for defense. in addition to have multiple functional roles, bacterially produced pga has found its way to serve as an important biodegradable component with multifarious potential applications in foods, pharmaceuticals, healthcare, water treatment and other fields. , a large commercial advantage of pga is that this although some amount of glutamic acid residues is crucial for the structure and function of ordered proteins/domains, when a protein or a peptide contains a large number of glutamic acid residues and, as a consequence, possesses a small number of hydrophobic residues, it is likely to be disordered at physiological ph due to strong charge-charge repulsion and weak hydrophobic attraction. an illustrative example of such charge-infused proteins is glurich human prothymosin α, in which out of residues are charged (there are asp, glu, arg and lys residues), the overall content of hydrophobic residues (leu, ile and val) is very low, and aromatic residues (trp, tyr, phe and his) and cystein are absent. based on this amino acid composition, it was not a big surprise to find that prothymosin α behaved as a highly disordered coil-like chain, since one cannot expect that a highly charged polypeptide (that contains % of glu+asp residues) will have a strong tendency to fold under physiological conditions. , the lack of stable structure also explains the extreme thermal and acid stability of prothymosin α, since one cannot break what is non-existent. the peculiar amino acid composition of prothymosin α, this biologically active random coil, was one of the defining factors behind the charge-hydropathy plot (ch-plot) development. in fact, based on the analysis of prothymosin α and of other non-globular proteins that lacked almost any ordered secondary structure under physiological conditions in vitro, it was concluded that a combination of high net charge and low hydropathy represents the necessary and sufficient factor for a polypeptide to behave as a natively unfolded protein. strategically positioned glutamic acid residues can modulate conformational stability and function of ordered proteins too. in fact, the role of a glutamic/aspartic acid cluster located outside the ca + -binding site, and of the n-terminal glu residue in destabilizing the structure and weakening the calcium-binding capabilities of α-lactabumin has been already discussed (see above). , therefore based on these observations, protein regions and whole proteins enriched in glutamic acids are expected to be substantially disordered. poly-γ-glutamate (pga) is a natural homopolymer synthesized by several bacteria, one archaea (natrialba aegyptiaca) and one eukaryote (cnidaria). one of the most known sources of pga is the japanese specialty natto, a fermentation product made by bacillus subtilis grown on soybean. pga is a highly soluble polyanionic polymer that sequesters water molecules and can be found in surface-bound and released forms. in structural studies, polyglutamic acid is traditionally used as a biopolymer with a well-characterized secondary structure response to changes in the environmental ph, where pga is in a random coil-like conformation at neutral ph, but gains monomeric α-helical structure at acidic ph and is transformed into a β-sheet structure at alkaline ph. [ ] [ ] [ ] curiously, the addition of polylysine to an aqueous solution of polyglutamic acid homopolypeptide at ebd is not a structurally stable entity in the conventional sense, since for this protein region there are no folded states that exist for any appreciable amount of time. instead, the ebd represents a time-average d region of a protein derived from the thermally driven motion of certain polypeptide chains, including those that are part of an otherwise stable folded protein. therefore, the ebd which is defined by the time-averaged occupancy of space by a polypeptide chain, can exclude lager molecules while allowing small molecules and water to move freely through it. it was proposed that since functions of ebd depend on the intrinsically rapid thermal motion of the polypeptide, and the free energy changes that result when that motion is confined, this domain can be used to control binding events, confer mechanical properties, and sterically control molecular interactions. obviously, to be able to serve as an ebd, a given fragment of a protein has to possess specific amino acid composition that would preclude it from folding. therefore, ebds are expected to possess low hydropathy and high net charge; i.e., in the ch-plot, they can be found well above the boundary separating compact and extended disordered proteins. one of the illustrative examples of biologically active ebds (which are not tightly folded, but expected to have a very extended conformation) is given by side-arms of neurofilament (nf) proteins. the side-arms of the nf heavy polypeptide, nf-h (which are ~ amino acids long), were shown by rotary shadow electron microscopy to be ~ nm long. since there was not enough mass to form a stiff folded structure to occupy such a volume, it was proposed that the side-arms were not folded but were in constant thermal motion. analysis of the amino acid sequence of the porcine nf medium polypeptide (nf-m, which has an apparent molecular mass of kda and is one of the two high molecular mass components of mammalian neurofilaments) revealed that this protein has several peculiar features. the n-terminal residues contain a non-α-helical arginine-rich headpiece (residues - ) with multiple β-turns followed by a highly α-helical rod domain that forms double-stranded coiled-coils (residues - ), followed by a c-terminal tailpiece extension (approximately residues) that represents an autonomous domain of unique amino acid composition, being characterized by a high content of lysines and particularly glutamic acids. in human nf-m, there are glutamic acids ( . %), most of which are concentrated within the c-terminal tail, where glutamate accounts for . % ( out of residues). similarly, human nf-h (a polypeptide comprising , residues) has glutamic acids, of which are found in the residues-long c-terminal tail of this protein, whereas in the human nf-l (nf light polypeptide which has residues), there are glutamic acids, with almost half of which ( ) being located within the acidic c-terminal subdomain (the last residues of the protein). in addition to neurofilament polypeptides, ebds were found in microtubuleassociated protein (map ) and numa. analysis of the amino acid compositions of these proteins revealed that they follow the trend established by nfs and contain significant amount of glutamic acid residues ( out of , residues in human map are glutamates and there are glutamic acids in the , residues-long human numa). natural biopolymer is nontoxic, biocompatible and nonimmunogenic. it can be produced by various bacterial strains in a controllable way. as a result, pga is commonly used in cosmetics/ skin care, bone care, nanoparticle for drug delivery system, hydrogel, etc. for example, the pga-based medusa system has been recently developed for slow release of therapeutic proteins and peptides. here, a poly l-glutamate backbone is grafted with hydrophobic α-tocopherol molecules, creating a colloidal suspension of nanoparticles in water that contain hydrophobic nanodomains suitable for the reversible binding of various drug molecules. the potential multifarious applications of pga in the areas of biomedical materials, drug delivery carriers, and biological adhesives have been studied extensively. in general, γ-pga is recognized now as an important biomaterial in drug delivery applications, with γ-pga-based nanoparticles being considered as promising delivery carriers for anticancer therapeutics. recently, a high molecular weight γ-pga was shown to be used as an immune-stimulating agent. finally, conjugation of paclitaxel, a widely used chemotherapeutic agent whose therapeutic index is limited by low tumor exposure and high systemic exposure, with biodegradable poly-lglutamic acid generates paclitaxel poliglumex (ppx, ct- ). this macromolecular drug conjugate enhances tumor exposure to the drug, since the release of paclitaxel from the polymeric backbone was shown to be dependent on the ppx degradation by the lysosomal protease cathepsin b, which is upregulated in many tumor types. glutamic acid as a part of the protein degradation targeting signals, pest motifs. pest sequences (i.e., sequences enriched in proline (p), glutamic acid (e), serine (s) and threonine (t)) are known to serve as specific degradation signals. [ ] [ ] [ ] [ ] these degradation signals define cellular instability of many proteins and direct them either to the ubiquitin-proteasome degradation or to the calpain cleavage. , this controlled protein degradation is important for activation and deactivation of regulatory proteins involved in signaling pathways that control cell growth, differentiation, stress responses and physiological cell death. [ ] [ ] [ ] [ ] pest-containing sequences were shown to be solvent exposed and conformationally flexible, which preclude them from been resolved in x-ray structures. based on the comprehensive bioinformatics analysis of experimentally characterized disordered and globular regions and of pdb chains containing pest regions, it has been concluded that the pest motif is most frequently located within idprs. furthermore, analysis of the prolinerich motif pro-x-pro-x-pro in pest sequences revealed that these sequences contain glutamic acids much more often than aspartic acids. in addition to this pro-x-pro-x-pro motif, many pest sequences are highly enriched in negatively charged residues and are characterized by a very specific distribution of negative charged patterns. glutamic acids in entropic bristle domains. the entropic bristle domain (ebd) concept was proposed to describe a characteristic behavior of some highly mobile protein regions. the several metals of the transition and main groups (ib-va, z = − ) of the periodic table of elements. phytochelatins are synthesized by a constitutive enzyme, γ-glutamylcysteine dipeptidyl transpeptidase, that uses glutathione (gsh) as a substrate and catalyzes the following reaction: γ-glu-cys-gly + (γ-glu-cys) n − gly→(γ-glu-cys) n+ − gly + gly. fertilization promoting peptide. another important glutamaterich peptide is fertilization promoting peptide (fpp; pglu-glu-pronh ), which is produced by the prostate gland and secreted into seminal plasma. fpp was shown to stimulate capacitation, which is the penultimate step in the maturation of mammalian spermatozoa required to render them competent to fertilize an oocyte. furthermore, although fpp inhibits spontaneous loss of acrosome (an organelle that develops over the anterior half of the head in the spermatozoa), cells retain high fertility in vitro. gala peptide. recently, a synthetic amino acid-long gala peptide with a glutamic acid-alanine-leucine-alanine (eala) repeat was designed to analyze how viral fusion protein sequences interact with membranes. this gala peptide was long enough to span a bilayer when in the α-helical state, and the eala repeat was adjusted so that the peptide would have a hydrophobic face of sufficient hydrophobicity to interact with the bilayer when the peptide was in an α-helix. glu residues were used in gala as a ph-responsive elements. when the ph is reduced from . to . , gala converts from a water soluble random coil conformation to an amphipathic α-helix that binds to bilayer membranes. functional analysis revealed that gala promoted fusion between small unilamellar vesicles and was able to form a transmembrane pore comprised of ~ gala α-helical monomers that were oriented perpendicularly to the plane of the membrane. based on these observations, it has been proposed that ph-controlled membrane permealization induced by gala can serve as a model for the design of environmentally responsive peptidic vehicles for drugs and genes delivery. other type of pests: ptp-pests. protein tyrosine phosphatases (ptp) with proline-, glutamate-, serine-and threoninerich sequence, ptps-pest, are a ubiquitously expressed critical regulators of cell adhesion and migration. , this family of ptps includes three intracellular phosphatases known as prolineenriched phosphatase (pep) in mice or lymphoid tyrosine phosphatase (lyp) in humans (also known as ptpn and ptpn ), ptp-pest (also referred to as ptpn ) and ptp-hematopoietic stem cell fraction (ptp-hscf, which is also known by several other names, such as also termed brain-derived phosphatase (bdp ), ptp , ptp-k , fetal liver phosphatase (flp ) and ptpn . all these phosphatases possess a common structural organization that includes an n-terminally located phosphatase domain, followed by a highly divergent central region that contains various motifs for interactions with other proteins, and a conserved c-terminal domain known as carboxyl-terminal homology (cth) domain. human ptp-lyp (ptpn / ptpn ) is a residues-long protein that contains and glutamic and aspartic acids and , and prolines, serines and threonines, respectively. human ptp-pest (ptpn ) consists of residues and has , , , and glutamates, aspartates, prolines, serines and threonines, respectively, most of recently, we proposed that ebds can be used as protein solubility enhancers. in fact, we showed that highly charged protein sequences (both natural and artificial) can act as ebds, and that translational fusion of such sequences to target proteins can serve as an effective solubilizing means by creating both large favorable surface area for water interactions and large excluded volumes around the partner. this suggests that intrinsically disordered ebds (which extend away from the partner and sweep out large molecules) can enable the target protein to fold free from interference. all artificial fusions used in our study had low sequence complexity and high net charge, but were diversified using distinctive amino acid compositions and lengths. among successful solubilizers were artificial ebds containing the most disorder-promoting residues (glu, pro, gln and ser) in the proportion glu:pro:gln:ser = : : : ; i.e., sequences containing > % glutamic acids. therefore, it seems that glutamic acid is crucial for the successful function of ebd-containing proteins. glutamic acids in intrinsically disordered chaperones. the high content of glutamic acids in artificial ebds designed as solubilization means was chosen because of the earlier observation that proteins with high net charge densities can function as effective intra-and intermolecular chaperones. - for example, polyglutamate among other polyanions was shown to act as a chaperone and to accelerate the in vitro refolding of the arc repressor protein. small heat shock proteins (hsps) have flexible c-terminal extensions that, although variable in length and sequence, are rich in acidic amino acids. the shsp α-crystallin can act as a chaperone on the fibroblast growth factor (fgf- ), and this chaperone action is mediated by electrostatic interactions between the basic regions of the growth factor and acidic regions of α-crystallin. nucleolar chaperone b ( residues, of which are glutamic acids) has two acidic regions (residues - and - ) that contain glutamic residues each and that are necessary for the b chaperone-like activity. tubulin has chaperone-like activity being able to suppress the aggregation of soluble lens proteins, equine liver alcohol dehydrogenase, malic dehydrogenase and insulin, but only if its acidic c-terminus (that contains % and . % of glutamic acid residuess in the porcine αand β-tubulins, respectively) was intact. [ ] [ ] [ ] many polyanionic propeptides were shown to serve as intramolecular chaperones to aid folding of the respective proteins. [ ] [ ] [ ] [ ] for example, propeptides of human neutrophil defensins contain up to . % glutamic acids. also, the c-terminal solubilizing domain of human α-synuclein (residues - ) contains . % glutamates, whereas erd ( residues) and erd dehydrins ( residues) from arabidopsis thaliana contain . % and . % glutamic acids respectively. some functions of glutamate-rich peptides. this section presents several illustrative examples of important biological functions attributed to glutamate-rich peptides. phytochelatins. heavy metal detoxification in higher plants is dependent on a set of heavy-metal-complexing peptides, phytochelatins, with structure of (γ-glutamic acid-cysteine) n -glycine (n = - ) [(γ-glu-cys) n -gly]. the longest of these peptides possesses a molecular mass of . kda, a pi . and a net charge of − . these peptides are induced by the exposure of plants to arglu . transcriptional activators and rna polymerase ii are bridged via the central transcriptional coactivator complex, the mediator complex. it has been recently shown that the arginine and glutamate rich protein (arglu ) colocalizes with the mediator subunit (med ) in the nucleus, being in contact with the far c-terminal region of med . this arglu -med interaction is crucial for the estrogen-dependent gene transcription and breast cancer cell growth. human arglu is a residues-long protein that contains arginines and glutamates. there are two regions with significant composition biases in this protein, an arginine-rich region (residues - ) that contains arginines and a glutamic acid-rich region (residues - ) containing glutamic acids. pelp . proline-, glutamic acid-and leucine-rich protein- (pelp ) plays an important role in mediation of genomic and nongenomic signaling of β-estradiol. this potential protooncogene functions as a co-regulator of estrogen receptor, and expression of pelp is deregulated during breast cancer progression. pelp contains ten nuclear receptor-interacting boxes (lxxll motifs), which allow it to interact with estrogen receptor and other nuclear hormone receptors, a zinc finger, a glutamic acid-rich domain and two proline-rich domains. there are several consensus pxxp motifs within the proline-rich regions, via which pelp couples the estrogen receptor (er) with sh domain-containing kinase signaling proteins, such as src and pi k p regulatory subunit. there are glutamic acids in pelp (which is , residues long), and the majority of them ( ) are concentrated within the glutamic acid-rich domain (residues - ). eif . eukaryotic translation initiation factor (eif ) is a monomeric protein of about kda that functions as a gtpaseactivating protein (gap) in translation initiation. eif is involved in initiation of protein synthesis in eukaryotic cells, where, after binding to the s initiation complex ( s-eif -mrna-met-trna f -eif -gtp) at the aug codon of an mrna, it promotes gtp hydrolysis. this initiates a cascade of events that starts from the release of bound initiation factors from the s subunit and ends with the joining of the s ribosomal subunit to the s complex to form the functional s initiation complex ( s-mrna-met-trna f ). although eif binds gtp and is able to promote gtp hydrolysis reaction, it does not hydrolyze gtp by itself acting as a typical gtpase-activating protein (gap). in fact, eif forms a complex with eif via its glutamic acidrich c-terminal region that binds to the lysine-rich n-terminal region of the β-subunit of eif thus activating the gtpase activity of eif . in human eif , the d structure is known for the n-terminal nucleotide binding domain (residues - , pdb id: e h) and for the w domain (residues - , pdb id: iu ). the linker region connecting these two domains is highly disordered and contains one of the functionally important glutamic acid-rich regions (residues [ ] [ ] [ ] [ ] [ ] [ ] [ ] . overall, there are . % glutamic acid residues in the residues-long amino acid sequence of human eif . histone-interacting proteins. since histones are polycations, they are known to be involved in interactions with several polyanionic proteins, particularly with proteins containing glutamic which are located outside the catalytic domain, with respectively , , , and glutamates, aspartates, prolines, serines and threonines being found in the non-catalytic region (residues - ). finally, among the residues of the human ptp-hscf (bdp /ptp /ptp-k /flp /ptpn ), there are glutamic acids, aspartic acids, prolines, serines and threonines. importantly, glutamate-rich, non-catalytic regions of all these ptps are known to be involved in interactions with multiple binding partners. for example, ptp-lyp is involved in interaction with grb , c-cbl, and the c-terminal src kinase (csk), which is the inhibitory protein tyrosine kinase (ptk). the interaction between the ptp-lyp and csk is mediated by the proline-rich motif in pep and by the src homology (sh ) domain of csk. ptp-pest promiscuously associates with various proteins involved in the organization of the cytoskeleton, such as cas (and cas-related proteins sin and casl), paxillin (and paxillin-related polypeptides hic- and leupaxin) and the ptks fak and pyk . this protein also associates with shc, grb and csk. finally, ptp-hscf is involved in association with csk and tec. multifarious functions of glutamic acid-rich proteins. delta factor. in addition to γ-pga, bacillus subtilis produces another important polyanion, delta factor, which is an important component of the bacterial rna polymerase. this delta factor is a . kda highly acidic (pi = . ) protein that contains two distinct regions, a kda n-terminal domain with uniform charge distribution and a glu-asp-rich c-terminal region. the overall contents of glutamic and aspartic acids in delta factor are . % and . % respectively, whereas these numbers increase to . % and . % in the glu-asp-rich c-terminal domain. the ordered n-terminal domain contains % α-helix and % β-sheet, whereas the c-terminal . kda domain is highly charged (net charge of − ) and therefore is largely unstructured. importantly, the c-terminal intrinsically disordered domain has an important biological function, since the ability of delta factor to displace rna from rna polymerase requires the activities of both the n-terminal core-binding domain and the polyanionic c-terminal region. marcks. myristoylated alanine-rich c kinase substrate (marcks) is an abundant kda protein which is unusually rich in alanine and glutamic acid, with glutamic acid and alanine in this proteins accounting for . % and . % residues, respectively. marcks is a very prominent cellular substrate for protein kinase c (pkc), and its serine residues and threonines are phosphorylated. human marcks is an acidic protein with a pi of . which in addition to ala-glu enriched n-and c-terminal domains possesses a compact "effector domain" (ed), which is responsible for interaction with calmodulin, is located near the middle of the sequence and is enriched in lysines, serines and phenylalanines. marcks is a typical idp with a labile conformation and little ordered structure. in addition to calmodulin this protein can interact with synapsin and actin, and can serve as filamentous actin (f-actin) cross-linking protein. furthermore, being myristoylated, marcks is able to interact with membrane and serves as a cytoskeleton-membrane linkage crucial for controlling cell shape changes. since although the molecular mass of the phosphoprotein was shown to be about kda by sedimentation equilibrium analysis, it runs on - % sds-page (sds-page) as a protein with a molecular mass of kda. later studies revealed that bsp is capable of nucleating the bone mineral hydroxyapatite and that this nucleation involves one or both of the glutamic acid-rich sequences suggesting that polycarboxylate sequences might represent a specific site for growth-modulating interactions between proteins and biological hydroxyapatite crystals. similarly, the ability of another acidic, non-collagenous protein of bone and dentin, osteonectin (also known as secreted protein, acidic, rich in cysteine), to bind to hydroxyapatite crystals is determined by its n-terminal region containing glutamic acid-rich sequences. sparc is a highly conserved acidic calcium-binding extracellular-matrix protein. this matricellular glycoprotein is composed of three functional domains that are evolutionarily conserved in organisms ranging from nematodes to mammals. starting from the n-terminus, these functional domains are: a ca + -binding glutamic acid-rich acidic domain (domain i), a follistatin-like module (domain ii), and an extracellular ca + -binding (ec) module that contains two ef-hands and two collagen-binding epitopes (domain iii). since domain i was not found in sparc isolated from the starlet anemone nematostella vectensis, it has been proposed that sparc first evolved as a collagen-binding matricellular glycoprotein. human sparc is a residueslong protein that contains glutamic acids, of which are located within the n-terminal calcium binding region (residues - ). although xenopus laevis sparc has a molecular mass of . kda, based on sds-page analysis this protein has a molecular mass of kda. nbp- . in nuclei of mice cells, there is a nuclear protein nbp- related to the nuclear proteins hmg- /- . nbp- can function as a transcriptional activator, binds specifically to nucleosome core particles, preferentially binds to euchromatin and modulates cellular transcription by counteracting linker histone-mediated chromatin compaction. nbp- is composed of amino acids and has several functional regions and domains: the n-terminal region (residues - ) contains three segments that are highly homologous to functionally important domains in the hmg- /- protein family, namely a nuclear localization signal, a nucleosome binding domain and a chromatin unfolding domain, whereas the c-terminal region (residues - ) has . % of negatively charged residues. in fact, of the glutamic acids and aspartic acids found in nbp- , glutamic and aspartic acids are located in this highly acidic region. garps in rod photoreceptors. glutamic acid-rich proteins (garps) are common in different organisms and have numerous biological functions. for example, rod photoreceptors contain three different glutamic acid-rich proteins (garps), two soluble forms, garp and garp , and the n-terminal cytoplasmic domain (garp part) of the b subunit of the cyclic gmp-gated channel (also known as cyclic nucleotide-gated cation channel β- , cngb ), that are involved in the control of the ca + propagation from the site of its entry at the cyclic nucleotidegated channel to the cytosol of the outer segment. the cyclic acid-rich domains or regions. for example, the non-epithelial intermediate filament (if) subunit protein (e.g., human vimentin, which is attached to the nucleus, endoplasmic reticulum and mitochondria, either laterally or terminally and that contains . % glutamic acids) can specifically bind core histones with a stoichiometry of core histones per a nonneuronal if protein dimer. glutamic acids clearly play a crucial role in this interaction since the kd neurofilament protein, which was already discussed in the ebd section and contains a glutamic acid-rich c-terminal extension, can bind more core histones per dimer ( molecules of core histones) than the dimer of the non-neuronal if proteins. in the nuclei of physarum polycephalum, there is an alanine, lysine and glutamic acid-rich nuclear protein (p ) with a molecular mass of ~ . kda that can specifically interact with histones and therefore is co-extracted with histones. based on amino acid sequence analysis, it has been concluded that p is a hmg-like protein, which, according to cd measurements, contains only % secondary structure and is, therefore, essentially unstructured under in vivo conditions. titin. the gigantic protein titin (there are , residues in the human protein) is a key component in the assembly and functioning of vertebrate striated muscles. among numerous cellular functions of titin (also known as connectin) are contribution to the fine balance of forces between the two halves of the sarcomere which is crucial for the elasticity of muscle cells, as well as participation in chromosome condensation and chromosome segregation during mitosis of non-muscle cells. the ability of titin to reversibly extend relies on a set of pevk segments, rich in proline (p), glutamate (e), valine (v) and lysine (k) residues. the single molecule analysis of the recombinant titin fragment, containing approximately -residue pevk repeats and glutamic acid-rich motifs, revealed that the bending rigidity of the pevk fragments can be reduced due to calcium-induced conformational changes. furthermore, the glutamic acid-rich motif was shown to be critical for this process. based on these observations, it has been concluded that the glutamic acid-rich motifs embedded into the pevk segments make titin a calcium-dependent molecular spring that can adapt to the physiological state of the cell. curiously, titin has , glutamic acids, of which are found in the glutamic acid-rich region (residues - ) that contains pevk motifs. glutamates are not evenly distributed within the glutamic acid-rich region; e.g., glutamic acids are concentrated within the first residues of this region (residues - , ). in other words, although the glutamic acid-rich region comprises just . % of the whole titin, it has . % of all the titin's glutamates. bone phosphoproteins. bone sialoprotein ii (bsp ii) is an important component of the bone mineralized matrix. this bone-specific glycoprotein contains phosphoserine and sulphotyrosine residues and two regions of contiguous glutamic acid residues (residues - and - ) . in one of the first studies dedicated to the analysis of bone phosphoprotein it has been shown that this glycoprotein can be purified from the mixture of proteins extracted by demineralization of rat bone with . m edta in m guanidinium chloride. it was also emphasized that this protein possessed an abnormal electrophoretic mobility this protein was shown to be involved in sensing infertile nutrient conditions in infected cells to promote a transfer from saprophytic to dormant microsclerotia for long-term survival. vdgarp is a short ( residues) extremely acidic protein with a pi of . that contains . % negatively charged residues ( glutamic acids and aspartic acids). there are also several other garps in various organisms, the functions of which are not known as of yet. small garp (a -amino acid protein, with a molecular mass of . kda and an isoelectric point of . , residues of which are glutamic acids) was found in euplotes octocarinatus. plasmodium falciparum garp consists of residues, of which are glutamic acids. rhox /tox. reproductive homeobox protein (rhox or tox) is a homeodomain protein which is distantly related to the members of the paired/pax family belonging to the pepp subfamily of paired-like homeobox proteins. in mice, tox is predominately transcribed in the testis and ovary and potentially plays an important role during gametogenesis. this residues-long protein contains glutamic acids organized in two poly-glutamic acid stretches (residues - and - ) and several glu-rich regions, which together with aspartic acids makes rhox highly acidic (pi . ). kibra. kidney and brain protein (kibra) is a large ( , residues) protein that serves as a potential regulator of the hippo/ swh (sav/wts/hpo or salvador/warts/hippo) signaling pathway that restricts proliferation and promotes apoptosis therefore being crucial for tumor suppression. , kibra has glutamic acids and possesses two n-terminal ww domains, an internal c -like domain and a c-terminal glu-rich stretch (residues - ). cellular functions of kibra are modulated via phosphorylation by protein kinase c zeta (prkcz). some cellular activities of kibra may be associated with memory performance. , furthermore, in mammalian cells, this protein co-activates functions of the dynein light chain , is involved in regulation of the collagen-stimulated activation of the erk/ mapk cascade and modulates directional migration of podocytes. kibra interacts with histone h via its glu-rich region, and this interaction might play an important role in conferring an optimal transactivation function to the estrogen receptor-α (er) and also may be involved in the proliferation of ligand-stimulated breast cancer cells. sh bgr. sh domain-binding glutamic acid-rich protein (sh bgr) is a highly acidic (pi . ) residues-long protein that possesses glutamates and aspartates and that is expressed in heart and skeletal muscles. the majority of glutamates are located within the c-terminal glu-rich region (residues - ), ~ % of which are glutamic acid residues. in addition to sh bgr, several other members of the sh bgr family were found in humans. these are the so-called sh bgr-like proteins, such as sh bgrl ( residues, glutamic acids), sh bgrl ( residues, glutamic acids) and sh bgrl ( residues, glutamic acids) encoded by chromosomes xq . q - , and p . - , respectively. it was shown that the sh domainbinding glutamic acid-rich-like protein is upregulated in glioblastoma. also, this protein was noticeably downregulated in nucleotide-gated (cng) cation channel of rod photoreceptors is a heterotetramer consisting of homologous subunits, α and β (also known as cnga and cngb a). cnga is known to be indispensable for channel activation, whereas cngb a plays mostly regulatory structural roles. in fact, the n-terminal glutamic acid-rich protein (garp) domain of cngb a and the soluble garp were shown to decrease the opening probability of the cng channel and therefore these garps serve as important autoinhibitors or molecular gate keepers that control the activation of heteromeric rod cng channels. furthermore, cngb and garp , in concert with a retinal tetraspanin (peripherin- or peripherin rds ), were shown to contribute to the organization of the specific organelle, outer segment (os), which possesses a characteristic membranous "stacked pancake" architecture that has to be partially renewed daily to maintain cell function and viability. in fact, a mouse knockout of cngb and garp attenuated rod function and caused structural alterations and slowly progressive retinal degeneration. bovine garp (or cngb ) is a , residues-long transmembrane protein which plays important roles in both visual and olfactory signal transduction. cngb has glutamic acids. garp is a -residues-long cngb splice variant that possesses glutamic acids. garp is another cngb splice variant that has residues of which are glutamic acids. native garp and garp purified from bovine rod photoreceptors were shown to be typical idps. mgarp. mitochondria-localized glutamic acid-rich protein [mgarp, which is also known as ovary-specific acidic protein (osap), corneal endothelium-specific protein (cesp- ) and hypoxia upregulated mitochondrial movement regulator protein (hummr)] is one of the highly expressed proteins in retina. mgarp is highly enriched in steroidogenic tissues and the visual system, and early in development, this protein is mainly detected in the retina and adrenal gland. during the estrous cycle, mgarp levels correlate with estrogen levels in the ovaries. furthermore, the expression of mgarp is regulated by estrogen in a tissue-specific manner and through a feedback regulatory mechanism. as it follows from a long list of names, this protein has numerous important functions. in fact, among functions listed for this protein in the uniprot are ( ) plays a role in the trafficking of mitochondria along microtubules, ( ) regulates the kinesin-mediated axonal transport of mitochondria to nerve terminals along microtubules during hypoxia, ( ) participates in the translocation of trak /grif from the cytoplasm to the mitochondrion and ( ) plays a role in steroidogenesis through maintenance of mitochondrial abundance and morphology. , there are residues in mouse mgarp, of which are glutamic acids exclusively located in the glu-rich region (residues - ). based on the spectroscopic analysis of this protein, it has been concluded that mouse garp is an idp. some other garps. the life cycle of the phytopathogenic fungus verticillium dahliae kleb causing wilt disease in a wide range of crops, including cotton, includes three vegetative phases: parasitic, saprophytic and dormant. one of the genes tagged in a pathogenicity encoded a glutamic acid-rich protein (vdgarp ), which shared no significant similarity to any known proteins. linked to a glutamic acid and alanine repeat (eg-ea repeat). there are , and glutamic acids, glycines and alanines in this latency associated nuclear antigen. although there is low sequence identity between lana , ebna and orf , all three proteins determine the poor recognition of viruses by cd + cytotoxic t lymphocytes (ctl). however, the mechanisms of their action are rather different. in the epstein-barr virus and kaposi's sarcoma-associated herpesvirus the repeat domains were shown to enhance the stability of ebna and lana and decrease their translation rates, whereas the eg-ea repeat has no effect on the stability of hvs orf or its rate of translation, but results in decreased steady-state levels of orf mrna. intriguingly, the motif eeaeeaeee of hvs orf was sufficient to cause a reduction in recognition of orf by cd + ctl, suggesting that the eg-ea repeat of hvs orf is crucial for the immune evasion. nsp a. the n-terminal domain of the severe acute respiratory syndrome coronavirus (sars-cov) nonstructural protein (nsp a) is a typical idp of residues characterized by the presence of an ubiquitin-like globular domain (residues - ) and a flexible, highly extended glu-rich domain (residues . nsp a is a highly acidic protein (ph . ) that contains glutamic acids, of which are located within the c-terminal glu-rich domain. ppe antigens. proline and glutamic acid rich proteins (or pperepeat containing proteins, or ppe proteins) are important t-cell antigens produced by mycobacterium avium subsp paratuberculosis (map). one of the ppes is a . kda protein ( residues, pi . ) which following recombinant expression in e. coli was shown to elicit significant delayed type hypersensitivity skin reaction in mice sensitized with map, suggesting that this recombinant ppe protein of map was definitely associated with cellular immune response. curiously, this ppe contains alanines, glycines, prolines, aspartic acids but just glutamic acids. pt l . cassava storage roots differentially produce an interesting pt l protein with low sequence complexity characterized by a reduced amino acid alphabet (just amino acids). this residue-long protein contains glutamic acids, alanines, valines, prolines, serines and lysines, but does not have any arginines, asparagines, cysteins, histidines, phenylalanines, tyrosines and tryptophanes. glutamic acid-rich protein from cassava roots. based on the analysis of changes in the cassava root proteome during physiological deterioration of cassava root after harvesting, it has been concluded that the glutamic acid-rich protein was one of the proteins that were upregulated after harvesting. cp . eukaryotic genomes contain a set of specific functional elements, chromatin insulators or boundary elements that regulate gene transcription by interfering with promoterenhancer communication. in drosophila melanogaster, the centrosome-associated zinc finger protein cp protein (cp ) is a component of the gypsy chromatin insulator complex, which is composed of cp , mod(mdg ) and su(hw) and is required for the function of the gypsy chromatin insulator and other endogenous chromatin insulators organized by su(hw), ctcf the hippocampus and cerebral cortex of app(e Δ)-transgenic mice that are used as a model to study the pathological effects of aβ oligomers in alzheimer's disease. abra. the acidic-basic repeat antigen (abra) is a -residues-long protein found in the vacuolar space surrounding merozoites in plasmodium falciparum-infected erythrocytes, being localized in the parasitophorous vacuole and associated with the merozoite surface at the time of schizont rupture. due to its surface location, abra is one of the potential vaccine candidates against erythrocytic stages of malaria. this protein is one of the antigens enriched in the clusters of merozoites formed with growth inhibitory immune serum and possesses chymotrypsinlike activity, which can be inhibited with serine protease inhibitors such as chymostatin and phenyl methyl sulfonyl fluoride (pmsf). it was shown that the n-terminal half of the protein is responsible for the protease activity, whereas the highly charged c-terminal part of the protein was not required for this activity. furthermore, the n-terminus contains an erythrocyte-binding domain located within the cysteine-rich n-proximal region of abra. there are glutamic acids and lysines in abra, and in agreement with its name, the amino acid sequence of this protein is characterized by the presence of eight tandem repeats of [vt]-n-d-[ed]-[ed]-d (residues - ) and by a lysinerich c-terminal region (residues - ). kerp . the parasite entamoeba histolytica that colonizes the large bowel and provokes an asymptomatic luminal gut infection contains a peculiar lysine and glutamic acid-rich protein (kerp ), which is associated to parasite surface, involved in the parasite adherence to host cells and plays a role in the entamoeba histolytica liver abscess pathogenesis. an interesting feature of kerp ( residues) is a very high content of lysines ( %) and glutamic acids ( %). proteins with long simple repeat elements from herpesviruses. one of the mechanisms employed by herpesviruses to evade the immune response, allowing them to persist life-long in their hosts, relies on the use of specific proteins that function as cis-acting inhibitors of antigen presentation. among these inhibitors are the nuclear antigen (ebna ) and pgzr in the epstein-barr virus (ebv) and the latency-associated nuclear antigen (lana ) of the kaposi sarcoma herpesvirus. the common feature of all these proteins is the presence of long simple repeat elements in their amino acid sequences. for example, pgzr is a amino-acids long glycine, glutamine, and glutamic acid-rich repeat ("gz" repeat) protein that which is encoded by a large nested open reading frame located in the ebna mrna and is highly similar ( % amino-acid identity) to the acidic repeat of lana . latent nuclear antigen of human herpesvirus (hhv- ) (kaposi's sarcoma-associated herpesvirus) is a large ( , residues) highly acidic protein (pi . ) that contains glutamic acids, glutamines, prolines and aspartic acids. in herpesvirus saimiri (hvs) that infects squirrel monkeys, the functional homolog of epstein-barr virus ebna and kaposi's sarcoma-associated herpesvirus lana proteins is the residues-long product of the open reading frame known as orf or latency associated nuclear antigen. orf contains a repeat domain composed of a glutamic acid and glycine repeat mutation affects the ability of hla-dpb to present beryllium to pathogenic cd + t cells. sickle cell anemia and glu val mutation in hemoglobin. sicklecell (sca) or drepanocytosis is an autosomal recessive genetic blood disease with over-dominance, characterized by red blood cells that assume an abnormal, rigid, sickle shape. the disease is caused by a single point mutation in the β-globin chain of hemoglobin where the hydrophilic and negatively charged amino acid glutamic acid is replaced by the hydrophobic amino acid valine at the sixth position. as a result of this substitution, sickle hemoglobin polymerizes inside the affected erythrocytes. it was pointed out that such sickle hemoglobin polymerization occurs by homogeneous and heterogeneous nucleation mechanisms, which are both highly sensitive to macromolecular crowding. in fact, the rates of homogeneous nucleation were shown to be enhanced by when the initial concentration was augmented by % nonpolymerizing hemoglobin. retinitis pigmentosa and mutations in a glu-rich domain of rpgr. retinitis pigmentosa (rp) is an inherited, degenerative eye disease associated with the progressive loss of photoreceptor genes that causes severe vision impairment and often blindness. among other factors, rp is caused by mutations in the retinitis pigmentosa gtpase regulator (rpgr) gene which accounts for - % of rp cases in caucasians. genetic analysis revealed that of rpgr mutations % are associated with x-linked retinitis pigmentosa (xlrp), % are found in cone, cone-rod dystrophy or atrophic macular atrophy, and % are related to syndromal retinal dystrophies with ciliary dyskinesia and hearing loss. importantly, all disease-causing mutations occur in one or more rpgr isoforms containing the c-terminal exon open reading frame (orf ), and % occur in a glu-rich domain within exon orf , which accounts for only % of the protein. rpgr ( , residues) contains glutamic acids, more than half of which ( ) are located within the c-terminal glu-rich domain (residues - ). pyoderma gangrenosum and glu gln mutation in pstpip . pyoderma gangrenosum is a condition that causes tissue to become necrotic, causing deep ulcers that usually occur on legs. pyoderma gangrenosum is one of the most common extra-intestinal manifestations of chronic inflammatory bowel disease. the disease is caused by the alterations in the pathway that links the members of the proline-rich, glutamic acid-rich, serine-rich and threonine-rich (pest) family of protein tyrosine phosphatases (which are critical regulators of adhesion and migration) to their substrates. a major player in this pathway is a cytoskeleton-associated adaptor protein, namely proline-serine-threonine phosphatase-interacting protein (pstpip , also known as cd -binding protein , cd bp ). defects in pstpip are the cause of papa syndrome (papas), also known as pyogenic sterile arthritis, pyoderma gangrenosum and acne or familial recurrent arthritis (fra). papas is characterized by an autosomal dominant inheritance of early onset, primarily affecting skin and joint tissues. missense mutations glu -gln and ala -thr in pstpip /cd bp were identified in two families. these mutations were shown to affect the ability of pstpip to interact with its natural partners. , and beaf . although cp is a large protein ( , residues) that possesses a complex multidomain structure, only three domains were shown to be essential for the insulator function and for the viability of flies: the btb/poz domain, an aspartic acid-rich (d-rich) region and a c-terminal glutamic acid-rich (e-rich) region. here, the n-terminal cp fragment containing the btb/poz domain and the d-rich region was shown to be involved in regulation of the cp interaction with insulator complexes, whereas the c-terminally located e-rich region was necessary for the cp dissociation from chromosomes during heat-shock. importantly, the glutamic acids are not equally distributed within the protein, with the n-terminal half containing just glutamic acids and with the remaining glutamates being concentrated within the c-terminal half of cp . therefore, although the overall glutamic acid content of this protein is %, its c-terminal half is especially enriched in these residues ( . %). also, this uneven distribution is seen not only for glu, but for all the charged residues. in fact, the n-terminal fragment (residues - ) has a net charge of + (asp + glu = + = ; arg + lys = + = ), whereas the c-terminal half of cp (residues - ) has a net charge of − (asp + glu = + = ; arg + lys = + = ). pcp l . purkinje cell produces two closely related proteins containing iq motifs, purkinje cell protein -like (pcp l ) and pcp /pep- . although pcp /pep- is able to interact with calmodulin and inhibit calmodulin-dependent enzymes, and although the synthetic peptide constituting only the iq motif of pcp l binds calmodulin and inhibits calmodulin-dependent kinase ii, the full-length pcp l does not interact with calmodulin. the lack of ability of the full length pcp l to interact with calmodulin was ascribed to its nine-residue glutamic acid-rich sequence that lies outside the iq motif in pcp l . mutational analysis showed that calmodulin binding can be restored not only by the deletion of this inhibitory motif, but also by exchanging it with the homologous region of pep- and by simple point mutation converting a single isoleucine (ile ) within this motif to phenylalanine or to other aromatic residues. therefore, although pep- and pcp l possess noticeable sequence similarities, their functional properties are very different due to the presence of the glu-rich element in pcp l that can functionally suppress an iq motif. glutamic acid mutations and human diseases. chronic beryllium disease and lys glu mutation in hla-dpb . chronic beryllium disease (cbd) is a hypersensitivity disorder that affects - % of workers professionally exposed to berillium in the workplace. cbd is characterized by a granulomatous inflammation and accumulation of beryllium-specific cd + t cells in the lung. the susceptibility to this disease depends on both genetic factors (genetic susceptibility) and the nature of the exposure. genetic analysis revealed that a single point mutation at the th position of the human leukocyte antigen (hla) class ii histocompatibility antigen dp β chain (hla-dpb ), where lysine is substituted by a glutamic acid, makes the carriers more susceptible to cbd. it has been proposed that the k→e point of enzymes, or be related to metal binding. in idps/idprs, overabundance of glutamic acids defines the extended conformation of native coils and native pre-molten globules. glutamic acid is an important part of the pest motif related to protein degradation. it is crucial for function of entropic bristle domains and several chaperones. stretches of glutamic acid residues have a lot of specific functions that range from unique metal binding properties of phytochelatins and bone phosphoproteins, to regulation of cell adhesion and migration, to defining specific immunochemical reactivity of several antigens. no potential conflicts of interest were disclosed. this review illustrates that glutamic acid is differently used in ordered proteins/domains and in idps/idprs. in ordered proteins, glutamic acid residues are crucial for protein solubility and, being strategically placed within protein structure, play several structure-forming and structure-stabilizing roles. here, glutamic acid is involved in electrostatic interactions and hydrogen bond formation, serves as an important α-helix former, and participates in the α-helix cap formation. glutamic acid is an important functional residue of ordered proteins, where it can be involved in the 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and function of bacterial nitric oxide reductase mono-adp-ribosylation: a reversible posttranslational modification of proteins structure-function relationships of na(+), k(+), atp, or mg( +) binding and energy transduction in na,k-atpase narberhaus f. alpha-crystallin-type heat shock proteins: socializing minichaperones in the context of a multichaperone network distinct roles of the n-terminal-binding domain and the c-terminal-solubilizing domain of alpha-synuclein, a molecular chaperone polyanions and the proteome physical stabilization of acidic fibroblast growth factor by polyanions acceleration of the refolding of arc repressor by nucleic acids and other polyanions complex effects of molecular chaperones on the aggregation and refolding of fibroblast growth factor- mapping the functional domains of nucleolar protein b chaperone-like activity of tubulin. binding and reactivation of unfolded substrate enzymes role of the carboxy-termini of tubulin on its chaperone-like activity chaperone-like activity of tubulin productive folding of human neutrophil alpha-defensins in vitro without the pro-peptide pro-sequence of subtilisin can guide the refolding of denatured subtilisin in an intermolecular process the alpha-lytic protease proregion does not require a physical linkage to activate the protease domain in vivo analysis of prepro-alpha-lytic protease expression in escherichia coli reveals that the pro region is required for activity heavy metal detoxification in higher plants--a review modulation of mammalian sperm function by fertilization promoting peptide (fpp) microbial biosynthesis of polyglutamic acid biopolymer and applications in the biopharmaceutical, biomedical and food industries new biological functions and applications of high-molecular-mass poly-gamma-glutamic acid review on medusa: a polymer-based sustained release technology for protein and peptide drugs biomedical applications of chemically and microbiologically synthesized poly(glutamic acid) and poly(lysine) production and characterization of gamma-polyglutamic acid nanoparticles for controlled anticancer drug release biological and clinical characterization of paclitaxel poliglumex (ppx, ct- ), a macromolecular polymer-drug conjugate pest sequences and regulation by proteolysis amino acid sequences common to rapidly degraded proteins: the pest hypothesis intrinsic unstructuredness and abundance of pest motifs in eukaryotic proteomes coupling caspase cleavage and proteasomal degradation of proteins carrying pest motif functional protein domains from the thermally driven motion of polypeptide chains: a proposal entropic exclusion by neurofilament sidearms: a mechanism for maintaining interfilament spacing hybrid character of a large neurofilament protein (nf-m): intermediate filament type sequence followed by a long and acidic carboxy-terminal extension electron microscopy of map (microtubule-associated protein ) a complex of numa and cytoplasmic dynein is essential for mitotic spindle assembly sweeping away protein aggregation with entropic bristles: intrinsically disordered protein fusions enhance soluble expression conformational studies on poly-l-glutamic acid and copolymers of l-glutamic acid and l-phenylalanine study of the "molten globule" intermediate state in protein folding by a hydrophobic fluorescent probe salt bridge induced changes in the secondary structure of ionic polypeptides glutamate racemase as a target for drug discovery cape, a -amino-acid peptide, is necessary for bacillus anthracis polyglutamate capsule synthesis fusobacterium nucleatum, the first gram-negative bacterium demonstrated to produce polyglutamate ueber ein spezifisches protein der kapsel des milzbrandbazillus key role of poly-gamma-dl-glutamic acid in immune evasion and virulence of staphylococcus epidermidis polyglutamic acid from bacillus anthracis grown in vivo; structure and aggressin activity metal-binding characteristics of the gamma-glutamyl capsular polymer of bacillus licheniformis atcc characterization of bacillus subtilis gamma-glutamyltransferase and its involvement in the degradation of capsule polygamma-glutamate occurrence of poly-γ-d-glutamic acid and poly-α-l-glutamine in the genera xanthobacter, flexithrix, sporosarcina and planococcus transfer of natrialba asiatica b t to natrialba taiwanensis sp. nov. and description of natrialba aegyptiaca sp. nov., a novel extremely halophilic, aerobic, nonpigmented member of the archaea from egypt that produces extracellular poly(glutamic acid) poly(gamma-glutamic acid)s are the major constituents of nematocysts in hydra (hydrozoa, cnidaria) microbial degradation of poly(amino acid)s poly (glutamic acid)--an emerging biopolymer of commercial interest cloning and characterization of a novel trinucleotide repeat-containing gene garp from euplotes octocarinatus structure of a plasmodium falciparum gene that encodes a glutamic acid-rich protein (garp) a novel pepp homeobox gene, tox, is highly glutamic acid rich and specifically expressed in murine testis and ovary kibra functions as a tumor suppressor protein that regulates hippo signaling in conjunction with merlin and expanded kibra is a regulator of the salvador/warts/ hippo signaling network characterization of kibra, a novel ww domain-containing protein kibra is a novel substrate for protein kinase czeta temporal-spatial expression and novel biochemical properties of the memory-related protein kibra essential role of kibra in co-activator function of dynein light chain in mammalian cells kibra interacts with discoidin domain receptor to modulate collagen-induced signalling kibra modulates directional migration of podocytes cloning a new human gene from chromosome q . encoding a glutamic acid-rich protein expressed in heart and skeletal muscle the identification of a novel human homologue of the sh binding glutamic acid-rich (sh bgr) gene establishes a new family of highly conserved small proteins related to thioredoxin 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highly expressed in retina that exhibits a large area of intrinsic disorder the expression of a mitochondria-localized glutamic acid-rich protein (mgarp/osap) is under the regulation of the hpg axis expression of ovary-specific acidic protein in steroidogenic tissues: a possible role in steroidogenesis hummr, a hypoxia-and hif- alpha-inducible protein, alters mitochondrial distribution and transport a glutamic acid-rich protein identified in verticillium dahliae from an insertional mutagenesis affects microsclerotial formation and pathogenicity gala: a designed synthetic ph-responsive amphipathic peptide with applications in drug and gene delivery pest family phosphatases in immunity, autoimmunity, and autoinflammatory disorders regulation of tumor cell migration by protein tyrosine phosphatase (ptp)-proline-, glutamate-, serine-, and threonine-rich sequence (pest) structural analysis of the bacillus subtilis delta factor: a protein polyanion which displaces rna from rna polymerase marcks: a case of molecular exaptation? arginine and glutamate-rich (arglu ) interacts with mediator subunit (med ) and is required for estrogen receptor-mediated gene transcription and breast cancer cell growth pelp --a novel estrogen receptorinteracting protein growth factor regulation of estrogen receptor coregulator pelp functions via protein kinase a pathway functional significance and mechanism of eif -promoted gtp hydrolysis in eukaryotic translation initiation interaction in vitro of non-epithelial intermediate filament proteins with histones isolation, characterisation and growth-related changes of an hmg-like protein from microplasmodia of physarum polycephalum calcium-dependent molecular spring elements in the giant protein titin isolation, characterization, and biosynthesis of a phosphorylated glycoprotein from rat bone modulation of crystal formation by bone phosphoproteins: role of glutamic acid-rich sequences in the nucleation of hydroxyapatite by bone sialoprotein acidic amino acid-rich sequences as binding sites of osteonectin to hydroxyapatite crystals molecular analysis of xenopus laevis sparc (secreted protein, acidic, rich in cysteine). a highly conserved acidic calciumbinding extracellular-matrix protein pcp l contains an autoinhibitory element that prevents its iq motif from binding to calmodulin genetic susceptibility and immune-mediated destruction in berylliuminduced disease crowding and the polymerization of sickle hemoglobin retinitis pigmentosa rpgr mutation analysis and disease: an update mutations in cd bp disrupt binding to ptp pest and are responsible for papa syndrome, an autoinflammatory disorder pyrin binds the pstpip / cd bp protein, defining familial mediterranean fever and papa syndrome as disorders in the same pathway reduction in rna levels rather than retardation of translation is responsible for the inhibition of major histocompatibility complex class i antigen presentation by the glutamic acid-rich repeat of herpesvirus saimiri open reading frame nuclear magnetic resonance structure of the n-terminal domain of nonstructural protein from the severe acute respiratory syndrome coronavirus expression of a gene encoding . ?kda ppe antigen of mycobacterium avium subsp isolation and characterization of the promoter sequence of a cassava gene coding for pt l , a glutamic acid-rich protein differentially expressed in storage roots itraq-based analysis of changes in the cassava root proteome reveals pathways associated with post-harvest physiological deterioration the chromosomal association/dissociation of the chromatin insulator protein cp of drosophila melanogaster is mediated by the btb/poz domain and two acidic regions proteomic analysis of the brain tissues from a transgenic mouse model of amyloid β oligomers amino terminus of plasmodium falciparum acidic basic repeat antigen interacts with the erythrocyte membrane through band protein immunogenicity of recombinant fragments of plasmodium falciparum acidic basic repeat antigen produced in escherichia coli plasmodium falciparum: chymotryptic-like proteolysis associated with a -kda acidic-basic repeat antigen expression and characterisation of plasmodium falciparum acidic basic repeat antigen expressed in escherichia coli the lysine-and glutamic acidrich protein kerp plays a role in entamoeba histolytica liver abscess pathogenesis the nested open reading frame in the epstein-barr virus nuclear antigen- mrna encodes a protein capable of inhibiting antigen presentation in cis key: cord- -vlk cebh authors: kolter, thomas title: ganglioside biochemistry date: - - journal: isrn biochem doi: . / / sha: doc_id: cord_uid: vlk cebh gangliosides are sialic acid-containing glycosphingolipids. they occur especially on the cellular surfaces of neuronal cells, where they form a complex pattern, but are also found in many other cell types. the paper provides a general overview on their structures, occurrence, and metabolism. key functional, biochemical, and pathobiochemical aspects are summarized. together with glycoproteins and glycosaminoglycans, glycosphingolipids (gsls) contribute to the glycocalyx that covers eukaryotic cell surfaces. gangliosides are sialic acidcontaining glycosphingolipids and provide a significant part of cell surface glycans on neuronal cells. gsls are lipids that contain a sphingoid base and one or more sugar residues [ ] . sialic acids ( figure ) are nine-carbon sugars biosynthetically formed from n-acetylmannosamine and phosphoenolpyruvate [ , ] . with a mean pk a value of around . , they are more acidic than the majority of carboxylic acids and negatively charged at most physiological ph values. the name "ganglioside" was coined by the german biochemist klenk ( klenk ( - and assigned to a group of acidic gsls that he isolated from ganglion cells [ , ] and from the brains of patients who suffered from the so-called amaurotic idiocy [ , ] . sialic acid was first isolated from submaxillary mucin in [ ] . its structure was elucidated in the nineteen fifties by different groups and it was found to be identical to that of the n-acetylneuraminic acid isolated by klenk and faillard. the first structure of a ganglioside was elucidated in by kuhn and wiegandt [ ] . in , svennerholm suggested a nomenclature of brain gangliosides [ , ] . the biochemical defects underlying the diseases formerly known as amaurotic idiocy, gm gangliosidosis [ ] , tay-sachs- [ ] , and sandhoff disease [ ] were identified by sandhoff and others in the s. in their structures, gangliosides combine a glycan and a lipid portion and contribute to both, the cellular lipidome and the glycome/sialome [ ] . a great variety of carbohydrate sequences are found within the gsls [ ] , including the gangliosides [ ] . although carbohydrate residues of different structure, linkage, and anomeric configuration occur in gsls, only a limited number of the so-called series with characteristic carbohydrate sequences are found within evolutionary related organisms (table ) . within the gangliosides, sialic acids can be attached only to a few of the gsl series, in adult mammals especially to the ganglio series. among the sialic acids, n-acetylneuraminic acid is the most frequently found member in humans, but also nglycolylneuraminic acid is abundant in many other species (figure ) . a total of more than different sialic acids have been described [ , ] . they can be o-acetylated in positions , , or [ ] , but also n-deacetylated, omethylated, sulfated, or modified by lactonization [ ] (see figure ). the nomenclature of gsls specifies the glycan part of these lipids. two ganglioside nomenclature systems are currently in use to assign names to the corresponding structures. most researches prefer the short-hand nomenclature according to svennerholm, which was initially based on the migration order of ganglio-series gangliosides in chromatography [ ] . later on, it has been extended to other root structures. the more comprehensive iupac system [ ] is less frequently applied. according to svennerholm, a core structure of neutral sugars define the name of a respective series, in which the pyranose forms of dgalactose (gal), d-n-acetyl-glucosamine (glcnac), or d-n-acetylgalactosamine (galnac) are attached in defined order and linkage to lactosylceramide (galβ , glcβ cer) or β-galactosylceramide (galβ cer). the names contain information about the series ("g" = ganglio, "l" = lacto), the number of sialic acids ("a" = , "m" = , "d" = , "t" = , "q" = , "p" = , "h" = , "s" = ), and, indirectly, on the number of uncharged carbohydrates: initially it has been assumed that this number cannot exceed , so that the name "ganglioside gm " indicates that this ganglioside contains ( − " " = ) neutral sugars of the ganglio series. this series is defined by the sequence galβ - galnacβ - galβ - glccer. sialic acids can be attached once, twice, or severalfold to different positions within the core structures. most often, they are found in α , -linkage to the "inner" or "outer" galactosyl residue, and in α , -linkage to other sialic acids. ganglioside gm bears one sialic acid moiety connected to the -oh-group of the galactosyl residue in position ii of the gangliotetraose moiety (see also figure ). the corresponding iupac-iubmb short-hand name is ii neu acgg cer. structures of ganglio-series gangliosides can also be derived from the scheme of ganglioside biosynthesis (see below; figure ). in general, ganglio-series gsls of the -series bear no sialic acids on the galactose in position ii, of the a-series bear one, of the b-series bear two, and of the c-series bear three sialic acid residues. however, gm b and gd c have a "b" and "c" in their names, although both are -series gangliosides (see the scheme of ganglioside biosynthesis, figure ). gm is a gala-series ganglioside, although the "g" suggests ganglio series. figure shows the structure of ganglioside gq b, one of the most abundant gangliosides in adult human brain (g = ganglio series, q = sialic acids, − = neutral carbohydrate residues, and bseries = sialic acids attached to the "inner" galactose). ganglioside core structures can be additionally modified; they can be elongated, such as in gd agalnac [ ] ( figure ). this ganglioside occurs, for example, on spinal neurons [ , ] and can give rise to autoantibodies as a cause of variant forms of the guillain-barré syndrome [ , ] and other neuropathies [ ] . a modified gm derivative that contains taurine in amide linkage to the sialic acid carboxyl group has been identified in the brain of patients with tay-sachs disease [ ] . hybrid-type gsls and gangliosides with postglycosylation modifications add further complexity to this substance class [ ] . as an example, lacto-ganglio hybrid-type gangliosides have been identified in bovine brain [ ] . most gangliosides found in adult mammals belong to the ganglio, gala, lacto, and neolacto series. ganglioside gm (figure ), a member of the gala series, has the structure neuacα , galβ cer and is often found with an α-hydroxyfatty acid within the ceramide moiety. during development, also gangliosides with other core structures are transiently formed, such as the stage-specific embryonic antigen ssea- , a ganglioside of the globo series [ ] (figure ). in adults, globo-series gangliosides occur on human erythrocytes [ ] , in human kidney [ ] , and on various stem cells [ ] . for example, ssea- , but not ssea- or globo-h (figure ), is expressed in cord blood-derived mesenchymal stem cells [ ] . with the exception of echinoderms (marine organisms of typically pentaradial symmetry), gangliosides are usually absent from invertebrates. arthropods, for example, form acidic gsls with a manβ , glcβ , cer core, which contain glucuronic acid instead of sialic acids. for gangliosides of echinoderms [ ] [ ] [ ] [ ] [ ] , there is no systematic short-hand nomenclature. they show structural features uncommon to mammalian gangliosides, such as sialic acid residues within the oligosaccharide moieties (e.g., lg- , figure ), α , linked sialic acids (e.g., llg- , figure ), sialic acid methylation or sulfation, or a glycosyl inositolphosphoceramide core, for example, [ , ] . in cultured neurons, echinodermal gangliosides show neuritogenic and growth-inhibitory activities. in this regard, they are more potent than other figure : structure of gq b, one of the most abundant gangliosides in adult human brain, which is involved in long term potentiation, synaptic plasticity, and improvement of cognitive function [ ] . gangliosides [ , ] and potentiate the neuritogenic effect of nerve growth factor. heterogeneity is not only found within the glycan part, but also within the ceramide moiety. this can consist of different sphingoid bases [ ] , sphinganine, sphingosine, and phytosphingosine of different chain lengths ( figure ), which can be further modified by o-acetylation [ ] . in higher animals, c -and c -sphingosine are the most abundant sphingoid bases of gangliosides. the fatty acids found in the ceramide part of gangliosides are mostly saturated. α-hydroxylated fatty acids [ ] are not frequently found in brain gangliosides, but are, for example, abundant in gangliosides from intestine, liver, or kidney, and in gm . to specify the lipoform of a ganglioside, designations such as (d : / : )gm are used for a ii neu aclaccer with a sphingosine (d = dihydroxy, = one double bond; see also figure ) of carbons and a stearoyl residue ( : ) within the ceramide portion. the functional consequences of the heterogeneities in the lipid component are largely unknown, but the lipid part can mask the receptor function of ganglioside glycans via interaction with membrane cholesterol [ , ] . as another example, the ceramide portion of gm dictates retrograde transport of cholera toxin bound to gm , and only gm with unsaturated acyl chains is sorted from the plasma membrane to the trans-golgi network and the er [ ] . ganglioside profiling with respect to glycan and ceramide structures is more and more in the focus of ganglioside analysis. gangliosides are especially abundant in the brain, where their occurrence in the grey matter is about -fold higher than in white matter. in adult human brain regions, the β galt-v β galnact figure : formation of ssea- from globotriaosylceramide (gb cer). ssea, stage-specific embryonic antigen; t, transferase; fut, fucosyltransferase (modified from [ ] figure : examples for gangliosides from echinoderms: lg- from the starfish astropecten latespinosus [ ] and llg- from the starfish linckia laevigata [ ] [ ] [ ] . d-ribo-phytosphingosine values range from to μg lipid-bound sialic acid/mg protein [ ] . in the brain, ganglioside expression correlates with neurogenesis, synaptogenesis, synaptic transmission, and cell proliferation [ , ] . in cultured murine hippocampal neurons, axonogenesis, but not dendritogenesis, is accompanied by an increase in the formation of complex gangliosides and by a shift from the a-to b-series [ ] . in extraneural tissues, the ganglioside content is one-to twoorders of magnitude lower than in the brain; relatively high concentrations of ganglio-series gangliosides are found in bone marrow, erythrocytes, intestine, liver, spleen, and testis, gm in kidney, and ssea- in embryonic stem cells. cellular gangliosides form in part complex, cell-type-and tissue-specific glycan patterns [ ] . these are not stable with time, but change with physiological and pathophysiological processes such as cell growth, differentiation, viral transformation, ontogenesis, oncogenesis, embryogenesis [ , ] , lactation, or tumor progression [ ] . gangliosides of the ganglio-series are especially found in the nervous system, where they contribute to - % of the lipid content [ ] . during brain development, the ganglioside pattern changes from the prevalence of the simple gangliosides gm and gd to more complex ones such as gd a and gt b [ ] (for structures, see figures and ) . ganglioside content and composition of the brain change also during aging: for example, the amount of lipid-bound sialic acid decreased from μg/g wet weight in a -year-old healthy proband to μg/g wet weight in a -year-old individual. despite this, the concentrations of gq b, gt b, and gd b increase with age at the expense of gm and gd a [ ] (for structures, see figures and ) . changes in ganglioside composition with age also occur in liver [ ] . there are only indications on the functional consequences of such changes [ ] . gangliosides are also found in serum. there, especially gm , gd , gd a, gm , gt b, sialylneolactotetraosylceramide ( figure ), gd b, and gq b are present, where about % of them are transported by serum lipoproteins, predominantly by ldl ( %), followed by hdl ( %) and vldl ( %) [ ] . after the discovery of extracellular microvesicles (formerly called microparticles) [ ] , which were not distinguished from lipoproteins in earlier experiments, it might turn out that these assignments have to be revised. experiments in rats have shown that after injection of [ c]sialic acid-labeled gangliosides gm and [ h]sphingosine-containing labeled ganglioside gm , the gm and gm probes had serum half-lives of . and . h, respectively. after three hours, % of the gm and % of the gm probes were taken up by the liver, and a smaller extent in the central nervous system, kidneys, and lung [ ] . subcellularly, the majority of gangliosides resides in the plasma membrane [ ] . however, gangliosides also occur in organellar membranes such as in mitochondria, where gd regulates apoptosis [ ] , and in the nucleus, where they are involved in ca + balance [ , ] . the glycans found in gangliosides are sometimes modified by the acylation of sialic acid residues in different positions [ ] . o-acetylated sialic acids in gangliosides occur especially in growing cells and tissues and are regarded as oncofetal markers present on different tumors [ ] . they also serve as receptors for influenza c viruses or coronaviruses [ ] . another modified sialic acid is n-glycolylneuraminic acid (neu gc) [ ] . with the exception of certain tumors and in fetuses, it is found only in trace amounts in human tissues [ ] . as a component of glycoconjugates, neu gc is known as the hanganutziu-deicher antigen [ ] . it is abundant in many species of the deuterostome lineage, including simians, mice, rat, beef, pork, or lamb, but is nearly absent from birds and reptiles [ ] . neu gc on glycoconjugates contributes to xenoantigenicity in pig-human xenotransplantation [ ] , and in cats, neu gc distinguishes the blood groups a and b: [neu gc] gd is found in feline blood group a erythrocytes, [neu ac] gd on blood group b, and feline blood group ab erythrocyte membranes contain [neugc] gd , [neu ac,neu gc]gd , and [neuac] gd [ ] . humans cannot synthesize neu gc due to an irreversible inactivation of the cmah gene on chromosome p . encoding cytidine monophosphate-n-acetylneuraminic acid hydroxylase [ ] . this enzyme converts cmpneu ac to cmpneu gc and its function is thought to be lost during a "sialoquake" in human evolution [ , ] . determination of neu gc and neu ac-containing gangliosides is either achieved by classical chromatographic techniques combined with antibody staining [ ] , or, with higher sensitivity, by combination of chromatography with esi-ms [ ] . a potential application is the immunochemical detection of [neu gc]gm as biomarker of nonsmall-cell lung cancer [ ] . also ganglioside lactones ( figure ) have been detected in various tissues, for example, gd lactone in mouse brain [ ] and gd b lactone in human brain [ ] . ganglioside lactones are more immunogenic than gangliosides [ ] and occur on tumor cells such as melanoma as tumor-associated antigens. in vitro, lactonization of gangliosides can be followed by a strong negative cotton effect at nm in cd spectroscopy [ ] . temporal and spatial differences are also observed for the ganglioside lipid part. in undifferentiated neuronal cell cultures, gangliosides with c sphingosine are present only in trace amounts, but their content increases with the onset of cell differentiation [ ] . in rat brain, the fraction of gangliosides containing c sphingosine increases with age [ ] in cerebellum [ ] or forebrain [ ] . spatial differences regarding the sphingoid base chain length have also been detected in mice: while gangliosides containing c species were widely distributed throughout the frontal brain, c species are selectively localized along the entorhinalhippocampus projections [ ] . fatty acid and sphingoid base composition is also different between human motor and sensory nerves [ ] . nutrition. since gangliosides are components of most vertebrate cell types, they are ingested with the nutrition, for example, with egg yolk (gm , gm , and gd ), meat, or in milk [ ] . milk contains gangliosides, especially gd and gm , in the membrane fraction of the fat globule. dietary gangliosides modify the intestinal microflora and prevent infections during early infancy [ ] . in infants, more than % of dietary gangliosides survive the passage through the stomach, in part with acid-catalyzed lactonization, and are absorbed in the intestine [ ] . ingestion of dietary gangliosides leads to an increase of gangliosides in serum. in human nutrition, sialic acid derived from gangliosides and other glycoconjugates is an essential nutrient for the rapidly growing brain in infants [ ] . the pathophysiological consequences of nutritional neu gc uptake are unknown. in the past, ganglioside structures and levels were obtained by comprehensive chemical analysis, while nowadays this is attempted within lipidomics using mass spectrometry as the key technology [ ] . in general, gangliosides are isolated from tissues and body fluids by chloroform-methanol extraction [ , ] . extraction efficiency can increase when small amounts of water are present in the extraction solvent [ ] , for example, using the solvent system chloroform : methanol : water ( : : ) [ ] . when extraction is followed by a partition step such as that developed by folch et al. [ ] , gangliosides-in contrast to the majority of other lipid classes-partition into the upper, aqueous phase. from there, they can be isolated by a solid phase extraction and separated from neutral gsls by anion exchange chromatography [ ] , such as with deae (=diethylaminoethyl) sephadex [ ] . o-acetylation of sialic acids and also ganglioside lactonization [ ] are modifications that are lost under alkaline conditions. these are often applied to remove glycerophospholipids that contain fatty acids in ester linkage [ ] . if information on these modifications is desired, gangliosides from tissues can be determined without alkaline treatment, for example, after chloroform/methanol extraction in a ratio of : and a subsequent partition step [ ] . separation of gangliosides according to their glycan composition is achieved by thin layer chromatography (tlc) [ ] and by hplc and other techniques that can be coupled to mass spectrometry [ ] . this facilitates their identification by mass spectrometry and is required for their characterization by staining with suitable antibodies [ , ] , lectins, or other binding proteins [ ] . although not required for mass-spectrometric profiling, separated ganglioside classes can also be further separated according to their ceramide structure by reversed phase chromatography [ ] [ ] [ ] . quantification can be achieved by staining and densitometry, or-if suitable standard substances are availableby mass spectrometry. since the biosynthetic machinery generates heterogeneities within both, the lipid and the glycan part, comprehensive ganglioside analysis is a highly demanding task within lipidomics [ ] . in addition to glycoforms that are also well known from glycoprotein analysis, "lipoforms" [ ] become increasingly important to understand ganglioside metabolism and function. various protocols for ganglioside determination by mass spectrometry have been developed [ ] . they are largely based on electrospray mass spectrometry as ionization technique; but also maldi plays a role. the available methods range from preanalytics to bioinformatic data handling and include imaging methods using maldi and secondary ion mass spectrometry (sims) to determine the spatial distribution of the analytes [ ] . in addition to their constitution, little is known about the conformation of gangliosides in their native, membrane-bound surroundings. x-ray data are not available for gangliosides, although isolated glycans have been investigated by various means. for a simulation of gm conformations in a bilayer, compare [ ] , which, for example, shows that the glucose moiety of gm is buried within phosphatidylcholine head groups. the diversity of cell surface glycans, including that of gangliosides, is generated within the golgi apparatus [ ] , and the heterogeneities within the ceramide part result from the biosynthesis of ceramide at the endoplasmic reticulum (er). de novo synthesis of gangliosides can be distinguished from salvage processes [ , ] , in which sialic acids, sugars, fatty acids, and sphingoid bases are recycled. the latter process can predominate by far in differentiated cells. ganglioside biosynthesis starts with the formation of ceramide ( figure ) at the cytoplasmic leaflet of the er membrane [ ] [ ] [ ] . the first step, the condensation of l-serine and a coenzyme a-activated fatty acid is catalyzed by the pyridoxal phosphate-dependent serine palmitoyltransferase (spt) [ ] . the incorporation of l-serine into gsls can be used to monitor their de novo biosynthesis using l-serine radiolabelled in the position isrn biochemistry (the carbon in position is lost as carbon dioxide). in the brain, the external supply of l-serine by astrocytes is essential for neuronal lipid biosynthesis and brain development [ ] . in agreement with this observation, genetically engineered rodents with deficient phosphoglycerate dehydrogenase required for l-serine formation from d-glucose show drastically reduced ganglioside levels, defects in brain morphogenesis, and drastically reduced lifespan [ , ] . the next step in sphingolipid biosynthesis is the nadph-dependent reduction of -ketosphinganine to sphinganine by -ketosphinganine reductase, followed by acylation of sphinganine to dihydroceramides of different chain lengths [ ] . during salvage, also other sphingoid bases are acylated by n-acyltransferases of the lass family. lass encodes ceramide synthase , which is expressed in the brain and involved in the formation of the membrane anchor of gangliosides. in mice, spontaneous recessive mutations in the lass gene are associated with cerebellar ataxia and purkinje cell degeneration [ ] . although the ceramide part of brain gangliosides contains mostly nonhydroxylated fatty acids, apparently all members of the lass family are also able to transfer the corresponding -hydroxy-fatty acids [ ] . dihydroceramides are dehydrogenated to ceramide by the dihydroceramide desaturase des [ ] , or hydroxylated to phytoceramides by des . ceramide is the common precursor of gsls and sphingomyelin and is transported to the golgi apparatus at least in part in a protein-dependent manner by the transport protein cert [ ] [ ] [ ] . formation. gsl synthesis continues by the stepwise transfer of nucleotide-activated monosaccharide units first on ceramide and then on gsls with growing glycan chains. glycosidation is coupled to exocytosis through the golgi apparatus to the plasma membrane [ ] at the rate of bulk vesicle flow [ ] . the complex ganglioside and gsl glycoforms on eukaryotic cell surfaces are generated by only a few enzymes that act within a combinatorial biosynthetic pathway [ , ] . the first glycosyltransferases involved in ganglioside biosynthesis have been characterized in the laboratories of roseman and basu [ ] . according to the number of sialic acids connected to the "inner" galactosyl residue, ganglioseries gangliosides are classified into members of the -, a-, b-, and c-series ( figure ). b-series gangliosides contain the neu acα , neu ac sequence, which is commonly not found in glycoproteins. higher members of these different subseries can be formed by the action of the same glycosyltransferases, which show less specificity than those acting early in the pathway [ ] [ ] [ ] [ ] [ ] . the glycosyltransferases and sialyltransferases [ , ] of the ganglioside biosynthetic pathway are expressed in a cell-type-and developmental-dependent fashion. ganglioside pattern changes during the development of the brain [ ] , and after differentiation, differences in glycolipid composition have even been found between different neuronal cell types [ ] . in addition, ganglioside patterns vary between different cell types and change with the differentiation of the cell. as an example, β , -n-acetylglucosaminyl-transferase expression, which leads to the formation of glycolipids of the lacto and neolacto series ( figure ), is high during murine embryonic development and decreases after birth to undetectable levels in most cell types [ , ] . in adult animals, expression is high in spleen [ ] , and in cerebellum, it is restricted to purkinje cells [ ] . gsls including gangliosides are formed biosynthetically at intracellular membranes from which they are transported to the plasma membrane by exocytotic membrane flow [ ] . while many human diseases are known that are due to defects in gsl and sphingolipid degradation, the only known human disease caused by a defect glycosyltransferase of ganglioside biosynthesis is the human autosomal recessive infantile-onset symptomatic epilepsy syndrome, which is caused by a nonsense mutation in the gene encoding gm synthase [ ] . a principal difference between ganglioside biosynthesis in the golgi apparatus and degradation in the endolysosomal compartment is that during gsl formation, membranebound glycosyltransferases interact with their membranebound glycolipid substrates by diffusion within the twodimensional plane of the lipid bilayer. therefore, reaction rates can become independent of the reaction volume and obey two-dimensional enzyme kinetics. this means that kinetic constants can be normalized on lipid surface area instead of reaction volume, for example, in terms of the amount of membrane protein [ ] . as a consequence, glycosyltransferases that lack their transmembrane domain lose most of their activity towards membrane bound substrates [ ] . during degradation in endosomes and lysosomes, the glycosidases are soluble enzymes, and the substrates are membrane-bound. this explains in part the requirement for endosomal and lysosomal lipid-transfer proteins for the degradation of gsls with short glycan chains, which is not the case in biosynthesis. in addition to ganglioside biosynthesis in the golgi apparatus, there are also indications ganglioside formation by plasma membrane-associated glycosyltransferases [ ] . in the monoglycosylceramides glucosylceramide (glccer) and galactosylceramide (galcer), which are also called cerebrosides, the hexosyl residues are present in βanomeric configuration. galcers with α-configuration occur only in lower organisms [ ] and are highly immunogenic for mammals [ ] . most gangliosides are biosynthetically derived from glccer; only ganglioside gm is derived from galcer. ganglioside gm has been discovered as a minor component of human brain gangliosides [ ] , where it is localized within myelin [ ] . it also occurs, for example, on erythrocytes, kidney, and in the intestine and is abundant in some fish species. however, the most frequently found members of the gala series are galcer and sulfatide (galcer- -sulfate) in oligodendrocytes, schwann cells, kidney, testis, and intestine. they are present in high concentrations in the multilamellar layers of the myelin where they are required for glial adhesion [ ] , apparently via interaction between the carbohydrate head groups of sulfatide and galcer on different myelin layers [ ] . myelin lipids contain the highest fraction of -hydroxy-fatty acids, which are formed by fatty acid hydroxylase- [ ] . their presence in gala-series gsls contributes to carbohydrate-carbohydrate interactions between the gsls [ ] . in contrast to galcer synthase, glccer synthase appears to be dispensable for oligodendrocytes [ ] . while ceramide galactosylation catalyzed by udp-glucose:ceramide galactosyltransferase (galt ) [ ] occurs at the er membrane, the later steps of gala-series gsl biosynthesis, formation of sulfatide [ ] , digalactosylceramide [ ] , and ganglioside gm take place in the lumen of the golgi apparatus. in contrast to most glycosyltransferases in ganglioside biosynthesis, which are type ii transmembrane proteins, ceramide galactosyltransferase is a type i transmembrane protein with the catalytic domain on the luminal side of the er [ ] . according to data obtained in zebrafish and mice, gm can be formed by st gal v, which can also make gm . therefore, gm and gm formation appear to depend on the availability of their precursors, galcer and laccer [ ] . little is known about the function of gm . it can interact with the myelin basic protein, shows immunosuppressive properties, and can prevent experimental allergic encephalomyelitis in guinea pigs [ ] . glucosylceramide. the first step in the biosynthesis of most gangliosides is the transfer of a glucose residue from udp glucose to ceramide catalyzed by udp-glucose:ceramide glucosyltransferase [ , ] . although glccer and galcer synthases catalyze similar reactions, their cdnas share no sequence homology. ceramide glucosyltransferase is a type iii transmembrane protein. it forms noncovalent dimers or oligomers [ ] with their cterminal catalytic domains in the cytosol [ ] . since the formation of glccer occurs on the cytoplasmic face [ ] and that of laccer on the luminal site of the golgi membrane [ ] , glucosylceramide has to be translocated across a membrane. this is mediated by a flippase of unknown identity: the abc-transporters, abc-b and -c , translocate short chain glccer analogs through the golgi membrane [ , ] . transversal translocation can be carried out after transport of glccer by the cytoplasmic lipid-transfer protein fapp (four-phosphate adaptor protein ) either to the er [ ] , where it might be translocated by an uncharacterized flippase [ ] , or at the trans-golgi [ ] . a part of the glccer pool can reach the cytosolic leaflet of the plasma membrane where it can be degraded by the β-glucosidase gba [ ] . candidate cytosolic glccertransporters are the glycolipid transfer protein gltp and fapp . the biosynthesis of higher gangliosides occurs on the luminal face of the golgi apparatus [ ] , so that their glycan chains are orientated extracytoplasmic. laccer is formed by galactosyltransferase i, which transfers a galactose residue from udp galactose to glucosylceramide [ ] . further carbohydrate residues are transferred in a stepwise manner to the growing glycan chains. laccer and its sialylated derivatives, the hematosides gm , gd , and gt ( figure ) serve as precursors for complex gangliosides of the -, a-, b-, and c-series. these different series ( figure ) are characterized by the presence of no ( -series), one (a-series), two (b-series) or three sialic acids residues linked to the position of the "inner" galactosyl residue. in adult mammalian brain, gangliosides from the -and c-series are found only in trace amounts, and gm b and gd α are transiently expressed during chick brain biogenesis [ ] . -series gangliosides (gm b, gd c, and gd α) are found in genetically engineered mice deficient in st gal v (gm synthase), where they are present in amounts that correspond to the total ganglioside content of normal animals [ ] . these mice are not able to form gm and higher gangliosides of the a-c-series. they display altered glucose homeostasis with an accelerated insulin receptor signalling pathway, a key finding that demonstrates the inhibition of the insulin receptor by gm or a higher ganglioside derived from it in vivo [ ] . c-series gangliosides (figures and ) are formed during mammalian brain development where they are thought to be involved in growth, differentiation, and migration of neuronal cells. they are abundant in fish brain, and in adult rats; they occur in liver, kidney, and pancreas [ ] and in tumors such as glioma. the transferases that catalyze the first steps in ganglioside biosynthesis show high specificity towards their glycolipid substrates. the relative amounts of laccer, gm , gd , and gt seem to determine the amount of -, a-, b-, and c-series gangliosides. the glycosyltransferases that act late in this pathway represent a kind of assembly line and transfer the respective carbohydrates to glycosyl acceptors that differ only in the number of sialic acid residues bound to the "inner" galactose residue. the complex "α-"gangliosides with sialic acid moieties in α , -glycosidic linkage to n-acetylgalactosamine residues is specific for cholinergic neurons [ ] and has been added later to the biosynthetic scheme [ ] . in mice, the sialyltransferases that form gangliosides gd a and gt b have been identified as st gal ii and st gal iii [ ] . a significant advance towards understanding the function of the complex ganglioside pattern found in eukaryotic cells is the development of mice with defects in distinct biosynthetic steps [ ] . a mouse melanoma cell line deficient in glccer and glccer-derived gsls was viable and showed only minor changes in cellular morphology and growth rate. from these observations it was concluded that gsls including gangliosides are not essential for animal survival [ ] . later, it was reported that mice with targeted disruption of the ceramide glucosyltransferase gene displayed no cellular differentiation beyond the primitive germ layers and died around day . of embryonic development [ ] . mice deficient in b galnt i (gm synthase) are not able to form gm , gd , and higher gangliosides derived from them. although these animals show only subtle impairment of brain function [ ] , they exhibit multiple defects, such as axonal degeneration, defects in myelination [ ] and motor function [ ] , or an impaired response of t cells to interleukin [ ] , only to mention a few. later studies showed that cd -and cd -positive t cells require different ganglioside subsets for activation [ ] . the mutant male mice are sterile and also show morphological and functional defects in the testis [ ] . further examination of galnac-transferase deficient mice revealed that gm -deficiency is accompanied by parkinsonlike symptoms, which could be rescued by l-dopa or the membrane permeable gm -analog liga (see figure ) [ ] . this is in agreement with a series of reports that gm can alleviate symptoms in models of parkinson disease, for example, [ ] . in parkinson disease, anionic lipids and especially gm inhibit aggregation of α-synuclein to cytotoxic fibrils [ ] . mice deficient in st sia i (gd -synthase) do not form gd and b-series gangliosides. they have a normal life span and are without detectable developmental defects [ ] . when these mice were crossbred with mice carrying a disrupted b galnt i gene, the resulting double mutant mice express only ganglioside gm as their major ganglioside. these "gm -only-mice" are extremely susceptible to sound stimuli, develop lethal seizures, and display a sudden death phenotype [ ] . double knockout mouse deficient in b galnt i and st gal v (gm -synthase) are not able to form any ganglioside of the ganglio-series. these animals are severely diseased and show elevated levels of laccer, laccersulfate, and traces of other gangliosides that are present also in normal brain [ ] . sphingolipid biosynthesis is a highly regulated process and also coordinated with sterol and glycerolipid biosynthesis. sphingolipids are major regulators of lipid metabolism and activate sterol-regulatory element binding proteins (srebps) [ ] . the sphingomyelin synthaserelated synthase, the ceramide transporter cert, and proteins of the orosomucoid-(orm)-familie seem to play key roles in sphingolipid homeostasis [ ] . ganglioside pattern are characteristic for a cell type in a certain differentiation state, and, for example, mice deficient in gm -synthase that cannot form the typical brain gangliosides show a ganglioside content similar to that of normal animals [ ] . how exactly the relative amounts of gangliosides are controlled is not clear [ ] , but the transcriptional regulation of transferase genes seems to be a key point [ ] . the picture gets more complicated by the fact that different transferase isoforms with different properties can be present: three murine gm -synthase isoforms that arise from two transcripts have been characterized. one is resident in the er membrane, the two others in the golgi, but with different half-life [ ] . in addition, the kinetic parameters of the transferases, their topological organization within the golgi apparatus, or spatial neighborhood to other transferases will influence the resulting ganglioside pattern. an attempt has been made to calculate glycolipid pattern on the bases of the kinetic constants of the transferases, that were estimated from the steady state concentrations of the glycolipid substrates in intact cells [ ] . contradictory results have been reported on the subcellular localization of the glycosyltransferases involved in the biosynthesis of ganglioseries gangliosides [ ] . an additional feature of ganglioside biosynthesis and its regulation [ ] is the formation of functional complexes, as predicted by roseman [ ] . in these complexes [ ] , the glycosyltransferases do not only form functional platforms, but can also show altered activity and suborganellar localization [ ] . one of these complexes characterized in certain cho cells [ ] comprises b galnt i and b galt iv (figure ), so that it can accept gm and release gm . this might explain why the brain contains large amounts of gm and gd a, but little gm . also galt i, st gal v, and st sia i can form such a complex [ ] . . . general. the constitutive degradation of gangliosides takes place in endosomes and lysosomes. in addition, also the plasma membrane-associated sialidase neu [ , ] can degrade gangliosides and is, for example, highly expressed on melanoma cells [ ] . even the nuclear envelope contains sialidases, with neu in the inner and neu in the outer nuclear membrane [ ] . lysosomal ganglioside degradation takes place after the endocytosis of parts of the plasma membrane at intraendosomal and intralysosomal membranes and related lipid aggregates. this requires the presence of suitable glycosidases [ ] , of an appropriate ph, in some cases also of lipid-transfer proteins, and of an appropriate composition of the ganglioside-containing membranes [ ] . as proposed in , two different membrane pools are present in endosomes and lysosomes [ ] (figure ). they differ in lipid-and protein composition and function. while the luminal membrane pool that is derived from the plasma membrane or by autophagy is degraded, the perimeter membrane ( figure ) is protected from degradation by various means [ ] . this ensures the integrity of the compartment, which can be abolished during apoptosis [ ] . a marker lipid that is exclusively found in luminal membranes [ ] is bis(monoacylglycero)phosphate (bmp; figure ) , chemically incorrect also named as lysobisphosphatidic acid (lbpa). bmp plays a key role for membrane degradation [ ] and is formed from phosphatidylglycerol [ ] . due to its sn , sn configuration, it is only slowly degraded by lipases and persists on inner membranes, in which it can amount up to % of total phospholipids [ ] . with a predicted pk a value of about , bmp is negatively charged even at lysosomal ph. in vitro studies show that negatively charged lipids are required for binding of lysosomal proteins to membranes. although other negatively charged lipids such as dolichol phosphate or phosphatidylinositol can be present on luminal membranes, bmp appears to be the key factor that distinguishes this membrane pool from the perimeter membrane. on the other hand, the perimeter membrane of endosomes and lysosomes shows an entirely different lipid and protein composition. it is protected by a glycocalyx formed by highly n-glycosylated integral membrane proteins [ , ] , and ganglioside gm present in this membrane is resistant to degradation [ ] . ganglioside degradation starts with the action of glycosidases that cleave off monosaccharide units from the non-reducing end of the ganglioside glycan chains. this happens in a sequential manner, which explains the different human diseases that are associated with defects in this pathway. the glycosidases are soluble enzymes in the lumen of endosomes and lysosomes. it turned out that their activity is not sufficient towards gsl substrates with cleavage sites in proximity to the intralysosomal membrane surface. although also other factors play a role, this can be attributed to steric hindrance by adjacent membrane components that impede the access of the soluble enzyme. for example, in wild-type and gm -activator deficient fibroblasts, radiolabelled gd agalnac (figure ), which has the same terminal trisaccharide as gm , is degraded in the absence of the gm -activator protein, while the degradation of gm itself is strictly dependent on the presence of the activator [ ] . as glycosidase substrates, gsls with four carbohydrate residues or less require the additional presence of small lipid binding glycoproteins, either the gm activator protein or one of the four saposins a-d. these act in part as lipid-transfer proteins that extract the membrane-bound substrates and present them to the hydrolases. they have different specificities and mechanisms of action [ ] . in the case of gangliosides, at least the gm -activator protein and saposin-b participate in the degradation of gm , gm , and gm ( figure ). in vitro, in addition to enzymes and activator proteins, also an appropriate membrane-lipid composition of the ganglioside-containing membrane is required for degradation [ ] . saposin-a [ ] and saposin-b [ ] extract membrane lipids much better from membranes that are rich in bmp and poor in cholesterol. bmp also increases the ability of the gm activator to solubilize lipids [ ] and stimulates the hydrolysis of membrane-bound gm by gm β-galactosidase [ ] and of ganglioside gm by β-hexosaminidase a [ ] . bmp also stimulates hydrolysis of the kidney sulfatide with ganglio-series gsl-core sm (gangliotriaosylceramide-ii sulfate) by β-hexosaminidases a and s in the presence of the gm activator [ , ] . cholesterol, which is known to stabilize lipid bilayers, has to be transported from intraendosomal membranes to the npc protein resident in the endosomal perimeter membrane by the soluble lipid-transfer protein npc . in vitro, this transfer is greatly stimulated by bmp and strongly inhibited by sphingomyelin [ ] . saposins are small, water-soluble lysosomal lipid-binding and -transfer proteins of about - kda molecular weight. they are derived from a common precursor protein, prosaposin, by proteolytic processing. saposins belong to a family of proteins with conserved three-dimensional fold [ ] and occur as homo-and heterodimers and -oligomers. the first saposin has been characterized in as the so-called sulfatide activator since it enables the degradation of sulfatide by arylsulfatase a [ ] . today this protein is known as saposin-b or sap-b. saposin-b has many functions: it is a lipid-binding protein with broad specificity [ ] and forms water-soluble lipidprotein complexes [ ] . with respect to gangliosides, it is able to stimulate the degradation of ganglioside gm by gm -β-galactosidase [ ] . studies in cultured human skin fibroblast derived from saposin-b and prosaposin-deficient patients show that it is also required for the degradation of gm [ , ] . it is important to note that glycosylation of saposin-b is essential for some of its functions and that human patients without this postranslational modification die, although the unglycosylated variant protein is present in lysosomes [ ] . mechanistically, saposin-b dimers seem to act similar to the gm activator: x-ray data indicate that they can adopt two conformations, an open one and a closed one [ ] . according to a model view, the open conformation interacts directly with the membrane and extracts the lipid ligand. this is accompanied by a change to the closed conformation in which the ligand is exposed to the degrading enzyme in a water-soluble activator-lipid complex. human patients with an inherited deficiency of saposin-b develop an atypical form of metachromatic leukodystrophy with the accumulation of sulfatides, digalactosylceramide, and globotriaosylceramide [ ] (see figure for structures). saposin-b knockout mice show enhanced levels of sulfatides especially in brain and kidney [ ] . activator. the gm activator is a small glycoprotein of . kda in its deglycosylated form and is required for the degradation of ganglioside gm by βhexosaminidase a in vivo [ ] . inherited deficiency of the gm -activator protein leads to the ab variant of gm gangliosidoses [ ] . based on the x-ray structure [ , ] and data from photoaffinity labeling [ ] , in some respects the gm -activator acts in a way similar to saposin-b. a more detailed picture of the binding mode was derived from binding studies using a spin-labelled gm activator to phosphatidylcholine bilayers [ ] . the protein can extract a variety of lipids, which has been exploited for assay development [ ] . however, its major function is to form a water-soluble gm -protein complex that is the native michaelis-menten substrate of β-hexosaminidase a [ ] . negatively charged lipids such as bmp, dolichol phosphate, or phosphatidylinositol increase the extraction efficiency towards gm [ ] , gm [ ] , and other lipids [ ] from liposomal membranes. binding characteristics of the gm activator are altered by the presence of a his tag [ ] . in langmuir experiments, the gm -activator protein is able to penetrate into a phospholipid monolayer, but only when the lateral pressure is below a critical value, which depend on the lipid composition and is in the range from to mn/m [ ] . in addition to its function as a ganglioside-transfer protein, the gm activator binds also other lipids like phosphatidylcholine [ ] and platelet activating factor (paf) and inhibits its action [ , ] . it is not clear whether the gm activator displays inherent hydrolytic activity towards lipid substrates such as platelet activating factor [ ] or phosphatidylcholine [ ] . apparently unrelated to its gm transfer property is the function of the gm activator as adipokine [ ] . gm activator orthologs might serve different functions in other organisms, for example as a pheromone-binding protein in drosophila [ ] , or an inhibitor of paf-induced chemotaxis in nematodes [ ] . the saposins and the gm -activator play major roles in the transfer of lipid antigens to membrane-resident cd proteins [ , ] . degradation. gm -β-galactosidase is a protein of kda, which is derived from an -kda precursor [ , ] . an alternatively spliced, enzymatically inactive β-galactosidase form of kda is an elastin/laminin-binding protein [ ] . gm -β-galactosidase is part of a lysosomal multienzyme complex, together with the so-called protective protein (carboxypeptidase a), sialidase, and n-acetylaminogalactose- -sulfate sulfatase [ ] . gm -β-galactosidase catalyzes the hydrolytic cleavage of several β-galactosides. the hydrolysis of ganglioside gm to gm requires the presence of either the gm -activator protein, or saposin-b [ ] , or, in vitro, of an appropriate detergent. degradation. gm is degraded by the cleavage of the n-acetylgalactosaminyl residue by β-hexosaminidases. in mice, the substrate specificity of the murine lysosomal sialidase allows for a significant cleavage also of the sialic moiety in gm (to yield ga ) [ ] . cleavage of the galnac residue requires the presence of the gm -activator protein in vivo, or of an appropriate detergent in vitro. three gene products participate in gm hydrolysis, the β-hexosaminidase αand β-chains, and the gm -activator protein. β-hexosaminidases are dimers that result from the combination of their αand β-subunits and differ in properties such as stability and substrate specificity. β-hexosaminidase a with subunit composition α,β cleaves terminal β-glycosidically linked n-acetyl-glucosamine and n-acetylgalactosamine residues from negatively charged and uncharged glycoconjugates by a retaining doubledisplacement mechanism. the enzyme has two active sites, one on the α-chain and the other on the β-chain [ ] . β-hexosaminidase b (ββ) [ , ] predominantly cleaves uncharged substrates such as ga and oligosaccharides with terminal n-acetyl-hexosamine residues (see also figure ). β-hexosaminidase s (αα) is thermolabile and of secondary significance for gm degradation, but it contributes to the degradation of glycosaminoglycans and sulfated glycolipids [ ] . defects in enzymes and other proteins required for lysosomal degradation of complex lipids and of oligomeric or polymeric biomolecules lead to inherited diseases, the lysosomal storage diseases [ ] . they can be classified according to the stored substances, as sphingolipidoses [ ] , mucopolysaccharidoses, mucolipidoses, glycoprotein-, and glycogen-storage diseases [ , ] . ganglioside degradation is impaired in the gangliosidoses and secondarily also in other sphingolipid storage diseases [ ] . the principles [ ] governing pathogenesis [ , ] and therapy of sphingolipidoses [ ] are also valid for the ganglioside storage diseases. key factors are the residual activity of the degrading system, which determines the course of the disease [ , ] , and the cell-type-specific expression of storage material. due to the cell-type-specific expression of gangliosides, the central nervous system is especially affected in the gangliosidoses. in sphingolipidoses in general, the storage lipids coprecipitate other hydrophobic substances present in the endolysosomal compartment, lipids and proteins, as secondary storage products [ ] . in niemann-pick disease, type c, which is a primary defect of endosomal cholesterol transport, a secondary accumulation of sphingomyelin (therefore the name niemann-pick) and of gangliosides is observed that is also of therapeutic relevance [ , ] ; for a remarkable treatment of niemann pick c fibroblasts with a histone deacetylase inhibitor, compare [ ] . secondary storage of gangliosides gm and gm occurs also in hurler disease [ ] (mucopolysaccharidosis type i; α-liduronidase deficiency). lipid storage produces a kind of traffic jam [ , ] , which interferes with lipid transport and lysosomal function. primary and secondary storages substances can impair nutrient delivery via the endolysosomal system: as demonstrated in mouse models of gm gangliosidoses and in a variant form of the gm gangliosidoses, sandhoff disease, iron homeostasis is impaired in the animals, and supplementation of the animals with iron ions increased their life expectancy by nearly % [ ] . since also autophagy can be impaired in lysosomal storage diseases [ ] , both pathways may lead to a shortage of nutrients. ganglioside degradation is impaired in the gangliosidoses. in another disease, galactosialidosis, the primary defect of carboxypeptidase a (protective protein), leads to a secondary loss of β-galactosidase and sialidase neu accompanied by gm storage [ ] . gangliosidoses are caused by defects in the genes encoding glycosidases or lipidtransfer proteins that are required for lysosomal ganglioside degradation. the theoretical basis for the therapeutic approaches towards gangliosidoses is the "threshold theory" [ ] , which predicts that the ratio of substrate influx into the lysosomes and the degradation capacity determine the course of the diseases. both parameters can be addressed by different therapeutic approaches [ ] . . . gm gangliosidosis. gm gangliosidosis is caused by an inherited deficiency of gm -β-galactosidase (acid βgalactosidase; glb ; ec . . . ) [ ] . after the description of the first patients [ ] it became also known as landing diseases [ ] . it is a rare disease with an autosomal recessive mode of inheritance and characterized by the accumulation of gm and ga (figure ) in neuronal cells [ ] . according to the substrate specificity of the variant enzyme in the patients, an inherited defect of the β-galactosidase can also lead to another disease, morquio disease, type b. three clinical forms of gm gangliosidosis can be distinguished, infantile (type ) gm gangliosidosis with the developmental arrest and progressive deterioration of the nervous system in early infancy and a life expectancy of about years, late infantile/juvenile form (type ), and an adult/chronic form (type ). dysmorphic changes characteristic for morquio disease type b are less prominent or completely absent in these clinical forms. in addition to gm , other enzyme substrates accumulate, such as ga (figure ) [ ] , oligosaccharides from glycoproteins, and intermediates of keratin sulfate degradation [ ] . these substances are stored in different organs, according to their major site of biosynthesis. lysosomal gm accumulation in neurons leads to the degeneration of the nervous system. like in other storage diseases, an inflammatory response [ ] , neurorestorative properties of excess ganglioside gm [ ] in the plasma membrane, and an unfolded protein response [ ] contribute to pathogenesis. such as in other sphingolipidoses [ ] , severity and progression of the disease correlate with the residual enzymatic activity in cells and body fluids. morquio type b disease clinically resembles a mild phenotype of morquio a disease, where keratan sulfate accumulates due to n-acetyl-galactosamine- -sulfatase deficiency. like gm gangliosidosis, morquio type b is due to the inherited defect of gm -β-galactosidase. it is characterized by the predominant storage of keratan sulfate and oligosaccharides with terminal β-galactosyl residues. patients show generalized skeletal dysplasia without involvement of the nervous system and without hepatosplenomegaly; for a clinical description, compare [ ] . differences between gm gangliosidosis and morquio b disease can be attributed to a lower affinity and activity of β-galactosidase variants towards substrates with gal-β , -glcnac motifs in morquio patients compared to the gal-β , -galnac motive present in ganglioside gm [ ] . there is no causal therapy available for gm -gangliosidosis; however, progress is made towards the development of pharmacological chaperones also for this lysosomal disease [ ] [ ] [ ] . the gm -gangliosidoses are caused by defects in degradation of ganglioside gm [ ] . the three variant forms of the gm -gangliosidoses are named according to the hexosaminidase isoenzyme that remains intact. the b-variant, in its infantile course better known as tay-sachs disease, is caused by the deficiency of hexosaminidases a and s, but with normal hexosaminidase b. the variant, or sandhoff disease, is caused by the deficiency of the β-chain and the resulting deficient activity of β-hexosaminidases a and b (therefore, none of the major enzymes is intact), however with the remaining activity of β-hexosaminidase s. the ab-variant-β-hexosaminidases a and b (and s) intact-results from mutations in the gm -activator gene; so that tissue samples from the patients are able to degrade gm in detergent-containing enzyme assays. clinically, the b variant of gm gangliosidoses can be subclassified into infantile, juvenile, chronic, and adult onset forms. the infantile form, tay-sachs disease, has a higher prevalence among ashkenazi jews with a heterozygote frequency of : . affected children are normal at birth and show first symptoms, such as mild motor weakness, a cherry red spot in the central retina, and increased startle reaction between and months of life. progressive deterioration with weakness, hypotonia, or poor head control leads to a vegetative state and death often between the second and fourth year of life. juvenile and adult course is observed in patients with a higher residual activity of the variant hexosaminidase a [ ] . symptoms are very heterogeneous; for a clinical description, compare [ ] . the b variant of gm gangliosidoses [ , ] was very difficult to elucidate: synthetic uncharged substrates used for diagnosis such as mufglcnac (figure ; for kinetic parameters see [ ] ) were cleaved, suggesting the presence of β-hexosaminidase, and also the gm activator was present. as it turned out, the b variant differs enzymatically from the b variant by an altered substrate specificity of the variant β-hexosaminidase a. while uncharged substrates are cleaved, no activity is detected towards gm and towards sulfated, negatively charged [ ] synthetic fluorogenic substrates. in the b variant, the function of the α-chain active site is defective, but subunit association, enzyme processing, and the activity of the β-chain are not impaired. homozygous patients with the b mutation show the course of the juvenile disease; compound heterozygotes with a b and a null allele show a late infantile course. disease. the variant of gm gangliosidosis was the first gangliosidosis for which the underlying enzymatic defect was identified [ ] . due to the deficiency of two enzyme activities, β-hexosaminidases a and b, storage of negatively charged glycolipids characteristic for tay-sachs disease and, in addition, of uncharged substrates such as ga in the brain and globoside in visceral organs (figure ) is observed. in infantile sandhoff disease, patients show clinical and pathological manifestations of tay-sachs disease (infantile b variant) and in addition also organomegaly and slight bone deformations. for further symptoms and the description of juvenile and adult forms, compare [ ] . the ab variant is due to the deficiency of the gm -activator protein [ ] , with intact β-hexosaminidases a and b (and s), therefore the name. the disease is characterized by accumulation of gm and ga (for structures, see figure ). the clinical picture [ ] resembles that of tay-sachs disease with a delayed appearance of symptoms; an animal model is available [ ] . although lysosomal gm as the major storage compound in gm gangliosidoses is neither toxic nor immunogenic, its accumulation induces inflammatory responses as demonstrated for glycoconjugates in the murine model of sandhoff disease [ ] . huge axon hillock enlargements, the so-called meganeurites, have been observed in neurons of patients with different lysosomal storage diseases, which might be attributed to the storage substance gm and contribute to synaptic dysfunction [ ] . as in other sphingolipidoses [ ] , the corresponding (more toxic) lysolipid, in this case lysogm (figure ), is elevated [ , ] and contributes to the pathogenesis. lysogm has been suggested as a biomarker for tay-sachs and sandhoff disease [ ] ; for occurrence and role of lysogsls in acquired diseases, compare [ ] . despite naturally occurring animal models of gm gangliosidoses in dogs, cats, and pig, murine models are used for therapy studies. since the mouse model of tay-sachs disease is largely asymptomatic, the mouse model of sandhoff disease is used for most studies [ ] . despite some success in the experimental treatment of juvenile and adult patients as well as in the animal models, there is no causal therapy available for the severe forms of the gm gangliosidoses. the limitations of the substrate reduction approach, which reduces the gm influx into the lysosomal compartment, have been evaluated by a genetic experiment: sandhoff-disease mice were crossbred with mice defective in gm synthase. the lifespan of these animals was much longer than that of sandhoff-disease mice, but instead of gm storage they developed a oligosaccharide storage, neurological disease [ ] . therapeutic approaches such as bone-marrow transplantation [ ] , enzyme-replacement therapy with recombinant highly phosphomannosylated β-hexosaminidase a [ ] , or transplantation of neural stem cells [ ] have been investigated in the animal model of the variant, substrate-reduction therapy with n-butyl deoxynojirimycin [ , ] , and with pyrimethamine as pharmacological chaperone [ , ] in adult patients and gene therapy in endothelial cells [ ] . treatment of the accompanying inflammation is beneficial [ ] . aspects. in addition to inherited diseases, ganglioside levels can also be altered in several acquired diseases [ ] . for example, gangliosides play roles in neurological diseases such as alzheimer's [ ] , parkinson, or huntington's disease [ ] . in cancer, ganglioside expression can also be altered in tumor cells with an impact on signalling and tumor-host interactions [ ] . [neu gc]gm [ ] , gd , gd , gm , and fucosylgm are regarded as tumor-associated antigens [ ] and are targets for the immunotherapy of cancer [ ] . also several neuropathies including variant forms of guillain-barré and miller-fisher syndrome are caused by serum antibodies against gangliosides [ ] . there are only a few therapeutic roles for gangliosides, especially since they can induce neuropathies. in the past, gangliosides isolated from bovine brain have been investigated and also applied to human patients to improve neural repair and for the treatment of stroke [ , ] . also the direct application of ganglioside gm into the brain of patients with alzheimer disease has been evaluated [ ] . as indirect roles, the inhibition of ganglioside biosynthesis for the treatment of insulin resistance [ ] , the interference with microbial binding to gangliosides [ ] , or the reduction of neurotoxicity with liga [ ] has to be mentioned. a plethora of functions has been attributed to gangliosides [ ] , for example, for gm [ ] , the most abundant ganglioside in most mammalian cell types, but not in neurons, or for gm [ ] . in general, gangliosides mediate their function via interaction with soluble or membranebound binding molecules outside the cell ("trans" interaction), or by influencing properties of proteins within the same membrane ("cis" interaction) [ , [ ] [ ] [ ] [ ] . "trans" interactions occur between the glycan part of gangliosides on the one side with lectins on the other side. also gangliosides contribute to the chemical high-density sugar code of cell surfaces [ ] . for example, gm can be recognized by galectin- [ ] and sialic acids in α , figure : structure of liga , a semitruncated and halogenated gm analog that can pass the blood-brain barrier. are recognized by the sialic acid binding immunoglobulin lectins siglec- , α , -sialosides by siglec- , and α , sialosides by siglec- [ ] . also carbohydrate-carbohydrate interactions can play a role [ , ] . although interactions between individual carbohydrate residues [ ] are weak, clustering of gangliosides offer the possibility for multivalent interactions, if they are not buried under glycoprotein glycans. apparently, gm on mouse melanoma b cells can mediate cell adhesion to mouse lymphoma l cells by binding to ga (gangliotriaosylceramide, galnacβ , galβ , glcβ , cer; see figure or figure ) [ ] . within the nervous system, gangliosides act in a "trans" manner with the myelin-associated glycoprotein mag. mag recognizes neuacα - galβ - galnactermini on axonal gangliosides, an interaction that is essential for axon-myelin stability and axon regeneration [ ] . "cis" interactions can happen via a direct interaction, or, indirectly, via the properties of the membrane or of putative-membrane domains [ ] . this way gangliosides influence the activities of receptor-tyrosine kinases in the plasma membrane, such as the receptors of epidermalgrowth factor, nerve growth factor, and insulin and therefore cell signaling [ ] . for example, gm enhances trka neurotrophin receptor-activation in a "cis"-manner [ ] ; for a brief overview of proteins affected by certain gangliosides, compare, for example, [ ] . since the characterization of lipid microdomains in living cells is difficult, some conclusions can be drawn from in vitro experiments. for example, gm inhibits the autophosphorylation of purified egfr reconstituted into the proteoliposomes of defined lipid compositions, but not the egf binding [ ] . there are indications that gangliosides may not act only in an autonomous manner, but might also support the formation of distinct membrane phases, although it is a matter of debate to which extent this operates in vivo. it is believed that gangliosides are not homogeneously distributed on the cell surface, but segregate into membrane domains together with gpi-anchored proteins, sphingomyelin and cholesterol. such rafts have been supposed to be the physiological surroundings of many membrane proteins, although no rigid proof for their existence has been provided. ganglioside plays a largely unexplored role for membrane structure [ ] . due to their large hydrated head groups, they stabilize membrane areas with positive curvature [ ] . a multitude of reports propose a segregation of gangliosides and other gsls with cholesterol and gpi-anchored proteins into the lipid platforms known as "rafts" in the membranes of living cells [ ] [ ] [ ] [ ] . since most of the applied methods (detergent extraction, antibody, and toxin staining) constitute a bias towards the formation of such domains [ ] , the existence, size, and lifetime of rafts are a matter of debate. from thermodynamic considerations, it is clear that procedures such as detergent extraction can (or have to) produce artificial results and are not suitable for raft characterization [ ] [ ] [ ] [ ] . although it has been demonstrated that the treatment of cellular membranes with detergents causes the redistribution of gangliosides and gpi-anchored proteins [ , ] , these techniques are still applied and not always critically examined. experiments in living cells using sted microscopy [ ] and other visualization techniques [ ] point to an upper limit of lifetime and size of such domains in the range of ms and nm. in addition, sialic acids and oligosialic acids present on gangliosides can modulate membrane surface charge density, the ph at the membrane surface, and membrane potentials [ ] . in planar lipid bilayers, ganglioside gd a can increase the excitability of voltage-dependent sodium channels [ ] . . . infection. "cis" and "trans" interactions of gangliosides play multiple roles in the immune system [ ] and in infectious diseases [ , ] , where gangliosides act as cellular receptors and coreceptors for viruses, bacteria, and microbial toxins. the most prominent example is gm as the receptor for cholera toxin [ ] ; other examples are the toxin of clostridium botulinum and the saba adhesin of helicobacter pylori [ ] that bind to cell surface gangliosides of the host [ ] . binding of sialylated cell surface glycoconjugates to siglecs [ ] on white blood cells is used within innate and adaptive immune responses to distinguish between self and nonself and to dampen autoimmune responses [ ] . many pathogens use sialic acids on cell surface glycoconjugates for cellular entry, for example, periodontal pathogens [ ] . recent examples include ganglioside gt b, which seems to be the host cell receptor for the merkel cell polyomavirus [ ] . this virus has been identified as the cause of merkel cell carcinoma, an aggressive type of skin cancer. also sialidase-insensitive rotaviruses recognize sialic acid, for example, on ganglioside gm , which is not substrate of all sialidases due to its branched structure [ ] and the glycan present in ganglioside gd a serves as host receptor for the adenoviruses that cause epidemic keratoconjunctivitis [ ] . gangliosides are secondary gene products. their function can be analyzed by knockout experiments, where in cells, tissues, or organisms their formation or degradation is interrupted by genomic, posttranscriptional, or chemical strategies. especially valuable were genetically engineered mice with defects in ganglioside biosynthesis [ ] , which revealed, for example, a role of gangliosides in calcium homeostasis [ ] , neural repair [ ] , or neurological diseases [ ] . also investigations in human patients [ ] , genetically engineered mammals [ ] , and other organisms [ ] allowed insight into various aspects of ganglioside metabolism and transport. also mutant and silenced cells have been applied for functional studies. in vitro systems such as liposomes or planar monolayers allow investigations that are to difficult to be carried out in cells. for example, when gm is incorporated into liposomes, a phase separation into gm -rich and gm -poor phases occurs above a certain gm content [ ] . this would fit to reports on gm -enriched microdomains in living cells. in experimental approaches, ganglioside biosynthesis can be modulated by inhibitors [ ] . also the enhancement of ganglioside biosynthesis can be used, for example, chemically, or by the introduction of glycosyltransferase encoding cdna in cultured cells [ , ] . an enantiomer of the glucosylceramide synthase inhibitor d-threo-pdmp (pdmp = -phenyl- -decanoylamino- -morpholino- -propanol), lthreo-pdmp, acts as an enhancer of ganglioside biosynthesis by upregulating glycosyltransferases. this was accompanied by increased neurite outgrowth [ ] . additional possibilities are the generation of mutant cells, for example for gm synthase [ ] or by posttranscriptional silencing like rna interference. while even complex systems like cultured cells can survive without gsls, they are required for the development of multicellular organisms [ ] . gangliosides. structurally homogenous gangliosides and ganglioside probes that are modified by isotopes, fluorescence, chemical reporter groups, photoaffinity, or affinity ligands are valuable tools for the analysis of ganglioside function, metabolism, and transport. these tools are available by total or partial chemical synthesis, or by biosynthetic incorporation of suitable-for example, photolabile-n-acylmannosamine precursors into gangliosides [ ] using the methodology for biosynthetic sialic acid modification developed by kayser et al. [ ] . for enzymological, transport, and crosslinking studies, tritium and c are incorporated into different positions of gangliosides, but also radioiodination is possible in the presence of aryl residues [ , ] . ganglioside total synthesis is a time-consuming and demanding task and usually performed only in specialized laboratories for examples, see [ , ] . it relies predominantly on the sequential glycosidation of a -o-protected azidosphingosine [ ] with suitably protected and activated glycosyl donors. this includes the trichloroacetimidates [ ] as well as methods for α-selective sialylation reactions [ ] . also chemoenzymatic procedures have been developed, where different glycosidation steps are catalyzed by glycosyltransferases [ ] or glycosidases [ ] , or where oligosaccharyl fluorides are coupled to native or fluorescent ceramide anchors using an engineered endoglycoceramidase (glycosynthase) [ , ] . ganglioside oligosaccharides, such as those of gm , gm , gm , gd , and gt , can be produced by genetically engineered bacterial strains. for the biotechnological production of ganglioside head groups, lactose can be internalized in e. coli as a precursor to be used as acceptor for glycosyltransferases [ ] [ ] [ ] [ ] [ ] [ ] . an application of the ganglioside biosynthetic machinery is the preparative production of neoglycolipids with ganglioside head groups [ ] figure : example of a gm analog [ ] spin labelled with a , -dimethyl-oxazolidine- -oxyl-(doxyl-) residue and a fluorescent gm analog used for sted microscopy [ ] . using a lung squamous-cell carcinoma line (rerf-lc-a) and -azidododecyl β-lactoside as a suitable primer [ ] . gsls isolated from natural sources can be used for the preparation of chemically modified derivatives [ ] like labelled gsls [ , ] , or those of enhanced metabolic stability [ ] . the chemical release of the ganglioside glycan chain can be achieved by osmium tetroxide/periodate treatment of protected gangliosides [ , ] , or by ozonolysis of native gangliosides [ ] . initially, both methods give rise to ganglioside aldehydes, which are not isolated but subsequently fragmented by alkaline treatment, or, if desired, are isolated for further applications [ ] . the glycan part can also be released enzymatically by ceramide glycanase [ , ] . lysogangliosides that lack the acyl moiety at the sphingosine nitrogen can be prepared by chemical procedures [ , ] , or by enzymatic treatment of gangliosides with sphingolipid n-deacylase [ ] [ ] [ ] . lysogangliosides (see figure ) can be used for the introduction of fluorescence, spin, or radiolabels or other modifications into the lipid backbone of gangliosides. a semitruncated, dihalogenated gm analog that is able to pass the blood-brain barrier is liga [ ] (figure ). there are also several approaches to the synthesis of photoactivatable gsl derivatives [ ] . it has to be noted that when gangliosides are added to the medium of cultured cells, they are largely present as oligomers in the form of micelles or vesicles, as monomers bound to proteins, and as free monomers. in aqueous surroundings, gangliosides form aggregates of different size and shape [ , ] . in most cases these are micellar structures of - kda [ ] , in the case of the gangliosides with small head groups, gm and gm also vesicles [ , , ] . the critical micellar concentrations of gsls are in the range of − - − m [ ] and depend on temperature, ph, and, in part, on the method of determination. typical values are . × − m for gm to . × − m for gt b [ ] . uptake of exogenously added gangliosides by cells in culture [ ] can proceed in different ways [ , ] ( figure ) . with the aid of radiolabelled [ ] and spinlabelled gangliosides [ ] (figure ), three modes of adherence have been distinguished: - % of the exogenous ganglioside consist in loosely associated micelles and also monomers, which can be removed by delipidated serum proteins. a second fraction is attached to cellular proteins in a trypsin-labile fashion, and, finally, a trypsin-stable fraction is presumably inserted into the plasma membrane of the cell. only the last fraction is in the topologically correct, native orientation. when bound to proteins, the offrate of gangliosides with native alkyl chain lengths can be very low. this is not the case for synthetic, semitruncated derivatives of higher solubility, which are frequently used, but show different intracellular transport behaviour compared to native gangliosides [ ] . fluorescently labelled glycosphingolipids [ ] have been applied, but also their properties can differ significantly from the ones of native glycosphingolipids. gangliosides can also be transferred from cultured donor to acceptor cells that are separated by a membrane [ ] . by this process known as "shedding" [ ] , tumor cells can release up to . % of their gangliosides per hour [ ] . fluorescent ganglioside probes that bear the fluorophore in at the membrane-water interphase ( figure ) 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liver identity of g(d c), g(t a) and g(q b) synthase in golgi vesicles from rat liver substrate specificity of α → -sialyltransferases in ganglioside biosynthesis of rat liver golgi the c-series gangliosides g(t ), g(t ) and g(p c) are formed in rat liver golgi by the same set of glycosyltransferases that catalyse the biosynthesis of asialo-, a-and b-series gangliosides current trends in the structureactivity relationships of sialyltransferases comprehensive analysis of sialyltransferases in vertebrate genomes gangliosides in human fetal brain differential distribution of major gangliosides in rat central nervous system detected by specific monoclonal antibodies cloning of a mouse β , n-acetylglucosaminyltrans-fer-ase glcnac(β , )gal(β , )glc-ceramide synthase gene encoding the key regulator of lacto-series glycolipid biosynthesis molecular cloning and characterization of udp-glcnac:lactosylceramide β , -n-acetylglucosaminyltransferase (β gn-t ), an essential enzyme for the expression of hnk- and lewis x epitopes on glycolipids multiple phenotypic changes in mice after knockout of the b gnt gene, encoding lc synthase-a key enzyme in lacto-neolacto ganglioside synthesis n-acetylglucosaminyl transferase regulates the expression of the sulfoglucuronyl glycolipids in specific cell types in cerebellum during development infantile-onset symptomatic epilepsy syndrome caused by a homozygous loss-of-function mutation of gm synthase model for the interaction of membrane-bound substrates and enzymes. hydrolysis of ganglioside g(d a) by sialidase of neuronal membranes isolated from calf brain two soluble glycosyltransferases glycosylate less efficiently in vivo than their membrane bound counterparts neobiosynthesis of glycosphingolipids by plasma membraneassociated glycosyltransferases the stimulating adventure of krn further gangliosides from the human brain gangliosides of human myelin: sialosylgalactosylceramide (g ) as a major component oligodendrocyte development and myelin biogenesis: parsing out the roles of glycosphingolipids participation of galactosylceramide and sulfatide in glycosynapses between oligodendrocyte or myelin membranes fa h is responsible for the formation of -hydroxy galactolipids in peripheral nervous system myelin a carbohydrate-carbohydrate interaction between galactosylceramide-containing liposomes and cerebroside sulfate-containing liposomes: dependence on the glycolipid ceramide composition absence of oligodendroglial glucosylceramide synthesis does not result in cns myelin abnormalities or alter the dysmyelinating phenotype of cgt-deficient mice ceramide udpgalactosyltransferase from myelinating rat brain: purification, cloning, and expression topography of cerebroside sulfotransferase in golgi-enriched vesicles from rat brain topology of sphingolipid galactosyltransferases in er and golgi: transbilayer movement of monohexosyl sphingolipids is required for higher glycosphingolipid biosynthesis udp-galactose: ceramide galactosyltransferase is a class i integral membrane protein of the endoplasmic reticulum zebrafish and mouse α , -sialyltransferases responsible for synthesizing gm ganglioside prevention of experimental allergic encephalomyelitis by ganglioside g(m ) purification and characterization of udp-glucose:ceramide glucosyltransferase from rat liver golgi membranes expression cloning of a cdna for human ceramide glucosyltransferase that catalyzes the first glycosylation step of glycosphingolipid synthesis oligomerization and topology of the golgi isrn biochemistry membrane protein glucosylceramide synthase identification of active site residues in glucosylceramide synthase: a nucleotide-binding/catalytic motif conserved with processive β-glycosyltransferases topology of glucosylceramide synthesis in golgi membranes from porcine submaxillary glands lactosylceramide is synthesized in the lumen of the golgi apparatus mdr pglycoprotein is a lipid translocase of broad specificity, while mdr p-glycoprotein specifically translocates phosphatidylcholine abc lipid transporters: extruders, flippases, or flopless activators? pre-and post-golgi translocation of glucosylceramide in glycosphingolipid synthesis reconstitution of glucosylceramide flip-flop across endoplasmic reticulum: implications for mechanism of glycosphingolipid biosynthesis glycosphingolipid synthesis requires fapp transfer of glucosylceramide mutation of βglucosidase causes glycolipid storage disease and impaired male fertility biosynthesis of glycolipids cdna cloning and expression of human lactosylceramide synthase isolation and characterization of extremely minor gangliosides, g(m b) and g(d α), in adult bovine brains as developmentally regulated antigens enhanced insulin sensitivity in mice lacking ganglioside gm tissue-specific expression of cseries gangliosides in the extraneural system structural characterization of a novel cholinergic neuron-specific ganglioside in bovine brain biosynthetic pathway for a new series of gangliosides, gt aα and gq bα biosynthesis of the major brain gangliosides gd a and gt b glycosphingolipid functions: insights from engineered mouse models a mouse b melanoma mutant deficient in glycolipids complex gangliosides are essential in spermatogenesis of mice: possible roles in the transport of testosterone mice lacking complex gangliosides develop wallerian degeneration and myelination defects a functional role for complex gangliosides: motor deficits in gm /gd synthase knockout mice attenuation of interleukin signal in the spleen cells of complex gangliosidelacking mice cd and cd t cells require different membrane gangliosides for activation mice lacking major brain gangliosides develop parkinsonism g(m ) ganglioside rescues substantia nigra pars compacta neurons and increases dopamine synthesis in residual nigrostriatal dopaminergic neurons in mptp-treated mice gm specifically interacts with α-synuclein and inhibits fibrillation mice expressing only monosialoganglioside gm exhibit lethal audiogenic seizures interruption of ganglioside synthesis produces central nervous system degeneration and altered axon-glial interactions sphingolipid synthetic pathways are major regulators of lipid homeostasis membranes in balance: mechanisms of sphingolipid homeostasis regulation of ganglioside biosynthesis in the nervous system the cytoplasmic tail of gm synthase defines its subcellular localization, stability, and in vivo activity multi-enzyme kinetic analysis of glycolipid biosynthesis golgi localization of glycosyltransferases involved in ganglioside biosynthesis regulation of ganglioside biosynthesis by enzyme complex formation of glycosyltransferases the synthesis of complex carbohydrates by multiglycosyltransferase systems and their potential function in intercellular adhesion glycosyltransferase complexes improve glycolipid synthesis physical and functional association of glycolipid n-acetylgalactosaminyl and galactosyl transferases in the golgi apparatus ganglioside glycosyltransferases organize in distinct multienzyme complexes in cho-k cells identification and expression of neu , a novel human sialidase associated to the plasma membrane a close association of the ganglioside-specific sialidase neu with caveolin in membrane microdomains membrane sialidase neu is highly expressed in human melanoma cells promoting cell growth with minimal changes in the composition of gangliosides sialidase occurs in both membranes of the nuclear envelope and hydrolyzes endogenous gd a degradation of glycolipids lysosomal degradation of membrane lipids activator proteins and topology of lysosomal sphingolipid catabolism regulation of apoptosis-associated lysosomal membrane permeabilization recycling compartments and the internal vesicles of multivesicular bodies harbor most of the cholesterol found in the endocytic pathway biological function of the cellular lipid bmp-bmp as a key activator for cholesterol sorting and membrane digestion metabolism, function and mass spectrometric analysis of bis(monoacylglycero)phosphate and cardiolipin separation and characterization of late endosomal membrane domains lysosome biogenesis and lysosomal membrane proteins: trafficking meets function lysosomal membrane proteins: life between acid and neutral conditions hoppe-seyler's zeitschrift für physiologische chemie the human g(m ) activator protein. a substrate specific cofactor of β-hexosaminidase a principles of lysosomal membrane digestion: stimulation of sphingolipid degradation by sphingolipid activator proteins and anionic lysosomal lipids saposin a mobilizes lipids from low cholesterol and high bis(monoacylglycerol)phosphate-containing membranes: patient variant saposin a lacks lipid extraction capacity saposin b mobilizes lipids from cholesterol-poor and bis(monoacylglycero) phosphate-rich membranes at acidic ph: unglycosylated patient variant saposin b lacks lipid-extraction capacity degradation of membrane-bound ganglioside gm by β-hexosaminidase a. stimulation by gm activator protein and lysosomal lipids degradation of membrane-bound ganglioside gm : stimulation by bis(monoacylglycero)phosphate and the activator proteins sap-b and gm -ap physiological substrates for human lysosomal βhexosaminidase s kidney sulfatides in mouse models of inherited glycosphingolipid disorders: determination by nano-electrospray ionization isrn biochemistry tandem mass spectrometry role of endosomal membrane lipids and npc in cholesterol transfer and membrane fusion a short guided tour through functional and structural features of saposin-like proteins hoppe-seyler's zeitschrift für physiologische chemie glycosphingolipid specificity of the human sulfatide activator protein the activator of cerebroside sulphatase. binding studies with enzyme and substrate demonstrating the detergent function of the activator protein the physiological role of activator proteins for lysosomal glycolipid degradation metabolism of g(m ) ganglioside in cultured skin fibroblasts: anomalies in gangliosidoses, sialidoses, and sphingolipid activator protein (sap, saposin) and prosaposin deficient disorders crystal structure of saposin b reveals a dimeric shell for lipid binding characterization of a mutation in a family with saposin b deficiency: a glycosylation site defect neurological deficits and glycosphingolipid accumulation in saposin b deficient mice purification and characterization of an activator protein for the degradation of glycolipids g(m ) and g(a ) by hexosaminidase a deficiency of a factor necessary for stimulation of hexosaminidase a-catalyzed degradation of ganglioside gm and glycolipid ga crystal structure of human gm -activator protein with a novel β-cup topology structural analysis of lipid complexes of gm -activator protein photoaffinity labelling of the human gm -activator protein mechanistic insight into ganglioside gm degradation interactions of the gm activator protein with phosphatidylcholine bilayers: a site-directed spin-labeling power saturation study a dansyl fluorescence-based assay for monitoring kinetics of lipid extraction and transfer ligand extraction properties of the gm activator protein and its interactions with lipid vesicles interaction of the gm -activator protein with phospholipid-ganglioside bilayer membranes and with monolayers at the air-water interface crystal structure analysis of phosphatidylcholine-gm -activator product complexes: evidence for hydrolase activity the gm activator protein, a novel inhibitor of platelet-activating factor gm activator protein inhibits platelet activating factor signaling in rats evidence for lipid packaging in the crystal structure of the gm -activator complex with platelet activating factor adipokine ganglioside gm activator protein stimulates insulin secretion a drosophila protein family implicated in pheromone perception is related to tay-sachs gm -activator protein functional characterisation of a nematode secreted gm -activator protein saposins utilize two strategies for lipid transfer and cd antigen presentation lipid-binding proteins in membrane digestion, antigen presentation, and antimicrobial defense β-galactosidase deficiency (β-galactosidosis): gm gangliosidosis and morquio b disease crystal structure of human beta-galactosidase: structural basis of gm gangliosidosis and morquio b diseases the -kd elastin/laminin-binding protein is related to an enzymatically inactive, alternatively spliced form of β-galactosidase molecular mechanisms of pathogenesis in a glycosphingolipid and a glycoprotein storage disease mouse models of tay-sachs and sandhoff diseases differ in neurologic phenotype and ganglioside metabolism evidence for two different active sites on human β-hexosaminidase a. interaction of g(m ) activator protein with β-hexosaminidase a the x-ray crystal structure of human β-hexosaminidase b provides new insights into sandhoff disease crystal structure of human β-hexosaminidase b: understanding the molecular basis of sandhoff and tay-sachs disease clarifying lysosomal storage diseases multi-system disorders of glycosphingolipid and ganglioside metabolism lysosomal disorders: from storage to cellular damage lysosomal storage disorders in the newborn secondary lipid accumulation in lysosomal disease sphingolipid metabolism diseases pathogenic cascades in lysosomal diseasewhy so complex? the cellular pathology of lysosomal diseases treating lysosomal storage disorders: current practice and future prospects quantitative correlation between the residual activity of βhexosaminidase a and arylsulfatase a and the severity of the resulting lysosomal storage disease partial enzyme deficiencies: residual activities and the development of neurological disorders neurons in niemann-pick disease type c accumulate gangliosides as well as unesterified cholesterol and undergo dendritic and axonal alterations critical role for glycosphingolipids in niemann-pick disease type c histone deacetylase inhibitor treatment dramatically reduces cholesterol accumulation in niemann-pick type c mutant human fibroblasts mucopolysaccharidosis type i: current knowledge on its pathophysiological mechanisms tarffic jam: a compendium of human diseases that affect intracellular transport processes traffic jams ii: an update of diseases of intracellular transport critical role of iron in the pathogenesis of the murine gangliosidoses lysosomal storage diseases as disorders of autophagy gm gangliosidosis: review of clinical, molecular, and therapeutic aspects gargoylism (hunter-hurler disease, dysostosis multiplex, lipochondrodystrophy); prenatal and neonatal bone lesions and their early postnatal evolution familial neurovisceral lipidosis. an analysis of eight cases of a syndrome previously reported as "hurler-variant," "pseudo-hurler," and "tay-sachs disease with visceral involvement central nervous system inflammation is a hallmark of pathogenesis in mouse models of gm and gm gangliosidosis enhanced susceptibility to kainate-induced seizures, neuronal apoptosis, and death in mice lacking gangliotetraose gangliosides: protection with liga , a membrane-permeant analog of gm gm -ganglioside-mediated activation of the unfolded protein isrn biochemistry response causes neuronal death in a neurodegenerative gangliosidosis imbalanced substrate specificity of mutant β-galactosidase in patients with morquio b disease chemical chaperone therapy for gm -gangliosidosis the potential action of galactose as a "chemical chaperone": increase of beta galactosidase activity in fibroblasts from an adult gm -gangliosidosis patient evaluation of nnonyl-deoxygalactonojirimycin as a pharmacological chaperone for human gm gangliosidosis leads to identification of a feline model suitable for testing enzyme enhancement therapy the gm gangliosidoses a review and predictive models of ganglioside uptake by biological membranes incorporation of ganglioside analogues into fibroblast cell membranes. a spin-label study direct observation of the nanoscale dynamics of membrane lipids in a living cell variant of gm -gangliosidosis with hexosaminidase a having a severely changed substrate specificity biochemical and molecular aspects of late-onset gm -gangliosidosis: b variant as a prototype diagnosis and carrier detection of tay-sachs disease: direct determination of hexosaminidase a using -methylumbelliferyl derivatives of β-n-acetylglucosamine- -sulfate and β-nacetylgalactosamine- -sulfate tay-sachs disease: one-step assay of β-n-acetylhexosaminidase in serum with a sulphated chromogenic substrate mouse model of gm activator deficiency manifests cerebellar pathology and motor impairment distortion of neuronal geometry and formation of aberrant synapses in neuronal storage disease occurrence of lysoganglioside lyso-g(m ) (ii -neu ac-gangliotriaosylsphingosine) in g(m ) gangliodosis brain accumulation of lysosphingolipids in tissues from patients with gm and gm gangliosidoses lyso-gm ganglioside: a possible biomarker of tay-sachs disease and sandhoff disease a view on sphingolipids and disease a genetic model of substrate deprivation therapy for a glycosphingolipid storage disorder bone marrow transplantation prolongs life span and ameliorates neurologic manifestations in sandhoff disease mice highly phosphomannosylated enzyme replacement therapy for gm gangliosidosis neural stem cell transplantation benefits a monogenic neurometabolic disorder during the symptomatic phase of disease substrate reduction therapy with miglustat in chronic gm gangliosidosis type sandhoff: results of a -year follow-up substrate reduction therapy an open-label phase i/ii clinical trial of pyrimethamine for the treatment of patients affected with chronic gm gangliosidosis (tay-sachs or sandhoff variants) crystal structure of β-hexosaminidase b in complex with pyrimethamine, a potential pharmacological chaperone induced secretion of β-hexosaminidase by human brain endothelial cells: a novel approach in sandhoff disease? nsaids increase survival in the sandhoff disease mouse: synergy with n-butyldeoxynojirimycin pathological significance of ganglioside clusters in alzheimer's disease functional roles of gangliosides in neurodevelopment: an overview of recent advances aberrant glycosphingolipid expression and membrane organization in tumor cells: consequences on tumor-host interactions ngcgm ganglioside: a privileged target for cancer vaccines cancer vaccines and carbohydrate epitopes immunology in the clinic review series, focus on cancer: glycolipids as targets for tumour immunotherapy a common mechanism and a new categorization for anti-ganglioside antibody-mediated neuropathies the therapeutic role of gangliosides in neurological disorders gangliosides, ngf, brain aging and disease: a mini-review with personal reflections alzheimer disease-effect of continuous intracerebroventricular treatment with gm ganglioside and a systematic activation programme inhibition of ganglioside biosynthesis as a novel therapeutic approach in insulin resistance receptor mimicry as novel therapeutic treatment for biothreat agents semisynthetic sphingoglycolipid liga is neuroprotective against human immunodeficiency virus-gp -mediated apoptosis gangliosides as modulators of cell function ganglioside gm and its biological functions in search of a solution to the sphinx-like riddle of gm functional role of glycosphingolipids and gangliosides in control of cell adhesion, motility, and growth, through glycosynaptic microdomains glycosynaptic microdomains controlling tumor cell phenotype through alteration of cell growth, adhesion, and motility gangliosides in cell recognition and membrane protein regulation glycolipid-mediated cell-cell recognition in inflammation and nerve regeneration beyond glycoproteins as galectin counterreceptors: tumor-effector t cell growth control via ganglioside gm galectin- is a major receptor for ganglioside gm , a product of the growth-controlling activity of a cell surface ganglioside sialidase, on human neuroblastoma cells in culture carbohydrate-to-carbohydrate interaction, through glycosynapse, as a basis of cell recognition and membrane organization carbohydrate-carbohydrate interaction in basic cell biology carbohydrate-carbohydrate interactions in cell recognition specific interaction between gangliotriaosylceramide (gg ) and sialosyllactosylceramide (g(m ) as a basis for specific cellular recognition between lymphoma and melanoma cells brain gangliosides in axon-myelin stability and axon regeneration gangliosides and the multiscale modulation of membrane structure receptor modifications in glycobiology neural functions of glycolipids lipid rafts: keys to neurodegeneration regulation of human egf receptor by lipids gangliosides as components of lipid membrane domains membrane organization and lipid rafts revitalizing membrane rafts: new tools and insights lipid rafts as a membraneorganizing principle lipids and cholesterol as regulators of traffic in the endomembrane system principles of microdomain formation in biological membranes-are there lipid liquid ordered domains in living cellular membranes? pip signaling in lipid domains: a critical reevaluation rafts defined: a report on the keystone symposium on lipid rafts and cell function detergentresistant membranes should not be identified with membrane rafts triton promotes domain formation in lipid raft mixtures membrane redistribution of gangliosides and glycosylphosphatidylinositol-anchored proteins in brain tissue sections under conditions of lipid raft isolation membrane redistribution of gangliosides and glycosylphosphatidylinositol-anchored proteins in brain tissue sections under conditions of lipid raft isolation imaging of mobile long-lived nanoplatforms in the live cell plasma membrane membrane oligo-and polysialic acids ganglioside gd a increases the excitability of voltage-dependent sodium channels multifarious roles of sialic acids in immunity sphingolipids in infectious diseases viruses and sialic acids: rules of engagement cholera toxin-a foe & a friend helicobacter pylori and complex gangliosides sialic acids in human health and disease siglecs and their roles in the immune system siglecs as sensors of self in innate and adaptive immune responses sialic acid, periodontal pathogens and tannerella forsythia: stick around and enjoy the feast! ganglioside gt b is a putative host cell receptor for the merkel cell polyomavirus sialic acid dependence in rotavirus host cell invasion the gd a glycan is a cellular receptor for adenoviruses causing epidemic keratoconjunctivitis knockout mice and glycolipids cerebellar neurons lacking complex gangliosides degenerate in the presence of depolarizing levels of potassium regulatory mechanisms of nervous systems with glycosphingolipids functions of sphingolipid metabolism in mammals -lessons from genetic defects sphingolipids and membrane biology as determined from genetic models phase behavior in multilamellar vesicles of dppc containing ganglioside gm with a c : sphingoid base and a : acyl chain (gm ( , )) observed by x-ray diffraction glycosphingolipids-nature, function, and pharmacological modulation enhancement of malignant properties of human osteosarcoma cells with disialyl gangliosides gd /gd gm /gd /ga synthase expression results in the reduced cancer phenotypes with modulation of composition and raft-localization of gangliosides in a melanoma cell line chapter neurotrophic and neuroprotective actions of an enhancer of ganglioside biosynthesis mutant ng - cells (ng-cr ) deficient in gm synthase respond aberrantly to axonogenic stimuli and are vulnerable to calcium-induced apoptosis: they are rescued with liga- biomolecule function: no reliable prediction from cell culture metabolism of diazirine-modified n-acetylmannosamine analogues to photocross-linking sialosides biosynthesis of a nonphysiological sialic acid in different rat organs, using n-propanoyl-d-hexosamines as precursors tubulin anchoring to glycolipid-enriched, detergent-resistant domains of the neuronal plasma membrane hplc-based procedure for the preparation of carbenegenerating photoreactive gm and gm ganglioside derivatives radioiodinated to high specific radioactivity with chloramine t as an oxidant azidosphingosine glycosylation in glycosphingolipid synthesis glycosyl trichloroacetimidates selective α-sialylation enzymatic approaches to o-glycoside introduction: glycosyltransferases enzymatic glycosylation by transferases glycosphingolipid synthesis employing a combination of recombinant glycosyltransferases and an endoglycoceramidase glycosynthase biosynthesis of conjugatable saccharidic moieties of gm and gm gangliosides by engineered e. coli the genetic bases for the variation in the lipo-oligosaccharide of the mucosal pathogen, campylobacter jejuni genetic engineering of escherichia coli for the economical production of sialylated oligosaccharides highly efficient biosynthesis of the oligosaccharide moiety of the gd ganglioside by using metabolically engineered escherichia coli a new fermentation process allows large-scale production of human milk oligosaccharides by metabolically engineered bacteria large-scale in vivo synthesis of the carbohydrate moieties of gangliosides gm and gm by metabolically engineered escherichia coli large scale biosynthesis of ganglioside analogues by rerf-lc-ai cells cultured in hyperflask use of sphingolipid analogs: benefits and risks a simple and novel method for tritium labeling of gangliosides and other sphingolipids preparation of radiolabeled gangliosides uptake and metabolism of exogenous glycosphingolipids by cultured cells structure and function of glycosphingolipids and sphingolipids: recollections and future trends release of carbohydrates from sphingoglycolipid by osmium-catalyzed periodate oxidation followed by treatment with mild alkali carbohydrate components of extraneuronal gangliosides from bovine and human spleen, and bovine kidney structural and functional glycosphingolipidomics by glycoblotting with an aminooxy-functionalized gold nanoparticle a unique glycosphingolipid-splitting enzyme (ceramide-glycanase from leech) cleaves the linkage between the oligosaccharide and the ceramide preparation of homogenous oligosaccharide chains from glycosphingolipids synthesis of lysogangliosides lysogangliosides: synthesis and use in preparing labeled gangliosides facile method for the preparation of lyso-gm and lyso-gm design of a covalently bonded glycosphingolipid microarray anti-ganglioside antibodies bind with enhanced affinity to gangliosides containing very long chain fatty acids sphingolipid photoaffinity labels thermodynamicgeometric correlations for the morphology of self-assembled structures of glycosphingolipids and their mixtures with dipalmitoylphosphatidylcholine aggregative properties of gangliosides in solution lipid membrane domains in glycobiology micellar properties of glycosphingolipids in aqueous media shedding and uptake of gangliosides and glycosylphosphatidylinositol-anchored proteins characterization of the cellular binding of exogenous gangliosides chemical synthesis of fluorescent glycero-and sphingolipids synthesis, shedding, and intercellular transfer of human medulloblastoma gangliosides: abrogation by a new inhibitor of glucosylceramide synthase shedding and immunoregulatory activity of yac- lymphoma cell gangliosides shedding of human neuroblastoma gangliosides synthesis of novel nbd-gm and nbd-gm for the transfer activity of gm -activator protein by a fret-based assay system support from professor dr. k. sandhoff, the dfg, and the european community ( th framework program "lipidomicnet", proposal no. ) is gratefully acknowledged. key: cord- - r e ijp authors: mandal, chitra; schwartz-albiez, reinhard; vlasak, reinhard title: functions and biosynthesis of o-acetylated sialic acids date: - - journal: sialoglyco chemistry and biology i doi: . / _ _ sha: doc_id: cord_uid: r e ijp sialic acids have a pivotal functional impact in many biological interactions such as virus attachment, cellular adhesion, regulation of proliferation, and apoptosis. a common modification of sialic acids is o-acetylation. o-acetylated sialic acids occur in bacteria and parasites and are also receptor determinants for a number of viruses. moreover, they have important functions in embryogenesis, development, and immunological processes. o-acetylated sialic acids represent cancer markers, as shown for acute lymphoblastic leukemia, and they are known to play significant roles in the regulation of ganglioside-mediated apoptosis. expression of o-acetylated sialoglycans is regulated by sialic acid-specific o-acetyltransferases and o-acetylesterases. recent developments in the identification of the enigmatic sialic acid-specific o-acetyltransferase are discussed. the generic term sialic acid defines a large family of -carbon monosaccharides which commonly occur as terminal residues of the oligosaccharide moiety of glycoconjugates. the diversity of sialic acids results from differential n-and o-substitutions of the two basic molecules, i.e., n-acetylneuraminic acid (neu ac) and n-glycolylneuraminic acid (neu gc). the most common modification of sialic acids is o-acetylation preferentially at the hydroxyl groups of carbon c , c , c , and c , whereas that at c seems to be the most frequent one. mab u [ ] , and mab h [ ] . these antibodies can also be used for immunodetection of o-ac gangliosides on hptlc plates using a method originally described by saito et al. [ ] . most recently, a method to determine the distribution of o-ac gangliosides in rat brain by matrix assisted laser desorption/ionization (maldi) mass spectrometry imaging was published [ ] . this procedure combines high resolution mass spectrometry with imaging software to map and image gangliosides with detailed structural information and histological accuracy. a highly sensitive method to analyze o-ac-sias relies on fluorescence detection of sialic acids derivatized with , -diamino- , -methylenedioxybenzene (dmb), followed by fluorometric high-performance liquid chromatography (hplc) [ ] . for analysis of o-ac-sias the acidic hydrolysis of glycosidically bound sialic acids should be performed with propionic instead of acetic acid [ ] . the presence of o-ac-sias can be determined by their respective rf values [ ] . in biological samples carboxyl groups of other compounds may result in additional peaks in chromatograms. therefore, the presence of o-ac-sias in hplc peaks should be confirmed by saponification, specific de-o-acetylation with influenza c virus esterase (for neu , ac ) or rat coronavirus esterase for neu , ac , or by mass spectrometry. the position of side groups of sialic acids can be determined by coupled gas chromatography/mass spectrometry (gc/ms) [ ] [ ] [ ] [ ] [ ] . very recently another highly sensitive method to identify different forms of o-acetylated sialic acids by electrospray ionization travelling wave ion mobility mass spectrometry coupled with low-energy collision-induced dissociation was described, allowing unambiguous assignment of the position of o-acetylation [ ] . sialic acids are constituents of different opportunistic human pathogens. bacteria can obtain sialic acids by either de novo biosynthesis or by acquisition from the environment. different gram negative bacteria like escherichia coli k , neisseria meningitidis, and campylobacter jejuni, and gram positive bacteria like group b streptococcus (gbs) can synthesize sialic acids [ ] . sialic acid uptake in other gram negative bacteria like haemophilus influenza, pasteurella multocida, haemophilus ducreyi, and pseudomonas aeruginosa from exogenous sources has recently been established [ , ] . these bacteria use their sialic acids for a variety of different purposes that play important roles in their ability to colonize and persist in organs of their hosts. for instance, sialic acids were shown to subvert immune clearance mechanisms by restricting complement c b deposition on its surface [ ] . once synthesized, sialic acid residues in the capsular polysaccharide or oligosaccharide of e. coli k , gbs serogroup iii, n. meningitidis, one or more of the hydroxyl groups in positions , , , and are substituted by acetyl groups [ ] . sia o-acetylation and de-o-acetylation is regulated by the gene neud and neua, respectively, to reach a final level of the surface expressed o-ac-sia modification [ , , ] . in addition to neud, which o-acetylates monomeric sialic acid, e. coli k strains harboring the prophage cus- express neuo, a polysialic acid-specific o-acetyltransferase [ , ] . expression of neuo is regulated by phase variation [ ] . in e. coli k strains o-acetylation increases immunogenicity of the k capsule and correlates with increased virulence in patients [ ] . o-acetylation of polysia increases desiccation resistance, which may favor survival in the environment [ ] . in n. meningitides the genes for o-acetyltransferases are located immediately downstream of the capsule synthesis genes siaa -siad [ ] and termed oatc and oatwy [ , , ] . most bacterial o-acetyltransferases are characterized by a hexapeptide repeat sequence folding into a left-handed b-helix [ , , ] . oatc, transferring acetyl groups exclusively onto polysialic acid joined by a , -linkages, apparently evolved separately and is characterized by an a/b hydrolase fold topology [ ] . the presence of the sialic acids and neu , ac a - galnac sialoglycotope has been demonstrated on p. aeruginosa [ ] . molecular analysis of gbs serogroup iii strain indicates that high levels of sia o-acetylation disrupt interactions with human siglec- present on neutrophils and block removal of capsular polysaccharide sia by bacterial sialidase, but do not alter deposition of complement on its surface [ , ] . blocking the interaction of gbs with neutrophils increases their activation followed by increasing bacterial killing. many viruses use sialic acids as receptors for binding to target cells, which is the critical first step of infection. influenza c viruses bind to cells via their surface glycoprotein termed hemagglutinin-esterase-fusion (hef) protein. these viruses bind to neu , ac via the hemagglutinin function of the hef protein [ , ] . in addition, the hef protein has a sialate- -o-acetylesterase activity [ , ] . infectious salmon anemia viruses, another genus of the orthomyxoviridae, encode a hemagglutinin-esterase (he) surface glycoprotein which binds to neu , ac [ ] . the esterase of these viruses is also specific for neu , ac [ ] . several coronaviruses and toroviruses also express he proteins which interact with o-ac-sias (table ) . a more comprehensive review of "sialovirology" will be published in another chapter of this issue. extensive research for the past decade has associated -o-acetylated sialic acids with promastigotes and amastigotes kinetoplastid parasites leishmania sp. [ ] [ ] [ ] [ ] [ ] [ ] [ ] . during the disease manifestation of visceral, cutaneous, and mucocutaneous functions and biosynthesis of o-acetylated sialic acids leishmaniasis, increased presence of -o-acetylated sialic acids is observed in virulent strains of leishmania sp., indicating their probable relevance in pathogenesis. in contrast minimal or undetectable presence of -o-acetylated sialic acids on a virulent strain of ur also signifies the role of -o-acetylated sialoglycotope as markers of virulence [ , ] . the function of parasite-associated -o-acetylated sialic acids for entry of promastigotes into macrophages has been demonstrated in comparison to the minimal internalization of de-o-acetylated promastigotes. analysis of the sialylation during the differentiation of internalized virulent promastigotes into amastigotes indicates that -o-acetylated sialic acids not only facilitate promastigote-entry but also play a probable role in differentiation and persistence of infection [ ] . additionally, increased presence of -o-acetylated sialic acids during metacyclic stages of virulent promastigotes has pointed to the direct correlation between the association of -o-acetylated sialic acids and virulence. apart from being a marker of virulence, -o-acetylated sialic acids also play an important role in conferring nitric oxide resistance in virulent leishmania sp. having enhanced distribution of the sialoglycotope. furthermore they also influence the intracellular survival of the parasite within macrophages and modulate the host responses in their favor as evidenced by decreased level of il- and ifn-g, the signature th cytokines [ ] . in contrast, macrophages show increased levels of these cytokines, when they are infected with de-o-acetylated promastigotes. this observation suggests that the parasite is capable of modulating the host responses via -o-acetylated sialic acids for the successful infection. apparently they act as effective ligands whose expression supports parasite internalization, intracellular differentiation. de novo synthesis of sialic acids usually occurs as a result of the fine-tuning of four enzymes, namely sialidase, trans-sialidase, esterase, and o-acetyltransferase. extensive work from the author's group (cm) has convincingly demonstrated that these parasites lack an active machinery for the biosynthesis of this unique sialoglycotope as corroborated by the absence of activity of udp-glcnac -epimerase which catalyzes the first step of sialic acid synthesis [ , ] . the presence of n-acetyltransferase in l. amazonensis has indicated the presence of enzymes for acetylation [ ] . however, any such claim for the presence of o-acetyltransferase requires the identification of the respective genes that, at present, is lacking. direct transfer of sialoglycoproteins from the serum demands extensive study of proteomic characterization of surface proteins on promastigotes and is a subject of future research. as there are multiple changes known to occur during the development of organs and tissues, glycosylation patterns are also subject to change [ ] . as an example, aberrant expression of sialyltransferases can result in displacement of cells, as shown for sialyltransferase st galnac , which is normally expressed only in brain. expression of this sialyltransferase in breast cancer cells alleviates their migration into the brain by mediating cell passage through the blood-brain barrier [ ] . in order to identify the functional impact of sialic acids, knockout mice with deleted sialyltransferase genes were created which exhibited a number of developmental changes. knockout of sialyltransferase st sia , also known as gd synthase, resulted in a complete absence of b and c series gangliosides. these mice appeared to undergo normal development and had a normal life span. double knockout mice with an additional disruption of the galnact gene encoding b , -n-acetyl-galactosaminyltransferase, thereby expressing gm as the sole ganglioside, were extremely sensitive to sound stimuli even leading to sudden death [ ] . mice with a knockout of the st gal gene exhibited tissue specific alterations in sialylation, concomitant with highly selective losses of -o-acetylation of sialic acid residues [ ] . knockout of polysialyltransferase st sia allowed for the first time a discrimination of the roles of neural cell adhesion molecule protein and polysialic acid in neural development and synaptic plasticity [ ] . addition of polysialic acid is an important modification of the neural cell adhesion molecule ncam, directing migration and differentiation of neuronal cells within the central nervous system [ ] . recent investigations point to a role of polysialic acids in the development of social interactions and aggression in mice [ ] . due to the fact that the understanding of the molecular functions of sialic acids in development and differentiation are just emerging, it is not surprising that the functions of o-acetylation of sialic acids are even less well understood. however, some examples pointing to specific roles of o-acsias are available. by expression of the influenza c virus sialate- -o-acetylesterase in transgenic mice, it was shown that o-acetylation is a prerequisite for normal development. mouse embryos constitutively expressing the esterase were arrested as early as in functions and biosynthesis of o-acetylated sialic acids the two cell stage [ ] . in the rat nervous system the -o-acetylated ganglioside gd ( -o-acgd ) was shown to exhibit discrete patterns during neuronal development [ ] . in the fetal mouse cortex -o-acetylated ganglioside gt is strongly expressed and decreases to undetectable levels after birth [ ] . in another study glycolipid-bound neu , ac was highest in embryonic mouse brain e , and gradually decreased until birth. significant amounts of neu , ac were found in adult mouse brain in the glycolipid fractions of the olfactory bulb, hippocampus, and telencephalon [ ] . in pig brain, an increase of neu , ac was observed during the maturation of the cortex and cerebellum [ ] . in patients with guillain-barré and fisher´s syndromes, which are manifested as acute inflammatory demyelinating polyneuropathies, antibodies against o-acetylated gangliosides were found [ ] . for -o-ac-gd , roles in neuronal motility were suggested [ , ] . antibodies to -o-ac-gd induce microtubule depolymerization in growing neurits [ ] , and -o-ac-gd was found in point contacts of neuronal growth cones [ ] . cerebellar granule neuron migration was blocked by the -o-ac-gd mab jones in live animals [ ] . this block was also observed in gd synthase knockout mice [ ] . results from the latter study indicated that the inhibitory effect of mab jones may be caused by binding to b -integrin. in chicken erythrocytes o-ac-sias represent a differentiation marker, which appears in -day-old birds and is fully developed in -day-old chickens [ ] . during the early stages of human development, -o-acgd is present in different tissues. in contrast, during the erythropoiesis, -o-acgd level is decreased during maturation in the erythroid progenitor cells in bone marrow. mature erythrocytes show lower -o-acgd levels than immature cells. alterations of membrane characteristics and morphology occur in mature erythrocytes via -o-acgd mediated signaling [ ] . such signaling via -o-acgd also induces membrane alterations, vesicularization, phosphatidyl serine exposure, and activation of cysteine proteases like caspase , suggesting a programmed cell death like pathway in mature erythrocytes. in contrast, enhanced level of -o-acgd is observed in lymphoblasts whereas gd expression is insignificant compared to normal lymphocytes. the anti-apoptotic role of -o-acgd in lymphoblasts in contrast to mature erythrocytes suggests a cell specific role of -o-acgd [ ] . the major task of the immune system is to fight against invading microorganisms, to differentiate between self and non-self, i.e., to control autoimmune reactivity, and to eliminate defective or mutated cells such as tumor cells. the immune detection of tumor cells is often hampered by the fact that they are able, by many mechanisms, to disguise themselves as being normal. in a wider sense repair mechanisms such as wound healing and angiogenesis as part of the inflammatory process should also be included within the range of immune reactions. in general, immune reactions can be divided into the branches of innate immunity as first line of defense and adaptive immunity for highly specific reactions including immunological memory. however, there are many molecular structures which bridge these two systems. during recent years it has become increasingly apparent that carbohydratelectin interactions play a vital role in many of these immune reactions regulated by the innate and the adaptive branch of the immune system [ ] . this includes both the recognition of microbial structures by immune cells and the intricate crosstalk between immune cells. it was hypothesized that attachment to and invasion of microorganisms into host cells taking advantage of carbohydrate-lectin interactions had induced a selective pressure to modulate their carbohydrate structures and the respective lectin receptors in order to prevent infection or on the side of the microorganisms to counteract these alterations on the host's side [ ] . by means of this putative evolutionary process the complexity of carbohydrate structures expressed on the cell surface might have developed. interestingly, a majority of these protein-carbohydrate interactions depends on the presence of terminal sialic acids as decisive recognition elements of the oligosaccharide ligand. accordingly there are at least two lectin families known, the selectins and the siglecs, which are specialized in the recognition of sialoglycans in various anomeric linkage patterns [ , ] . within the evolutionary development of carbohydrate complexity, o-acetylation of sialic acids seems to play a major role in infections and possibly also in defense mechanisms of immune cells. for example, coronaviruses have adapted specialized hemagglutinins to attach to cell surface expressed o-acetylated sialoglycans and to destroy further the linkage using sialic acid specific o-acetylesterases by removing the respective o-acetyl group [ , [ ] [ ] [ ] ] . when analyzing o-acetylation of lymphocytes it became evident that these immune cells preferentially synthesize -o-acetyl-and -o-acetyl sialoglycans [ , , [ ] [ ] [ ] . o-acetylated sialic acids resulted in increased susceptibility to alternate complement pathway-mediated lysis of murine erythrocytes. progressive loss of neu , ac -gps with differentiation is concomitantly associated with an increasing resistance to alternate complement pathway activation. increased presence of neu , ac -gps on erythrocytes of patients with visceral leishmaniasis may be responsible for~two-to threefold greater susceptibility to alternate complementmediated hemolysis as compared to healthy individuals [ , ] . although theoretically o-acetyl sialoglycans can occur on glycoproteins and glycosphingolipids, the majority of studies focused on the disialo-ganglioside gd as the major carrier of terminal o-acetylated sialic acid. gd itself has been described to be involved in several immune reactions. these results were obtained to a large extent by application of specific monoclonal antibodies against gd and functions and biosynthesis of o-acetylated sialic acids its -o-and -o-acetylated variants (designated as cd a (gd ), cd b ( -o-acetyl gd ), and cd c ( -o-acetyl-gd ) [ ] . by means of these cd antibodies the expression of these gangliosides can be observed in the sterical context of the cell surface of live cells. in addition these anti-ganglioside antibodies are useful tools to study the content of gd and its o-acetylated variants in distinct cellular compartments. in earlier in vitro studies it was shown that gangliosides such as gd shed by tumors can inhibit the activity of natural killer (nk) cells [ , ] . it was proposed that anti-tumor cytotoxicity of nk cells was blocked, thereby alleviating undisturbed tumor growth. while at that time the mechanism of this possible inhibition was not clear, consecutive work may explain the mechanism. nicoll et al. described that gd expressed on target cells is able to modulate nk cell cytotoxicity by interaction with siglec- expressed on nk cells. siglec- has a preference for binding of a , linked disialo glycans and seems to be one of the inhibitory nk cell receptors [ ] . in the case of melanoma cells the inhibitory function of gd could not be verified since gd expressed on melanoma cells can induce another class of cytotoxic cells, the nkt cells [ ] . it was further elucidated that the fine specificity of gd reactive nkt cells is mediated by binding to cd d, a surface molecule structurally related to the major histocompatibility antigen (mhc) which is expressed on t cells. cd d has a preference for carbohydrate ligands. it is not clear at the moment whether in the in vivo situation there may be a balance between anti-melanoma nk and nkt cellmediated immunity which affects the outcome of an effective immune surveillance in melanoma patients. another mechanism to explain immune escape of melanoma cells may be that gd seems to be able to impair dendritic cell differentiation from monocytes and may induce their apoptosis [ ] . on the other hand it is known that melanoma cells not only express gd but also its o-acetylated variants in various degrees [ ] . one may speculate that o-acetylation of gd can provide another protective mechanism of melanoma cells against cytotoxic attack of immune cells. o-acetylated sialoglycans may also be part of glycoproteins as shown for the mucin family of o-glycosylated glycoproteins. in the colon, sialyl lewis x (cd s) moieties of muc and muc were found to be differentially o-acetylated [ , ] . it may be that o-acetylated cd s is a negative regulator of the metastasis process because it may block the recognition of cd s by e-selectin (cd e) expressed on vascular endothelial cells and thereby inhibit the attachment of metastasizing cells to the vessel wall as a prerequisite to invade the host tissue. krishna and varki described the presence of -o-acetylated sialomucins on murine cd t lymphocytes [ ] . whether sialic acid o-acetylation has an effect on the binding capacity of siglec proteins is still not unequivocally resolved. in an earlier report sjoberg et al. stated that semisynthetic n-linked oligosaccharides with terminal o-acetylation have reduced binding to cd (siglec ) and, further, treatment of murine lymphocytes with a chimeric influenza c esterase (che-fc) clipping off o-acetyl residues increased binding of cd to various murine lymphocyte subsets [ ] . cd is a b lymphocyte specific lectin which recognizes preferentially terminally a , sialylated lactosaminyl oligosaccharides. it does not react with a , sialylated structures. in the murine system cd has a preference for neu gc whereas in humans cd solely recognizes neu ac. whether neu gc is synthesized and expressed in the human system is most unlikely although some reports described its presence though in small quantities [ ] . the conceptional problem with the above-mentioned results of sjoberg et al. is that natural o-acetylated a , sialylated lactosaminyl ligands have not yet been identified. effects of the influenza esterase on cd binding as described may be a result of steric alterations in the composition of surface oligosaccharides. to clarify the influence of -o-acetylated sialoglycans on the intracellular functions of cd as a negative regulator of b cell activation, cariappa et al. used murine mutants with a defect in the cellular sialate o-acetylesterase and investigated the function of cd in b cell signaling [ ] . indeed, they found increased -o-acetylation in b cells of these mouse mutants and subsequently also enhanced b cell receptor signaling, pointing to a possible suspension of cd control. although these results prove a regulatory role of the sialate o-esterase towards b cell activation, the direct effect of -o-acetylation of cd ligands is still to be shown. interestingly, mutations in the sialate o-esterase gene can be linked to certain human autoimmune disorders [ ] . the overall expression of o-acetylated sialoglycoconjugates at a given stage of lymphocyte differentiation depends on the intricate balance of enzymes involved both in synthesis and degradation of these oligosaccharides. wipfler et al. recently measured the transcription of gd synthase st sia , the putative sialic acidspecific o-acetyltransferase casd , the human sialidases neu and neu , and the sialic acid o-esterase siae in various human lymphocyte subsets representing various differentiation and activation stages in comparison to the expression of gd and its -o-acetylated and -o-acetylated variants (cd a,b,c) [ ] . it became apparent that the transcription of anabolic and catabolic enzymes was different in lymphocytes of various stages which had an impact on the intracellular and surface expression of cd structures. reduced o-acetylation may help tumor cells to escape from complementmediated lysis because recognition of carbohydrates by elements of the alternative complement pathway may be inhibited by o-acetylation [ ] . in malignant cells glycosylation is often altered in different ways. examples are changes in glycosaminoglycans, altered branching on n-glycans, changes in mucin o-glycans, and in many instances elevated expression of sialic acids [ ] . changes in o-acetylation are also observed regularly in cancer cells. the identification of the disialo ganglioside -o-acetyl gd , considered as an oncofetal marker, has been achieved using a lectin derived from the crab cancer antennarius that recognizes sialic acids which are o-acetylated at both c and c positions and have been shown to be a biomarker in human melanoma [ ] . enhanced presence of o-acetylated gd has been reported in breast cancer, basaliomas, tumors of neuroectodermal origin [ , ] , childhood lymphoblastic leukemia (all) [ ] , and glioblastoma [ ] . ravindranath et al. [ ] found that in one melanoma patient only metastatic lesions expressed the o-acetylated forms of gd and gd whereas the primary tumor expressed exclusively the non-o-acetylated gangliosides. the same was observed in basalioma. in basalioma the expression of this antigen was generally up to -fold higher than in surrounding normal skin [ ] . however, in breast carcinomas the situation seems to be more complex. in normal ducts and in benign lesions -o-acetylated sialoglycans were present in golgi regions and at the plasma membrane as detected by reaction with a cd b antibody. this is in contrast to carcinomas of the breast where -o-acetylated sialoglycans were distributed in the cytoplasm in a disorderly fashion [ ] . cell surface expression of cd b structures was only observed in well-differentiated carcinomas and overall expression decreased with progression of malignancy. similar changes in distribution of sialoglycans and the sialyltransferase st gal responsible for a , sialylation have been found in hepatocellular carcinomas in which the disorder of sialoglycan distribution was correlated to the grade of malignancy [ ] . it is still unclear whether loss of o-acetylation in malignant colon carcinomas is an advantage for tumor progression. surface-expressed -o-and -o-acetylated gd are abundantly expressed on human t lymphocytes [ ] . expression of both cd b and cd c ( -oacetylated gd ) has also been detected on small resting lymphocytes of peripheral blood and on mature, activated t lymphocytes in lymph nodes [ , ] . tonsillar b lymphocytes, though to a smaller extent than t lymphocytes, express cd b and c [ , ] . additionally the occurrence of cd c was described on cd + nk cells, monocytes, and granulocytes to various extents [ ] and on human cd hematopoietic progenitor cells derived from bone marrow (schwartz-albiez, unpublished). cell surface-expressed cd b and c on t and b lymphocytes may have a functional role in the lymphocytic activation process because anti-cd b and c monoclonal antibodies can influence lymphocytic proliferation [ , , ]. an interesting observation was that t and b lymphocytes react towards stimulation with cd b and c antibodies in a different way. while in t lymphocytes cd c antibodies alone, like a mitogen, can stimulate proliferation in b cells, additional signals such as addition of the cytokine il- and triggering of the b cell receptor (surface expressed immunoglobulin) are required. for stimulation with antibodies against cd b in t and b lymphocytes, additional signals are required [ ] . this differential behavior may have a basis in a different surface distribution of the c. mandal et al. antigens. while cd b structures are found in dot-like formations, possibly as components of rafts, both in t and b cells, cd c on t cells showed a more homogenous distribution on the cell surface [ ] . it is most likely that cd b is a cno-stimulatory signal for raft-concentrated receptors while cd c in t cells acts as a mitogen. it is rather unlikely that gangliosides themselves confer transmembrane signaling because they do have neither a transmembrane nor an intracellular domain. an explanation may be that distinct glycosphingolipids crosslinked by antibodies can contribute to raft formation by pulling together a certain array of receptors. the disialoganglioside gd is a well-known inducer of the apoptotic-program and its proapoptotic-effects can be counteracted by o-acetylation. exogenous addition of gd to lymphoblasts promotes the apoptotic program whereas -o-acetyl-gd has anti-apoptotic effects. unlike gd , -o-acetyl-gd fails to depolarize mitochondrial membranes followed by the release of cytochrome c and caspase and . the removal of o-acetyl groups by sodium salicylate in lymphoblasts re-establishes the gd -responsiveness to apoptotic signals. thus, the balance of de novo synthesized gd and -o-acetyl-gd plays important roles in the survival of lymphoblasts in leukemia [ ] . recently, differential expression and possible function of -o-and -oacetylated gd during apoptosis of human tonsillar b and t lymphocytes has also been reported [ ] . malisan et al. [ ] also demonstrated that acetylation suppresses the proapoptotic activity of ganglioside gd . interestingly, this ganglioside and its o-acetylated variants also have a function inside the cell, breaking the dogma that the final destiny for gangliosides is the cell surface. intracellular gd is involved in cd -and ceramide-mediated apoptosis [ ] . upon receptor-triggering, de novo synthesized gd accumulates intracellularly which can be demonstrated by increased activity of gd synthase. gd as a rule is restricted to be present in the golgi network and at the plasma membrane it is transferred to mitochondria via endosomal transport [ ] to raft-like mitochondrial membrane domains [ ] . the mechanisms of gd -induced apoptosis can be traced back to a gd -mediated change in the mitochondrial membrane potential and consequently increased production of reactive oxygen species [ , ] . oxidation of gd to gd - -aldehyde was shown to increase the apoptotic effect by targeting adenine nucleotide translocase [ ] . o-acetylation of gd suppresses this pro-apoptotic function of non-acetylated gd and does not have the deleterious effects of gd on mitochondria membranes [ ] . cells which are resistant to overexpression of gd convert existing gd more readily to -o-acetylated gd [ ] . in further confirmation of the anti-apoptotic effects of o-acetylated gd , kniep et al. found that exogenous o-acetylated gd given to cells in vitro is internalized and can prevent apoptosis [ ] . it was also observed that a t leukemia cell line resistant to apoptosis induced by n-acetyl-sphingosine or daunorubicin functions and biosynthesis of o-acetylated sialic acids transferred gd more readily into -o-acetyl gd [ ] . targetting -oacetylated gd with sialate- -o-acetylesterase results in apoptosis of biopsyderived human glioblastoma cells. compared to treatment of cells with exogenous o-acetylesterase, the effect of de-o-acetylation is more pronounced when the esterase is expressed within the cells from a recombinant baculovirus vector [ ] . thus, these data strongly point to an anti-apoptototic function of intracellular -o-acetyl gd that may protect tumor cells from apoptosis. while data have been gathered on the anti-apoptotic effect of -o-acetyl gd , no data are available on possible effects of -o-acetyl gd . we have observed that in t and b cells -oacetyl gd followed in its expression intensity by -o-acetyl gd is present in an intracellular pool [ ] . given that intracellular -o-acetyl gd confers antiapoptotic capacity, can this protective effect possibly be accelerated by conversion of -o-acetyl-into -o-acetyl gd ? we also have no knowledge of what regulates the apparently differential transport of both acetylated forms of gd to the cell surface and what mechanisms with regard to the balance between the intracellular pool and cell surface expression of cd b and c are decisive for regulation of either anti-apoptotic or proliferative effects. it was shown that human tonsillar b lymphocytes undergoing in vitro either spontaneous or staurosporine-induced apoptosis are characterized by surface expression of cd b but not cd c [ ] . it may be that o-acetylated gangliosides fulfil different tasks at the cell surface and in intracellular compartments. in the gastrointestinal tract, the concentration of o-acetylated sialic acids of colonic mucin decreases in colorectal carcinomas, colonic adenomas, ulcerative colitis, and hirschsprung's disease, suggesting its reversal to the embryonic form. however, human skin contains very little o-acetylated sialic acids. this decrease in o-acetylated sialic acids is associated with a concomitant increase in expression of the sialylated antigens, sialyl tn, sialyl lewis (a), and sialyl lewis (x), which are considered to be adverse prognostic indicators. an increased amount of o-acetylated sialic acids (neu , ac -gps all ) on erythrocytes [ ] and peripheral blood mononuclear cells (pbmc) of patients suffering from childhood acute lymphoblastic leukemia (all) [ ] [ ] [ ] [ ] [ ] has been demonstrated using the preferential specificity of a lectin, achatinin-h, towards neu , ac -a , -galnac [ , ] . the absence of neu , ac -gps and corresponding anti-neu , ac -gps antibodies in corresponding cells of healthy children or in patients with other cross-reactive hematological disorders such as acute myelogenous leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, non-hodgkin's lymphoma, thalassemia, and aplastic anemia confirmed the specificity of these biomarkers [ ] [ ] [ ] [ ] [ ] . the binding of neu , ac -gps all with achatinin-h in the presence of several synthetic sialic acid analogs further confirmed the presence of this sialoglycotope on lymphoblasts in leukemia [ ] . c. mandal et al. in spite of successful treatment, patients may retain small numbers of malignant cells which is referred to as minimal residual disease (mrd) responsible for relapse. mrd is the main cause of a relapse of the disease. neu , ac -gps are strongly expressed in childhood all at the onset of disease, then they decrease with chemotherapy and reappear with relapse. this observation makes neu , ac -gps a potential biomarker for diagnosis and monitoring the disease status in childhood all (fig. ) [ , [ ] [ ] [ ] [ ] [ ] . a -year longitudinal follow-up study reveals that the expression of three newly induced leukemia-associated neu , ac -gps all ( , , and kda) disappears after treatment in patients who have disease free survival [ , ] . the -kda band persists in a few patients who subsequently relapse with the re-expression of the -kda band. early clearance of neu , ac -gps all + cells, during - weeks of treatment, shows a good correlation with low risk of relapse [ ] . therefore, close monitoring of -and -kda -o-acsgs may serve as a reliable index for longterm management of these children and merits therapeutic consideration. subsequently, a suitable template has been established by using the differential expression of neu , ac -gps all along with other known cd antigens to monitor on lymphoblasts, soat in microsomes [ ] , and anti-neu , ac gps antibodies [ ] [ ] [ ] [ ] in serum as signature molecules useful for diagnosis and monitoring childhood all functions and biosynthesis of o-acetylated sialic acids mrd [ ] . a -year longitudinal follow-up study of patients [b-(n ¼ ) or t-(n ¼ ) all], from the onset of the disease until the end of chemotherapy, reveals the sensitivity of mrd detection reaching . % for a patient in clinical remission using flow cytometry. presence of enhanced mrd due to failure in early clearance of lymphoblasts is implicated in an elevated risk of relapse. elevated mrd during the chemotherapeutic regime predicts clinical relapse, at least weeks before clinical manifestation. therefore, these templates can function for mrd detection, during and post-chemotherapy for proper patient management strategies, thereby helping in designing tailor-made chemotherapy. the role of o-acetylated sias for the survival of lymphoblasts has been reported ( fig. ) [ , , [ ] [ ] [ ] ]. enhanced levels of antibodies against neu , ac -gps in leukemia patients as compared to normal individuals have also been used for monitoring disease status [ - , , ]. an enhanced amount of neu , ac -gps specific igg in leukemia was unable to trigger the complement cascade, activation of fcgr and cell-mediated cytotoxicity, although its sialoglycotope binding ability remains unaffected [ ] . interestingly, only neu , ac -gps specific igg n purified from normal human serum emerged as the potent mediator of cell-mediated cytotoxicity, complement fixation, and activator of effector cells through fcgr. therefore, the generation of customized o-acetylated sialic acid specific chimeric anti- -neu , ac -gps-igg antibody constructs bearing functional normal fc domain, having a homogenous glycoform and a pre-determined profile of functional potential would be ideal for therapeutic applications. such customized antibodies might lead to their proper functioning and therefore possibly being useful along with cytokine therapy to activate in vivo anti-cancer pathways for proper immune-surveillance in leukemia. subclass switching of anti-neu , ac -gps specific to igg , alteration in their fc-glycosylation profile, along with impairment of a few fc-glycosylation-sensitive effector functions hint towards an unbalanced homeostasis helpful for evading the host's immune defense, suggesting a possible mechanism for functional unresponsiveness of tumor antibodies in general [ ] . an interesting phenomenon is that patients suffering from certain tumors, for instance medullablastomas, a neural tumor disease, carry antibodies of the igm subtype against o-acetylated gd in their serum [ ] . appearance of o-acetylated sialoglycoproteins or glycosphingolipids is cell type specific and developmentally regulated. their synthesis and turnover is a finely tuned phenomenon. following the translocation of cytidine monophosphate (cmp)-sialic acid residues into the golgi apparatus, sialyltransferases catalyze the transfer of sialic acid onto an acceptor like galactose or n-acetylgalactosamine or less commonly n-acetylglucosamine or -n-acetyl neuraminic acid of an appropriate oligosaccharide chain as part of a nascent glycoconjugate in a , , a , , a , , or a , linkages. subsequently, sialate o-acetyltransferases (soat) transfer the acetyl group from acetyl-coa onto sialoglycoconjugates at the c- / / positions, generating o-acetylated sialoglycoconjugates. the primary insertion site for the o-acetyl group may well be the c -oh group, from where it can non-enzymatically migrate to the c- position, presumably via the c -oh group, leaving the c -oh group available for a new transfer [ , ] . these o-acetyl esters are removed by a family of other important enzymes in sialic acid metabolism, the sialate-o-acetylesterases (siae). both soat and siae fig. immune escape of lymphoblasts possibly due to enhanced sialylation, o-acetylation of glycoproteins or disialo gangliosides gd [ ] [ ] [ ] , soat [ ] , and reduced membrane bound sialidase (neu ) [ ] in all. subclass switching of anti-neu , ac gps antibodies from igg to igg , modulation of fc-glycosylation, and weakening a few fc-glycosylation-sensitive effector functions seem to be responsible for evading the host's immune response [ ] are the two main enzymes responsible for the quantity of the o-acetyl ester groups on sialic acids. therefore the activities of sialyltransferases and soat at one end of the spectrum, and the siae and a group of another key catabolic enzyme (sialidases), responsible for cleaving sialic acid residues from glycoproteins and glycolipids, at the other end of the spectrum, regulate the expression of o-acetylated sialoglycoconjugates. cancer cells frequently alter the regulation of sialylation processes leading to the appearance of characteristic sialoglycoproteins and sialoglycosphingolipids. a reduced soat enzyme activity in human colon and colorectal carcinoma is corroborated with decreased o-acetylation in the course of tumor development [ , , ] . in contrast, enhanced soat in microsomes of lymphoblasts from bone marrow of children with leukemia, irrespective of their lineage, is corroborated with increased neu , ac -gps [ ] . the o-acetylation of exogenously added gd by all-microsomes extends the specificity of this soat towards gangliosides. enhanced activity of soat with higher v max in leukemia is one of the few descriptions of an enzyme of this type in human. however, a higher acetylation rate may also be partly due to differences in transporters and natural acceptors. besides endogenous acceptors, exogenous substrates like different sialoglycoproteins, cmp-neu ac and gd , are substrates for the enzyme. however, it is difficult to discover the selectivity of the soat in vivo. the reaction products are mainly neu , ac and neu , ac , suggesting the primary insertion site of the o-acetyl group to be at c- , followed by c- of neu ac. the acetyl group possibly migrates from the seven position to the primary alcohol group of sialic acid at c- presumably via c- . this is corroborated by enhanced neu , ac exclusively in isolated microsomes of these lymphoblasts. accordingly, the leukemia soat was denoted as sialate- ( )-o-acetyltransferase [ ] . the possibility that a number of distinct soats are controlling o-acetylation of sialic acids attached to glycans via different linkages cannot be ruled out, suggesting another level at which o-acetylation is possibly controlled in cancer and normal tissue. this has been supported by the observations wherein soat activity with high specificity for terminal a , -linked sialic acid residues and no detectable activity for a , -linked sialic acids is reported [ ] . interestingly, expression of -o-acgd is higher in leukemic cells, which gives the plausible answer that increased amounts of gd might be converted to -o-acgd by means of enhanced soat and reduced membrane-bound sialidase (neu ) [ ] , thereby reducing the gd content. complex regulations of the overall metabolism of sphingolipids through different activation of other enzymes are involved in association with sialyltransferases in leukemia. c. mandal et al. augmented lactosylceramide might contribute to the increased resistance of malignant lymphocytes towards apoptosis. the soat activities increase rapidly with the onset of disease, decrease with clinical remission, and increase sharply again with clinical relapse and correlate well with high levels of cell surface neu , ac -gps and -o-acgd on lymphoblasts. thus understanding the mechanisms of o-acetylation of sialic acids will enhance our knowledge of the functions of sialic acids in animals and humans in general and not only in cancer. analysis of soat may provide insight into the pathogenesis of disease and its progression, and may even provide clues for designing new drugs. clearly, further studies are needed to unravel the sialic acid linkage specificity of soat in cancer. as already indicated, o-acetylation depends on multiple factors, including the origin of tissues or the type of cell lines used for soat assays. attempts to isolate the enzyme by biochemical procedures [ , [ ] [ ] [ ] [ ] led to the identification of at least two different soat activities: partially purified soat from bovine submandibular glands transfers acetyl groups to c of sias. it was proposed that migration of acetyl groups from c to c might be enzymatically catalyzed [ ] . in a later publication the existence of a "migrase" could not be substantiated [ ] . a second type of soat was found in golgi-enriched fractions of guinea pig liver, which transfers acetyl groups to c [ ] . soat activity isolated from guinea pig liver preferred gangliosides as substrate. in addition, a heat-stable low molecular weight cofactor, which could be separated from soat activity by ultrafiltration, was proposed to enhance -o-soat activity [ ] . in vitro several substrates, including free sialic acid, cmp-sia, gangliosides, and glycoprotein-bound sias could be acetylated by soat derived from different sources. in vivo free sias most likely do not represent natural substrates for soat, because sia is transferred into the golgi as cmp-sia by a nucleotide-sugar transporter [ , ] . higa and paulson isolated cmp-sia synthase and used this enzyme to prepare -o-ac-cmp-sia [ ] . interestingly, -o-ac-cmp-sia could not be synthesized with this enzyme. sialyltransferases were able to transfer neu , ac from the donor -o-ac-cmp-sia to glycoproteins, but at lower rates than neu ac or neu gc. it was concluded that a direct transfer of o-ac-sias to glycoproteins like bovine mucin from -o-ac-cmp-sia could account only for a fraction of the total o-ac-sias found. moreover, it was proposed that -o-acetylation would result from the action of an o-acetyltransferase on the glycosidically-bound sia [ ] . later it was found that both the cmp-sia and acetyl-coa transporters are critical components for the o-acetylation of cmp-sia in the golgi lumen. in addition, it was also suggested that a sialyltransferase exists that preferentially utilizes cmp-neu , ac as the donor substrate to sialylate galb , ( )r-residues [ ] . this finding was in contrast to earlier observations that acetyl-coa does not enter isolated rat liver golgi vesicles which are able to perform the acetylation of a , linked sialic acids on n-glycans [ ] . additional data had led to the proposal that o-acetylation is the product of a trans-membrane reaction, involving a membrane protein with essential histidine and lysine residues [ ] . in summary, it has not yet been possible to obtain a purified eukaryotic soat preparation suitable to determine its amino acid sequence and the gene(s) encoding soat. other laboratories have tried to identify soat by expression cloning. expression of different cdnas was found to stimulate o-acetylation of sialic acids. with such experiments, ogura and coworkers reported the cloning of an o-acetyl ganglioside synthase with a significant homology to milk fat globule membrane protein [ ] . kanamori et al. isolated a trans-membrane protein that most likely represents an acetyl-coa transporter. interestingly, they found that expression of this transporter induced the formation of o-ac-gd [ ] . during the search for the soat gene shi et al. also isolated cdnas which most likely are not directly involved in transfer of o-acetyl groups: a cdna clone encoding a chimeric protein composed of a bacterial tetracycline resistance gene repressor and a plasmid sequence was found to enhance o-acetylation. also, a clone encoding a truncated form of vitamin d binding protein was isolated. in both cases, expression of the recombinant proteins was required to observe increased o-acetylation [ ] . this finding may indicate that the expressed mrnas or proteins are recognized by pattern recognition receptors that then induce an "alarm" pathway, finally resulting in o-acetylation of sialic acids. binding to the bacterial tetracycline resistance gene repressor may have triggered the initiation of cells to become pre-apoptotic by inducing the expression of -o-ac-gd . to speculate further, induction of apoptosis would then just require activation of the cellular siae to generate the proapoptotic ganglioside gd . another molecule possibly involved in o-acetylation of gd was identified as tis , a cell cycle regulator and cell death molecule [ ] . it is possible that tis may be a mediator of o-acetylation, but it appears unlikely that tis represents the elusive soat. in gm /gd knockout mice high amounts of -o-ac-gd were found to accumulate in nerve tissue [ ] . in this publication expression levels of the previously reported inducers of o-acetylation were also examined. no up-regulation was found for vitamin d binding protein, acetyl-coa transporter, or the putative o-acetyl ganglioside synthase, while tis was partially down regulated. recently, a new player in the field was identified. a screening of the human genome database revealed the casd gene as a candidate to encode a key enzyme involved in sialic acid o-acetylation [ ] . when the cas protein was expressed in cos cells together with st sia , a substantial increase of synthesis of -o-ac-gd was observed. human cas p was shown to co-localize with the golgi marker st gal . at the mrna level, elevated casd expression was concomitant with increased levels of o-ac-gd in primary human cells and in cell lines derived from human melanoma and liver cancers. transfection of casd specific sirna resulted in a reduction of o-ac-gd expression. on the other hand, expression of cas p in cos cells is not sufficient to direct o-acetylation of sialic acids on n-or o-linked glycans on the model glycoprotein erythropoietin (manuscript in preparation). cas p is encoded by the casd gene located on chromosome q . . the gene is homologous to the cas gene of cryptococcus neoformans. the designation is derived from the function of its encoded protein in the formation of the fungal c. mandal et al. capsule structure. genetic evidence strongly indicates that the fungal cas protein is involved in the o-acetylation of glucorono-xylomannans [ ] . deletion of the cas gene resulted in a loss of o-acetylation on c- of either man or man glca residues. o-acetylation could be restored by expression of cas from a plasmid. the human and fungal cas proteins are composed of an n-terminal serine-glycine-asparagine-histidine (sgnh) domain and a c-terminal trans-membrane domain with - trans-membrane regions (fig. ) . homologs of cas are also present in plants. they were shown to be directly involved in o-acetylation of plant cell walls. this modification is present in high amounts in different plant polysaccharides. o-acetylation is a hurdle in the processing of plant material into biofuels, because glycosidases used for degradation of plant polysaccharides are negatively affected by o-acetylation [ ] . on top of that, acetate and its conversion products are inhibitory to microorganisms used for fermentation [ ] . during the screening of arabidopsis mutants, plants were identified with insertional mutations in the plant cas genes, which were termed rwa (reduced wall acetylation) [ ] . in arabidopsis four rwa genes are present, and their expression in different plant tissues partially overlaps. the rwa mutant exhibited a - % reduction in cell wall acetylation. different polysaccharides were affected at similar rates. therefore, it was speculated that rwa proteins act immediately upstream of the transfer of acetyl groups to different acceptors. interestingly, the plant cas proteins lack the n-terminal sgnh domain. instead, a large number of proteins with similarity to the n-terminal domain of cas p was identified in a bioinformatics approach [ ] . in plants, the duf (domain of unknown function) family of proteins may be involved in the transfer of o-acetyl groups to specific acceptors, possibly by a trans-esterase mechanism [ ] . in mammals, other proteins, including members of the fam and fam families, and c orf , exhibit similarities to the sgnh domain of cas p [ ] . thus, it may be speculated that these proteins are candidates to direct o-acetyl groups to sialic acids in different glycosidic linkages and/or to different carbons of sialic acids (fig. ). in addition, other yet unidentified components of the cellular soat activities may be required for the fine specificity of o-acetylation. moreover, it is also unclear how soat and siae work in concert to regulate acetylation. this regulation may be at transcriptional level, but interactions at the protein level may also play a role. siae hydrolyses either c -or c -o-acetyl groups of glycosidically-linked or free sialic acid released from glycoconjugates by sialidases [ , [ ] [ ] [ ] [ ] . at least two forms of cellular -o-acetyl-siae, one in the cytoplasm and the other in the lysosomal compartment (i.e., membrane-bound), exist in mammals. the secreted form, originally termed luminal sialic acid esterase [ , ] , was later termed lysosomal sialic acid o-acetylesterase (lse) [ ] , because it was shown by immuno-electron microscopy to co-localize with acid hydrolases and lysosomal membrane glycoproteins [ ] . this siae contains a cleavable n-terminal signal sequence and is secreted from cos cells as a glycoprotein with an apparent molecular mass of kda [ ] , whereas in rat liver it is found predominantly as a heterodimer composed of a small ( -kda) and large ( -kda) subunit connected by disulfide bridges. the small subunit could be labeled with the serine hydrolase inhibitor diisopropyl fluorophosphate, indicating that siae is a serine esterase. no sequence similarities to other known serine esterases were found [ ] . amino acid residues, important for catalytic activity and secretion, were determined by expression of mutated cdna. the mutations tested were found in the siae gene of patients with autoimmune disease [ ] . both subunits are encoded by the same mrna in the order signal sequence -small subunit -large subunit. the precursor is presumably cleaved in lysosomal compartments [ , ] . siae was identified independently by another research team as cdna derived from a gene that is upregulated during b cell maturation. several cdnas were isolated which apparently are derived from differentially spliced siae mrna [ ] . one of the spliced mrnas is lacking the exon for the signal sequence and encodes the cytosolic siae. while the mrna for the secreted siae is widely expressed in different adult tissues, the expression of the cytosolic siae is restricted. elevated expression of the latter was found mainly in liver, ovary, and brain [ ] . siae regulates b cell antigen receptor signal strength and peripheral b cell development in mice [ ] , presumably by regulating recognition of a , -linked sialic acids by cd [ ] , a negative regulator of the b cell receptor [ ] [ ] [ ] [ ] and toll-like receptors [ ] . mutations in the siae gene resulting in a loss of function are strongly linked to autoimmune disease [ ] , while overexpression of siae is linked to preeclampsia [ ] , a condition of pregnant women who exhibit high blood pressure and proteinurea. concerning the function of siae, many open questions remain. while it can be envisaged that cytosolic siae is involved in de-o-acetylation of o-ac-sias delivered from lysosomes, the function of the "lysosomal" siae remains controversial due to the unfavorable ph in lysosomes. the ph optimum of siae is in the neutral to alkaline range. data indicate that intracellular vesicles exist which contain mannose- -phosphate positive glycoproteins [ ] . such entities possibly represent transport vesicles on the way from the golgi to lysosomes. in any case, precise models of how siae is involved in tuning the balance of o-acetylation of sialic acids are currently not available. consequently, a large field is open for future investigations. another question concerns the enzymatic activity of secreted siae, which was shown to be increased upon proteolytic cleavage [ ] . the protease required for cleavage activation was proposed to be a lysosomal enzyme, but the nature of this protease remains to be determined. moreover, it is apparently not a common protease. cos cells were shown to express and secrete the uncleaved siae [ ] . another puzzle remains concerning the distribution of the secreted siae. available data indicate that it is intracellularly concentrated in lysosomal compartments [ ] . on the other hand it is efficiently secreted into the culture supernatant of cells over-expressing siae, and therefore it has access to o-ac-sias at the cell surface [ , ] . furthermore, the substrate specificity was shown for o-acetyl groups on free sialic acids [ ] . in siae knockout mice, an increase in a , -linked o-ac-sias was observed by using the influenza c virus lectin [ ] . strictly speaking, the influenza c virus lectin binds to any accessible o-ac-sia regardless of the underlying linkage. thus, it remains an open question whether a , or a , linked sialic acids are also de-o-acetylated by siae. furthermore, no siae hydrolyzing neu , ac has been purified to homogeneity allowing the functions and biosynthesis of o-acetylated sialic acids determination of its relationship to siae specific for neu , ac . in summary, the expression of -o-acetylated sialoglycoproteins seems to be controlled by the relative activities of soat and siae. many details on o-acetylation and de-o-acetylation still require clarification. the existence of diverse o-acetylated sias together with the enormous variations in sialoglycotopes having o-acetylated sias in different linkages, with different subterminal sugars and their regulative functions in proliferation, and their controlled expression may be explored in view of possible applications in cancer therapy. despite huge efforts to elucidate the factors mediating the escape of cancer cells from immunological surveillance, our knowledge in this regard is still rather limited. exploring the function of o-acetylated sialic acids on the immune cells may contribute to our understanding of this problem. in this direction, much emphasis of current research is invested in development of target-oriented anti-cancer drugs. future investigation using both enzymological and molecular biology approaches of some key enzymes like soat, siae, sialyltranferases, and sialidases as drug targets may also lead into the direction of therapeutic interventions. for instance, deeper understanding of their functioning could be explored in a more practical direction for pharmacological manipulation of the apoptotic pathways. glycoproteins ii gabius hj (ed) the sugar code. fundamentals of glycosciences leukocyte typing vii functions and biosynthesis of o-acetylated sialic acids etzler me (eds) essentials in glycobiology functions and biosynthesis of o-acetylated sialic acids key: cord- -keo gv authors: duarte, carolina; akkaoui, juliet; yamada, chiaki; ho, anny; mao, cungui; movila, alexandru title: elusive roles of the different ceramidases in human health, pathophysiology, and tissue regeneration date: - - journal: cells doi: . /cells sha: doc_id: cord_uid: keo gv ceramide and sphingosine are important interconvertible sphingolipid metabolites which govern various signaling pathways related to different aspects of cell survival and senescence. the conversion of ceramide into sphingosine is mediated by ceramidases. altogether, five human ceramidases—named acid ceramidase, neutral ceramidase, alkaline ceramidase , alkaline ceramidase , and alkaline ceramidase —have been identified as having maximal activities in acidic, neutral, and alkaline environments, respectively. all five ceramidases have received increased attention for their implications in various diseases, including cancer, alzheimer’s disease, and farber disease. furthermore, the potential anti-inflammatory and anti-apoptotic effects of ceramidases in host cells exposed to pathogenic bacteria and viruses have also been demonstrated. while ceramidases have been a subject of study in recent decades, our knowledge of their pathophysiology remains limited. thus, this review provides a critical evaluation and interpretive analysis of existing literature on the role of acid, neutral, and alkaline ceramidases in relation to human health and various diseases, including cancer, neurodegenerative diseases, and infectious diseases. in addition, the essential impact of ceramidases on tissue regeneration, as well as their usefulness in enzyme replacement therapy, is also discussed. ceramides are bioactive sphingolipids responsible for cell apoptosis, senescence, and autophagy [ ] . they are the precursors of other bioactive sphingolipids, including sphingosine (sph), sphingosine- -phosphate (s p), and ceramide- -phosphate, which play specific roles in signal transduction pathways ( figure ) [ ] . figure . role of ceramidases in ceramide metabolism. ceramide in mammalian cells may be generated: ( ) via the de novo synthesis pathway, which begins with the condensation of l-serine and palmitoyl-coa); ( ) by the hydrolysis of sphingomyelin and glucosylceramide; or ( ) from the dephosphorylation of ceramide- -phosphate. ceramidase is an enzyme that cleaves fatty acids from ceramide, producing sphingosine. sphingosine may then be phosphorylated by a sphingosine kinase to form sphingosine- -phosphate. smase-sphingomyelinase; gcs-glucosylceramide synthase. the de novo anabolic pathway for the biosynthesis of ceramide begins with the condensation of the amino acid, l-serine, and palmitoyl-coa, producing -ketosphingonine. then, -ketosphingonine is quickly converted into dihydrosphingosine (dhsph) by -ketosphinganine reductase. the subsequent acylation of dhsph by (dihydro)ceramide synthases gives rise to dihydroceramides. finally, the removal of two hydrogens from a fatty acid chain of the dihydroceramides by the enzyme desaturase results in the formation of ceramides ( figure ) [ ] . ceramides can be further hydrolyzed into sphingosine (sph) and free fatty acids by ceramidases ( figure ). sph is the most common sphingolipid base molecule in mammalian cells and is the precursor of s p [ ] . the bioactive lipid mediator s p is involved in cell proliferation, differentiation, and survival, whilst ceramides and sph mediate cell death [ , ] . notably, sph is exclusively generated from the catabolism of ceramides by ceramidases [ ] . ceramidases control the balance between s p and ceramides/sph concentration, which leads to either cell survival or cell death [ , ] . hence, a ceramidase-based enzyme replacement therapy that simultaneously achieves ceramide reduction and sph elevation has been recently examined [ ] . this therapeutic approach intends to reduce the negative pathophysiological impact of cell death mediated by ceramides [ ] . to date, five human ceramidases have been identified and classified according to their optimal ph for catalytic activity: one acid ceramidase (acdase) encoded by the gene asah , one neutral ceramidase (ncdase) encoded by asah , and three alkaline ceramidases (alkcdase) encoded by the genes acer , acer and acer [ ] . acdase is the most widely studied ceramidase, and its effects on pathophysiology are variable. while ncdase and alkcdases - have been the subject of studies in recent decades, our knowledge about their roles in pathophysiology remains limited. thus, the aim of this review was to summarize the most recent knowledge of the biology of ceramidases and their role in the pathology of various common human diseases, including cancer, diabetes, and neurodegenerative diseases ( figure ). in addition, the roles of the ceramidases in infectious diseases, tissue regeneration, and healing were also addressed. acdase (asah ) is synthesized from a - kda polypeptide precursor which is proteolytically processed into the enzyme's kda α-subunit and kda β-subunit inside the lysosomes [ , ] . acdase is a lipid hydrolase found in the lysosomal compartment of cells which catalyzes the hydrolysis of c : -c : ceramides to sph [ ] . it is ubiquitously expressed in human tissues, with a particularly high expression in the heart and kidneys and is known for its role in senescence and apoptosis [ ] . ncdase (asah ) is synthesized as kda and kda isoforms in humans and as a kda molecule in mice [ ] . ncdase is a transmembrane glycoprotein that is highly expressed in the human intestinal system and uses various ceramides and dihydroceramides as a substrate, with a reported preference for c : and c : ceramides [ , ] . it is localized to different cellular compartments, including the plasma membrane of cells; regulates the conversion of ceramide into sph and s p; and is important for the metabolism of dietary sphingolipids [ ] . alkcdases - (acer , acer , acer ) are the smallest proteins among the ceramidases, with molecular weights of - . kda [ , ] . alkcdases are predominantly located in the golgi complex and endoplasmic reticulum and play a role in cell differentiation [ ] . acer hydrolyses c : -c : ceramides [ ] . it is predominantly expressed by skin cells and is involved in their acdase (asah ) is synthesized from a - kda polypeptide precursor which is proteolytically processed into the enzyme's kda α-subunit and kda β-subunit inside the lysosomes [ , ] . acdase is a lipid hydrolase found in the lysosomal compartment of cells which catalyzes the hydrolysis of c : -c : ceramides to sph [ ] . it is ubiquitously expressed in human tissues, with a particularly high expression in the heart and kidneys and is known for its role in senescence and apoptosis [ ] . ncdase (asah ) is synthesized as kda and kda isoforms in humans and as a kda molecule in mice [ ] . ncdase is a transmembrane glycoprotein that is highly expressed in the human intestinal system and uses various ceramides and dihydroceramides as a substrate, with a reported preference for c : and c : ceramides [ , ] . it is localized to different cellular compartments, including the plasma membrane of cells; regulates the conversion of ceramide into sph and s p; and is important for the metabolism of dietary sphingolipids [ ] . alkcdases - (acer , acer , acer ) are the smallest proteins among the ceramidases, with molecular weights of - . kda [ , ] . alkcdases are predominantly located in the golgi complex and endoplasmic reticulum and play a role in cell differentiation [ ] . acer hydrolyses c : -c : ceramides [ ] . it is predominantly expressed by skin cells and is involved in their differentiation, as well as in the viability of hair follicle stem cells [ , [ ] [ ] [ ] . acer hydrolyses ceramides and dihydroceramides c : and c : [ ] . it is upregulated during dna damage and induces programmed cell death through an sph-dependent pathway [ ] . acer hydrolyses ceramides, dihydroceramides, and phytoceramides with long unsaturated acyl chains [ ] . it has been described as a seven-transmembrane protein, much like the adipocyte receptor (adipor), and is associated with cytokine upregulation [ ] . this brief overview of the general characteristics of ceramidases indicates that they have been classified according to their optimal ph. however, ceramidases also differ in molecular weight and expression patterns. importantly, all three groups of ceramidases have a specific ceramide affinity and reported cellular functions. it is important to highlight that the alkcdases, although classified together, differ in ceramide affinity and function. moreover, alkcdase- , like ncdase, is a transmembrane protein and not a soluble enzyme. thus, further consideration should be given to the classification of ceramidases and, particularly, the alkcdases. the activation of acdase induces a pro-survival state, while its inhibition leads to cell death through a variety of apoptotic pathways mediated by caspases (casp), poly (adp-ribose) polymerase (parp), or cathepsins (cts) [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . cathepsin b and cathepsin d are activated during ceramide-induced apoptosis but are inhibited by acdase activity [ , ] . interestingly, the downregulation of cathepsin b by acdase increases acdase's own activation, triggering a feedback mechanism through which acdase prolongs its own activation through cathepsin b inhibition [ ] . additionally, acdase activity can be regulated by ceramide synthase (cers ) [ ] . cers increases the levels of c : , which, in turn, activate acdase through jnk-ap -dependent mechanisms. however, this same mechanism mediates the inhibition of the gene expression of ncdase and alkcdases in colorectal adenocarcinoma [ ] . an age-dependent inhibition of acdase leads to ceramide accumulation, an increase in oxidative stress, and the death of retinal cells and erythrocytes [ , ] . by contrast, it was reported that kidney cells collected from aged mice show an elevated expression of asah mrna compared to that of young mice [ ] . thus, this published evidence suggests a tissue-specific acdase activity in relation to cellular senescence and aging. the activity and gene expression of ncdase have been linked to cell-cycle arrest and growth regulation [ ] . biochemically, ncdase is a lipid amidase with a mechanistic similarity to a bacterial ncdase [ ] . ncdase activates nitric oxide (no), the wnt/β-catenin pathway, caspase apoptotic pathways, and autophagosomal activity in vivo and is associated with mitochondrial integrity [ ] [ ] [ ] [ ] . its gene expression and activity are regulated by c-jun/ap- signaling, no, all-trans retinoic acid, and ultra-violet radiation [ , ] . the alkcdases - are regulated through markedly different mechanisms. acer is upregulated by extracellular calcium, through which it contributes to the regulation of cell differentiation and growth arrest [ ] . meanwhile, acer is induced by p and activates p mapk and ap- signaling to mediate dna damage response, autophagy, and apoptosis [ , , ] . acer is associated with the akt/bax pathway and activates the s p phosphorylation of akt through s pr and pi k in cancer cells [ , ] . frbrl is an autosomal recessive lysosomal disorder with a broad spectrum of phenotypes caused by identified mutations of asah [ ] . it is characterized by a substantial neurologic deficit, subcutaneous nodules, progressive arthritis with joint deformities, laryngeal hoarseness, and an accumulation of storage-laden cd + macroglia/macrophages in white matter, periventricular zones, and meninges of the brain [ ] . animal models of acdase deletion present hematopoietic organ hypertrophy, characterized by a foamy macrophage infiltration and increased myeloid progenitor colonies [ ] . these myeloid progenitor colonies are comprised of cells that can develop normally when treated with acdase [ ] . additionally, acdase deletion causes an impaired airway resistance, elastance, and compliance; reduced blood oxygenation; lung edema; and increased immune cell infiltration of the lungs by foamy macrophages and neutrophils [ ] . furthermore, an increased vascular permeability of the lungs, heart, thymus, liver and spleen, as well as neurologic problems, including decreased deambulation, anxiety, and impaired motor coordination, are also observed [ ] . these neurologic problems are caused by abnormal sphingolipid profiles in the brain and cd + microglia [ ] . changes in the asah gene expression in frbrl patients result in the upregulation of the inflammatory cytokines interleukin (il- ), il- , tumor necrosis factor alpha (tnfα), and macrophage colony stimulating factor (m-csf) in addition to the angiogenic marker, vascular endothelial growth factor (vegf) [ , ] . likewise, the expressions of the chemo-attractants, monocyte chemotactic protein- (mcp- ), and interferon gamma-induced protein (ip ), are inversely correlated with the level of acdase activity [ ] . these mediators of inflammation, angiogenesis, and insulin resistance may be associated with the immune cell infiltration found in the organ tissues of frbrl animal models [ ] . mcp- deletion can partially rescue frbrl phenotypes by improving organomegaly, blood cell counts, and liver and lung damage by inflammatory infiltrates, as well as the behavioral and neurologic aspects of the disease. however, hematopoiesis is not improved [ ] . similarly, the overexpression of mcp- , ip- , and il- can be partially corrected by hematopoietic stem cell transplants [ ] . moreover, treatment with acdase induces a dose-dependent decrease in hematopoietic organ weight, macrophage infiltration, and mcp- expression, as well as increased expression of collagen type (col ), aggrecan, and sox- by chondrocytes [ ] . sma-pme is a rare autosomal recessive disorder that is frequently associated with frbrl and is caused by two identified mutations of asah [ ] . this disorder is characterized by motor neuron disease and progressive myoclonic epilepsy, with a variable occurrence of sensorineural hearing loss, action tremor, cognitive dysfunction, and cerebral/cerebellar atrophy. patients with sma-pme present a - % reduction in acdase activity, a low acdase/β-galactosidase ratio, and increased creatine kinase levels [ ] . additionally, the muscle atrophy associated with sma can be accompanied by cyclooxygenase deficiency [ ] . the consequence of acdase gene overexpression during gestation and its therapeutic effect on associated genetic disorders has also been described. iugr can result from the tgfβ/alk -mediated overexpression of asah mrna and increased acdase activity, which upregulates sph but not s p concentrations during pregnancy [ ] . s p is not upregulated at the same rate as sph in iugr due to the inactivation of sph kinase through the alk -smad / pathway [ ] . this suggests that acdase may induce embryonic cell death through sph rather than affect embryonic cell proliferation and differentiation through s p in iugr. globoid cell leukodystrophy, or krabbe disease, is a congenital disorder caused by mutations in the galactosylceramidase gene, galc, and is characterized by psychomotor regression, muscular hypertonia, muscular spasticity, truncal hypotonia, irritability, seizures, and nystagmus [ ] . this disorder is caused by an accumulation of psychosine, a by-product of the deacylation of galc by acdase [ ] . therefore, the inhibition of acdase activity, as observed in frbrl or after treatment with acdase inhibitors, can rescue the krabbe disease phenotype by preventing psychosine accumulation [ ] . there are no reports demonstrating the association of the point genetic mutations of asah with inheritable diseases in humans. it is important to mention that asah −/− mice are viable and appear without severe defects [ ] . progressive leukodystrophy is a group of disorders that affect the white matter of the brain and can occur as a consequence of acer deficiency [ ] . this condition is caused by a loss of function mutation in p.e g which inactivates the catalytic activity of acer and leads to an accumulation of sphingolipids in the blood [ ] . the clinical phenotype associated with acer mutations is caused by incorrect central nervous system myelination due to abnormal levels of ceramides in the brain [ ] . while the study reporting the loss of function mutation in p.e g did not report a sphingolipid accumulation or pattern in the brain, it is reasonable to assume that sphingolipid accumulation due to acer inactivation results in abnormal sphingolipid patterns in the brain. the overexpression of ceramidases have been identified in various cancer cell types, and growing evidence suggests that they can be considered molecular markers and/or therapeutic targets for cancer [ ] (figure ) . . . . acid ceramidase asah has been identified in cancer cells and is associated with radiotherapy/chemotherapyresistant tumors [ , [ ] [ ] [ ] , metastatic cell lines [ ] , and estrogen/progesterone/androgen receptorpositive cells [ , ] . while acdase gene overexpression has been identified in low-survival-rate colorectal adenocarcinoma and glioblastoma [ , ] , it has also been observed in node-negative melanoma and breast cancer [ , ] , which makes it a questionable marker for the aggressiveness or invasiveness of the disease. the asah mrna expression in cancer cells can be increased by radiotherapy, thereby generating resistance [ ] . likewise, the overexpression of asah can be driven by the oncogene microphthalmia-associated transcription factor (mitf) [ ] . acdase activity is increased by the androgen receptor activation by dihydrotestosterone in prostate cancer, leading to decreased c : levels and reduced cell apoptosis [ ] . incidentally, the acdase activity is significantly more upregulated than the asah expression in melanoma cells [ ] . this may suggest that gene expression alone should not be the determining factor in the use of acdase as a marker for cancer; acdase activity should also be assessed. multiple molecular mechanisms by which acdase activation regulates cancer development and progression have been identified. for instance, drug resistance in leukemia is mediated by the acdase activation of the drug transporter molecule atp-binding cassette, subfamily b, member (abcb ), through nuclear factor kappa b (nf-κb) [ ] , whilst leukemic cancer cell survival is increased by the acdase-mediated upregulation of the myeloid cell leukemia sequence (mcl- ) [ ] . furthermore, cancer cell necrosis is mediated by acdase gene overexpression in polynuclear giant cancer cells that undergo asymmetric cell division [ ] . acdase also regulates cancer cell motility through the activation of the itgαvβ /fak signaling cascade [ ] . additionally, the significant roles of acdase in angiogenesis, chronic inflammation, and tumorigenesis may contribute to cancer development and progression [ , ] . acdase affects multiple factors in cancer pathogenicity, which adds to the complexity of the enzyme in the diagnosis and treatment of the disease. . . . acid ceramidase asah has been identified in cancer cells and is associated with radiotherapy/chemotherapyresistant tumors [ , [ ] [ ] [ ] , metastatic cell lines [ ] , and estrogen/progesterone/androgen receptor-positive cells [ , ] . while acdase gene overexpression has been identified in low-survival-rate colorectal adenocarcinoma and glioblastoma [ , ] , it has also been observed in node-negative melanoma and breast cancer [ , ] , which makes it a questionable marker for the aggressiveness or invasiveness of the disease. the asah mrna expression in cancer cells can be increased by radiotherapy, thereby generating resistance [ ] . likewise, the overexpression of asah can be driven by the oncogene microphthalmia-associated transcription factor (mitf) [ ] . acdase activity is increased by the androgen receptor activation by dihydrotestosterone in prostate cancer, leading to decreased c : levels and reduced cell apoptosis [ ] . incidentally, the acdase activity is significantly more upregulated than the asah expression in melanoma cells [ ] . this may suggest that gene expression alone should not be the determining factor in the use of acdase as a marker for cancer; acdase activity should also be assessed. multiple molecular mechanisms by which acdase activation regulates cancer development and progression have been identified. for instance, drug resistance in leukemia is mediated by the acdase activation of the drug transporter molecule atp-binding cassette, subfamily b, member (abcb ), through nuclear factor kappa b (nf-κb) [ ] , whilst leukemic cancer cell survival is increased by the acdase-mediated upregulation of the myeloid cell leukemia sequence (mcl- ) [ ] . furthermore, cancer cell necrosis is mediated by acdase gene overexpression in polynuclear giant cancer cells that undergo asymmetric cell division [ ] . acdase also regulates cancer cell motility through the activation of the itgαvβ /fak signaling cascade [ ] . additionally, the significant roles of acdase in angiogenesis, chronic inflammation, and tumorigenesis may contribute to cancer development and progression [ , ] . acdase affects multiple factors in cancer pathogenicity, which adds to the complexity of the enzyme in the diagnosis and treatment of the disease. a variety of acdase inhibitors have been developed and successfully tested in different cancer cell types. acdase deletion blocks the cell cycle at g /s, promotes senescence through the β-galactosidase/mitf pathway, induces apoptosis, reduces tumorigenesis, increases growth arrest, and decreases malignancy [ ] . it was demonstrated that the activity of acdase was significantly inhibited by carmofur [ , ] , lcl [ , , , ] , ceranib [ , [ ] [ ] [ ] , n-oleocylethanolamine (noe) [ , ] , arn [ ] , lcl [ , ] , monascus purperus (mp) [ ] , hesperetin (hst) [ ] , hesperetine- -o-acetate (hta) [ ] , silibinin [ ] , curcumin [ ] , and sanguinarine [ , ] , leading to an increased accumulation of intracellular ceramide and apoptosis in various types of cancer cells, including glioblastoma; squamous cell carcinoma; acute myeloid leukemia; colorectal adenocarcinoma; and breast, prostate, lung, gastric, and kidney cancer. furthermore, carmofur, noe, lcl , and ceranib have been used in combination with chemotherapeutic drugs or photodynamic therapy to either overcome cancer cell resistance to treatment, increase cell sensitivity to specific drugs, or increase the overall effectiveness of cancer cell apoptosis [ , , , , , ] . ceranib treatment leads to an abnormal cell and mitochondria morphology and decreases the ability of cells to cluster [ , ] . it activates parp and casp / / / -mediated cell apoptosis; increases the expression of the pro-apoptotic markers bid, bcl -associated agonist of cell death (bad), and bcl -associated x protein (bax); and decreases the expression of anti-apoptotic protein b-cell cll/lymphoma (bcl- ) [ , ] . furthermore, mp, hst, hta, curcumin, and sanguinarine activate the apoptotic pathways dependent on casp / or reactive oxygen species (ros) [ , , , , ] . sanguinarine induces peroxide-dependent ceramide generation and the inhibition of the akt activation pathway [ ] . noe and lcl induce parpand casp -mediated apoptosis [ , ] , whereas lcl increases c : levels, autophagosome accumulation, er stress, and cathepsin b-or cathepsin d-mediated apoptosis [ ] . altogether, acdase inhibitors are effective promoters of cancer cell death through different apoptotic pathways and have been shown to affect not only apoptosis but also cancer treatment resistance and cancer cell adhesion. an elevated gene expression of ncdase has been identified in both the plasma membrane and golgi apparatus of colorectal cancer (crc) cells, where its overexpression inhibits ceramide c -mediated cell death [ ] . meanwhile, its deletion induces caspase and autophagosome-mediated apoptosis in the presence of c [ ] . ncdase regulates crc cell proliferation through the wnt/β-catenin pathway and by increasing the accumulation of sph and s p [ , ] . ncdase inhibition may affect cell-to-cell adhesion by reducing the β-catenin levels through akt phosphorylation and, subsequently, gsk β activation [ ] . it also significantly reduces azoxymethane-induced colon carcinogenesis by inhibiting aberrant crypt foci formation and transformation [ ] . ncdase inhibition does not affect non-cancerous cell function, which makes it a suitable target for colon cancer therapy [ ] . we can conclude that ncdase inhibition, like that of acdase, activates apoptosis and affects adhesion in cancer cells. in addition, it may be a contributing factor in cancerous transformation. alkcdases can also affect cancer development and treatment. acer is upregulated by the tumor suppressor gene p [ , ] . it was demonstrated that a moderate upregulation of acer increases the levels of sph and s p and inhibits cell cycle arrest and senescence. however, when overexpressed, acer mediates programmed cell death, autophagy, and apoptosis through ros [ , , ] . acer also contributes to the effects of ionizing radiation treatment [ ] . it also increases the phosphorylation of ezrin-radixin-moesin through intracellular s p production, hereby inactivating this group of proteins that regulate cell shape and motility and have been associated with cancer progression and metastasis [ ] . acer is expressed in low-survival hepatocellular carcinomas and acute myeloid leukemia [ , ] . it induces the s p phosphorylation of akt through the s p receptor and pi k and cells , , of inhibits the akt/bax apoptotic pathway in cancer cells [ , ] . therefore, the inhibition of acer reduces cell growth and increases cancer cell apoptosis. these published observations indicate that alkcdases are also associated with the regulation of cancer cell apoptosis. however, the observations of acer overexpression reflect molecular effects contrary to those expected of ceramidases. nonetheless, alkcdases are associated with drug resistance and cancer metastasis, like the previously described acdase. ceramidases are involved in myelin and fatty acid metabolism and are associated with changes in the brain during aging [ ] . for instance, acer is upregulated with age and leads to a decrease in the brain levels of c : and c : ceramide, and its deletion results in purkinje cell degeneration and impaired motor coordination and balance in mice [ ] . it has been reported that the overexpression of acdase has implications for the onset and progression of neurodegenerative diseases, including alzheimer's disease (ad) and gaucher disease. furthermore, treatment with acdase inhibitors can control ad and gaucher disease, as well as type iv mucolipidosis. acid ceramidase ad is a multifactorial, highly heterogeneous, and complex disorder that affects the memory and cognitive functions of patients to the extent that they are completely dependent upon nursing care. it is now estimated that nearly . million patients are affected by ad worldwide and that about . million new cases are added each year, causing enormous societal and economic burdens, with the estimated cost reaching $ trillion/year [ ] . ad is caused by an accumulation of derivates from the amyloid precursor protein (app), which can be modulated by the atp-binding cassette transporter- (abca ). abca is a phospholipid transporter which increases the transcription of app by activating the acdase-mediated production of sph [ ] . furthermore, acdase inhibition by ceranib decreases sph concentration and, subsequently, app production in abca -overexpressing cells [ ] . gaucher disease is a disorder caused by a loss of function mutations in the glucocerebrosidase (gcase)-encoding gene, gba . in a gcase deficiency, the breakdown of glucocylceramide (glccer) into ceramide and glucose by gcase is replaced by the acdase deacylation of glccer into glucocylsph (glc-sph), a cytotoxic compound [ ] . the inhibition of acdase by carmofur corrects the lipid abnormalities in the gcase deficiency by reducing the accumulation of glc-sph [ , ] . gba mutations are also a risk factor for parkinson's disease, a neurodegenerative disorder characterized by lewy body inclusions containing α-synuclein. treatment with acdase inhibitors decreases the accumulation of α-synuclein in cases of gba mutation [ ] . similar lipid patterns are observed in the optic nerves of glaucoma patients, where asah and asah genes are overexpressed, but non-lysosomal gcase-gba is inhibited, resulting in a lower total lipid content and significantly higher concentrations of glc-sph [ ] . type iv mucolipidosis is a neurodegenerative disease caused by a loss-of-function mutation of human transient receptor potential-mucolipin- (trpml- ). treatment with the acdase inhibitor, carmofur, induces the activity of trpml- tunnels by increasing the sph concentration in kidney cells and acting as a mediator of lysosome fusion and trafficking in multivesicular bodies, which can potentially compensate for the loss of function of trpml- [ ] . elevated levels of ceramide are known to be correlated with adverse cardiac events, whereas sph has been shown to increase intracellular no levels and maintain the mitochondrial integrity of the cardiovascular system [ ] . conversely, increased blood s p levels are associated with the pathogenesis of inflammatory and cardiovascular diseases [ ] . hence, an association between ceramidase and cardiopulmonary events is expected. the inhibition of acdase activity is associated with cystic fibrosis (cf), which is caused by a dysregulation of the epithelial fluid transport in the lungs, resulting in a sticky dry mucous accumulation [ ] . in cf, β -integrins are ectopically expressed in the luminal pole of epithelial cells and downregulate acdase, leading to an increased ceramide accumulation. however, treatment with recombinant acdase internalizes the β-integrins and regulates ceramide accumulation, rescuing the cf phenotype [ ] . ncdase is inhibited in coronary artery disease vessels. ncdase and adipor mediate the no-dependent flow-induced dilation (fid) through s p. meanwhile, ncdase inhibition induces the damaging peroxide-dependent fid [ ] . in addition, the inhibition of ncdase also leads to mitochondrial dysfunction in diabetic hearts through a lactocylceramide accumulation [ ] . a high expression of alkcdase genes, particularly acer , has been observed in cardiac tissue during hypoxia, where it plays a protective role [ ] . however, an overexpression of acer has been associated with chronic obstructive pulmonary disease (copd) [ ] . acer inhibition contributes to a reduction in the circulating s p and its analogue, dhs p, as well as their precursors, sph and dhsph, in hematopoietic cells and reduces the concentration of dhs p in the lungs [ , ] . these data indicate that acdase and alkcdase are increased in cf and copd, respectively, whereas ncdase is decreased in coronary artery disease. multiple factors are involved in the onset and progression of metabolic disease, including the activities of ceramidases. genetic variations of asah have been associated with exercise tolerance and skeletal/cardiac muscle adaptation to exercise, which can condition adherence to physical activity regimens necessary for a healthy lifestyle, thereby increasing the individual risk of metabolic diseases [ ] . after onset, metabolic disorders affect the physiology of the cardiovascular system, kidneys, and liver. hyperglycemia inhibits the unc -like autophagy-activating kinase (ulk ) phosphorylation in aortic endothelial cells, which leads to a dysregulation of autophagy and atherogenesis. however, acdase activity can increase the phosphorylation of ulk and restore its function even in nutrient-rich conditions, thus preventing atherogenesis [ ] . in addition, obesity-induced kidney damage is caused by hyperglycemic conditions that stimulate the nlr family pyrin domain-containing (nlrp ) inflammasomes to release il- β in podocytes, but the treatment of podocytes with acdase decreases the nlpr -induced cytokine release through extracellular vesicles [ ] . acdase reduces the activity of pannexin- (panx ), a transmembrane channel glycoprotein that activates nlrp through s p accumulation [ ] . animal models of acdase deficiency show significant damage to the liver and change to lipid profiles and metabolism, including hepatomegaly with higher serum levels of aspartate, aminotransferase, alanine aminotransferase, and alkaline phosphatase and decreased levels of free fatty acids, triglycerides, and cholesterol [ ] . the inducible liver-specific overexpression of acdase in the alb-ac transgenic mice, results in significantly reduced c : ceramide in the liver and improved total body glucose homeostasis and insulin sensitivity under a high-fat diet [ ] . however, aberrant acdase overexpression in very low-density lipoprotein (vldl) deficiency may result in non-alcoholic fatty liver disease, which can be normalized by supplementation with vitamin e [ ] . adipocyte-specific acdase overexpression improves glucose metabolism by white adipose tissue, reverses insulin resistance, reduces lipid accumulation in the liver, and reduces adipose inflammation and fibrosis [ ] . this could be due to an acdase-mediated activation of the adiponectin receptor that triggers an amp-dependent kinase pathway, which subsequently inhibits adipogenesis and induces fatty acid oxidation [ ] . palmitate is a precursor of palmitoyl-coa, a thioester used in the de novo biosynthesis of ceramide that is associated with pancreatic β-cell apoptosis and insulin resistance. palmitate inhibits ncdase gene expression and activity in pancreatic β cells, which, in turn, exacerbates apoptosis through ceramide accumulation [ ] . pancreatic β cells secrete ncdase via exosomes that reduce palmitate-induced ros and act as a protective mechanism against free fatty acid-induced apoptosis [ , ] . an overexpression of ncdase inhibits palmitate-induced apoptosis and may be a therapeutic target for type diabetes mellitus and lipotoxicity [ ] . furthermore, asah is one of the four genes related to sphingolipid metabolism that are deregulated in animal models of type maturity-onset diabetes of the young [ ] . this pathology is characterized by increased ceramide and sph levels as well as hypochromic microcytic anemia, with abnormally-shaped and osmotically fragile red blood cells characterized by an accumulation of sph [ ] . non-alcoholic fatty liver disease is associated with an increased expression of acer , which reduces the accumulation of c : -ceramide in the liver [ ] . acer deletion reduces inflammation, fibrosis, oxidative stress, and apoptosis of hepatocytes through a palmitic acid-induced increase in c : -ceramide [ ] . altogether, we can conclude that the holistic beneficial effects of acdase in metabolic disease have been demonstrated. acdase activity controls atherogenesis, kidney damage, and liver damage, while improving glucose and lipid metabolism. in addition, ncdase also appears to improve metabolic conditions via a protective effect on pancreatic β cells, while alkcdase mediates liver damage. ceramidases have been identified as contributors to bacterial infection and mediators of the immune response and inflammation. the α-toxin released by staphylococcus aureus inhibits acdase gene expression, causing decreased levels of sph that contribute to bacterial infection susceptibility [ ] . moreover, this mechanism further increases the risk of infection by s. aureus and pseudomonas aeruginosa in already acdase-and sph-deficient cf patients [ , ] . porphyromonas gingivalis, an etiological factor for periodontitis, downregulates acdase in periodontal tissues, thereby increasing its own apoptotic potential and inhibiting the host's inflammatory response [ ] . the inhibition of acdase by bacteria increases host cell apoptosis and reduces the production of inflammatory cytokines, such as tnf-α, il- β, il- , and il- a, which delay the immune response [ , ] . conversely, acdase overexpression upregulates the inflammatory cytokines involved in the recruitment of neutrophils and macrophages, as demonstrated in ulcerative colitis, where acdase mediates the associated histopathological characteristics of the disease [ ] . ceramide accumulation is increased after burn injuries and may be associated with bacterial infections that frequently lead to death. ncdase treatment protects against pseudomonas aeruginosa infection after burn injuries by controlling ceramide accumulation and inducing the accumulation of sph, which directly kills bacteria [ ] . bacterial lipopolysaccharides may also downregulate the expression and activity of acer and increase c : ceramide accumulation in mice [ ] . a loss of acer expression leads to the production of pro-inflammatory il- β, il- , il- α, and tnf-α cytokines from peritoneal macrophages, bone mononuclear cells, and colonic epithelial cells isolated from acer -/mice [ ] . overall, the bacterial species inhibit ceramidase activity to reduce the concentration of sph in the host cells, which results in a reduced immune response. viruses have the potential to spread among individuals, resulting in epidemics that cause loss of human life and heavy burdens to healthcare systems [ ] . the influenza, ebola, and zika epidemics are recent examples of the effects of broad viral infection and of the mechanisms by which viruses can be studied and controlled [ ] [ ] [ ] . a recent mutation of the coronavirus, named sars-cov- , has caused a pandemic of unprecedented magnitude. this virus has a lower mortality rate but is exponentially more contagious than the closely related sars-cov and mers-cov [ ] . however, our knowledge of the potential role of host ceramidases in viral pathology remains elusive. it was reported that the overall inhibition of ceramidase activity in host peripheral blood lymphocytes using ceranib and ceranib significantly reduces the replication of the rhinovirus and measles virus, respectively [ , ] . furthermore, the inhibition of acdase activity in macrophages significantly increases the propagation of herpes simplex virus- , which, in turn, elevates the mortality rate in asah −/− mice [ ] . collectively, these published observations indicate that ceramidases may have an important antiviral effector role that should further studied. ceramidases are expressed in epithelial cells and fibroblasts and may be involved in their response through s p [ , , ] . however, only a limited number of studies have demonstrated the effects of these enzymes in tissue regeneration and healing. acdase activity contributes to physiological processes involving collagen turnover. asah is associated with familial keloid healing and is overexpressed in keloid scar tissue and hypertrophic scars caused by excessive collagen deposition during epidermal healing [ ] . in the liver, hepatic stellate cells (hsc) are activated during normal wound healing but can, after multiple activations, cause hepatic fibrosis. however, the inhibition of acdase by tricyclic antidepressants leads to ceramide accumulation, which inactivates hscs and prevents hepatic fibrosis [ ] . in vivo studies have also demonstrated a positive effect of acdase in chondrocyte differentiation. in cartilage replacement therapy, pre-treatment with acdase induces chondrocyte proliferation, the production of glycosaminoglycan, the expression of col , the adhesion of chondrocytes to a scaffold, a reduced resorption after implantation, and an improved differentiation to cartilage [ ] . furthermore, a variation of frbrl characterized by peripheral osteolysis not associated with mmp- and mmp- was found, suggesting the involvement of asah in bone remodeling [ ] . various studies have focused on the use of exosomes for tissue repair and regeneration [ , ] . the results of a recent study indicated that hepatocyte exosomes show significant ncdase activity and promote hepatocyte proliferation in vitro and liver regeneration in vivo [ ] . this suggests a role of ncdase in tissue regeneration. the ceramidase has also been identified as an antagonist of cell necrosis caused by dg/aa-dependent ceramide accumulation and mitochondrial damage [ ] . furthermore, ncdase increases autophagy and protects cells from er stress-mediated cell death [ ] . acer inhibition leads to abnormal hair, alopecia, hyperproliferation, inflammation, an abnormal differentiation of the epidermis, sebaceous gland abnormalities, and infundibulum expansion, as well as an increased trans-epidermal water loss and hypermetabolism with an associated reduction in fat content during aging [ ] . its inhibition gradually depletes the number of hair follicle stems and causes alopecia through decreased hair follicle activity [ ] . the specific mechanisms through which these effects of alkcdase occur are still not detailed in the literature. however, its expression has been associated with keratinocyte growth arrest and differentiation [ ] . altogether, these data suggest that acdase is involved in collagen matrix metabolism, whereas ncdase and alkcdase appear to affect tissue regeneration and healing through their anti-apoptotic effects. ceramidases (acid, neutral, alkaline) are key enzymes that maintain the intracellular homeostasis of ceramide/sph and are critical regulators of signals that tilt the balance between cell survival and death. various studies have demonstrated the involvement and potential therapeutic role of these enzymes in a diverse set of common human diseases, including bacterial-induced infectious diseases, neurodegenerative diseases, cancer, diabetes, and others ( figure ) . therefore, the clinical applicability of studies examining the versatility of the effects of ceramidases in health and disease deserves further examination. cells , , x for peer review of expansion, as well as an increased trans-epidermal water loss and hypermetabolism with an associated reduction in fat content during aging [ ] . its inhibition gradually depletes the number of hair follicle stems and causes alopecia through decreased hair follicle activity [ ] . the specific mechanisms through which these effects of alkcdase occur are still not detailed in the literature. however, its expression has been associated with keratinocyte growth arrest and differentiation [ ] . altogether, these data suggest that acdase is involved in collagen matrix metabolism, whereas ncdase and alkcdase appear to affect tissue regeneration and healing through their anti-apoptotic effects. ceramidases (acid, neutral, alkaline) are key enzymes that maintain the intracellular homeostasis of ceramide/sph and are critical regulators of signals that tilt the balance between cell survival and death. various studies have demonstrated the involvement and potential therapeutic role of these enzymes in a diverse set of common human diseases, including bacterial-induced infectious diseases, neurodegenerative diseases, cancer, diabetes, and others ( figure ) . therefore, the clinical applicability of studies examining the versatility of the effects of ceramidases in health and disease deserves further examination. 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homeostasis and regulating whole-body energy expenditure use of liposome preparation to treat mycobacterial infections alkaline ceramidase protects mice from premature hair loss by maintaining the homeostasis of hair follicle stem cells alkaline ceramidase and its bioactive product sphingosine are novel regulators of the dna damage response structure of a human intramembrane ceramidase explains enzymatic dysfunction found in leukodystrophy bahrehbar, i. data on cell survival, apoptosis, ceramide metabolism and oxidative stress in a- renal cell carcinoma cell line treated with hesperetin and hesperetin- -o-acetate monascus purpureus induced apoptosis on gastric cancer cell by scavenging mitochondrial reactive oxygen species ceramide activates lysosomal cathepsin b and cathepsin d to attenuate autophagy and induces er stress to suppress myeloid-derived suppressor cells new aspects of silibinin stereoisomers and their -o-galloyl derivatives on cytotoxicity and ceramide metabolism in hep g 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recognition and hydrolysis by human neutral ceramidase akt as a key target for growth promoting functions of neutral ceramidase in colon cancer cells role of neutral ceramidase in colon cancer manipulation of the sphingolipid rheostat influences the mediator of flow-induced dilation in the human microvasculature loss of neutral ceramidase protects cells from nutrient-and energy -deprivation-induced cell death neutral ceramidase: advances in mechanisms, cell regulation, and roles in cancer new insight into the structure, reaction mechanism, and biological functions of neutral ceramidase upregulation of the human alkaline ceramidase and acid ceramidase mediates calcium-induced differentiation of epidermal keratinocytes alkaline ceramidase is a novel direct target of p and induces autophagy and apoptosis through ros generation tumor suppressor p links ceramide metabolism to dna damage response through alkaline ceramidase acer supports development of acute myeloid leukemia alkaline ceramidase promotes growth of hepatocellular carcinoma cells via regulating s p/s pr /pi k/akt signaling acid ceramidase deficiency in mice results in a broad range of central nervous system abnormalities markedly perturbed hematopoiesis in acid ceramidase deficient mice chronic lung injury and impaired pulmonary function in a mouse model of acid ceramidase deficiency acid ceramidase deficiency is characterized by a unique plasma cytokine and ceramide profile that is altered by therapy deletion of mcp- impedes pathogenesis of acid ceramidase deficiency acid ceramidase deficiency associated with spinal muscular atrophy with progressive myoclonic epilepsy spinal muscular atrophy associated with progressive myoclonic epilepsy: a rare condition caused by mutations in asah aberrant tgfbeta signalling contributes to dysregulation of sphingolipid metabolism in intrauterine growth restriction identification and characterization of novel galc gene mutations causing krabbe disease genetic ablation of acid ceramidase in krabbe disease confirms the psychosine hypothesis and identifies a new therapeutic target neutral ceramidase encoded by theasah gene is essential for the intestinal degradation of sphingolipids deficiency of the alkaline ceramidase acer manifests in early childhood by progressive leukodystrophy role of ceramidases in sphingolipid metabolism and human diseases acid ceramidase is a novel drug target for pediatric brain tumors acid ceramidase confers radioresistance to glioblastoma cells acid ceramidase and its inhibitors: a de novo drug target and a new class of drugs for killing glioblastoma cancer stem cells with high efficiency acid ceramidase inhibition sensitizes human colon cancer cells to oxaliplatin through downregulation of transglutaminase and beta integrin/fak-mediated signalling acid ceramidase is associated with an improved prognosis in both dcis and invasive breast cancer increased acid ceramidase expression depends on upregulation of androgen-dependent deubiquitinases, usp , in a human prostate cancer cell line proteomic profiling of rectal cancer reveals acid ceramidase is implicated in radiation response acid ceramidase in melanoma: expression, localization, and effects of pharmacological inhibition lysosomal acid ceramidase asah controls the transition between invasive and proliferative phenotype in melanoma cells acid ceramidase promotes drug resistance in acute myeloid leukemia through nf-κb-dependent p-glycoprotein upregulation genetic and pharmacological inhibition of acid ceramidase prevents asymmetric cell division by neosis a guanidine-based synthetic compound suppresses angiogenesis via inhibition of acid ceramidase loss of acid ceramidase in myeloid cells suppresses intestinal neutrophil recruitment complete acid ceramidase ablation prevents cancer-initiating cell formation in melanoma cells dose dependent actions of lcl on acid ceramidase and key sphingolipid metabolites anticancer actions of lysosomally targeted inhibitor, lcl , of acid ceramidase effects of ceranib- on cell survival and tnf-alpha in colon cancer cell line anticancer effect of acid ceramidase inhibitor ceranib- in human breast cancer cell lines mcf- , mda mb- by the activation of sapk/jnk, p mapk apoptotic pathways, inhibition of the akt pathway targeting ceramide metabolic pathway induces apoptosis in human breast cancer cell lines examining impacts of ceranib- on the proliferation, morphology and ultrastructure of human breast cancer cells hydrogen peroxide/ceramide/akt signaling axis play a critical role in the antileukemic potential of sanguinarine intracellular sphingosine kinase -derived sphingosine- -phosphate mediates epidermal growth factor-induced ezrin-radixin-moesin phosphorylation and cancer cell invasion white matter lipids as a ketogenic fuel supply in aging female brain: implications for alzheimer's disease alkaline ceramidase deficiency results in purkinje cell degeneration and cerebellar ataxia due to dyshomeostasis of sphingolipids in the brain the worldwide societal costs of dementia: estimates for the atp-binding cassette transporter- (abca ) overexpression modulates sphingosine levels and transcription of the amyloid precursor protein (app) gene acid ceramidase inhibition ameliorates α-synuclein accumulation upon loss of gba function lysosomal glycosphingolipid catabolism by acid ceramidase: formation of glycosphingoid bases during deficiency of glycosidases optic nerve lipidomics reveal impaired glucosylsphingosine lipids pathway in glaucoma control of lysosomal trpml channel activity and exosome release by acid ceramidase in mouse podocytes alkaline ceramidase is essential for the homeostasis of plasma sphingoid bases and their phosphates beta -integrin accumulates in cystic fibrosis luminal airway epithelial membranes and decreases sphingosine, promoting bacterial infections lactosylceramide contributes to mitochondrial dysfunction in diabetes cardioprotective regimen of adaptation to chronic hypoxia diversely alters myocardial gene expression in shr and shr-mtbn conplastic rat strains gene expression profile of human lung in a relatively early stage of copd with emphysema genetic variation in acid ceramidase predicts non-completion of an exercise intervention glucose and palmitate uncouple ampk from autophagy in human aortic endothelial cells lysosomal regulation of extracellular vesicle excretion during d-ribose-induced nlrp inflammasome activation in podocytes inhibition of pannexin- channel activity by adiponectin in podocytes: role of acid ceramidase activation targeted induction of ceramide degradation leads to improved systemic metabolism and reduced hepatic steatosis vitamin e alleviates non-alcoholic fatty liver disease in phosphatidylethanolamine n-methyltransferase deficient mice adiponectin receptor agonist adiporon decreased ceramide, and lipotoxicity, and ameliorated diabetic nephropathy neutral ceramidase activity inhibition is involved in palmitate-induced apoptosis in ins- cells oral administration of the endocannabinoid anandamide during lactation: effects on hypothalamic cannabinoid type receptor and food intake in adult mice neutral ceramidase-enriched exosomes prevent palmitic acid-induced insulin resistance in h iiec hepatocytes hepatocyte nuclear factor a deficiency causes hemolytic anemia in mice by altering erythrocyte sphingolipid homeostasis targeting alkaline ceramidase alleviates the severity of nonalcoholic steatohepatitis by reducing oxidative stress pulmonary infection of cystic fibrosis mice with staphylococcus aureus requires expression of α-toxin endogenous acid ceramidase protects epithelial cells from porphyromonas gingivalis-induced inflammation in vitro frontline science: sphingosine rescues burn-injured mice from pulmonary pseudomonas aeruginosa infection alkaline ceramidase deficiency aggravates colitis and colitis-associated tumorigenesis in mice by hyperactivating the innate immune system the ghost of pandemics past: revisiting two centuries of influenza in sweden the evolution of ebola virus: insights from the - epidemic zika virus: history, emergence, biology, and prospects for control immune responses in covid- and potential vaccines: lessons learned from sars and mers epidemic. asian pac host lipidome analysis during rhinovirus replication in hbecs identifies potential therapeutic targets use of acid ceramidase and sphingosine kinase inhibitors as antiviral compounds against measles virus infection of lymphocytes in vitro acid ceramidase of macrophages traps herpes simplex virus in multivesicular bodies and protects from severe disease berchner-pfannschmidt, u. cd enhances sphingolipids in orbital fibroblasts: potential role of sphingosine- -phosphate in inflammatory t-cell migration in graves identification of asah as a susceptibility gene for familial keloids tricyclic antidepressants promote ceramide accumulation to regulate collagen production in human hepatic stellate cells acid ceramidase treatment enhances the outcome of autologous chondrocyte implantation in a rat osteochondral defect model brief report: peripheral osteolysis in adults linked to asah (acid ceramidase) mutations: a new presentation of farber's disease hepatocyte exosomes mediate liver repair and regeneration via sphingosine- -phosphate exosomes in extracellular matrix bone biology the authors declare no conflict of interest. key: cord- -okmkgsbr authors: ohno, marumi; sekiya, toshiki; nomura, naoki; daito, taku ji; shingai, masashi; kida, hiroshi title: influenza virus infection affects insulin signaling, fatty acid-metabolizing enzyme expressions, and the tricarboxylic acid cycle in mice date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: okmkgsbr although the severity of influenza virus infections has been associated with host energy metabolism, the related mechanisms have not yet been clarified. here we examined the effects of influenza virus infection on host energy metabolism in mice. after infecting mice with intranasal applications of plaque-forming units of a/puerto rico/ / (h n ; pr ) virus, the serum levels of most intermediates in the tricarboxylic acid (tca) cycle and related metabolic pathways were significantly reduced. these data suggest that substrate supply to the tca cycle is reduced under these conditions, rather than specific metabolic reactions being inhibited. then, we focused on glucose and fatty acid metabolism that supply substrates to the tca cycle. akt phosphorylation following insulin injections was attenuated in the livers of pr virus-infected mice. furthermore, glucose tolerance tests revealed that the pr virus-infected mice showed higher blood glucose levels than the vehicle-inoculated control mice. these results suggest that influenza virus infection impairs insulin signaling, which regulates glucose uptake. however, increases in the hepatic expressions of fatty acid-metabolizing enzymes suggest that fatty acids accumulate in liver cells of infected mice. collectively, our data indicate that influenza virus infection dysregulates host energy metabolism. this line of investigation provides novel insights into the pathogenesis of influenza. www.nature.com/scientificreports/ investigated metabolic changes by determining the serum levels of metabolites, insulin sensitivity in the liver, glucose availability, and hepatic gene expressions in the early stages of symptom onset as well as the lethal phase of influenza in a mouse model. the results of this study indicate that influenza virus infection dysregulates glucose and fatty acid metabolism and decreases tricarboxylic acid (tca) cycle activity, leading to enhanced degradation of adenosine triphosphate (atp) and guanosine triphosphate (gtp). we believe that this research provides novel insights into the pathogenesis of influenza and contributes to the development of novel influenza drugs. metabolome analysis indicated reduced tca cycle activity and enhanced purine degradation in mice with severe influenza. upon infection of plaque-forming units of pr virus, the mice showed significant body weight loss starting at day post infection ( dpi, . % ± . % in control mice, . % ± . % in pr virus-infected mice; dpi, . % ± . % in control, . % ± . % in pr virus-infected mice; dpi, . % ± . % in control mice, . % ± . % in pr virus-infected mice). mice were sacrificed when weight loss reached a humane endpoint ( % at dpi). to investigate the systemic effect of influenza virus infection on the host metabolic system, metabolome analyses were performed with serum samples collected at , , and dpi for samples at very early-stage, onset of symptoms, and lethal phase during influenza, respectively. metabolome analyses identified metabolites in the serum samples of the control and pr virus-infected mice at all time points. peak areas and relative ratios of all metabolites are provided in supplemental tables s and s . samples collected at dpi showed the greatest effect of pr virus infection on serum metabolomes, as indicated by principle component analyses with high contribution rates of principle component (pc ; . %; supplemental fig. s ). moreover, pc scores for the control and pr virus-infected mice were negative and positive, respectively, suggesting that the pc score was positively related to the effects of pr virus infection. accordingly, metabolites with high positive and negative factor loading in pc tended to increase and decrease with pr virus infection, respectively (table ) . relative increases in the serum levels of hypoxanthine, xanthine, and inosine were . -, . -, and . -fold, respectively, in the pr virus-infected mice. these molecules are intermediates of purine degradation, and their increased serum levels are considered indicative of enhanced adenosine triphosphate (atp) and guanosine triphosphate (gtp) catabolism. in agreement, the relative serum levels of the upstream purine metabolites adenosine monophosphate (amp), adenosine, guanosine monophosphate (gmp), and guanosine, were decreased by . -, . -, . -, and . -fold, respectively, at dpi. among them, the serum levels of amp and adenosine did not differ significantly between the control and infected groups at any time point owing to large individual differences in the control groups (fig. ) . thus, the lethal phase of influenza is considered to be characterized by increased degradation of atp and gtp. metabolites that were present at reduced levels in the sera of pr virus-infected mice were mainly related to the tca cycle, urea cycle, and amino acid metabolism, as indicated by the serum levels of metabolite in these pathways at , , and dpi (fig. ) . the effects of -day pr virus infections on the serum levels of metabolite are summarized in fig. . the levels of tca cycle metabolites pyruvic acid, -ketoglutaric acid, fumaric acid, and malic acid were significantly reduced in pr virus-infected mice at dpi ( . -, . -, . -, and . -fold, respectively). succinic acid levels were significantly increased by . -fold at dpi but decreased by . -fold at dpi in pr virus-infected mice. in addition, -aminobutyric acid, which is synthesized from glutamic acid and converted to succinic acid, was present at reduced in pr virus-infected mice at and dpi ( . -and . -fold, respectively). the levels of urea cycle metabolites argininosuccinic acid, ornithine, and citrulline were decreased by . -, . -, and . -fold, respectively, in pr virus-infected mice at dpi, whereas the level of arginine was not altered significantly at any time point. given that the levels of most metabolites were significantly reduced in the tca cycle and related pathways, we suggest that pr virus infection reduces flux through these pathways, particularly through the tca cycle. as for amino acids, while the serum levels of ketogenic amino acids did not show any apparent decreases, those of glucogenic amino acids were significantly reduced in pr virus-infected mice at dpi. specifically, the serum levels of asparagine, glycine, proline, serine, and tyrosine were significantly decreased by . -, . -, . -, . -, and . -fold, respectively. furthermore, to estimate the effect of pr virus infection on glutaminolysis in which glutamine is converted to -ketoglutaric acid via glutamic acid and enters the tca cycle, we compared the ratio of the peak areas of glutamic acid to those of glutamine and the ratio of the peak areas of ketoglutaric acid to those of glutamic acid. while the ratio of glutamic acid to glutamine was not changed by the infection, the ratio of -ketoglutaric acid to glutamic acid was significantly decreased by . -and . -fold, respectively, at and dpi (supplemental table s ). therefore, it was indicated that glutaminolysis contribution to the tca cycle was attenuated by influenza virus infection. pathway analysis using metaboanalyst further confirmed that the tca cycle and related pathways were significantly affected by pr virus infection at dpi. the top pathways are listed in table . given that the tca cycle generates gtp and contributes electrons to mitochondrial oxidative phosphorylation to generate a substantial amount of atp, suppression of the tca cycle in pr virus-infected mice is considered to cause imbalanced synthesis and degradation of atp and gtp. moreover, these data suggest that suppression of the tca cycle was due to reduced substrate supply to the pathway due to pr virus infection, rather than inhibition of specific metabolic reactions. tca cycle flux is associated with glucose and fatty acid metabolism because both these energy sources are eventually converted to acetyl-coa, which enters the tca cycle. because the liver plays a predominant role in whole-body energy metabolism, we further investigated the effect of pr virus infection on liver function in terms of glucose and fatty acid metabolism. www.nature.com/scientificreports/ insulin regulates cellular glucose uptake from the blood and intracellular glycolysis , , which supplies substrates to the tca cycle. upon insulin binding to the insulin receptor on the cell surface, the signal is transduced to downstream molecules and phosphorylates akt. phosphorylated akt activates glucose transporters to increase glucose uptake. therefore, phosphorylation of akt is a good indicator of activation of insulin signaling. here we examined the effect of influenza virus infection on insulin sensitivity in the livers according to ratios of insulin-induced phosphoryla- table . increased or decreased serum levels of metabolites in pr virus-infected mice at dpi. metabolites with high factor loading (> . ) and significant differences (p < . , t-test) were selected as significant metabolites and are listed here. the relative serum levels of each metabolite from pr virus-infected mice are presented as fold changes relative to those from control mice. factor loading was defined for each metabolite as correlation coefficients between pc scores and metabolite levels that were normalized by autoscaling in each sample. www.nature.com/scientificreports/ tion of akt. western blotting analyses ( fig. a ) of phosphorylated and total akt in whole liver lysates of control and pr virus-infected mice at dpi revealed that the ratios of phosphorylated akt to total akt were increased by . -fold after insulin treatments in control mice, but this ratio was increased by only . -fold in pr virusinfected mice (fig. b) . similar experiments were performed with the livers collected at other time points. at dpi, akt phosphorylation was clearly inhibited by pr virus infection, but no clear differences were observed in samples collected at dpi (supplemental fig. s ). taken together, these results demonstrated that influenza virus infection impairs insulin actions in the liver. we also performed glucose tolerance tests (gtt) in control and pr virus-infected mice at dpi to investigate whether pr virus infection affected glucose uptake in response to high doses of glucose (fig. c) . we found no significant differences in fasting blood glucose levels between the groups. moreover, at min after glucose injections, the control and pr virus-infected mice showed similar blood glucose levels ( . ± . mg/dl vs. . ± . mg/dl). subsequently, blood glucose levels were rapidly decreased in control mice ( min, . ± . mg/dl; min, . ± . mg/dl; min, . ± . mg/dl). conversely, decreases in blood glucose levels were delayed and higher blood glucose levels were observed in pr virus-infected mice, compared with the control mice, at all time points ( min, . ± . mg/dl; min, . ± . mg/dl; min, . ± . mg/dl). significant differences in blood glucose levels were identified at and min between the groups (p < . , two-way anova). these results indicate that pr virus infection impairs insulin signaling in the liver and induces a tendency toward glucose intolerance, potentially reflecting reduced glucose uptake. fatty acid accumulation in the liver of infected mice was suggested by gene expression assays. we also investigated the effects of pr virus infection on the hepatic expressions of genes that are transcriptionally regulated by insulin signaling (fig. ) . pck , which encodes a gluconeogenic enzyme, was expressed dpi. mice were intranasally inoculated with pbs alone or pbs comprising pr virus, and serum samples were collected for metabolome analysis at , , and dpi. the serum levels of purine metabolites in pr virusinfected mice were expressed relative to those in control mice at each time point. bars represent mean ± sem of or animals. in each panel, white, gray, and black bars indicate data from pr virus-infecetd mice at , , and dpi, respectively; *p < . , unpaired t-test using the holm-sidak correction method with an alpha value of . , control vs. pr virus-infected mice at each time point. the right panel shows a schematic of the effects of pr virus infection on purine metabolism at dpi. upward and downward black arrows indicate significant increases and decreases, respectively. downward gray arrows indicate observed but not significant decreases. www.nature.com/scientificreports/ at slightly but significantly increased ( . -fold) levels in the liver of infected mice at and dpi (fig. a ). this gene is negatively regulated by insulin signaling through inactivation of a transcriptional factor forkhead boxcontaining protein o sub-family (foxo ) which reduces glycolysis and activates gluconeogenesis . therefore, the increase in pck expression in the present study also suggests reduced insulin activity or sensitivity in pr virus-infected mice. in addition to glucose, fatty acids are important sources of acetyl-coa for the tca cycle. fatty acid metabolism was previously reported to be inhibited by influenza virus infection , . therefore, we investigated whether pr virus infection altered the hepatic expressions of genes encoding fatty acid-metabolizing enzymes (fig. b) . cd and pnpla play important roles in fatty acid transport and lipolysis, respectively, and their gene expressions are downregulated by insulin signaling , . here, cd was expressed at significantly increased levels in the livers of pr virus-infected mice, with . -and . -fold increases at and dpi, respectively. in addition, pnpla was significantly induced by . -and . -fold at and dpi, respectively. significant increases in the expressions of figure . relative serum levels of intermediates of the tca cycle and related pathways in control vs. pr virusinfected mice at , , and dpi. mice were intranasally inoculated with pbs alone or pbs comprising pr virus, and serum samples were collected for metabolome analysis at , , and dpi. the serum levels of tca cycle metabolites in pr virus-infected mice are presented relative to those in control mice at each time point. each panels show metabolites of the tca cycle, urea cycle, and amino acid metabolism, respectively. bars represent mean ± sem of or animals. in each panel, white, gray, and black bars indicate data from pr virus-infected mice at , , and dpi, respectively; *p < . , unpaired t test using the holm-sidak correction method with an alpha value of . , control vs. pr virus-infected mice at each time point. www.nature.com/scientificreports/ cd and pnpla as well as of pck suggested the attenuation of insulin signaling in the liver of pr virus-infected mice. conversely, we observed no changes in the expressions of acox and cpt β, which encode key enzymes of fatty acid β-oxidation. in addition, the serum levels of carnitine were significantly decreased by . -fold at dpi (fig. ) . because carnitine is involved in fatty acid transport into mitochondria, the observed decrease would possibly reduce fatty acid β-oxidation in mitochondria. these changes suggest fatty acid accumulation in the liver cells of pr virus-infected mice, although further analyses on protein expressions and enzymatic activities are warranted in the future. in the present study, metabolome analyses demonstrated that pr virus infection decreases the serum levels of most tca cycle intermediates and metabolites in the related metabolic pathways, suggesting reduced flux through the tca cycle. because the tca cycle greatly contributes electrons for mitochondrial oxidative phosphorylation, suppression of the tca cycle leads to reduced atp synthesis. hence, the rates of atp and gtp degradation were possibly greater than the respective rates of their synthesis in pr virus-infected mice, leading to significant increases in the serum levels of metabolites, such as xanthine, hypoxanthine, and inosine. energy depletion indicated by the reduced levels of atp and gtp has been described in several studies on infectious diseases and endotoxemia [ ] [ ] [ ] . reduced atp levels in blood and various organs, including the liver, have been previously associated with influenza virus infection in a mouse model . decreased gtp levels have been shown www.nature.com/scientificreports/ in the lung tissues of rabbits treated with bacterial endotoxin lipopolysaccharide . increases in the serum levels of hypoxanthine and inosine have also been reported in patients with primary dengue virus infection at the febrile stage, suggesting an imbalance of atp and/or gtp synthesis and degradation during the acute stage of the dengue fever . these studies and ours indicate that regulatory energy depletion is a common symptom of infectious diseases. tca cycle flux is associated with glucose and fatty acid metabolism because these energy sources are eventually converted to acetyl-coa for entry into the tca cycle. if either pathway is inhibited, the other pathway becomes activated to compensate for the reduced substrate supply to the tca cycle. in the case of hfd-induced obese mice with insulin resistance, fatty acid oxidation is increased in a leptin-dependent manner, and tca cycle activity is enhanced , . conversely, db/db mice, a well-known mouse model of type diabetes, reportedly developed insulin resistance without activation of fatty acid oxidation due to lack of leptin receptor and showed reduced serum levels of the tca cycle intermediates citric acid, -ketoglutaric acid, malic acid, and fumaric acid with disease progression . in the present study, no changes in the expression of cpt or acox in the liver suggest that fatty acid oxidation was not enhanced in pr virus-infected mice despite insulin resistance. although the effect of pr virus infection on fatty acid oxidation was not evaluated in the present study, reduced fatty acid oxidation during influenza has been demonstrated previously , . if both insulin signaling and fatty acid oxidation are inhibited during influenza, it might result in suppression of the tca cycle due to reduced substrate supply. given significant body weight loss in pr virus-infected mice, however, decrease in food intake during influenza could affect the serum levels of the tca cycle and related pathways. further investigations, such as studies with pair-fed control and lower titer of virus, will provide helpful information to confirm the underlying association. here we demonstrated for the first time that influenza virus infection impairs insulin signaling in liver tissues, which critically regulates glucose metabolism , , and induces a tendency toward glucose intolerance. importantly, nagao et al., reported a high blood glucose level (over mg/dl) as one of the worst prognostic factors in pediatric patients with influenza-associated encephalopathy . in addition to the elevated glucocorticoid levels, as speculated by nagao et al., our study suggests the impairment of insulin signaling as a mechanism of elevated blood glucose levels in children with severe influenza. conversely, another study reported that glucose uptake was activated in the lungs of patients with respiratory viral infections, including influenza virus infection . this discrepancy may be associated with tissue-specific metabolic changes in response to influenza virus infection. however, given the direct activating effect of influenza virus proliferation on glucose metabolism in cultured mice were intranasally inoculated with pbs alone or pbs comprising pr virus. at dpi, the mice were intraperitoneally injected with pbs or insulin after overnight fasting, and liver samples were collected after min for whole lysate preparation. western blotting was performed to quantitate phosphorylated and total akt protein levels in lysates. (a) a representative western blotting analysis shows total akt and akt phosphorylated at ser on the same membrane that was sequentially immunoblotted with corresponding antibodies. (b) relative akt phosphorylation levels were calculated from band densities and expressed relative to data from pbs-treated control mice. bars represent mean ± sem of animals. white and black bars indicate data from pbs-and insulin-treated mice, respectively; p < . , two-way anova, pbs vs. insulin (*), control vs. pr virus-infected mice (#). (c) mice were intranasally inoculated with pbs alone or pbs comprising pr virus. at dpi, gtt was performed after overnight fasting. to this end, mice were intraperitoneally injected with glucose, and their blood glucose levels were sequentially measured at , , , and min post injection. dots represents mean ± sem of animals; *p < . , unpaired t test using the holm-sidak correction method with an alpha value of . , control vs. pr virus-infected mice at each time point. www.nature.com/scientificreports/ madin-darby canine kidney cells , the hepatic insulin resistance demonstrated here could have been induced by host factors and not the virus itself. upon intranasal infection of pr virus, proliferation of the virus occurs mainly in the lung and not in the liver of mice . hence, the hepatic insulin resistance observed herein is possibly induced by host factors, such as systemically secreted cytokines. the direct roles of cytokines on insulin signaling have been demonstrated in a study showing that the proinflammatory cytokine interleukin- (il- ) inhibited insulin-dependent phosphorylation of akt in hepg cells and human primary hepatocytes . furthermore, il- treatments reportedly impaired insulin signaling and inhibited insulin-induced decreases in blood glucose levels in mice ; in this previous experiment, the serum levels of il- reached approximately pg/ml, which was comparable to that observed herein in pr virus-infected mice at and dpi when reduced insulin-induced phosphorylation of akt was observed (supplemental fig. s , . and . pg/ml at and dpi, respectively). thus, we suggest that this cytokine is one of host factor candidates that affect insulin resistance, particularly in the liver. in addition to il- , interferon-gamma (ifn-γ) was reportedly induced by murine cytomegalovirus infections and was recently shown to induce insulin resistance in skeletal muscle . besides il- , the serum levels of ifn-γ were elevated in pr virus-infected mice at dpi but were very low at dpi in our study (supplemental fig. s ; . and . pg/ml at and dpi, respectively). therefore, we propose that influenza virus infection induces hepatic insulin resistance through systemic cytokine secretion. as stated above, in addition to glucose, fatty acids are important sources of the tca cycle substrate acetyl-coa. reduced fatty acid metabolism during influenza has been reported in previous reports showing mitochondrial abnormalities, decreases in the expressions of relevant enzymes, and hepatic steatosis in mice , . consistently, our gene expression data indicate altered fatty acid metabolism in the livers of pr virus-infected mice. in particular, the transcription levels of the fatty acid transporter cd and the lipolytic enzyme pnpla were upregulated at and dpi, possibly elevating extracellular fatty acid delivery and stored triglyceride lysis in liver cells. however, no changes were observed in the mrna expressions of acox and cpt b. these encoded enzymes are rate limiting factors for fatty acid β-oxidation in peroxisomes and mitochondria, respectively. taken together, our data suggest that fatty acids are accumulated in the cytoplasm of liver cells in influenza virus-infected mice. although anorexia due to influenza virus infection may induce similar alterations in gene expressions and subsequent hepatic steatosis, the findings of pair-fed studies on influenza eliminated the possibility that starvation www.nature.com/scientificreports/ is solely responsible for these changes , . moreover, fasting generally increases the mrna expressions of acox and cpt b, thereby discriminating the effects of influenza virus infection on fatty acid metabolism from those of starvation , . fatty acid accumulation can induce mitochondrial dysfunction, as reported previously in cultured hepatocytes . mitochondrial dysfunction increases oxidative stress via reactive oxygen species production. reactive oxygen species and diacylglycerol, converted from triacylglycerol by adipose triglyceride lipase (encoded by pnpla ), inhibit insulin signaling . it is suggested that accumulation of intracellular fatty acids triggers and complicates insulin resistance in the liver of infected mice. given the higher influenza-associated mortality and morbidity rates in patients and mice with energy metabolism disorders - , energy metabolism dysregulation induced by influenza virus infection is possibly associated with pathogenicity. increased mortality rates with influenza have been reported in diet-induced obese mice which probably had insulin resistance as well as in long-chain acyl-coa dehydrogenase-knockout mice, which had reduced mitochondrial fatty acid oxidation , . interestingly, both these mouse models presented with subdued immune responses to virus infection and similar or lower lung virus titers compared with the control mice. hence, increased influenza severity in animals and patients with energy metabolism disorders can be explained neither by the increased levels of inflammatory cytokines nor by the reduced clearance of pathogens , . as described above, dysregulation of energy metabolism appears to be a downstream response to cytokine and inflammatory signaling and thus could be more directly associated with the pathogenesis and severity of influenza. previous cohort studies have demonstrated that antiinflammatory corticosteroids have no beneficial effects and that these corticosteroids can exacerbate outcomes in patients with severe influenza . given that glucocorticoids inhibit insulin signaling as well as cytokine-dependent pathogen clearance , , corticosteroid treatments could confound pathogenicity. thus, improvements in host energy metabolism would be a novel therapeutic target for severe influenza rather than immune suppression. moreover, this concept could be expanded to other infectious diseases because energy metabolism dysregulation is possibly induced by host factors, such as proinflammatory cytokines, and not by the viruses themselves. given the recent emerging infectious diseases such as the pandemic influenza in and severe pneumonia by the novel corona virus in , the importance of developing novel therapeutic drugs that target host factors against common symptoms of various diseases is important. infants and children younger than years of age are generally at high risks of mortality and morbidity due to influenza compared with middle-aged adults . previously, a negative correlation of nasal lavage and plasma inflammatory cytokine levels with age has been reported and thought to be a reason for the high mortality and severe illness caused by influenza virus infection in young children , . in addition to cytokine responses, age-specific features of energy metabolism could provide insights into the pathogenesis of influenza at different ages. in pediatric patients with type i diabetes, the youngest population ( - years of age) reportedly showed the highest prevalence of diabetic ketoacidosis, which has an impact on morbidity and mortality , indicating that attenuation of insulin signaling by virus infection could result in more severe symptoms in such populations. the present study provides novel insights into host responses during influenza. based on the findings of the present and previous studies, we conclude that influenza virus infection induces dysregulation of both insulinregulating glucose metabolism and fatty acid oxidation, leading to decreased tca activity. in future studies, we aim to investigate the effect of influenza virus infection at lethal and sublethal doses of various virus strains for further understanding of influenza pathogenesis and examine the therapeutic effects of interventions that improve host energy metabolism. materials. phosphate-buffered saline (pbs) was purchased from gibco/life technologies (carlsbad, ca). tris-buffered saline (tbs) tablets were purchased from takara bio (otsu, japan). urea, tween , and glucose were purchased from sigma-aldrich (st louis, mo). sodium dodecyl sulfate (sds) was purchased from wako (tokyo, japan). mouse. male c bl/ mice were purchased from hokudo (sapporo, japan) and were kept at a bsl- laboratory at the research center for zoonosis control, hokkaido university, under standard laboratory conditions (room temperature °c ± °c, relative humidity % ± %) and a / -h light/dark cycle. the mice were administered a standard ce- chow diet purchased from clea japan (sapporo, japan) with water ad libtum. experiments were performed on - week-old mice. virus infection and sample collection. pr virus particles at plaque-forming units in µl of pbs or pbs only (control) were intranasally inoculated into the mice under inhalation anesthesia with isoflurane. at , , or dpi, the mice were euthanized, and their liver and blood samples were collected. blood samples were incubated at room temperature for h to clot and were then centrifuged at , g for min. supernatants were collected as serum and were stored at − °c until further analysis. tissue samples were stored at − °c until further analysis. to evaluate insulin sensitivity, the mice were administered insulin (humulin r, eli lilly, indianapolis, in) at u/kg body weight intraperitoneally at dpi after overnight fasting. after min, the mice were euthanized, and their liver samples were collected and frozen immediately in liquid nitrogen. liver samples were stored at − °c until further analysis. research (kyoto, japan) using liquid chromatography-tandem mass spectrometry (lc-ms/ms). briefly, after the addition of an internal standard ( -isopropylmalic acid) to the serum samples, sample preparation was conducted using a series of hydrochloride and acetonitrile extractions. after centrifugation at , rpm for min at room temperature, the supernatants were divided into two tubes: one was used for analyses of amp, gmp, aconitic acid, citric acid, and fumaric acid, whereas in the other one, the supernatant was mixed with hydrochloride and used for analysis of other compounds. chromatographic separations were performed on a discovery hs f - column ( . mm × mm, μm, sigma-aldrich). the oven temperature for the column was maintained at °c. mobile phases a and b were . % formic acid-water solution and . % formic acid-acetonitrile, respectively. separation was performed using gradient elution at a flow rate of . ml/min with a nexera uhplc system (shimadzu). compounds were eluted by changing proportions of mobile phase b as follows: % ( - min), %- % b ( - min), %- % b ( - min), %- % ( - min), % ( - min), %- % ( - . min), and % ( . - min). compounds were detected using an lcms- (shimadzu) instrument with electrospray ionization. the peak areas of each compound were measured using labsolutions (shimadzu) and were normalized to that of an internal standard. the metabolome analyses identified molecules in our sample sets. among them, cysteine, cytosine, and methionine sulfoxide were not detected in some samples from pr virus-infected mice at and dpi (supplemental table s ). to examine the effects of pr virus infection on the serum levels of various compounds, the relative peak areas for pr virus-infected mice were expressed as fold changes relative to the average peak areas for the control mice at corresponding time points. the peak areas and relative ratios are provided in supplemental table s and s . serum metabolome data analysis using metaboanalyst. utilizing peak areas of molecules, principal component analysis and pathway analysis were performed with metaboanalyst (https ://www.metab oanal yst.ca). the values were normalized by autoscaling function. missing values in cysteine, cytosine, and methionine sulfoxide were replaced by small values (a half of the minimum positive values in the original data). factor loading was defined for each metabolite as the correlation coefficient between the pc score and the level of the metabolite after normalizing by autoscaling for each sample. metabolites with high factor loading (> . ) and significant differences (p < . , t-test) were selected as significant metabolites and are listed in table . pathway analyses were performed based on the kyoto encyclopedia of genes and genomes (https ://www.genom e.jp/ kegg/), and the most significantly altered metabolic pathways in pr virus-infected mice were identified. the top pathways identified are listed in table . evaluation of phosphorylated akt by western blotting. liver samples were homogenized in tbs comprising m urea and % sds using an ultrasonic homogenizer (q sonicator, qsonica, newtown, ct) and centrifuged at , g for min at °c to obtain supernatants as whole liver lysates. protein levels were measured using pierce bca protein assay kits (thermo fisher scientific, waltham, ma). samples for western blotting were prepared by mixing whole liver lysates with the nupage lds sample buffer (thermo fisher scientific) and heating at °c for min. subsequently, μg aliquots of total protein were separated using % sds-polyacrylamide gel electrophoresis (sds-page) and were transferred to polyvinylidene difluoride membranes. after blocking with % nonfat dry milk in tbs buffer comprising . % tween (tbst), the membranes were probed with an anti-akt phosphorylated at ser antibody ( : , , # , cell signaling technology, beverly, ma) or an anti-akt antibody ( : , , cell signaling technology) in tbst buffer comprising % bovine serum albumin overnight at °c. the membranes were then treated with secondary horseradish peroxidase-conjugated antirabbit antibody ( : , , sc- , santa cruz) for h at room temperature. protein bands on membranes were detected using supersignal west femto maximum sensitivity substrate (thermo fisher scientific) and an imagequant las system (ge, buckinghamshire, uk). the bands were quantified using the imagequant tl analysis system (ge). full-length images are provided in supplemental fig. s . pbs at g/kg body weight after overnight fasting. blood was collected from tail veins at , , , and min after injections, and plasma glucose concentrations were measured using an accu-chek glucose meter (roche diagnostic, mannheim, germany). www.nature.com/scientificreports/ amount of assay buffer for serum samples or serum matrix for standards and controls. magnetic beads coated with antibodies against the target cytokines were added to each well, and the plates were incubated on a plate shaker overnight at °c. after washing with washing buffer in the kit, the samples were reacted with biotinylated detection antibodies for h and then with streptavidin-phycoerythrin for min. after washing and addition of loading buffer from the kit, the samples were analyzed by the magpix system (luminex, austin, tx, usa). the results are presented in supplemental fig. s . statistical analysis. statistical analyses were performed using prism (graphpad software, san diego, ca, usa). differences were identified using unpaired t-tests or two-way anova and were considered significant when p < . . data are presented as mean ± standard error of the mean (sem). all experiments were performed at least twice to confirm the reproducibility. ethical statement. all mouse experiments were performed with approval from the animal care and use committee of hokkaido university following the fundamental guidelines for proper conduct of animal experiment and related activities in academic research institutions under the jurisdiction of the ministry of education, culture, sports, science and technology in japan. body weight losses were monitored daily after infection, and mice were humanely euthanized when weight loss reached %. all data generated or analyzed during this study are included in this published article and its supplementary information files. received: december ; accepted: june influenza-who cares diet-induced obese mice have increased mortality and altered immune responses when infected with influenza virus impact of diabetes mellitus on mortality 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inflammation and lipid signaling in the etiology of insulin resistance corticosteroids for severe influenza pneumonia: a critical appraisal glucocorticoid-induced insulin resistance: dexamethasone inhibits the activation of glucose transport in rat skeletal muscle by both insulin-and non-insulin-related stimuli interleukin- limits influenza-induced inflammation and protects against fatal lung pathology age-specific mortality risk from pandemic influenza mucosal immune responses predict clinical outcomes during influenza infection independently of age and viral load cytokine and chemokine response in children with the pandemic influenza a (h n ) virus infection ketoacidosis at diagnosis of type diabetes in french children and adolescents we thank the national institute of infectious disease in japan for kindly providing influenza virus a/puerto rico/ / (h n ). this work was supported by japan science and technology agency basic research programs, jsps kakenhi (grant number k ), and northern advancement center for science and technology, hokkaido japan (grant number k ). the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to h.k. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -x g bnk authors: zheng, yutong; yan, meitian; wang, lan; luan, liang; liu, jing; tian, xiao; wan, nan title: analysis of the application value of serum antibody detection for staging of covid‐ infection date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: x g bnk coronavirus disease (covid‐ ) has now spread all over the world. the national health commission of the people's republic of china reported , cured and discharged cases, deaths, , confirmed cases and , close contacts as of june , . joint detection of nucleic acids and antibodies has become an important laboratory diagnostic for covid‐ patients. disease progression and infection stage can be established based on the biological characteristics of these tests. however, there have been few studies of the different infection stages of covid‐ . we conducted a retrospective analysis to explore the clinical characteristics of covid‐ patients at different infection stages and to characterize the characteristics of specific serum antibodies at each stage. these data will provide a theoretical basis for clinical diagnosis and treatment. this article is protected by copyright. all rights reserved. patients. in this study we explored the clinical value of specific serum antibody detection in covid- patients. nucleic acid and specific antibody detection sars-cov- nucleic acids in nasopharyngeal swab samples were detected using a model pcr gene amplification instrument (abi, foster city, ca, usa). a cycle threshold value of > was considered negative and a value of < was considered positive. fasting venous blood ( - ml) was collected and centrifuged. the serum was separated and stored at - °c. specific serum igm and igg antibodies were quantitatively detected using an axceed magnetic particle-based chemiluminescence immunoanalyzer (bioscience, tianjin, china). a chemiluminescence signal cutoff value (s/co) of < was considered negative and > was considered positive. this article is protected by copyright. all rights reserved. statistical methods spss statistics software version . (ibm, armonk, ny, usa) was used for all statistical analyses. count data were expressed as frequency (%) and the chi-square test was used to assess differences between groups. continuous data with normal distributions were expressed as x ±s, and differences between two groups or among multiple groups were assessed using the student's t test and analysis of variance, respectively. non-normally distributed variables were expressed as medians and interquartile ranges and differences between groups were assessed using the mann-whitney u test. graphpad prism . ( graphpad software,san diego,ca)was used to produce all figures. a total of covid- patients were enrolled, comprising male patients and female patients with an average age of . ± . years. moderate cases made up the largest number of patients, accounting for . % of the total. mild cases were rarest, accounting for only . % of the total, while severe and critical cases accounted for . % and . % of the total, respectively. according to the biological characteristics of nucleic acids and specific serum igm and igg antibodies, the covid- cases were classified into infection stages ( table ) . analyses of the characteristics of different periods were performed ( table ). patients who were nucleic acid negative at admission could be divided into two categories:① the nucleic this article is protected by copyright. all rights reserved. acid test had a positive record, but was negative more than twice before admission, and remained negative many times after admission; or ② nucleic acid test results were negative since the onset of the disease, with diagnosis made based on lung imaging, symptoms, epidemiological history and serum antibody results. because most patients had a long disease course when they were admitted to hospital, there were more cases in the active, middle/late and convalescent stages of infection (p< . ). figure a ). this article is protected by copyright. all rights reserved. on january , , the national health commission decided to include covid- pneumonia in statutory infectious disease category b and to take preventive and control measures for category a infectious diseases . on january , the who called the epidemic "an emergency of international concern". sars-cov- is a novel enveloped β coronavirus with an rna genome. in the early stages of the epidemic, nucleic acid detection was taken as direct evidence of infection. however, the accuracy of nucleic acid detection was affected by the quality of detection kits, sample collection methods, operator ability, rna stability, patient condition and concurrent drug use this article is protected by copyright. all rights reserved. in addition, we found that levels of igg were persistently high after the active stage of infection, indicating that disease progression coincided with emergence of these antibodies. we found that levels of igg in patients with different clinical severity were higher than those of igm. this finding may be related to the high affinity and easy detection of igg antibodies and the longer course of disease of the patients included in the study following their admission to hospital. however, levels of serum specific igm tables table . combined analysis of nucleic acid and serum antibody testing. ① the nucleic acid test had a positive record, but was negative more than twice before admission, and remained negative many times after admission.② the nucleic acid test result was negative since the onset of the disease. table . analysis of covid- infection stages. combined detection of two detection methods to determine the stage of infection nucleic acid+ igm-igg-①window period ②igm begins to appear but is still below the detection limit in the early stage of infection. this article is protected by copyright. all rights reserved. igm+igg-the early stage of infection. the middle and late stage of infection (the result of early antibody test of the patient is unknown, the possibility of recurrent infection can not be ruled out). the active stage of infection, but at this time the body has developed a certain immune capacity. igm-igg-it may be in the infection recovery stage where the nucleic acid turns negative, the igm antibody disappears and the igg antibody begins to appear but is still below the detection limit. igm-igg+ previous infection. the convalescent stage of infection, igm decreased but still above the detection limit. nucleic acid-② igm-igg-this type of patient was diagnosed by pulmonary ct, symptoms and epidemiological history on admission. ( ) the possible "window period" of false negative nucleic acid. ( ) the convalescent stage in which the nucleic acid turned negative, the igm antibody disappeared and the igg antibody began to appear but was still below the detection limit. igm+igg-may be in the acute stage of infection, consider the possibility of false negative nucleic acid. consider the possible active stage of infection with false negative nucleic acid. ① the nucleic acid test had a positive record, but was negative more than twice before admission, and remained negative many times after admission. ② the nucleic acid test result was negative since the onset of the disease. as of : on june th, the latest situation of covid- epidemic situation there are confirmed cases by novel coronavirus outside china consensus of -ncov nucleic acid testing experts on the issuance of novel coronavirus's diagnosis and treatment plan for pneumonia (seventh edition for trial implementation):health office medical care administration file〔 〕 [s].beijing:national health commission of the people's republic of national health commission of the people's republic of china.pneumonia infected by novel coronavirus is included in the management of legal infectious diseases cause analysis and countermeasures of false negative results of novel this article is protected by copyright. all rights reserved. accepted article coronavirus nucleic acid test false positive analysis of chemiluminescence microparticle immunoassay in hiv detection ping'an zhang.the diagnostic value of joint detection of serum igm and igg antibodies to -ncov in -ncov infection research progress on mechanism of antibodydependent enhancement is covid- receiving ade from other coronaviruses? immunoregulation with mtor inhibitors to prevent covid- severity: a novel intervention strategy beyond vaccines and specific antiviral medicines dengue fever, covid- (sars-cov- ), and antibody-dependent enhancement (ade): a perspective is covid- receiving ade from other coronaviruses susceptibility of the elderly to sars-cov- infection: ace- overexpression, shedding, and antibody-dependent enhancement (ade) similarities and evolutionary relationships of covid- and related viruses. preprints identification of epitopes on sars-cov nucleocapsid protein that induce the cross-or specific-reactivity among sars-cov, hcov-oc and hcov- e effectiveness of convalescent plasma therapy in severe covid- patients proc. natl. acad. sci first infection by all four non-severe acute respiratory syndrome human coronaviruses takes place during childhood this study was supported by research grants from the natural science the authors declare that there are no conflict of interests. yz and nw contributed to data analysis, graphics and writing the paper. yz, nw, my, lw and ll contributed to data collection and graphics development. jl and xt contributed to editing the paper. no date are available.this article is protected by copyright. all rights reserved. key: cord- -e bs e authors: arming, sigrid; wipfler, dirk; mayr, juliane; merling, anette; vilas, ulrike; schauer, roland; schwartz-albiez, reinhard; vlasak, reinhard title: the human cas protein: a sialic acid-specific o-acetyltransferase? date: - - journal: glycobiology doi: . /glycob/cwq sha: doc_id: cord_uid: e bs e sialic acids are important sugars at the reducing end of glycoproteins and glycolipids. they are among many other functions involved in cell–cell interactions, host–pathogen recognition and the regulation of serum half-life of glycoproteins. an important modification of sialic acids is o-acetylation, which can alter or mask the biological properties of the parent sialic acid molecule. the nature of mammalian sialate-o-acetyltransferases (ec . . . ) involved in their biosynthesis is still unknown. we have identified the human casd (capsule structure domain containing ) gene as a candidate to encode the elusive enzyme. the human casd gene encodes a protein with a serine–glycine–asparagine–histidine hydrolase domain and a hydrophobic transmembrane domain. expression of the cas protein tagged with enhanced green fluorescent protein in mammalian and insect cells directed the protein to the medial and trans-cisternae of the golgi. overexpression of the cas protein in combination with α-n-acetyl-neuraminide α- , -sialyltransferase (gd synthase) resulted in an up to % increased biosynthesis of -o-acetylated ganglioside gd . by quantitative real-time polymerase chain reaction, we found up to -fold increase in casd mrna in tumor cells overexpressing o-ac-gd . casd -specific small interfering rna reduced o-acetylation in tumor cells. these results suggest that the human cas protein is directly involved in o-acetylation of α - -linked sialic acids. sialic acids are a family of acidic amino sugars derived from -n-acetyl-neuraminic acid, typically found in n-and o-linked glycans of glycoproteins and in glycolipids. they are mostly present at the nonreducing end of glycan chains. they are capping sugars which prevent further elongation of glycan chains. an exception is the further elongation by specific sialyltransferases leading to the formation of oligo-or polysialic acids. structural variations occur at carbon , which result in the formation of either -n-glycolyl-neuraminic acid or -keto- -deoxynononic acid. in addition to modifications at carbon , substitutions of the hydroxyl groups at carbons , , and are known. substituents are o-acetyl, sulfate, methyl and lactyl groups, which occur at one or more of these positions. until now, approximately different sialic acid derivatives have been isolated and characterized from natural sources. an overview on the biosynthesis, biology and diversity of sialic acids is given in several reviews (schauer and kamerling ; varki varki , schauer schauer , varki and schauer ). among many other functions, sialic acids are known to mask glycan epitopes. as an example, human erythrocytes are covered with sialic acids. upon aging, sialidases cleave off sialic acids, and the modified erythrocytes are then sequestered by galactose-specific receptors (müller et al. ; bratosin et al. ) , e.g. the asialoglycoprotein receptor in the liver (ashwell and harford ) or similar receptors on macrophages. different glycoforms of therapeutica are also cleared from the bloodstream at different rates. in several instances, therapeutic glycoproteins lacking sialic acids are rapidly removed from the blood stream by the asialoglycoprotein receptor and/or the mannose receptors (ashwell and harford ; lee et al. ) . the serum half-lives of desialylated glycoproteins decrease in average from hours to minutes. this has been reported for pro-urokinase (henkin et al. ) , chorionic gonadotropin (martinuk et al. ) , thyrotropin (szkudlinski et al. a (szkudlinski et al. , b , luteinizing hormone (burgon et al. ) and erythropoietin (fukuda et al. ) . in contrast, the introduction of additional glycosylation sites into the protein backbone of erythropoietin or follicle-stimulating hormone results in artificial hypersialylation concomitant with extended half-life and improved pharmacokinetics (egrie et al. ; perlman et al. ) . the best studied o-acetyl esterification is the substitution of the hydroxyl group at carbon . this modification arises by enzymatic transfer of o-acetyl groups to carbon of glycosidically bound sialic acids, followed by the migration of the acetyl group to carbon (kamerling et al. ; vandamme-feldhaus and schauer ) . the biosynthesis of -n-acetyl- -o-acetyl-neuraminic acid is tightly regulated during brain development (constantine-paton et al. ; levine et al. ; blum and barnstable ) and the activation of b and t cells (kniep et al. ; erdmann et al. ) . aberrant expression coincides with malignant transformation and metastasis. different types of changes in the expression of o-acetylated sialic acids exist: in cancer cells derived from the neuroectoderm and in acute lymphoblastic leukemia, the expression levels are significantly increased varki et al. ; sjoberg et al. ; pal et al. ; kohla et al. ; ghosh et al. ) , whereas in colon cancer the amount of o-acetylated sialic acids, primarily on mucin-type molecules, is reduced (corfield et al. ; byrd and bresalier ; shen et al. ) . current models indicate that overexpression of the ganglioside gd results in the activation of apoptosis (de maria et al. ; rippo et al. ) , whereas o-acetylation of the α , -linked sialic acid of gd contributes to the inhibition of apoptosis (chen and varki ; malisan et al. ; erdmann et al. ; kniep et al. ) . moreover, o-acetylation regulates the activation of the innate immune system (crocker and varki ; crocker et al. ; schauer et al. ) and the alternate complement pathway (shi et al. c) . most recently, it was shown that a cellular sialate o-acetyl esterase regulates the function of cd , a siglec which negatively regulates the b cell receptor ). loss of function of the cellular acetylesterase results in autoimmune disease surolia et al. ) . modifications of sialic acids result in altered recognition by bacterial sialidases (corfield et al. (corfield et al. , (corfield et al. , ) and viral pathogens, including influenza c viruses (herrler et al. ; vlasak et al. ) , coronaviruses (vlasak et al. ; schultze et al. ; regl et al. ; smits et al. ) , infectious salmon anemia viruses (hellebo et al. ) and toroviruses (smits et al. ; de groot ) . the search for the enzyme catalyzing the transfer of o-acetyl groups to sialic acids started almost years ago. the latest status on the purification was described recently (lrhorfi et al. ; srinivasan and schauer ) . two types of transferases apparently exist: the bovine enzyme transfers acetyl groups to carbon , whereas the transferase isolated from equine and guinea pig tissues preferentially adds o-acetyl groups to carbon . the expression of the -o-acetyltransferase is developmentally regulated (varki and kornfeld ; shi et al. b; krishna and varki ) . attempts to identify the gene by expression cloning failed in the past (shi et al. ; satake et al. ). other laboratories also described efforts toward identifying the gene (ogura et al. ; kanamori et al. ) . in summary, the nature of the sialic-acid-specific o-acetyltransferase remains enigmatic. we have used a rational approach to identify the gene encoding the mammalian sialate-o-acetyltransferase (soat) (ec . . . ). taking into account the diversity of o-acetylated sialic acids on n-and o-glycans as well as on glycosphingolipids and possibly also on polysialic acids, more than one gene may exist. alternatively, different specificities may be derived from the action of modulating factors. genetic evidence suggested the presence of a single human o-acetyl transferase gene related to o-acetylation of mucin sialic acids in the intestine (campbell et al. ) . therefore, we screened the human genome databases for genes with unknown functions, which are predicted to possibly encode acetyl transferases and potentially be located in the golgi membrane. this search finally resolved the candidate gene casd (capsule structure domain containing ), which is similar to a gene of the fungus cryptococcus neoformans. our results indicate a direct involvement of the human cas protein (cas p) in the o-acetylation of sialic acids. data mining of the human genome in order to detect candidate genes, we used the search function of the ensembl programme (www.ensembl.org) to screen for genes with unknown functions, which are predicted to possibly encode acetyl transferases. the first search for "transferase" in the human genome resulted in hits. when the search was narrowed to "o-acetyltransferase", entries with a matching term were found. they were then further screened for a potential golgi location of the encoded protein. this search finally resolved a single candidate gene, which is similar to a gene of the yeast strain c. neoformans. strong genetic evidence exists that the yeast cas p is an o-acetyl transferase, which adds o-acetyl groups at the c position of mannose in a capsid structure composed of glucuronoxylomannans (janbon et al. ) . genes homologous to the human casd gene are present in plants and throughout the animal kingdom. the human casd gene is predicted to consist of exons. in the human and chimpanzee genome, the gene is located on chromosome q . . casd is a maternally expressed imprinted gene in mice (babak et al. ) , and it is located next to the paternally expressed imprinted genes sgce (grabowski et al. ) and peg (ono et al. ). in the drug-resistant human neuroblastoma cell line igrn- -r, its expression is significantly upregulated (flahaut et al. ). the predicted length of the transcript is nucleotides, which encodes a protein of amino acid residues. analysis of the potential full-length transcript revealed a signal sequence, usually found in proteins which are delivered to the endoplasmic reticulum (er), the golgi or the plasma membrane. the protein consists of a serine-glycine-asparagine-histidine (sgnh) hydrolase domain and a c-terminal transmembrane domain. the human cas p shares some sequence similarity with viral sialic acid-specific o-acetylesterases, particularly around the active site residues (figure ). the amino acid sequence flanking the catalytic serine was originally identified for the influenza c virus esterase (vlasak et al. s arming et al. ) and for cellular sialic acid-specific o-acetylesterases (hayes and varki ) . additional features include an er export signal (d-x-e) and several trans-golgi network (tgn)-endosome export signals (y-xx-hydrophobic amino acid or d/e-xxx-l/i). the amino acid sequences connecting the hydrophobic regions exhibit a high content of basic and acidic amino acid residues. to allow the expression of cas p in mammalian cells, the coding region of the casd cdna was amplified by polymerase chain reaction (pcr) and cloned into the expression vector pegfp-n . this vector facilitates the expression of chimeric recombinant proteins with a c-terminal enhanced green fluorescent protein (egfp) tag. two types of clones were created: pcasd egfp allows the detection of the expressed protein by fluorescence microscopy and western blot analysis, whereas pcas stop encodes the authentic human protein without c-terminal extension ( figure ). when we expressed the gfp-tagged protein in insect sf cells, the protein was found predominantly in a golgi-enriched microsomal fraction. it migrated on an % sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) with the predicted molecular mass of approximately kda. in addition to the monomeric protein, two other bands which presumably represent trimers and tetramers were also detected with the gfp-specific antiserum ( figure ). we also wanted to determine the intracellular location of cas p. therefore, we transfected cos cells with plasmid pcasd egfp and monitored expression by fluorescence microscopy. the gfp-tagged cas p was detected in intracellular compartments close to the nucleus, as shown by counterstaining of nuclei. in addition, cas p-egfp was regularly found as bright spots, which sialic acid-specific o-acetyltransferase presumably represent the protein within transport vesicles ( figure a -c). to further localize cas p, we also transfected cos cells with plasmids pcasd egfp and pst gal . the latter plasmid directs expression of sialyltransferase st gal , a marker of the tgn ). cas p-egfp was detected directly by fluorescence microscopy, and st gal was detected with mab stg (cao et al. ) . analysis revealed a partial colocalization in cells expressing both cas p and st gal (β-galactosamide α- , -sialyltransferase ) ( figure d -f). we then tested whether the cas protein was involved in the o-acetylation of ganglioside gd . we therefore expressed α-n-acetyl-neuraminide α- , -sialyltransferase (st sia ), which directs the formation of gd from ganglioside gm . to allow the expression of st sia , we constructed plasmid pgd synthhistag. when we cotransfected this plasmid with pcas stop, we detected -o-acetylated ganglioside gd ( -o-ac-gd ) by immunostaining with mab u in a patched distribution at the cell surface, possibly located in "lipid rafts" ( figure a ). for quantitative determination of the expression of -o-ac-gd in cos cells, we used flow cytometric analysis. control cells and cells transfected with either plasmid pgd synthhistag alone or double transfected with pgd synthhistag and pcas stop were labeled with either the gd -specific mab r or mab u , which is specific for -o-ac-gd . a background expression of gd (cd a) and -o-ac-gd (cd c) was found in control cells. it should be mentioned that we selected cos cells for these experiments, because it was shown earlier that in contrast to chinese hamster ovary (cho) cells these cells do not express significant amounts of intrinsic soat (shi et al. a) . as shown in figure b , the expression of st sia (gd synthase) resulted in an % increase in gd and a small increase ( %) in -o-ac-gd . the increase in the -o-ac-gd may be due to the activation of a pre-existing intrinsic o-acetyltransferase. co-expression of gd synthase and cas p led to an increase in both gd ( %) and -o-ac-gd ( %). in another experiment ( figure c ), we also determined the expression of -o-ac-gd (cd b) with mab mt . whereas the expression of -o-ac-gd (cd c) was increased in the presence of cas p, no significant change in -o-ac-gd could be detected. this experiment indicates that cas p transfers o-acetyl groups exclusively to carbon of sialic acids. we also determined the expression of casd mrna in a number of primary b and t lymphocytes and in cell lines derived from human melanomas and lymphomas. t and b cells of tonsillar and peripheral blood lymphocytes were subjected to d flow cytometric analysis, where b cells were characterized by an anti-cd + mab conjugated to pe and t cells by an anti-cd secondary mab conjugated to cy . as shown in figure , all freshly extracted normal lymphocytes of various stages and lineages express the casd gene albeit in different quantities. non-stimulated b and t lymphocytes from peripheral blood had the highest values followed by tonsillar t and b cells which represent lymphocytes undergoing various activation stages and thymocytes which represent t cells of early t cell differentiation. human leukemia cell lines express the casd gene, and the erythroleukemia cell line k had the lowest values. the highest values were observed by the t cell leukemia cell line jurkat, followed cell lines kg and kg a derived from malignant acute myeloblastic leukemia. interestingly, the cell line kg a which was found to have a higher state of surface sialylation when com- downregulation of o-acetylation by casd -specific small interfering rna in order to determine whether the casd gene product is directly involved in the o-acetylation of sialic acids, we used small interfering rna (sirna) to downregulate the intrinsic casd mrna. in this assay, we used ma-mel cells, which express high levels of casd mrna ( figure ) and cd c (data not shown). first we determined whether transfection of casd -specific sirna resulted in decreased amounts of detectable mrna. by reverse transcription (rt)-pcr, we found that the transfection procedure alone or with unspecific sirna caused a drop in casd mrna expression by approximately %, compared with untreated cells. in contrast, transfection of specific sirna resulted in a more than % reduction of detectable casd mrna ( figure a ). we then measured the amount of cd b-expressing cells by fluorescence-activated cell sorter (facs). compared with cells sialic acid-specific o-acetyltransferase transfected with unspecific sirna, a significant reduction in cd b + cells was observed h after transfection of casd specific sirna. o-acetylation gradually re-appeared in a timedependent manner and approached original values after h ( figure b ). o-acetylation is a common modification of sialic acids. it is found from bacteria to man. human-pathogenic bacteria such as escherichia coli k , neissera meningitidis and streptococci known to infect the central nervous system express different forms of o-acetylated sialic acids. recently, several bacterial genes encoding o-acetyl transferases were identified in human-pathogenic e. coli k (deszo et al. ; steenbergen et al. ; vimr and steenbergen ) , campylobacter jejuni (houliston et al. ) , group b streptococci (lewis et al. (lewis et al. , and neissera meningitides (bergfeld et al. ; lee et al. ). the bacterial genes and their encoded proteins do not exhibit significant similarities to animal genes or gene products. despite numerous attempts to identify the specific o-acetyltransferase(s) responsible for the transfer of acetyl groups, the nature of the enzyme up to now remained elusive. forty years ago, the first reports on a specific o-acetyltransferase in bovine and equine submandibulary glands were published. whereas the bovine enzyme catalyzed the transfer to positions and (schauer b) , the equine transferase delivered the acetyl groups to position of the sialic acids (schauer a) . then it was shown with rat liver that incubation of purified golgi vesicles with h-acetyl-coenzyme a (accoa) resulted in a rapid accumulation of radioactivity within the lumen of the golgi ). apparently, no transport of accoa into the golgi was required, only the labeled free acetate was found inside the vesicles. the finding that solubilization of the golgi membrane with detergents immediately abolished acetyltransferase activity turned out as a major hurdle towards future expression of casd in untreated cells was set to %, and reduction in specific mrna levels was determined following the transfection of unspecific and casd -specific sirna. as a negative control, transfection reagent without sirna was used. (b) reduction (%) of cd c ( -o-ac-gd ) by casd sirna, when compared with cells transfected with unspecific sirna was monitored by facs analysis. isolation of the intact enzyme. ) in a further study, it was shown that histidine and lysine residues are essential for the transmembrane transfer of acetyl groups . it was also shown that the o-acetyltransferase activity is located in golgi subcompartments which are beyond the block caused by brefeldin, indicating a location in late golgi vesicles. in addition, the data indicated that o-acetylated ganglioside gd is synthesized either from the precursor ganglioside o-ac-gd or by direct transfer of the o-acetyl group to gd (sjoberg et al. ; sjoberg and varki ) . later, cho cell lines were created which stably expressed either gd synthase (st sia ) or st gal (shi et al. a ). most surprisingly, the expression of each of these sialyltransferases alone was sufficient to allow o-acetylation of gd or α , -linked sialic acids on n-glycans, respectively. thus, an endogenous o-acetyltransferase was presumably activated by the expression of the heterologous sialyltransferases. when the same sialyltransferases were expressed in cos cells, no acetylation of sialic acids could be detected, indicating that these cells did not express the endogenous o-acetyltransferase present in cho cells. interestingly, the expression of st gal , which transfers sialic acids in an α , linkage to lactosamine, did not result in the formation of o-acetylated sialic acids in cho cells. in other publications, it was shown that the ganglioside gd can induce its own o-acetylation (chen et al. ; kniep et al. ) . vandamme-feldhaus and schauer ( ) described the partial purification of a -o-acetyl transferase from bovine submandibulary glands and proposed the existence of a "migrase", which directs the transfer of acetyl groups from carbon to carbon . a partially purified enzyme was also prepared from guinea pig liver and equine submandibular glands, which preferentially directs o-acetylation at carbon (iwersen et al. (iwersen et al. , tiralongo et al. ) . the most recent results on the characterization of the mammalian o-acetyl transferase are summarized in three publications (lrhorfi et al. ; mandal et al. ; srinivasan and schauer ) . in this study, we describe new approaches toward identifying the mammalian enzyme catalyzing the transfer of o-acetyl groups from acetyl-coa to sialic acids. by data mining, we identified the product of the human casd gene as a candidate protein which might represent the elusive sialic acidspecific o-acetyltransferase. the cas protein is a protein with a predicted molecular mass of . kda composed of up to transmembrane domains. the expression of cas p with a c-terminal egfp domain resulted in the formation of monomeric and oligomeric proteins. the peptides connecting the transmembrane domains contain histidine and lysine residues. thus, the human cas p fulfills the requirements for an acetyl-coa transporter/antiporter. our data indicate that it is located in specific intracellular compartments, most likely in the tgn as shown by partial colocalization with st gal , a marker enzyme for tgn . furthermore, cas p possesses an sgnh hydrolase domain with sequence similarity to viral sialic acid-specific o-acetyl esterases. co-expression of cas p and st sia (gd synthase) resulted in the formation of -o-ac-gd . we observed a slight increase in the presence of -o-ac-gd in cos cells transfected with st sia alone, indicating that these cells also express a low background level of intrinsic soats. however, the co-expression of st sia and cas p resulted in significantly increased amounts of -o-ac-gd . it should also be noted that we did not observe a significant increase in o-acetylation of gd in all experiments. the reasons are currently unclear. they may be a result of variations of transfection efficiencies. on the other hand, our data cannot completely exclude the possibility that the expression of cas p stimulates the expression of intrinsic soats, while cas p might have other functions. in previous work, it was hypothesized that o-acetyltransferase activity may be associated within a membrane-bound complex composed of the acetyl-coa transporter, sialyltransferase and acetyltransferase activities and possibly an acetylated intermediate lrhorfi et al. ; schauer et al. ). in addition, a soluble cofactor may also be required for activity ). thus, the metabolic status of cells may contribute to the observed differences in the amount of o-acetylation of gd . on the other hand, the transfection of casd -specific sirna into melanoma cell line ma-mel resulted in an approximately % reduction of casd mrna and a concomitant % reduction of -o-ac-gd within h of incubation. in conclusion, we hypothesize that the human cas p represents a sialic acid-specific o-acetyltransferase. the results indicate that the expression of cas p in cos cells directs acetyl groups to carbon of sialic acid. upon co-expression of cas p and st sia increased amounts of -o-ac-gd were detected, whereas -o-ac-gd levels remained essentially unchanged. this observation is in accordance with published data, which indicate that acetylation at carbon results from the migration of the acetyl group at carbon (vandamme-feldhaus and schauer ). the cd c antigen ( -o-ac-gd ) was shown to be differently expressed from cd b ( -o-ac-gd ) in human b and t lymphoblasts, indicating differences in their biosynthesis and function (erdmann et al. ) . most recently, the soat activity in lymphoblasts derived from patients with acute lymphoblastic leukemia was characterized, and again this enzyme predominantly catalyzed the formation of -o-acetylated sialic acid (mandal et al. ). we therefore suggest that the human cas p may represent a sialic acid-specific o-acetyltransferase, which transfers acetyl groups to carbon . most interestingly, the casd gene is imprinted in mice. imprinted genes are expressed from one allele derived either maternally or paternally. several imprinted genes are essential to mammalian embryogenesis. genomic imprinting influences mammalian development, growth and behavior. a number of human genetic diseases are related to imprinted genes that exist in different chromosomal regions. aspects on the evolution of imprinting and the impact for human health have been reviewed recently (das et al. ). casd exhibits equal biallelic expression in neonatal mouse brain. in extraembryonic tissues, the gene is expressed ubiquitously, and a weak maternal bias was observed (ono et al. ) . the finding that casd is a maternally expressed imprinted gene was substantiated by transcriptome sequencing (babak et al. ) . for humans, no evidence of imprinting of casd has sialic acid-specific o-acetyltransferase been published, and no human genetic disease has yet been described to be associated. in summary, current evidence indicates that casd in postnatal mice is predominantly expressed from the maternal allele. mapping of the adult mouse brain showed high expression levels of casd in the hippocampus [allen mouse brain atlas (internet), seattle (wa): allen institute for brain science © . available from http ://mouse.brain-map.org]. in this region of the mouse brain, a number of sialyltransferases, including st gal , st gal , st gal , st gal , st gal , st galnac , st sia , st sia , st sia and st sia are also highly expressed. in the future, we want to perform enzyme tests with the purified cas p. preliminary data indicate that purification of cas p alone may be not sufficient to determine enzymatic activity (data not shown), suggesting that cas p may require interactions with other cellular components in order to become active. interaction partners may either be the substrates themselves, other proteins, e.g. sialyltransferases or other glycosyltransferases within the golgi, or cytoplasmic proteins that may modulate the transport of accoa into the golgi. we preferentially want to test the role of sialyltransferases, specific substrates and the putative cofactor/activator. cloning and expression of the human genes encoding casd , sialyl- , -gal-transferase and sialyl- , -sia-transferase (gd synthase) the clones with the casd cdna (image clone ) and the cdna for st sia (image clone ) were obtained from geneservice ltd. (uk), the cdna clone for st gal (iratp a d) was purchased from rzpd deutsches resourcenzentrum für genomforschung gmbh (berlin). plasmid pcasd -egfp. from the plasmid pimage , two pcr fragments were generated. fragment was obtained with primers casd fwd and casd -rev-esp i. fragment was generated with primers casd -fwd -esp i and casd -bamhi rev (table i) . both pcr products were digested with esp i, ligated and digested with acc i and bamhi. the digested ligation product was inserted into the acc i/bamhi window of plasmid pegfp-n (takara bio europe/clontech, france), resulting in plasmid pcas -egfp. plasmid pcas stop. from the plasmid pimage a pcr product encoding the entire orf and the stop codon was generated with primers cas if fwd and cas stop if rev. the resulting pcr product was digested with acc i and bamhi and ligated into the acc i/bamhi window of plasmid pegfp-n (takara bio europe/clontech, france), resulting in plasmid pcas stop. plasmid pgd synthhistag. from plasmid pimage , a pcr product covering the entire orf for gd synthase was generated with primers gd fwd and gd - his rev. the resulting pcr product was digested with acc i and sali and inserted into the acc i/sali window of plasmid pci (promega, germany), resulting in plasmid pgd synthhistag. heidelberg-mannheim. non-inflammatory tonsillar lymphocytes were extracted from tissue after tonsillectomy and purified as described (erdmann et al. ) , and lymphocytes from peripheral blood were separated by standard ficoll-paque centrifugation. thymocytes were prepared from thymic tissue obtained in the course of corrective cardiac surgery. all biological material from patients was obtained after having received informal consent by the patients or their parents and after approval of the ethical committee on the use of human tissue in research at the universities of heidelberg and heidelberg-mannheim. plasmid pcasd -egfp was cleaved with acc i and noti to obtain a fragment representing casd -egfp. this fragment was ligated into the baculovirus transfer vector pbacpak (takara bio europe/clontech, france.), resulting in the construct pbacpak casd -egfp. the integrity of the fusion site was confirmed by dna sequencing on both strands. recombinant baculoviruses were prepared by the transfection of ng of pbacpak casd -egfp with ng of baculo gold dna (bd bioscienes pharmingen, germany) into sf cells using cellfectin (invitrogen, karlsruhe, germany) according to the for flow cytometric analysis of surface expression of gd (cd a) and its o-acetylated variants -o-ac-gd (cd b) and -o-ac-gd (cd c), the following monoclonal antibodies (mabs) were used: for cd a: mab r (igg isotype), for cd b mab mt (igm isotype) and cd c: u (gd isotype). mabs r and u were a kind gift of dr. c. claus, university of mainz, germany, and were purified in our laboratory (rsa), mab mt was kindly donated by dr. b. kniep, university of dresden, germany. preparation and the binding capacity of the mabs have been described elsewhere (erdmann et al., ) . cells ( × cells/ml) were resuspended after careful washing in pbs + % bsa + . % nan and incubated with the respective mabs for min on ice. purified mabs (r , u , mg/ml) were diluted : for the staining procedure and hybridoma supernatants (mab mt ) were applied undiluted ( μl/cell preparation). cells were washed three times in pbs + % bsa + . % nan and incubated for further min on ice with secondary anti-mouse igg/igm antibody conjugated to fluorescein isothiocyanate (fitc) in a dilution of : . then, the preparations were washed again three times, and cells were resuspended in μl of pbs. for the exclusion of dead cells, viaprobe © (bd biosciences pharmingen, germany) was added to the cell preparations according to the manufacturer's instructions shortly before cytometric measurement. viaprobe-stained dead cells were then determined at fl and excluded from staining with the respective mabs. flow cytometric analysis was performed using an facs canto ii (bd biosciences pharmingen, germany). transfection of plasmid dna into cos cells was performed using lipofectamine (invitrogen, germany) according to the manufacturer's recommendations. for transfection of cells μl of lipofectamine and μg of plasmid dna were used. after transfection, cells were incubated for - days at °c/ % co until further investigations were performed. transfection efficiency was determined by fluorescence microscopy. for immunofluorescence, mab u was used in a : dilution and mab um d was diluted : . mab stg against the human st gal was produced by one of the authors (cao et al. ) . rna isolation and cdna synthesis cellular rna was isolated using the high pure rna isolation kit (roche, basel, switzerland). total rna ( ng) was oligo(dt)-primed and first-strand cdna synthesis was performed according to the manufacturer's guidelines (super script tm first-strand synthesis system for rt-pcr; invitrogen). for the quantification of casd mrna expression, cdna samples were analyzed by real-time quantitative pcr. a total of ng of cdna was amplified in μl using sybr green pcr master mix (applied biosystems, foster city, ca) in the presence of nmol of the specific casd primers (fwd: gtggattttctgtggcatcc, rev: aagcgcttcactgctaccat) using the real-time pcr system (applied biosystems, foster city, ca). for gd synthase, pcr was performed with primers st sia fw (gcgatgcaatctccctcct) and st sia rev (ttgccgaattatgctgggat). oligonucleotide specificity, synthesized by mwg biotech (ebersberg,germany), was computer-tested (blast, ncbi) by homology search with the human genome. samples were run in triplicate and experiments were repeated twice. the thermal profile for the reaction was min at °c, followed by min at °c and then cycles of s at °c and min at °c. in order to exclude unspecific amplification, dissociation curve analysis and agarose gel electrophoresis of the pcr products were performed at the end of the run. the endogenous reference gene β-actin was chosen for normalization. primers were β-actin fw (gctcctcctgagcgcaag) and β-actin rev (catctgc tggaaggtggaca). relative gene expression was calculated using the comparative c t method (livak and schmittgen ) . for transient sirna transfection, we used the on-targetplus smartpool sirna system of dharmacon (bonn, germany) containing four target sequences (gauggagguuagaccg uua; cguaaugcucaucggaaga; uagagaacaaa cagacgaa; ggauaugcccguucaguuu) and an on-targetplus negative-control non-targeting sirna. before transfection, cells were cultured for days and brought to approximately % confluency. transfection was performed using the amaxa nucleofector kit v, programme t according to the manufacturer's instructions (amaxa, cologne, germany). after h of cultivation, rt-pcr analysis for the suppression of the casd gene was performed. in addition, effective inhibition of cd c expression as a consequence of casd gene inhibition was monitored by flow cytometric analysis as described in results. this work was funded by the austrian science fund ( project number l -b ), salzburg research fellowship ( project number p - ) and by friendly financial support of the deutsche josé carreras leukämie-stiftung (djcls) to r.s.a. ( project no. djcls r / ). none declared. accoa, acetyl-coenzyme a, casd , capsule structure domain containing ; cas p, cas protein; egfp, enhanced green fluorescent protein; er, endoplasmic reticulum; facs, fluorescence-activated cell sorter; mab, monoclonal antibody; o-ac-gd , o-acetylated ganglioside gd ; -o-ac-gd , sialic acid-specific o-acetyltransferase -o-acetylated ganglioside gd ; -o-ac-gd , -o-acetylated ganglioside gd ; rt-pcr, reverse transcription-polymerase chain reaction; sgnh, serine-glycine-asparagine-histidine; sirna, small interfering rna; soat, sialate-o-acetyltransferase; st gal , β-galactosamide α- , -sialyltransferase ; st sia , α-n-acetyl-neuraminide α- , -sialyltransferase ; tgn, trans-golgi network carbohydrate-specific receptors of the liver global survey of genomic imprinting by transcriptome sequencing the polysialic acid-specific o-acetyltransferase oatc from neisseria meningitidis serogroup c evolved apart from other bacterial sialate o-acetyltransferases 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activity and intra-golgi localization o-acetylation of gd : an enigmatic modification regulating apoptosis? o-acetylation of disialoganglioside gd by human melanoma cells creates a unique antigenic determinant a monoclonal antibody recognizes an o-acylated sialic acid in a human melanoma-associated ganglioside a cell surface molecule distributed in a dorsoventral gradient in the perinatal rat retina reduction of sialic acid o-acetylation in human colonic mucins in the adenoma-carcinoma sequence the action of sialidases on substrates containing o-acetylsialic acids mucin degradation in the human colon: production of sialidase, sialate o-acetylesterase, n-acetylneuraminate lyase, arylesterase, and glycosulfatase activities by strains of fecal bacteria the roles of enteric bacterial sialidase, sialate o-acetyl esterase and glycosulfatase in the degradation of human colonic mucin siglecs and their roles in the immune system siglecs in the immune system imprinting evolution and human health structure, function and evolution of the hemagglutinin-esterase proteins of corona-and toroviruses requirement for gd ganglioside in cd -and ceramide-induced apoptosis escherichia coli k polysialic acid o-acetyltransferase gene, neuo, and the mechanism of capsule form variation involving a mobile contingency locus o-acetyltransferase from rat liver golgi vesicles darbepoetin alfa has a longer circulating half-life and greater in vivo potency than recombinant human erythropoietin differential surface expression and possible function of -o-and -o-acetylated gd (cd b and c) during activation and apoptosis of human tonsillar b and t lymphocytes the wnt receptor fzd mediates chemoresistance in neuroblastoma through activation of the wnt/beta-catenin pathway survival of recombinant erythropoietin in the circulation: the role of carbohydrates interferon gamma promotes survival of lymphoblasts overexpressing -o-acetylated sialoglycoconjugates in childhood acute lymphoblastic leukaemia (all) the epsilon-sarcoglycan gene (sgce), mutated in myoclonus-dystonia syndrome, is maternally imprinted sialic acid esterases of diverse evolutionary origins have serine active sites and essential arginine residues infectious salmon anemia virus specifically binds to and hydrolyzes -o-acetylated sialic acids high sialic acid content slows prourokinase turnover in rabbits the receptor-destroying enzyme of influenza c virus is neuraminate-oacetylesterase o-acetylation and de-o-acetylation of sialic acids. o-acetylation of sialic acids in the rat liver golgi apparatus involves an acetyl intermediate and essential histidine and lysine residues-a transmembrane reaction identification of a sialate o-acetyltransferase from campylobacter jejuni: demonstration of direct transfer to the c- position of terminalalpha- , -linked sialic acid solubilisation and properties of the sialate- -o-acetyltransferase from guinea pig liver enzymatic -oacetylation of n-acetylneuraminic acid in guinea-pig liver cas p is a membrane protein necessary for the o-acetylation of the cryptococcus neoformans capsular polysaccharide migration of o-acetyl groups in n,o-acetylneuraminic acids expression cloning and characterization of a cdna encoding a novel membrane protein required for the formation of o-acetylated ganglioside: a putative acetyl-coa transporter -o-acetyl-gd in human t-lymphocytes is detected by a specific t-cell-activating monoclonal antibody -o-acetyl gd protects tumor cells from apoptosis gangliosides with o-acetylated sialic acids in tumors of neuroectodermal origin -o-acetylation of sialomucins: a novel marker of murine cd t cells that is regulated during maturation and activation mannose receptor-mediated regulation of serum glycoprotein homeostasis structural and kinetic characterizations of the polysialic acid o-acetyltransferase oatwy from neisseria meningitidis localization of a neurectoderm-associated cell surface antigen in the developing and adult rat the group b streptococcal sialic acid o-acetyltransferase is encoded by neud, a conserved component of bacterial sialic acid biosynthetic gene clusters discovery and characterization of sialic acid o-acetylation in group b streptococcus analysis of relative gene expression data using real-time quantitative pcr and the (-delta delta c(t)) method properties and partial purification of sialate-o-acetyltransferase from bovine submandibular glands acetylation suppresses the proapoptotic activity of gd ganglioside high level of sialate-o-acetyltransferase activity in lymphoblasts of childhood acute lymphoblastic leukaemia (all): enzyme characterization and correlation with disease status effects of carbohydrates on the pharmacokinetics and biological activity of equine chorionic gonadotropin in vivo selective inactivation of influenza c esterase: a probe for detecting -o-acetylated sialic acids developmental regulation of sialic acid modifications in rat and human colon involvement of membrane galactose in the in vivo and in vitro sequestration of desialylated erythrocytes cloning and expression of cdna for o-acetylation of gd ganglioside a retrotransposon-derived gene, peg , is a novel imprinted gene located on human chromosome q identification of a large novel imprinted gene cluster on mouse proximal chromosome o-acetyl sialic acid specific igm in childhood acute lymphoblastic leukaemia glycosylation of an n-terminal extension prolongs the half-life and increases the in vivo activity of follicle stimulating hormone esterases and autoimmunity: the sialic acid acetylesterase pathway and the regulation of peripheral b cell tolerance the hemagglutinin-esterase of mouse hepatitis virus strain s is a sialate- -o-acetylesterase gd ganglioside directly targets mitochondria in a bcl- -controlled fashion genes modulated by expression of gd synthase in chinese hamster ovary cells. evidence that the tis gene is involved in the induction of gd -o-acetylation biosynthesis of n-acetyl-o-acetylneuraminic acids. i. incorporation of ( c) acetate into sections of the submaxillary salivary gland of ox and horse biosynthesis of n-acetyl-o-acetylneuraminic acids. ii. substrate and intracellular localization of bovine acetyl-coenzyme a: n-acetylneuraminate- -and -o-acetyltransferase sialic acids: fascinating sugars in higher animals and man sialic acids as regulators of molecular and cellular interactions chemistry, biochemistry and biology of sialic acids o-acetylated sialic acids and their role in immune defence the s protein of bovine coronavirus is a hemagglutinin recognizing -o-acetylated sialic acid as a receptor determinant cell surface sialylation and ecto-sialyltransferase activity of human cd progenitors from peripheral blood and bone marrow o-acetylation and de-o-acetylation of sialic acids in human colorectal carcinoma linkage-specific action of endogenous sialic acid o-acetyltransferase in chinese hamster ovary cells regulation of sialic acid -o-acetylation during the growth and differentiation of murine erythroleukemia cells induction of sialic acid -o-acetylation by diverse gene products: implications for the expression cloning of sialic acid o-acetyltransferases sialic acid -o-acetylation on murine erythroleukemia cells affects complement activation, binding to i-type lectins, and tissue homing structural and immunological characterization of o-acetylated gd . evidence that gd is an acceptor for ganglioside o-acetyltransferase in human melanoma cells kinetic and spatial interrelationships between ganglioside glycosyltransferases and o-acetyltransferase(s) in human melanoma cells sialic acid-specific o-acetyltransferase nidovirus sialate-o-acetylesterases: evolution and substrate specificity of coronaviral and toroviral receptordestroying enzymes assays of sialate-o-acetyltransferases and sialate-o-acetylesterases separate pathways for o acetylation of polymeric and monomeric sialic acids and identification of sialyl o-acetyl esterase in escherichia coli k functionally defective germline variants of sialic acid acetylesterase in autoimmunity asparagine-linked oligosaccharide structures determine clearance and organ distribution of pituitary and recombinant thyrotropin subunit-specific functions of n-linked oligosaccharides in human thyrotropin: role of terminal residues of alpha-and beta-subunit oligosaccharides in metabolic clearance and bioactivity characterisation of the enzymatic -o-acetylation of sialic acids in microsomes from equine submandibular glands characterization of the enzymatic -o-acetylation of sialic acids and evidence for enzymatic o-acetyl migration from c- to c- in bovine submandibular gland sialic acids as ligands in recognition phenomena glycan-based interactions involving vertebrate sialic-acid-recognizing proteins developmental abnormalities in transgenic mice expressing a sialic acidspecific -o-acetylesterase an autosomal dominant gene regulates the extent of -o-acetylation of murine erythrocyte sialic acids. a probable explanation for the variation in capacity to activate the human alternate complement pathway sialic acids mobile contingency locus controlling escherichia coli k polysialic acid capsule acetylation the influenza c virus glycoprotein (he) exhibits receptor-binding (hemagglutinin) and receptordestroying (esterase) activities human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza c viruses influenza c virus esterase: analysis of catalytic site, inhibition, and possible function key: cord- - vkzci authors: li, hao; shi, yongmin title: study on the performance degradation of sandstone under acidification date: - - journal: acs omega doi: . /acsomega. c sha: doc_id: cord_uid: vkzci [image: see text] in most oilfields, acid fracturing is widely used for oil production. understanding the relationship between the individual factors (i.e., carbonate rock types, acid rock reaction kinetics, and deterioration of rock mechanical properties) can provide practical guidelines that can be used for the design and optimization of acid fracturing operation. this paper takes hydrochloric acid, acetic acid, and citric acid as the main research objects and carries out acidification experiments on sandstone in changqing oilfield, china. in addition, the effects of tribasic, dibasic, and monobasic acids on the mechanical properties of sandstone were studied. results show that in this study area, the most obvious effect was seen with the use of dibasic acids (hydrochloric acid + acetic acid), which effectively reduced the sample quality, uniaxial compressive strength, and elastic modulus. citric acid and mg promote the conversion of amorphous calcium carbonate to high-crystallinity calcite, forming a white precipitate. furthermore, it is found by scanning electron microscopy analysis that experimental group (hydrochloric acid + acetic acid) has the most ideal rock erosion effect. inductively coupled plasma emission spectrometry analysis shows that the acid rock is present in the solution. x-ray diffraction qualitative analysis of the composition and concentration of ions shows that the formation of white precipitates is citric acid and mg promotes the conversion of amorphous calcium carbonate to high-crystallinity calcite, forming a white precipitate. the findings of this study can help to better understand the erosion, failure state, and failure mechanism of different acid types on sandstone, which may provide certain references and guidelines for sandstone acid fracturing oil production. the focus of the development of low-permeability reservoirs is mainly to maximize the output of a single well while reducing development costs and improving the development benefits of using low-permeability reservoirs. acid fracturing is currently widely used in oilfield mining to significantly improve recovery. while acidification can be used in both carbonate reservoirs and sandstone reservoirs, the rock mineral structure and mineral chemical composition of sandstone reservoirs are more complicated than carbonate rocks. in and , labrind and lund et al. proposed that the dissolution rate of dolomite at °c will be limited by the surface reaction; − however, under the condition of °c, the dissolution rate of dolomite will reach the limit regardless of whether the experiment uses high speed or low speed. fogler et al. found that the dissolution of terranic acid and feldspar is caused by hcl, which has a catalytic effect on hydrogen fluoride (hf) and can improve the dissolution of minerals. fogler and hekim found that minerals in sandstone have different degrees of solubility in an acidic soil system. in addition, because hf has the dual nature of ionization and association, other forms such as h + , f − , hf, and hf − can be found in solution. thus, fogler et al. proposed that the first step in the melting of hf and siliceous minerals is f − chemistry adsorbed on the siliceous surface. , zhao et al. proposed the exact opposite of the study of fogler et al. this study reported that when hf reacts with siliceous minerals, it is not the fluoride ion that plays the leading role but the hf molecule. hartman et al. studied the reaction of various minerals with hcl system and hcl/hf system acid solutions. by detecting the concentration of aluminum, iron, magnesium, sodium, and other metal ions in solution after the reaction of various minerals with an acid solution, the corresponding results were obtained. the dissolution kinetic parameters of minerals and the experimental study of kaolinite were conducted under the same conditions. after comparing the experimental results, the obtained parameters are applied to the sandstone acidification simulation using the geochemical model. zhao et al. designed a variety of acid solutions to dissolve single minerals and found that % hcl + . % hf system acid solution has a higher corrosion rate of chlorite at °c, . %: polyhydrogen acid. the corrosion rates of the system to kaolinite, montmorillonite, and chlorite are relatively high, respectively, . , . , and . %. among them, the corrosion rate of montmorillonite increases obviously with the increase in polyhydrogen acid concentration, with an average that is greater than %. , li et al. concluded that high temperature, high pressure, and a strong acidic environment will accelerate sandstone destruction. the research period of this study ranged from h to days. due to the influence of the corona virus disease (covid- ), the research period was extended to days, and it was unexpectedly concluded that the citric acid action promoted the precipitation of regular carbonate in the sandstone, resulting in calcium crystals, and an experimental analysis of the reasons for precipitation. simple hydraulic fracturing has limited damage to the rock, resulting in a relatively single fracture initiation and a fixed direction. the use of acid for fracturing will destroy the internal structure of the rock through chemical reactions, thereby creating more cracks, and the cracks are complex and not fixed in direction so that more oil can be extracted ( figure ). for rock samples in different blocks, different acid types are needed. in this study, seven experimental groups and a control group were set up to analyze the chemical reaction between acid and rock. finally, it is concluded that the mixed use of hydrochloric acid and acetic acid is most suitable for this block. when compared to the previous work, this work, at first, studied the influence of monobasic acid, dibasic acid, and tribasic acid on the acidification of sandstone and concluded that dibasic acid (hydrochloric acid + acetic acid) has provided the best effect. further, icp-oes analysis of the ion composition and concentration in the solution after the reaction solidly revealed the trend of the acid rock reaction. however, this analysis method is not generally used. finally, it was found that the coexistence of citric acid and magnesium will promote the formation of amorphous calcium carbonate (acc). further, it is transformed into calcite with high crystallinity and forms a white precipitate, which is irreversible due to underground environmental pollution factors. thus, this method is not proposed for the acidification in oilfields. first, the sandstone used in this study is taken from a depth of m in china changqing oilfield, yuan block. the acid solution used in the experiment consists of hydrochloric acid, acetic acid, and citric acid. further, seven experimental groups and one control group are set (see table ). corrosion of a core with a diameter of mm and a length of mm at room temperature for days. change in the mass of sandstone samples subjected to acid treatment. the calculation equation based on the mass loss rate was used and is as follows where m is the mass loss rate, m is the mass before acid etching, and m is the mass after acid etching. the calculated mass loss for all samples is shown in figure a . acid etching can be clearly seen from the appearance of the cores before and after etching. there is no obvious change in groups − , , and , but the cores in groups and have been obviously damaged and white substances have been precipitated. from the appearance analysis, the greatest degree of damage to the core can be seen in the treatment of acetic acid + citric acid and in the presence of citric acid alone. as shown in figure b , the two groups with the most mass loss were and followed by , , , and . group resulted in a reddish brown color. this is because h + in the acid liquid reacted with illite and chlorite to form fe + , which further reacted with oxygen to form fe + , and finally reacted with acetic acid to form a colloid with fe + , resulting in a reddish brown color. the acid etching reaction was fully confirmed by the absence of a precipitate in the presence of strong acid, and only ca + will be consumed quickly. in the presence of citric acid, calcium carbonate crystals (aragonite) will be precipitated, causing the core to burst. analysis of mechanical properties, specific surface area, and porosity. the uniaxial compressive strength of the samples after acid etching was tested, and the stress−strain curves of sandstone samples exhibited three typical stages: compaction, elastic, and yielding ( figure a ). the strength and stiffness of the samples after acid etching are significantly smaller than those of the untreated samples. it can be found that the stress−strain curves of the samples decreased after different acid etching. compared with the untreated samples, the compaction phase becomes longer, the elastic phase becomes shorter, and the strain value at the peak load point is also not the same. sample was damaged by complete acid etching, and thus, the uniaxial compressive strength was not measured. in addition, the bottom of sample had been damaged by acid etching and the compressive strength was low. the results indicate that group (hydrochloric acid + acetic acid) showed the most obvious decrease in mechanical properties. as shown in figure b , the bet test results show that group has the highest specific surface area and porosity after (hydrochloric acid + acetic acid) treatment, which proves that the acid etching effect is the most pronounced. the second most pronounced effect was found in group , which proves that the erosion effect of acetic acid alone on the core is also ideal. groups and , which contained citric acid, show no obvious changes in the specific surface area and porosity. the results indicate that in acidic solutions, protons promote the displacement reaction of intergranular cations, thereby inducing the formation of larger pores in the mineral and loosening the structure. overall, the main process of water− rock chemical interactions is the gradual dissolution of clay minerals, which increases the porosity of the rock mass. it can be seen from figure that the physical adsorption and desorption curves of group are the most obvious, indicating that the porosity is the largest. sem analysis. an obvious imon mixed layer can be seen for the control group from the sem results ( figure a ). as shown in figure b , the surface is covered with more citric acid, which hinders the acid rock reaction to proceed further. calcium carbonate whiskers that have been generated can be clearly seen in figure e , and after figure h was analyzed, it the experiments of rodriguez-blanco et al. showed that ph and mg have an important influence on the pathway and mechanism of the conversion of amorphous calcium carbonate (acc) to crystalline caco . the neutral initial ph or the presence of mg in the solution will prompt the system to develop in the direction of acc directly crystallizing into calcite. , conversely, a higher initial ph value promotes the formation of metastable intermediate spherulites, which drives the system toward calcite through the second dissolution− recrystallization step. mg can improve the stability of acc and inhibit the crystallization of ball stone, which is beneficial to the direct conversion of acc to calcite. , analysis of icp-oes. the icp-oes elemental composition and content of the acid solution after the core erosion were measured. as shown in figure a , the newly added group was the white material precipitated from core samples and . concentrations of mg, al, fe, and k all show that group has the least content, the order is > > and > > , and the si element content is basically the same. the related reaction of feldspar mineral and acidic solution is as follows: calcite easily reacts with the acidic solutions as follows: the si element content was measured in all samples and showed that quartz exhibits a slight hydrolysis effect: a small amount of biotite and muscovite may react with acidic solution as follows: elemental analysis showed that feldspar, mica, and calcite in the sandstone reacted with h + in acidic solution, indicating that the sandstone mineral composition used in this article reacted with h + in acidic solution in chlorite than the chemical reaction in quartz and acidic solution more obvious. as shown in figure b , the ca + content of group is relatively low because the citric acid is limitedly attached to the rock surface, preventing further the reaction of h + . group shows a relatively high ion content, indicating that hydrochloric acid and acetic acid both react with minerals in the sandstone, although the cores used in groups and are the most damaged due to the formation of precipitated substances by weak acids. cracks and pipeline blockages are caused by the construction of a site, and thus, it is not recommended to use them separately in construction. the results show that the sandstone damage is causally related to the acidity of the infiltrating fluid, and similar results have been obtained under various acidic conditions. , solution acidification promotes chemical corrosion in water−rock interactions. in terms of sandstone composition, it contains a high proportion of clay minerals (montmorillonite, illite, chlorite, etc.), which expand and contract with hydration and water loss and are easy to develop in combination with structural and weathered cracks. in addition, the three dominant clay minerals in the sandstone are silicate minerals; the acidification of sandstone in this block is suitable for the joint use of hydrochloric acid and acetic acid. xrd analysis. xrd analysis resulted in groups − all showing a significant decrease in the peak intensity corresponding to the chlorite, which proved that the acid had effectively eroded the core (figure ) . group had the smallest decrease in sensitive minerals. it has also been verified that in the monobasic acid, group has the least remaining sensitive minerals, but at the same time, it also generates new white precipitated materials. the dibasic acid performs best in group followed by groups and . the same xrd analysis was performed on white precipitated materials (group in figure ) , and a more obvious aragonite crystal peak appeared at °, proving that the precipitated crystal was a calcium carbonate crystal. in this study, a series of uniaxial compression, bet, sem-eds, icp-oes, and xrd tests were performed on tight sandstone samples corroded by different types of acids. destruction characteristics including mass, porosity, specific surface area, uniaxial compressive strength, ionic components in acid solution, and acid rock reaction failure mechanisms were investigated. the microstructure and damage mechanism of sandstone after multiacid erosion were systematically studied. the damage to morphology and the mechanism depend on the type of acid and the rock mineral composition. according to the experimental results, the following three major conclusions can be drawn. ( ) the sandstone samples treated with different acid solutions showed a decrease in quality and mechanical properties. the hydrochloric acid and acetic acid test group having the highest porosity, specific surface area, and mechanical properties was significantly weaker than the untreated samples. ( ) as the strength of the acid solution changes from strong to weak, the leaching amount of alkaline cations in the sandstone increases. this is mainly because h + not only acts as a reactant but also provides energy for physical and chemical weathering. compared with monovalent cations, the leaching of divalent cations (ca + and mg + ) in sandstones is largely limited by the environmental ph. the calcium and magnesium contents in group are . and . times those of the control group, respectively, and the aluminum and iron contents are . and . times those of the control group, respectively. however, the k + in the sandstone does not change much, mainly because the potassium feldspar is difficult to be acidified and destroyed. − ( ) sem-eds results show that citric acid promotes the precipitation of regular aragonite crystals of calcium carbonate with a length of − micrometers. it is recommended not to add citric acid in acid pressure treatment. it is recommended to use hydrochloric acid and acetic acid in the acid pressure of this test block. the main advantage of this study is that the most commonly used acid is compounded and optimized, and the acid solution suitable for the acid fracturing system used in the yuan block of changqing oilfield is obtained. further, it is aimed at the ion exchange of different acid solutions and rock reactions. performed icp-oes, xrd, and sem-eds characterization and analyses revealed that the best option is the mixture of hydrochloric acid and acetic acid. moreover, the presence of citric acid will promote the formation of white precipitates, pollute the underground environment extensively, and block the crude oil pipelines. the limitations of this study are as follows: ( ) the obtained sandstone samples did not reflect the entire area as they are not taken from all the areas due to the difficulty of coring in the entire changqing oilfield. ( ) the developed (indoor) experimental pressure does not correspond (not able to attain) fully to the underground confining pressure. thus, the number of samples is increased and tests are carried out under high temperature and high pressure. the test is repeated. the acid rock reaction mainly produces precipitation materials such as iron precipitation, calcium precipitation, and na salt and k salt precipitation. when the reservoir contains minerals such as chlorite, the acid will chemically react with the minerals to form a precipitate. for example, in experimental group , calcareous precipitation is formed mainly from carbonate minerals such as dolomite and calcite, but it will be dissolved with the further addition of strong acid (hydrochloric acid), and because of this, precipitation will not affect the entire acid fracturing process. the white precipitates obtained in experimental groups and are amorphous calcium carbonate (acc) precipitates, which will seriously threaten the underground environment and block the wellbore. therefore, citric acid should not be used for acid fracturing during field application. when hydrochloric acid, acetic acid, and citric acid are compared, citric acid has the strongest calcium ion chelating ability. the reaction temperature in this study is room temperature, which is highly favorable for the chelation of calcium ions. it is found that the white precipitates generated in experimental groups and will cause great damage to the formation after acid pressure and will severely affect the future work. therefore, citric acid should not be used in field applications. acetic acid also has the same complexation effect on iron ions, so experimental group (hydrochloric acid + acetic acid) has the best effect without precipitation and will not cause any damage to the optimal group. ■ materials and methods materials used. rock samples. the core samples were taken from the yuan block of changqing oilfield, china. the lithology is mainly fine-fine feldspar sandstone. the detrital components are mainly feldspar (average, . %) and quartz (average, . %). the cement type was made mainly of clays, carbonates, and siliceous sensitive minerals: clay minerals are mainly illite (average . %) and chlorite ( . %); carbonates are mainly iron calcite (average . %) and iron dolomite ( . %); and siliceous materials are mainly quartz ( . %), occasionally pyrite ( . %) and ridge iron ore ( . %). the samples were prepared in a cylinder with a diameter of mm and a height of mm for convenient use in the experiment. the main parameters are shown in table . acidizing fluids. in this paper, the impact of polyacids on the performance of sandstone was studied. triacids (hydrochloric acid, acetic acid, and citric acid correspond to experimental group ), dibasic acids (hydrochloric acid + acetic acid, hydrochloric acid + citric acid, and acetic acid + citric acid correspond to groups − , respectively), and monobasic acids (hydrochloric acid, acetic acid, and citric acid correspond to the experimental groups − , respectively) and the control group for a total of eight experimental groups. the specific acid ratio and concentration are shown in table . table lists the main mineral content of the samples used in the main experiments. testing equipment and test procedures. acid erosion. the seven cores were dried in an oven at °c for h, and the mass was weighed and recorded. the samples were then added them to wide-mouth jars with the corresponding acid concentration, capped with stoppers, and placed in a dark place at room temperature. in the liquid erosion experiment, because of the corona virus disease (covid- ), the research period was extended to days. after days, the core was taken out and placed in an oven to dry at °c for h. the weight was weighed again and recorded. mechanical property tests. the mechanical properties of the carbonate rocks were measured by using the rtr- rock mechanics servo testing system manufactured by gcts company. uniaxial compression tests were run at room temperature, and the deterioration degree of the mechanical properties of carbonate rocks was determined under acidified conditions. to analyze the degradation mechanism of sandstone mechanical properties under acidizing conditions, scanning electron microscopy (sem-eds) images were used to identify the composition and type of sandstone microcrack fillers and cement. a surface scan analysis was conducted, as well as the composition of precipitated materials. after the core erosion was diluted with water, the acid solution had a solvent ratio of : , and the elemental composition and content determined by inductively coupled plasma emission spectrometry (icp-oes) were used to further analyze the impact of different acids on the composition of sandstone. a bet analysis of the change in the specific surface area and porosity after acid etching was conducted, and finally, an x-ray diffraction (xrd) analysis of the crystalline form of the core after acid etching was conducted to further corroborate the degradation mechanism. thermodynamic and kinetic aspects of argillaceous sandstone acidizing acidization-i. the dissolution of dolomite in hydrochloric acid acidization-ii. the dissolution of calcite in hydrochloric acid acidization the kinetics of the dissolution of sodium and potassium feldspar in hf/hcl acid mixture on the movement of multiple reaction zones in porous media dissolution kinetics: the nature of the particle attack of layered silicates in hf acid-sensitive aluminosilicates: dissolution kinetics and fluid selection for matrix-stimulation treatments research and performance evaluation on an ha integrated acid system for sandstone acidizing effect of acid-temperature-pressure on the damage characteristics of sandstone citrate effects on amorphous calcium carbonate (acc) structure, stability, and crystallization a non-classical view on calcium oxalate precipitation and the role of citrate the role of ph and mg on the stability and crystallization of amorphous calcium carbonate the kinetics and mechanisms of amorphous calcium carbonate (acc) crystallization to calcite, via vaterite the effect of additives on amorphous calcium carbonate (acc): janus behavior in solution and the solid state mechanistic insights into the crystallization of amorphous calcium carbonate (acc) enhancing the permeability of a carbonate rock core by dissolution/ precipitation treatment with organophosphorus additives evaluated by sem/eds and icp-oes physical and mechanical properties of sandstone containing a single fissure after exposure to high temperatures strength and post-peak response of colorado shale at high pressure and temperature mechanical properties of qinling biotite granite after high temperature treatment an experimental study of fractured sandstone permeability after hightemperature treatment under different confining pressures experimental investigation on triaxial mechanicaland permeability behavior of sandstone after exposure to different high temperature treatments temperature and pressure effect on permeability of chinese sandstone: a review both authors contributed equally to this work. the authors declare no competing financial interest. key: cord- -i gpxcyo authors: zhang, jianguo; chen, deyu; liang, guoxin; xu, wenrong; tao, zhimin title: biosynthetic polymalic acid as a delivery nanoplatform for translational cancer medicine date: - - journal: trends biochem sci doi: . /j.tibs. . . sha: doc_id: cord_uid: i gpxcyo poly(β-l-malic acid) (pmla) is a natural polyester produced by numerous microorganisms. regarding its biosynthetic machinery, a nonribosomal peptide synthetase (nrps) is proposed to direct polymerization of l-malic acid in vivo. chemically versatile and biologically compatible, pmla can be used as an ideal carrier for several molecules, including nucleotides, proteins, chemotherapeutic drugs, and imaging agents, and can deliver multimodal theranostics through biological barriers such as the blood–brain barrier. we focus on pmla biosynthesis in microorganisms, summarize the physicochemical and physiochemical characteristics of pmla as a naturally derived polymeric delivery platform at nanoscale, and highlight the attachment of functional groups to enhance cancer detection and treatment. high-grade production of pmla from fermentation by fungi or myxomycetes enables increasing applications of this biodegradable polymer in medical research. pmla-based nanoconjugates can successfully penetrate the blood-brain barrier in rodent models, thus delivering imaging and/or therapeutic reagents to intrabrain targets and showing great potential for treating neurological disorders in human. with unmatched compatibility and resorbability, biosynthetic pmlas are good examples of future macromolecular compounds generated by a green and sustainable approach, eventually benefiting human health. given its great sustainability because it is produced from natural sources, pmla serves as a platform for potential multimodal conjugates with unmatched bioavailability, biocompatibility, and biodegradability. for this reason, pmla has been intensively and extensively used in biomedical and medicinal research, particularly in drug delivery and in bioimaging for cancer theranostics [ , ] . following its discovery in p. cyclopium as an acidic substance containing no nitrogen (molar mass m =~ g/mol), pmla was also found in the myxomycete p. polycephalum (m = - g/mol) as an inhibitor to dna polymerases, and in the yeast-like fungi aureobasidium sp. (m = - g/mol) and a. pullulan (m = - g/mol) as an extracellular secretion in culture broth [ , ] . bacteria producing pmla have not yet been identified. a study that examined pmla bioproduction from strains of a. pullulan glossary antisense oligonucleotides (aons): synthetic, short, single-stranded nucleotides that target dna or mrna. crispr/cas : clustered regularly interspaced short palindromic repeats, dna fragments originally derived from bacteriophages, whereas crisprassociated protein (cas ) is an endonuclease that specifically recognizes and cleaves doublestranded dna at a sequence complementary to the crispr sequence. endosome escape: release of entrapped molecules by endosomes to avoid further degradation. ester bond: a chemical bond between an acid and an alcohol via the elimination of water. first-order kinetics: a reaction that proceeds at a rate proportional to the reactant concentration. glioblastoma (gbm): one of the most aggressive brain tumors; has a poor prognosis. hyperosmolarity: fluid of abnormally high osmolarity. immunogenicity: the ability of a molecule to provoke an immune response in the host. ld : lethal dose %, a dose that kills a half the group of animals studied. malyl unit: the repeating unit in polymalic acid. mitochondrial pyruvate carrier (mpc): a protein gatekeeper that governs the transport of pyruvate into mitochondria to provide oxidative fuel. molecular chaperone: a class of proteins that assist in the folding and translocation of newly synthesized polypeptides. myxomycetes: mostly known as slime molds, these are fungus-like microorganisms whose life cycle includes spore, plasmodium, and fruiting body stages. nonribosomal peptide synthetase (nrps): microbial enzymes that synthesize peptides independently of ribosomes and mrnas. nucleophilic: a substance that can provide an electron pair to generate a chemical bond. oligomers: intermediate products generated by polymer breakdown or by condensation that contain only a few repeating units. phylogenetic clade: a group of lineages that share a common ancestor in their phylogenetic tree. (a) structures of α, β, and α,β types of polymalic acid. (b) pmla hydrolases break down the first ester bond from oh-terminus while binding to the th malyl unit along the polymer chain towards the c-terminus [ ] . (c) intracellular trafficking of pmla hydrolases and their extracellular activation. in typical eukaryotes, pmla hydrolase binds to pmla and remains inactive, carrying nuclear proteins into the nucleus while pmla hydrolase remains at the surface of nuclear envelope. cargo-discharged pmla exits from the nucleus into the cytoplasm via nuclear pores; it binds to pmla hydrolases that then translocate across the cell membrane where they are phosphorylated by membrane-bound tyrosine kinases, restoring their activity to degrade pmlas extracellularly [ , ] . of a diversity of phylogenetic clades indicated high productivity but low molar mass ( - g/mol), where pmla was bound to polysaccharides of varying molar mass depending on the exact strain type [ ] . by screening various aureobasidium spp. and optimizing the culture conditions, efforts have been made to obtain efficient biosynthesis of pmla with high molar mass (up to g/mol) [ ] . using a highly productive aureobasidium sp. strain isolated from mangrove systems, purified pmla with m = g/mol was reported [ ] . by contrast, pmla produced from the plasmodial stage of p. polycephalum sustains a much elongated linear chain in its pure form, with m = - g/mol [ , ] . using d-glucose as the most efficient carbon source, and relying on nutrients available in the extracellular culture medium, independent extramitochondrial and intramitochondrial metabolic routes for generating l-malate (the immediate precursor to pmla) in vivo were unraveled [ ] ( figure ). on the one hand, in the presence of exogenous carbonates (e.g., caco or na co ), l-malate is produced via a reductive pathway in the cytoplasm; because of the high level of co in the cytoplasm, pyruvate is prioritized for carboxylation to oxaloacetate by pyruvate carboxylase, and this is further reduced by malate dehydrogenase to malate [ ] . alternatively, in a similar manner, phosphoenolpyruvic acid is directly converted by phosphoenolpyruvate carboxylase to oxaloacetate, forming malate [ ] . therefore, phosphoenolpyruvic acid and pyruvate from the glycolysis pathway are indispensable molecules for malate synthesis in vivo, whereas their carboxylases govern carbon flux for malate biosynthesis because regulated carboxylase activity influences the ultimate level of pmla bioproduction [ , ] . on the other hand, if exogenous carbonates are absent, malate formation via the oxidative pathway is mostly achieved in the mitochondrial matrix through either the tricarboxylic acid (tca) cycle or the glyoxylate bypass [ ] . glucose or another sugar is transformed into pyruvate through glycolysis, followed by import into mitochondria by mitochondrial pyruvate carrier (mpc) proteins and subsequent decarboxylation by pyruvate decarboxylase complex to produce acetyl coenzyme a (acetyl-coa), thus entering the tca cycle and being converted to four-carbon oxaloacetate and subsequently to six-carbon citrate [ , ] . the tca cycle continues as a series of biochemical transformations that take place in an enzyme-mediated cascade, generating products including cis-aconitate, isocitrate, α-ketoglutarate, succinate, fumarate, malate, and plasmodial: related to the plasmodium, a single cell that contains multiple nuclei but a single cytoplasm. polyhydroxyalkanoates (phas): a group of plastic polyesters produced by various microorganisms in nature. polyhydroxybutyrate (phb): a bacteria-generated plastic that is biodegradable and water-insoluble. protein homeostasis: biological pathways for balanced protein synthesis, modification, trafficking, and degradation in living organisms. renal tubular reabsorption: a process in which functional units within the kidney remove water and solutes from pre-urine tubular fluid, and these are reabsorbed into the bloodstream for reuse. serine protease: a proteolytic enzyme that contains a catalytic serine residue within its active site t / : half-life, the time needed to reduce the concentration of a drug administered to half its original level. tyrosine kinase: an enzyme that covalently adds a phosphate group to tyrosine residues of proteins to modify their conformation and function. pmla hydrolases (also called pmla depolymerases or polymalatases) have been purified and characterized from both eukaryotic and prokaryotic microorganisms [ , ] . in eukaryotes, particularly pmla-producing myxomycetes, soluble pmla hydrolase serves as a molecular chaperone of pmla and binds to its hydroxyl terminus at the penultimate malyl residue for catalytic cleavage, although another binding site malyl residues down the polymeric chain was also proposed to govern stability and carriage function ( figure b ) [ ] . intracellular pmla hydrolase remains inactive and forms complexes with pmla that may carry other nuclear proteins, assisting the translocation of pmla into the nucleus while leaving pmla hydrolase on the surface of nuclear envelope, possibly because of either its binding to envelope proteins and/or the outer nuclear membrane, or disruptive variation in local ionic strength ( figure c ) [ ] . after discharging its nuclear protein cargo, pmla exits through nuclear pores and to join newly synthesized pmla in the cytoplasm for the next deliveries [ ] . pmla thus shuttles between the nucleus and the cytoplasm to maintain homeostasis in different phases of the cell cycle. excess pmlas bound to hydrolases are excreted through the cell membrane, where a membrane-bound tyrosine kinase then phosphorylates pmla hydrolases and activates their hydrolytic activity, leading to degradation of concomitantly released pmlas in the culture medium (peak activity at ph - ) [ ] . however, the hydrolases may differ across different eukaryotes such as pmla-producing myxomycetes and fungi: pmla isolated from p. polycephalum had higher molar mass and polydispersity than that from a. pullulan [ ] . in prokaryotes such as bacteria that are devoid of pmla, insoluble pmla hydrolases are confined to the outer cell membrane where they hydrolyze outsourced pmla into malic acid monomers that are further taken up for cellular metabolism [ ] . unlike phb depolymerase, pmla hydrolase does not exhibit serine esterase activity in which a nucleophilic serine in the active site initiates substrate hydrolysis, and is not inhibited by serine protease inhibitors [ ] . eventually oxaloacetate again. in parallel, taking a shortcut in the tca cycle, a process known as the glyoxylate shunt, isocitrate is directly converted into glyoxylate and succinate; glyoxylate then reacts with acetyl-coa to produce malate, and succinate generates malate catalyzed by succinate dehydrogenase and fumarase [ ] . the glyoxylate pathway could thus be driven to produce malate and pmla if key enzymes (e.g., fumarase, succinate dehydrogenase) in the tca cycle are blocked [ ] , or if malate synthase is upregulated by exogenous ethanol stress [ ] . because the inner membrane of mitochondria remains a barrier to most molecules, malate and oxaloacetate from the oxidative tca cycle or glyoxylate shunt interchange with their counterparts in the cytosolic reductive pathway via the malate/aspartate shuttle, where oxaloacetate is reduced to malate both extramitohchondrially and intramitochondrially to permit transportation and avoid oxaloacetate accumulation in the cytoplasm [ ] . in cultures of p. polycephalum and a. pullulan, the addition of exogenous carbonates augments co fixation and pyruvate carboxylation into oxaloacetate by pyruvate carboxylase in the cytoplasm, abolishing the intramitochondrial pathways for l-malate production and ensuing pmla synthesis ( figure ) [ , ] . under these conditions, the figure . generic biosynthetic pathways of poly(β-l-malic acids) (pmlas) in eukaryotic cells. glucose is transported into the cytoplasm where it is metabolized by glycolysis to produce pyruvate (green). excess co activates the reductive extramitochondrial pathway to generate malate (blue). when exogenous carbonates are absent, intramitochondrial malate formation via the oxidative pathway takes place in the mitochondrial matrix through either the tricarboxylic acid (tca) cycle or the glyoxylate shunt (orange). through either pathway, malates are polymerized to form pmla in the cytoplasm (gray). a pmla synthetase has been proposed that possibly combines the activities of a malyl-amp ligase and an unknown pmla polymerase, in conjunction with an auxiliary peptide or enzyme (e.g., spherulin b in p. polycephalum) [ , ] . addition of tca cycle metabolites into the cell culture might not promote pmla production [ ] . in other words, environmental carbonate works as a switch between oxidative and reductive pathways to produce malate. furthermore, carbonate adjusts the acidity of the culture medium, noting that low ph values accelerate pmla hydrolysis. in contrast to p. polycephalum, that merely uses d-glucose as a carbon source in malate and pmla bioproduction, a. pullulan takes up a broad spectrum of saccharides (e.g., sucrose, fructose, and maltose) because it expresses a remarkable diversity of polysaccharide lyases, glycoside hydrolases, carbohydrate esterases, glycosyltransferases, and sugar transporters, enabling its efficient utilization of diverse carbohydrates to produce malate [ ] [ ] [ ] . by depleting the nitrogen supply or augmenting the carbon/nitrogen ratio in the cell medium, pmla bioproduction is significantly enhanced per unit of cell mass because nitrogen starvation upregulates the expression of key enzymes in pmla biosynthetic pathways, uncoupling cell growth from pmla production [ , ] . at the cost of atp probably derived from glycolysis, malates are polymerized to form pmla in the cytoplasm [ ] . the acellular slime mold p. polycephalum only synthesizes pmla in its multinucleated plasmodia, whereas the yeast-like fungus a. pullulan spends its entire life cycle (except hyphae form) producing pmla [ , ] . although the search for pmla synthetase is still underway, a nonribosomal peptide synthetase (nrps) machinery was proposed that first forms malyl-amp via the action of malyl-amp ligase, and this is then assembled into a polymeric chain by an unidentified polymerase [ ] . in addition, a plasmodium-specific polypeptide spherulin b (i.e., nka ) was found to assist pmla synthesis, although similar enzymes have not been identified in fungi [ ] . alternatively, in a. pullulan, it was proposed that cytosolic malate may be polymerized into pmla using malyl-coa as the precursor, followed by the action of malate-coa ligase and pmla synthetase [ ] . in one variety of a. pullulan, namely aureobasidium melanogenum, a characteristic nrsp was recently reported to be a putative pmla synthetase that contains an adenylation domain for atp binding and malyl-amp formation, an activatable thiolation domain for phosphopantetheine attachment and polymerization of malyl-amp into pmla, and a hexatransmembrane region for transport of pmla out of the cytoplasm [ ] . because the whole-genome sequences of several pmla-producing fungal strains have been determined, a conserved pmla synthetase across species is expected to be unmasked in the near future [ , ] . biosynthetic pmla in aqueous solution ( % w/v) has a ph of . but a pk a of . - . (average m = - g/mol) [ ] . at acidic ph less than the pk a (e.g., ph - ), pmla remains protonated, promoting the formation of intramolecular double hydrogen bonding between side-chain carboxylic acids and the construction of dense, inflexible, double-stranded segments [ ] . in phosphate buffer (ph . ) at °c, pmla with fully ionized carboxylic groups retains an opencoil conformation owing to the negatively charged neighboring side-chains, and undergoes hydrolysis with a half-life of h, initially following first-order kinetics, whereas elevated temperature and acidic ph dramatically accelerate its hydrolytic degradation [ , , ] . moreover, random hydrolysis prioritizes the breakdown of intrachain ester bonds over those at the ends of the molecule, producing oligomers instead of malates until hydrolysis proceeds to completion, whereas hydrophobic substitution (e.g., alkylation) of pmla side-chains delays this hydrolytic degradation, possibly because of limited water access to ester bonds in the backbone as the polymer conformation alters [ , ] . in addition, a larger substituent group or a higher degree of substitution in the sidechain that increases hydrophobicity, leads to the slower degradation in aqueous solutions [ , ] . in a pilot study, repeated intraperitoneal injection of synthetic pmla into rabbits returned no detectable immune response, demonstrating non-immunogenicity, whereas the same injection into trends in biochemical sciences mice revealed nearly no acute toxicity (ld = . g/kg body weight) [ ] . intravenous (i.v.) injection of pmla sodium salt into mouse tail vein, with repetitive administration at low dosage, showed no mortality or adverse effects at doses up to g/kg body weight; however, mice only tolerated a one-time i.v. high-dose injection (up to~ g/kg body weight or otherwise) toxicity was due to the hyperosmolarity of the concentrated polymer solution injected rather than to the polymer concentration per se [ ] . the elimination of injected pmla from blood was very fast, with a t / of min or much less, and injection might not even be complete before the polymer is exported into urine [ , ] . indeed, % of injected pmla was excreted after h, and % after h, although there was low but persistent liver accumulation h after injection, yet no substantial accumulation in other organs, including kidney, lung, intestine, spleen, heart, muscle, and brain [ , ] . notably, these early studies on the pharmacokinetics and biodistribution of synthetic pmla laid a solid foundation for recent preclinical research using natural pmla as a pharmaceutical carrier [ ] . bioproduced pmlas possess a similar or even superior biocompatibility profile to synthetic pmlas, and their end-product is only l-malic acid. intriguingly, l-malate administered i.v. into the tail vein in mice had a half-life of only min, and one third of injected dose ended up in exhaled co , whereas the majority of the remainder accumulated in tissues via renal tubular reabsorption, participating in the tca cycle [ ] . pmla is negatively charged at ph . , and hence poses no disruptive threat to phospholipid membranes because they have the same charge. given that there is no specific cell-surface receptor of pmla, water-soluble pmla is transported into the cytoplasm through the invagination of cell membrane via a process of non-specific endocytosis, although the efficiency of transmembrane transport can be very low. in contrast to pmla, its copolymers with lipophilic ligands undergo hydrophobic interactions in aqueous solution and assemble into lipophilic patches, and these can interact with the cell membrane, leading to anchoring of lipophilic patches in close proximity to lipid bilayers [ ] . depending on the attached hydrophobic ligands, three distinct mechanisms have been proposed by which different pmla-conjugated copolymers can induce membrane permeation ( figure ); these are discussed in the following text: (i) the carpet model is typified by pmla leucine ethyl ester (pmla-loet x h −x ) [ ] . at physiological ph, esterification of carboxylic acid groups in pmla side chains leads to permanent charge neutralization, excluding further protonation even when the environmental ph drops. upon binding to the cell membrane, pmla-loet x h −x orients itself to insert the hydrophobic loet side chain into the phospholipid layer, leaving the outer membrane surface expansively covered by the hydrophilic backbone of the polymeric chain. the hydrophobic binding energy is sufficient to strongly bend the plasma membrane into a curved structure, creating a transient pore that enables membrane permeation [ , ] . this process is independent of ph and its membranolytic efficiency varies according to the ratio of hydrophobic/hydrophilic moieties (box ) in the polymer (i.e., x −x ). (ii) the belt model is typified by pmla tritryptophan (pmla-www x h −x ) [ ] . tritryptophan contains three side-chain indoles and one terminal α-carboxylic acid, constituting a nonpolar hydrophobic tripeptide. at ph . , the terminal α-carboxylic acid in the side chain is deprotonated and ionized; this would be repelled from the cell membrane, but, because of strong hydrophobic interactions, indole in the side chain can attract and intercalate into phospholipids, generating pmla tritryptophan-lipid complexes and releasing binding energy to stabilize the structure. in another scenario, ph reduction from neutral to acidic may protonate (and neutralize) the end-group carboxylate in the side chain, whereas protonation of the indole moieties is constrained because this would lead to loss of aromatic stabilization. under both circumstances, the pmla backbone is sandwiched between two layers of phospholipids inserted with outflanking tryptophans, forming a 'belt-like' or 'dental brace' configuration [ ] . the ph-dependent charge neutralization of the carboxylic acid endgroups does not hamper strong hydrophobic interactions between indole and membrane lipids, thereby leading to ph-independent membrane permeation. this permeation thus resembles the 'boomerang model' that was proposed to mediate viral membrane fusion with the host cell [ ] . a highly conserved tryptophan-rich domain has been found in many human viruses, including coronavirus, influenza virus, and hiv, and has been proposed to be a key determinant of viral entry through strong interactions between the aromatic rings of the tryptophan-rich domain and lipids in the target membrane lipid, thus perturbing the lipid bilayer and mediating membrane fusion [ ] . (iii) the barrel-stave model typified by pmla trileucine (pmla-lll x h −x ) [ ] . trileucine in the side chain of pmla-lll x h −x has three hydrophobic isobutyl groups and one α-carboxylic acid end-group that is subject to ph-dependent protonation. at neutral ph, ionized pmla-lll x h −x is likely to generate a random-coil conformation, similarly to pmla, and is largely unable to penetrate the cell membrane owing to its negative charge. as the ph was decreased below , pmla trileucine was found to form aggregates via oligomerization that vertically pierce the membrane core and tentatively form a transmembrane pore to allow entry. in this manner, an increase in the fraction of hydrophobic substituents or the molar mass of the amphiphilic polymer could augment its membranolytic activity [ , ] . importantly, acidic ph-triggered membranolysis would enable selective disruption of intracellular membranes by ionizable polymeric carriers, including endosomes that are of particular interest for drug delivery (ph~ . ), thus escaping endosomal capture and lysosomal degradation, leading to release of drug payloads in the cytosol, thereby promoting intracellular drug trafficking and targeting. in addition to pmla conjugation with hydrophobic ligands through covalent coupling, methods to regulate its membrane permeability have been developed by promoting non-covalent interactions between the pendant carboxylic acids of pmla and attaching moieties to the polymer. given that protonated pmla can only form hydrogen bonds with functional groups containing electronegative atoms at acidic ph [ ] , and these are much weaker than covalent or ionic bonds, pmla indole moieties in the side chain and membrane phospholipids induces the pmla backbone to be sandwiched between two layers of phospholipids, resulting in a 'belt-like' configuration. (c) the barrel-stave model: pmla polymers first self-aggregate to form oligomers and then vertically penetrate the membrane core, generating a transmembrane pore. (d) endocytosis: cellular engulfment of pmla. once pmla comes into contact with the cell surface, the cell membrane forms vesicles that wrap around the random-coil polymers to generate early endosomes; these can mediate secretion from the cell or develop into late endosomes. in view of its hydrophilicity and negative charge at physiological ph, pmla is a polyanion that has little affinity for negatively charged lipid bilayers and does not translocate through cell membranes. to increase the interaction between the biopolymer and the plasma membrane, methylation of carboxylic acid groups with different levels of diazomethane was used to generate a pmla-me x h −x copolymer (where x is the percentage of methyl units) [ , ] . as x increases, the hydrophobicity of the molecule increases in the order pmla < pmla-me h < pmla-me h < pmla-me h < pmla-me. both pmla-me h and pmla-me were completely insoluble in water, similarly to pmla benzyl esterification. the same order was observed for hydrolysis in saline and plasma, rupture of liposome membranes, and cytotoxicity [ ] . in addition, hydrophobic amino acids or peptides can be conjugated to pmla side-chain carboxylic acids to modulate its hydrophilicity and net charge, thus tuning the interplay with cellular/subcellular membranes in a ph-responsive manner. adjacent carboxylic acid pendants on the pmla backbone are five atoms apart, equal to their distance in poly(aspartic acid) that has a similar membranolytic profile [ ] . importantly, this spacing dictates an optimized combination of physicochemical parameters for side-chain substituents, including their individual hydrophobicity, charge-neutralizing capacity, ligand length, and density, thus determining the optimal molecular geometry and charge distribution for effective membrane disruption [ , ] . complexes generated by hydrogen bonding are unsuitable for membranolytic modification or pharmaceutical loading. nonetheless, the generation of pmla ionic complexes via electrostatic interactions offers an alternative strategy for enhanced cellular uptake. at neutral ph, pmla is a polyanion in which negatively charged carboxylates can form stable complexes with positively charged compounds [ ] . depending on the stoichiometry and chemistry of the attached cations, the polyelectrolyte complexes generated can have a variety of sizes, surface charges, water solubilities, molecular structures, and morphologies, each modulating their membrane penetration. in a scenario where anionic pmla segments are preferentially situated on the surface of polyelectrolyte complexes, they are internalized via non-specific endocytosis. conversely, cationized pmla complexes translocate into cells in a similar manner to polycations, which first adsorb onto the hydrophilic outer surface of the cell membrane and then induce the formation of aqueous pores or defects in the membrane hydrophobic core, and subsequently integrate into or permeabilize the cell membrane [ ] . once transported into the cytoplasm, ph reduction or, to a lesser extent an increase in ionic strength, would accelerate hydrolytic degradation of the pmla backbone and dissociation of ionic bonds, liberating the complexed moiety for intracellular utility [ ] . a novel type of pmla-based nanoconjugate has been developed, termed polycefin [ ] . through step-by-step chemical synthesis, pmla was activated by n-hydroxysuccinimidyl ester to enable direct conjugation or further modification by linker molecules, permitting linkage with a variety of chemical and biological ligands, including polyethylene glycol (peg), monoclonal antibody against transferrin receptor (tfr), an endosome escape unit (lll or loet), and two antisense oligonucleotides (aons) [ ] , to synergistically inhibit the α and β chains of laminin- that are overexpressed by human glioblastoma (glioblastoma multiforme, gbm); a fluorescent reporter was also covalently bonded with the pmla backbone to visualize and localize the biodistribution of polycefin (figure ) [ ] . the attachment of pendant functionality groups established a new hydrophobic-hydrophilic balance (box ) in the macromolecular structure, as the sizes of pure pmla and variant polycefins were determined to be < and~ nm, respectively [ ] . these pmla-based nanoconjugates provide a multifunctional delivery system that can effectively pass through the blood-brain and blood-tumor barriers, leading to enhanced accumulation in brain tumors following i.v. injection into the mouse tail, thus allowing visualization of cancerous lesions and liberating medicinal agents to inhibit tumor angiogenesis or growth [ ] . since this invention, variant polycefin biopolymers have been made by combining them with different therapeutic antibodies [ , ] , penetrating peptides [ , ] , and fluorescent or magnetic contrast agents [ ] [ ] [ ] that are linked to the pmla chain through direct or indirect conjugation chemistry, and these have demonstrated improved targeting and accumulation in specific tumors such as breast and brain cancers. notably, by targeting and inhibiting the laminin α and β subunits, treatment of mice bearing intracranial human gbm xenografts with pmla nanoconjugates led to significantly prolonged survival and reduced tumor sizes relative to mice bearing gbms in which laminins had been knocked out using crispr/cas [ ] . the minute physical size, potential for multifunctional conjugation, and outstanding water solubility of these pmla conjugates give them enormous advantages over many other drug delivery systems in terms of finding their way through a labyrinth of cancers, highlighting their huge potential for clinical translation. microbial production of pmla currently remains a challenge because of its low yield, high cost, and the difficulty in defining the length of the biopolymer produced (see outstanding questions). novel gene-editing techniques are urgently needed that would allow efficient delivery of given the complications of scalability from bench to industrial pilot plant production, how best to scale up pmla microbial production and further functionalization for medicinal purposes? what is the best way to uncover the genes that govern pmla synthesis in relevant microorganisms such that they can be engineered in industrial bacteria or yeasts for large-scale pmla bioproduction? how can we tune the length of biosynthesized pmla? what is the precise relationship between the molar mass of pmla and its suitability as a delivery platform for cancer medicine? what are the potential applications of pmla-based biopolymers other than as delivery platforms? trends in biochemical sciences exogenous gene sequences into physarum plasmodia or filament fungal cells to upregulate pmla production. crispr/cas -mediated gene modification was recently successfully applied in a. pullulan, leading to a mutation rate nearly ten-fold higher than that obtained by traditional homologous recombination [ ] . it is likely that further genes involved in pmla biopolymerization will soon be identified, with the prospect of producing pmla in a controlled manner by using novel gene-editing tools that preselect high-yielding strains. indeed, pmla per se could be used as an effective transmembrane gene-delivery shuttle. future genetic modifications will be necessary not only to increase pmla yield but also to predetermine the molecular weight of the polymer. furthermore, suppression of polymalatase to inhibit the decomposition of pmla could also be achieved through gene editing, thus increasing control over both polymer yield and length. therefore, to harness the potential of biopolymer production, further elucidation of the genes and molecular machinery that mediate the biosynthesis of pmla and its decomposition will be essential. because pmlas represent a versatile platform for both disease diagnosis and treatment, further research is required into the pmla biosynthetic machinery, the biosafety of bioproduced pmla, and clinical translation of pmla for the detection and therapy of diseases (particularly at early stage) including cancers. there is also an urgent need to scale-up pmla production from the laboratory to the industrial pilot plant. in addition, multiple new uses of biodegradable pmla in daily life can be envisaged, and this would not only stimulate industrial interest in pmla bioproduction and increase the popularity of pmla as a natural biomaterial, but would also promote wider medical applications of these remarkable biopolymers. poly-(l)-malic acid; a new protease inhibitor from penicilliumcyclopium poly(beta-l-malic acid) (pmla) from aureobasidium spp. and its current proceedings microbial polyhydroxyalkanoates and nonnatural polyesters screening for beta-poly(l-malate) binding proteins by affinity chromatography her -positive breast cancer targeting and treatment by a peptide-conjugated mini nanodrug enhanced endocytic and ph-sensitive poly (malic acid) micelles 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pullulans gxl- strain and its techno-economic analysis effects of nitrogen availability on polymalic acid biosynthesis in the yeast-like fungus aureobasidium pullulans. microb gata-type transcriptional factor gat regulates nitrogen uptake and polymalic acid biosynthesis in polyextremotolerant fungus aureobasidium pullulans is beta-poly(l-malate) synthesis catalysed by a combination of beta-l-malyl-amp-ligase and beta-poly(l-malate) polymerase? water-soluble aliphatic polyesters: poly (malic acid)s. biopolymers online published online comparative synthesis and hydrolytic degradation of poly (l-malate) by myxomycetes and fungi stage specific expression of poly(malic acid)-affiliated genes in the life cycle of physarum polycephalum spherulin b and polymalatase biosynthesis of polymalic acid in fermentation: advances and prospects for industrial application a novel pma synthetase is the key enzyme for polymalate biosynthesis and its gene is regulated by a calcium signaling pathway in 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of nanoparticle cellular endocytosis we thank jiangsu university for financial support. key: cord- -lqul ho authors: kaczmarek, beata title: tannic acid with antiviral and antibacterial activity as a promising component of biomaterials—a minireview date: - - journal: materials (basel) doi: . /ma sha: doc_id: cord_uid: lqul ho as a phenolic acid, tannic acid can be classified into a polyphenolic group. it has been widely studied in the biomedical field of science because it presents unique antiviral as well as antibacterial properties. tannic acid has been reported to present the activity against influeneza a virus, papilloma viruses, noroviruses, herpes simplex virus type and , and human immunodeficiency virus (hiv) as well as activity against both gram-positive and gram-negative bacteria as staphylococcus aureus, escherichia coli, streptococcus pyogenes, enterococcus faecalis, pseudomonas aeruginosa, yersinia enterocolitica, listeria innocua. nowadays, compounds of natural origin constitute fundaments of material science, and the trend is called “from nature to nature”. although biopolymers have found a broad range of applications in biomedical sciences, they do not present anti-microbial activity, and their physicochemical properties are rather poor. biopolymers, however, may be modified with organic and inorganic additives which enhance their properties. tannic acid, like phenolic acid, is classified into a polyphenolic group and can be isolated from natural sources, e.g., a pure compound or a component of a plant extract. numerous studies have been carried out over the application of tannic acid as an additive to biopolymer materials due to its unique properties. on the one hand, it shows antimicrobial and antiviral activity, while on the other hand, it reveals promising biological properties, i.e., enhances the cell proliferation, tissue regeneration and wound healing processes. tannic acid is added to different biopolymers, collagen and polysaccharides as chitosan, agarose and starch. its activity has been proven by the determination of physicochemical properties, as well as the performance of in vitro and in vivo studies. this systematics review is a summary of current studies on tannic acid properties. it presents tannic acid as an excellent natural compound which can be used to eliminate pathogenic factors as well as a revision of current studies on tannic acid composed with biopolymers and active properties of the resulting complexes. human organisms are exposed to different external factors which may cause diseases and which pose a threat to people's lives. according to us national institute of health reports, % of all microbial infections in the human body are associated with pathogen biofilm formation. there is an increased need to search for effective compounds which would enable protection against pathogens. since protection against viruses and bacteria is a crucial issue for humans, one of the main healthcare problems is to find effective compounds demonstrating antiviral and antibacterial properties [ ] [ ] [ ] . moreover, the evaluation of their effectiveness in real-time use and the examination of their exact application conditions is also essential. one strategy is related to inhibiting the microbes' adhesion to surfaces which have been specially modified to repel pathogens [ ] . another strategy is to add antimicrobial compounds as additives materials , , of during material fabrication or as coatings. moreover, increasing the material roughness can be effective, as it prevents bacteria attachment to the cell wall. to protect the surface against pathogens, different drugs may be added to the material during fabrication and then released to surroundings. such a strategy is more effective than traditional drug treatment, i.e., administering pills or injections; however, the problem of bacteria resistance to antibiotics has recently become serious [ ] [ ] [ ] . there is a growing interest in compounds which may be extracted from natural sources and which have unique antimicrobial properties. their application has been considered in the form of diet supplements with or as raw materials, i.e., for medical or packaging purposes. within the last few years, polyphenols have also attracted significant attention and they are being tested for their antiviral and antibacterial properties. biomaterials are one of the most rapidly developing fields of science [ ] [ ] [ ] . there is a growing interest in naturally derived compounds which may be isolated from natural sources and then used as raw compounds for biomaterials preparation. materials based on both types of natural polymers, proteins, and polysaccharides have been found as biocompatible and non-toxic for the human body [ ] [ ] [ ] . thereby, they may be applied as implants, wound dressings, metal coatings, etc. despite their excellent biocompatibility, the main issue is that the physicochemical properties of biopolymers are rather poor. moreover, natural polymers may be easily infected by microbes, as they do not possess antimicrobial activity themselves [ ] . therefore, it is necessary to improve biopolymers' physicochemical properties as well as provide antimicrobial activity by their modification. special interest in the biopolymers cross-linkers has been focused on natural compounds. if they contain hydrophilic groups able to form hydrogen bonds, they may be potentially studied as biopolymers cross-linkers. effective and safe modifiers such as polyphenolic acids have been studied in recent years [ ] . polyphenols constitute a large group of organic compounds, covering a wide range of complex structures. they contain numerous phenolic rings in their structures, with prevalent carboxylic and hydroxyl groups. they may be divided into two groups-phenolic acids and phenolic alcohols. over seven hundred polyphenolic compounds have already been identified as derivatives from natural sources. they are biosynthesized naturally by plants and marine organisms, from which they are commonly extracted. polyphenols include flavonoids, phenolic acids, stilbenes, and lignans. as a group, they hold a special position in biological science for their unique biological properties [ ] . phenolic acids support human health protection against chronic degenerative ailments [ ] . they are known to present preventive properties against many diseases, e.g., cardiovascular disease, osteoporosis, neurogenerative disease, diabetes mellitus, and even against cancers. moreover, polyphenols demonstrate active properties against the cells' metabolic process, as they may block cell propagation and apoptosis [ , ] . the use of natural compounds such as polyphenolic acids is a novel, economical, simple to use, and environmentally friendly approach to health issues. tannins are a group of polyphenols which commonly occur in nature. they may be easily extracted from plants. the extraction method which is employed in the case of tannins influences the chemical nature of the compounds, their molecular weight, and contamination. analyzing the isolation conditions, choosing the type of a plant (as well as a plant part i.e., leaves or roots, season), as well as a solvent, deciding on the number of repeating series for final extraction, etc. are also essential. therefore, it is difficult to find extraction methods which would give the same resulting compounds. it suggests that each extraction method should be followed by the final product characterization [ , ] . tannic acid (ta; figure ) is a natural tannin from the phenolic acid group and consists of a central glucose unit and ten gallic acid molecules attached to it [ ] . it may be isolated from both herbaceous and woody types of plants [ ] . ta is one of the main examples of tannins which can be efficiently extracted from natural sources with high efficiency, and thus, it attracts much scientific interest. moreover, it has a higher molecular weight than, for instance, gallic acid. as a result, it has been studied as a biopolymer cross-linker, or an active additive to metals coatings and nanoparticles. nowadays, it is especially important to search for natural compounds which are biocompatible and show antiviral and antibacterial activity to protect human organisms against pathogenic factors. natural compounds may be considered as promising ones to support the fight against many diseases. tannic acid can be offered as a valuable component of supplements as well as different types of useful materials. in this review, however, i would like to emphasize the antiviral and antibacterial properties of tannic acid, which seem to be of great significance, especially in the time of the covid- pandemic, which has adversely affected human lives, and its consequences show how important it is to carry out studies aimed at health protection. various tannins are often found in plant extracts, and they may differ in their antiviral activity [ ] . such activity has been demonstrated in the case of tannic acid, for which it depends on its molecular weight as well as the extraction method and conditions during the process. there are several studies which prove the antiviral activity of tannic acid (table ) . tannic acid activity against influenza a virus is times higher than another phenolic acid, gallic acid. the total number of galloyl residues determines its antiviral activity. ta (high molecular weight tannin) activity is related to the inhibition of both the influenza a virus (iav) receptor binding and neuraminidase activity. gallic acid, as a low molecular weight tannin, inhibits neuraminidase but not hemagglutination. thereby, tannic acid exhibits higher activity against iav [ ] . it has been reported that a tannic acid-enriched extract inhibits human papilloma virus (hpv) type infection. hpv is a non-enveloped type of virus which can cause genital warts or cervical carcinoma. a vaccine against hpv is available; however, it protects only against a minor fraction of over serotypes. high vaccination cost considerably limits the frequency of its use by humans. thereby, there is a huge demand for cheap and effective active compounds against hpv. ta may bind the hpv host cell receptor and, as a result, inhibit its attachment [ ] . tannic acid is a component of many traditional chinese medicaments. its effectiveness has long been known against noroviruses (novs). ta inhibits norovirus binding to hbga receptors. in this tannic acid has many unique properties. it has antimutagenic and antitumor properties. tannic acid shows activity against microorganisms (bacteria and viruses). it acts also as an antioxidant and homeostatic agent. moreover, tannic acid can neutralize free radicals which cause different diseases' development such as allergies, diabetes, parkinson's, alzheimer's, and cardiovascular. also, it has been proved that tannic acid has anticancer activity. currently, tannic acid is also being studied as an organic polymer additive, because it reveals bioactive properties and enhances the properties of materials for biomedical applications. thereby, it is an interesting active compound which may be used as an ingredient in nutritional products, and also various types of consumables [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . nowadays, it is especially important to search for natural compounds which are biocompatible and show antiviral and antibacterial activity to protect human organisms against pathogenic factors. natural compounds may be considered as promising ones to support the fight against many diseases. tannic acid can be offered as a valuable component of supplements as well as different types of useful materials. in this review, however, i would like to emphasize the antiviral and antibacterial properties of tannic acid, which seem to be of great significance, especially in the time of the covid- pandemic, which has adversely affected human lives, and its consequences show how important it is to carry out studies aimed at health protection. various tannins are often found in plant extracts, and they may differ in their antiviral activity [ ] . such activity has been demonstrated in the case of tannic acid, for which it depends on its molecular weight as well as the extraction method and conditions during the process. there are several studies which prove the antiviral activity of tannic acid (table ) . tannic acid activity against influenza a virus is times higher than another phenolic acid, gallic acid. the total number of galloyl residues determines its antiviral activity. ta (high molecular weight tannin) activity is related to the inhibition of both the influenza a virus (iav) receptor binding and neuraminidase activity. gallic acid, as a low molecular weight tannin, inhibits neuraminidase but not hemagglutination. thereby, tannic acid exhibits higher activity against iav [ ] . it has been reported that a tannic acid-enriched extract inhibits human papilloma virus (hpv) type infection. hpv is a non-enveloped type of virus which can cause genital warts or cervical carcinoma. a vaccine against hpv is available; however, it protects only against a minor fraction of over serotypes. high vaccination cost considerably limits the frequency of its use by humans. thereby, there is a huge demand for cheap and effective active compounds against hpv. ta may bind the hpv host cell receptor and, as a result, inhibit its attachment [ ] . tannic acid is a component of many traditional chinese medicaments. its effectiveness has long been known against noroviruses (novs). ta inhibits norovirus binding to hbga receptors. in this study, different forms of hydrolysable tannins were tested. as research shows, ta has the strongest inhibitor which limits the novs proteins binding to their hbga receptors [ ] . herpes simplex virus type (hsv- ) infections are very common, and the virus is an important human pathogen. medicinal plants have been used for many years for treating human diseases. it was proven that herbal extracts containing tannic acid show activity against hsv- in in vitro studies and have low cytotoxicity. tannic acid inhibits hsv- replication, as indicated by the relative absence or reduction of cpe. most antiviral drugs are toxic, and hence the benefits connected with tannic acid application should be emphasized [ ] . tannic acid was also tested as a silver nanoparticle modifier in effective herpes virus infection treatment [ ] . the results confirmed the ability of hydrogels with silver nanoparticles modified by ta to affect viral attachment, impede penetration and cell-to-cell transmission, although profound differences in the activity displayed by the tested preparations toward herpes simplex virus type (hsv- ) and type (hsv- ) were noted. the effectiveness was also tested in in vivo conditions [ ] . such studies provide pre-clinical assumptions that tannic acid-based materials may be applied against viruses. the antiviral effects of tannic acid-modified silver/copper nanoparticles against hsv- by in vitro and in vivo methods were also confirmed, using a murine model of a hsv- genital infection [ , ] . tannins were also studied against some viruses at various stages of infection by determining their influence on the viruses' replication. ta was studied against human immunodeficiency virus (hiv) as it inhibits hiv replication in h lymphocytes [ ] . hydrolysable tannins were studied against hiv by xu et al. [ ] the inhibition effect against human immunodeficiency virus (hiv)- protease was confirmed [ ] . uchiumi et al. [ ] also studied reaumuria hirtella and quercus coccifera extracts which contain tannic acid. tannic acid shows high antiviral effectiveness. it has been studied as a component of extracts isolated from natural sources, but also as a pure compound. its antiviral activity is based on the virus cell membrane adsorption, which results in inhibiting the virus activity and the ability to attack human cells. however, tannic acid is not commercially registered as a supplement or a drug. it suggests that further studies have to be carried out. in vivo studies on animals showed promising results, however, there is a lack of clinical evidence concerning tannic acid activity. it is difficult to carry out phenolic acid studies because it has a high ability to bind proteins. thereby, before tannic acid is considered as a potential antiviral compound, its influence on various proteins present in the human body has to be determined. tannic acid has drawn significant interest, owing to its broad spectrum of chemical and biological properties. the rapid spread of multidrug-resistant bacteria has influenced demand for effective antimicrobial agents which reveal more direct bactericidal mechanisms [ ] . antibiotic resistance is one of the main challenges in antibacterial testing. it leads to higher medical costs, prolonged hospitalization, and increased mortality rate. an expanding list of infections (i.e., pneumonia, tuberculosis, blood poisoning, gonorrhea, and foodborne diseases) shows that antibiotic treatment is becoming more difficult, and sometimes impossible, as antibiotics are becoming less effective. there is a need for new natural antibacterial compounds; also, a better understanding of the mechanism of their actions on bacteria is important. the antimicrobial activity has been demonstrated for many tannins extracted from plants ( table ) . tannin-rich plant extracts have shown high antimicrobial effects. their antibacterial activity depends on conditions such as ph, temperature, type of solvent/matrix, and action time [ , ] . tannins are multidentate ligands which may bind to proteins, mainly by hydrophobic interactions and hydrogen bonds [ , ] . as a result, the inhibition of bacteria metabolism is achieved. dabbaghi et al. [ ] have reported the tannic acid activity against staphylococcus aureus and escherichia coli which dependend on the phenolic hydroxyl groups content. tannic acid was used as a polymer cross-linker, and final hydrogels showed antibacterial activity against both types of bacteria in the case of which the increased activity was related to increasing content of ta in the material. anti-infectious properties have also been demonstrated for tannins isolated from green tea extract. they are active against streptococcus pyogenes, which was discussed in the paper by hull vance et al. the results obtained during studies indicated that the extract addition inhibited the attachment of the bacteria to the kidney epithelial cells in a dose dependent manner [ ] . the antibacterial activity of tannins obtained by extraction from anthemis praecox link was studied by belhaoues et al. the tannic acid showed a broad spectrum of activity, especially against staphylococcus aureus and enterococcus faecalis, which suggests that gram-positive bacteria were most susceptible to tannic acid than gram-negative ones. tannic acid functions as an inhibitor of the nora efflux pump, which is considered as the main mechanism responsible for its antibacterial activity [ ] . an antibacterial effect was also detected for quercus infectoria galls extract, which contains tannic acid as the main phenolic compound. a gel containing the extract was prepared by mixing it with cholesterol and soy lecithin. the obtained forms showed antibacterial efficiency against p. aeruginosa and s. aureus. however, the results of long-term preclinical studies need to be confirmed by further investigation [ ] . tannic acid is a component of neolamarckia cadamba fruits extracts, which demonstrate antibacterial effectiveness against many types bacteria, such as e. coli, p. aeruginosa, y. enterocolitica, s. aureus, b. cereus, and l. innocua. tannic acid content was determined as one of the main components in the obtained extracts. the study results suggested that at higher concentrations, the extracts inhibited the sugar and amino acid uptake, which is one of the main mechanisms of bacterial growth inhibition [ ] . tannic acid antibacterial activity has been proven on gram-positive and gram-negative bacteria. as gram-negative bacteria have been a great challenge to modern medicine, the reported ta activity against them has been of most significance. however, there is a lack of preclinical and clinical studies of the effectiveness of tannins against bacteria. only such research would provide complete data concerning their influence on bacteria cells in the presence of normal human cells. in case the compound showed antibacterial effectiveness, a risk would exist that it would show cytotoxicity to somatic cells. therefore, it is important to search for compounds presenting antibacterial effectiveness which would not be toxic to human cells. in vivo studies would show a wide range of living cells and tissues responses to tannic acid. moreover, the examination of bacteria strains which pose the highest risk of infection is important. tannic acid activity against viruses is related to the inhibition of receptor binding and the influence on their activity. as it is binds to the cell receptor, it inhibits viruses' attachment to the different types of surfaces. moreover, it inhibits the attachment of proteins to the cells which are necessary for the metabolite processes [ , , ] . most bacteria can be broadly classified as gram-positive or gram-negative. gram-positive bacteria have cell walls composed of thick layers of peptidoglycan. gram-positive cells stain purple when subject to a gram stain procedure. gram-negative bacteria cell walls have a thin layer of peptidoglycan. gram-positive bacteria are easier to kill. gram-negative bacteria are not destroyed by certain detergents which easily kill gram-positive bacteria [ ] . the antibacterial effectiveness of tannins is explained by their ability to pass through the bacterial cell wall up to the internal membrane, interference with the metabolism of the cell, and -as a result-their destruction. in gram-positive bacteria, the activity of tannins is rapid. however, in gram-negative bacteria, it is slower as a result of the bilayered membrane presence. gram-negative bacteria are more harmful and cause certain diseases; so, the examination of this group of bacteria is especially required. tannic acid has been studied against different types of bacteria so far, both, gram-positive (mainly staphylococcus aureus) as well as gram-negative ones (mainly escherichia coli) [ , , [ ] [ ] [ ] . tannic acid inhibits the bacteria attachment to the surfaces [ ] . a lack of bacteria adhesion to the surface results in bacteria cell death. moreover, the sugar and amino acid uptake are inhibited by tannic acid what limits the bacteria growth [ ] . however, phenolic acid activity against bacteria depends on its concentration, ph, temperature, and type of matrices to which tannic acid was added. therefore, all types of composites have to be examined in antimicrobial studies [ , ] . tannic acid has been study as additive to produce biomaterials (table ). rheological measurements of tannic acid-collagen complexes have been reported as hydrogel formation studies [ ] . hydrogels revealed overall pseudoplastic rheological behavior. the formed hydrogels showed viscoelastic behavior that prevails over the viscous contribution, as shown from oscillatory rheometric results. their high viscosity ensures adhesion to wound and their elasticity prevents against the material damage during application. tannic acid-collagen hydrogels studies also presented a significant increase in the antioxidant activity of hydrogels with ta, in comparison to pure collagen. tannic acid release from such hydrogels was examined. in the case of matrices, the release is faster, which is typical of porous structures. ta released from hydrogels is delivered into the direct spot from the material [ ] . tannic acid-collagen hydrogels were studied with estrogen receptor-positive breast cancer cells, triple-negative breast cancer cells, and normal breast epithelial cells [ ] . recepto-positive breast cancer cells were characterized as more sensitive to ta influence. the fact that released ta induced caspase-mediated apoptosis makes another interesting observation. tannic acid-collagen complexes have also been successfully studied against a melanoma cancer cells [ ] . the presence of tannic acid inhibits the melanoma cancer cells growth. such studies provide a potential for further studying the anticancer properties of tannic acid incorporated into biopolymers. ta-collagen complexes may also be formed by microwave heating. a thermal analysis was used to determine the tannic acid influence on collagen-based materials. the results showed the higher hydrothermal stability of the cross-linked collagen than that without ta which results from stronger ta to collagen bonding [ ] . tannic acid was studied as a cross-linker for a cell-laden collagen scaffold fabricated via cell-printing. ta addition resulted in both improved scaffold mechanical properties as well as its cellular preosteoblasts activity. such studies provide evidence that materials based on tannic acid-collagen complexes can be obtained by bio-printing [ ] . tannic acid contains many hydroxyl groups which may cause hydrogen interactions with amine groups of chitosan [ ] . it is the basis for the cross-linking process which leads to the improvement of chitosan-based materials properties. ta-chitosan complexes are used to obtain thin solid films. after tannic acid addition, the material's structure is changed into an anhydrous crystalline conformation when compared to a neat chitosan film. the presence of tannic acid improves the mechanical properties of the films and decreases the degradation rate [ , ] . moreover, ta added to chitosan improves cell viability, which was determined by carrying out tests by seeding cells on the thin film surface [ ] . tannic acid addition to chitosan results in the decrease of bacteria adhesion, as well as the therapeutic release of phenolic acid into its surrounding at the ph = . [ ] . thereby, such mixtures may be proposed as coatings, e.g., to cover a metal surface or act as antibacterial protection during implantation. tannic acid-chitosan films were tested as drug delivery systems (i.e., doxorubicin hydrochloride) for anticancer treatment [ ] . the abundant carboxylate groups in such a mixture increased the loading amount of the drug and decreased its rapid release. the concentration of the released compounds may thereby be modified by the addition of tannic acid, which acts as a cross-linker. tannic acid was also tested as a chitosan cross-linker in the hydrogel form [ ] . chitosan molecular weight is an important factor. hydrogels based on medium molecular weight chitosan showed a reduced degree of swelling when compared to those containing high molecular weight. high molecular weight chitosan has polymeric chains longer than the medium weight one. in such a case, tannic acid has a lower ability to bind functional groups which are more distant in the chain. as a result, the chitosan of medium molecular weight is vulnerable to the cross-linking process. moreover, the higher tannic acid content in the hydrogel composition results in a higher crosslinking density in the hydrogels and reduces the swelling degree. agarose/tannic acid hydrogel scaffolds were fabricated for drug delivery purposes [ ] . tannic acid was studied in release tests, where its concentration was determined in the dependence on the medium ph. the prepared hydrogels showed anti-microbial and anti-inflammatory properties as well as a lack of cytotoxicity. as a result, the proposed hydrogels may be studied by in vivo methods, as they are promising for wound dressing applications. agarose-based hydrogels with tannic acid addition show improved mechanical properties than those without ta addition. the wound healing process is stimulated by the tannic acid presence. moreover, hydrogels are characterized by high biocompatibility [ ] . agarose functionalized by tannic acid was tested as titanium, stainless steel, and silicon coating via direct adsorption [ ] . such coating effectively reduces the adsorption of bovine serum albumin and the adhesion of escherichia coli and t fibroblasts. it is a promising modification of metal surfaces, aimed at the enhancement of their biological properties. starch may interact with phenolic acids by non-covalent bonding formation. a detailed mechanism of interactions has been suggested by zhu [ ] . the complexes were formed by hydrogen interactions between phenolic acid and a polymeric chain. the obtained forms had highly intermolecular, cross-linked, and gel-like network structures; also, the ta content was lower inside and higher outside the material. such complexes are not homogeneous, which is caused by competing interactions with water molecules. studies of the ph dependence should be carried out, since it would allow fabricating homogeneous structures for potential biomedical applications [ ] . within the past years, few novel papers of tannic acid-starch complexes have been published. starch is difficult to modify because of its low solubility in polar solvents. it has significant biological properties; however, more studies of starch-based materials cross-linked by tannic acid should be carried out. such materials present great potential for biomedical applications, but they also need to be studied in in vitro and in vivo tests. hyaluronic acid-based hydrogels undergo rapid degradation processes which limit the range of their applications. tannic acid has been studied as a hyaluronic acid physical cross-linker in a hydrogel form [ ] . it interacts by hydrogen bonding and enhances the material physicochemical properties. the inhibition of degradation by hyaluronidase was noticed. moreover, for such hydrogels, an increase in cell adhesion to the surface and their proliferation was observed, with no sign of cytotoxicity. the prepared hydrogels possess also antioxidant properties. significant enhancement of hyaluronic-acid based hydrogels by tannic acid addition may provide great potential for extending the scope of their biomedical applications [ ] . tannic acid was used to form a complex with silk by non-covalent interactions. gel-like forms based on silk fibroin cross-linked by tannic acid showed improved wet-adhesive properties and stability [ , ] . ta and silk sericin may be conjugated via hydrogen bonding interactions. the mixture may be then deposited on the titanium (ti) surfaces through surface adhesive trihydroxyphenyl groups in ta [ ] . the modified ti surface showed good protein repellent as well as platelet, bacterial anti-adhesive properties, and low cytotoxicity. tannic acid-silk hydrogels presented antibacterial efficiency against s. aureus, candida albicans, cornebacterium, and e. coli. in vivo studies confirmed that applying such hydrogels significantly accelerates the wound healing process. tannic acid is a naturally derived compound which has attracted scientific interest owing to its unique antimicrobial properties. tannic acid is an interesting compound studied due to its antiviral as well as antibacterial effectiveness. activity against different viruses, i.e. influeneza a, papilloma, noroviruses, herpes simplex type and , and human immunodeficiency virus (hiv) were also reported. moreover, ta showed activity against gram-positive and gram-negative bacteria e.g., staphylococcus aureus, escherichia coli, streptococcus pyogenes, enterococcus faecalis, pseudomonas aeruginosa, yersinia enterocolitica, listeria innocua. to sum up, tannic acid is an interesting natural compound with noteworthy antiviral and antibacterial properties confirmed by in vitro methods. however, there is a lack of preclinical and clinical examination results of ta effectiveness in real-time studies. if in vivo studies confirmed its activity, a great opportunity for a large scale industrial use would emerge, mainly for biomaterials fabrication. so far, tannic acid has been tested in combination with collagen, chitosan, starch, agarose, hyaluronic acid, and silk. in each case, it acts as a cross-linker; however, it is also released from material and may influence the course of medical treatment. tannic acid has antimicrobial, antioxidant, and anticancer properties. however, its release rate has to be studied to exclude its toxicity, which is correlated with its concentration. further in vivo studies are required to exhibit multifunctional response on implanted materials with tannic acid, where the local release occurs. novel studies showed an excellent ability of tannic acid to bind to biopolymers, but what is more important, is that its biological properties have been proven. recent studies put much attention to its potential application in anticancer treatment; however, such tests should also be performed on cancer tissues. tannic acid may be called the cross-linker of the future because of its significant novel properties. funding: 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enhanced gelation and antibacterial activities tannic acid-assisted deposition of silk sericin on the titanium surfaces for antifouling application key: cord- -w spqqt authors: huan, yuchen; kong, qing; mou, haijin; yi, huaxi title: antimicrobial peptides: classification, design, application and research progress in multiple fields date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: w spqqt antimicrobial peptides (amps) are a class of small peptides that widely exist in nature and they are an important part of the innate immune system of different organisms. amps have a wide range of inhibitory effects against bacteria, fungi, parasites and viruses. the emergence of antibiotic-resistant microorganisms and the increasing of concerns about the use of antibiotics resulted in the development of amps, which have a good application prospect in medicine, food, animal husbandry, agriculture and aquaculture. this review introduces the progress of research on amps comprehensively and systematically, including their classification, mechanism of action, design methods, environmental factors affecting their activity, application status, prospects in various fields and problems to be solved. the research progress on antivirus peptides, especially anti-coronavirus (covid- ) peptides, has been introduced given the covid- pandemic worldwide in . alexander fleming discovered lysozyme in , and this discovery marked the birth of modern innate immunity. since then, antibiotics and antimicrobial peptides (amps) have been discovered. a total of , amps have been reported in the antimicrobial peptide database (apd ) updated on august , . different types of amps have the following commonalities: their number of amino acid residues is between and (average: . ), and almost all amps are cationic (average net charge: . ). however, several anionic amps also exist, and they have several acidic amino acids like aspartic acid and glutamic acid (malkoski et al., ; schittek et al., ; lai et al., ) . the anti-microbial resistance of microorganisms is becoming increasingly serious with the abuse of antibiotics in medicine, agriculture and animal husbandry, especially in developing countries. research from kenya has detected substantial amounts of antibiotic residues in edible meat (ayukekbong et al., ) . the prevalence of vancomycin-resistant enterococcus (vre) and methicillin-resistant staphylococcus aureus (mrsa) is increasing in clinical medicine, so the countermeasures are urgently needed to address these bacterial infections. however, from the http://aps.unmc.edu/ap/ perspective of pharmaceutical companies, the development of new antibiotic drugs results in low profit. thus, replacing antibiotics has become a consideration in the pharmaceutical, agricultural, animal husbandry, and food industries. research on amps is continuously developing and considerable amounts of data on amps have been stored in amp databases. however, the mechanism of amps remains incompletely understood, and further work needs to be performed to determine the relationship between different physicochemical properties to obtain low-cost and highly safe amps with remarkable antimicrobial effects and the specificity and a high capacity for synergies of amps should also be further developed (lazzaro et al., ) . the diversity of natural amps causes difficulty in their classification. amps are classified based on ( ) source, ( ) activity, ( ) structural characteristics, and ( ) amino acid-rich species (figure ). the sources of amps can be divided into mammals (human host defense peptides account for a large proportion), amphibians, microorganisms, and insects according to statistical data in apd . the amps found in oceans have also attracted widespread attention. mammalian antimicrobial peptides are found in human, sheep, cattle, and other vertebrates. cathelicidins and defensins are the main families of amps. defensins can be divided into α-, β-, and θ-defensins depending on the position of disulfide bonds (reddy et al., ) . human host defense peptides (hdps) can protect human from microbial infections but show different expressions in every stage of human growth. for example, cathelicidin ll- , a famous amp derived from the human body, is usually detected in the skin of newborn infants, whereas human betadefensin (hbd- ) is often expressed in the elderly instead of the young (gschwandtner et al., ) . hdps can be identified in many parts of the body such as skin, eyes, ears, mouth, respiratory tract, lung, intestine, and urethra. besides, amps in human breast milk also play an important role in breastfeeding because it can decrease the morbidity and mortality of breastfeeding infants (field, ) . what's interesting is that casein (peptide derived from β-casein - aa), identified in colostrum, shows different levels in preterm human colostrum and term human colostrums . dairy is an important source of amps, which are generated through milk enzymatic hydrolysis. several amps have been identified from α-lactalbumin, β-lactoglobulin, lactoferrin, and casein fractions, and the most famous peptide obtained is lactoferricin b (lfcinb) (sibel akalın, ) . furthermore, whether the amps derived from dairy products can be used for dairy preservation is also an interesting subject to develop. in addition to antimicrobial activity, hdps, such as cathelicidins and defensins, also affect immune regulation, apoptosis, and wound healing (wang, ) . antimicrobial peptides from amphibians play an important role in the protection of amphibians from the pathogens that have induced the global amphibian population decline (rollins-smith, ). frogs are the main source of amphibian amps and the most famous amp from frogs is magainin; the skin secretions of frogs from genera xenopus, silurana, hymenochirus, and pseudhymenochirus under the pipidae family are rich in amps (conlon and mechkarska, ) . furthermore, cancrin, which has an amino acid sequence of gsaqpykqlhkvvnwdpyg, has been reported as the first amp from the sea amphibian rana cancrivora (lu et al., ) . this marks a broader source of amps of amphibians. figure | classification of antimicrobial peptides. (semreen et al., ) . myticusin-beta is an immune-related amp of mytilus coruscus and a promising alternative to antibiotics (oh et al., ) . moreover, ge , known as pardaxin, is a marine amp and the ge -based vaccine has shown the ability to enhance antitumor immunity in mice (huang et al., ) . the activity of amps can be divided into categories according to the statistics of the adp database. these categories can be summarized as antibacterial, antiviral, antifungal, antiparasitic, anti-human immunodeficiency virus (hiv), and anti-tumor peptides (figure ). antibacterial peptides account for a large part of amps and have a broad inhibitory effect on common pathogenic bacteria, such as vre, acinetobacter baumannii, and mrsa in clinical medicine and s. aureus, listeria monocytogenes, e. coli in food and salmonella, vibrio parahaemolyticus in aquatic products. many natural and synthetic amps like nisin, cecropins and defensins have shown good inhibition activity to gram-positive bacteria and gram-negative bacteria. in recent research, amps p (yirkirrffkklkkilkk-nh ) and p (syerkinrhfktlkknlkkk-nh ), which are designed based on aristicluthys nobilia interferon-i, inhibit mrsa and show a low cytotoxicity . antifungal peptides are a subclass of amps that address fungal infections with enhanced drug resistance. many afps have shown excellent anti-fungal activities against common pathogenic fungi, such as aspergillus and candida albicans in clinical medicine, yeast, filamentous fungi (e.g., aspergillus flavus), mold in food and agriculture. except for brevinin, ranatuerin, cecropins, many synthetic peptides also show good antifungal activity. for example, aurh , derived from aurein . , can effectively treat c. albicans infection, which has a lethal rate up to % (madanchi et al., ) . aflatoxin, which is a carcinogen produced by a. flavus, is harmful to the human body. many afps can inhibit the growth of a. flavus. for example, an afp with a sequence of fpshtgmsvppp can inhibit the growth of a. flavus md . a total of antifungal peptides isolated from lactobacillus plantarum te and their mixture can reduce a. flavus spore formation in fresh maize seeds (muhialdin et al., ) . moreover, two chemically synthesized radish amps show a good inhibitory effect against different yeast species, such as zygosaccharomyces bailii and zygosaccharomyces rouxii (shwaiki et al., ) . viruses cause serious harm to human life and huge economic losses to the animal husbandry. the covid- , which is the recent outbreak, has caused great loss of lives and properties. furthermore, foot-and-mouth disease virus, avian influenza virus (aiv), and hiv are long-term threats to human life. so, it is extremely urgent to solve these problems, and antiviral peptides provide new ways. antiviral peptides show a strong killing effect on viruses mainly by ( ) inhibiting virus attachment and virus cell membrane fusion, ( ) destroying the virus envelope, or ( ) inhibiting virus replication (jung et al., ) (shown in figure ). a recent report has shown that amp epi- mediates the inactivation of virus particles and has good inhibitory activity against foot-and-mouth disease virus . moreover, infectious bronchitis virus (ibv) is the pathogen of infectious bronchitis and the inoculation of swine intestinal amp (siamp)-ibv mixed solution remarkably reduced the mortality of chicken embryos compared with the ibv infection group, showing the good inhibitory activity of siamp on ibv (sun et al., ) . anti-hiv peptides are a subclass of anti-viral peptides. the most important examples of these peptides include defensins (including αand β-defensins, which have different mechanisms), ll- , gramicidin d, caerin , maximin , magainin , dermaseptin-s , dermaseptin-s , siamycin-i, siamycin-ii, and rp (madanchi et al., ) and antiviral peptide fuzeon tm (enfuvirtide) has been commercialized as an anti-hiv drug (ashkenazi et al., ) . due to the global spread of the covid- (figure a ), the antiviral peptides against the coronavirus will be discussed in more detail. coronaviruses (covs) belong to the family coronaviridae; they are enveloped viruses with a positive-sense single-stranded rna genome and have a helical symmetry (franks and galvin, ) . covs, including severe acute respiratory syndrome cov (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) (mustafa et al., ) , and the recent outbreak of covid- have caused serious threats to human life and property. covs can cause lifethreatening respiratory diseases and the viral particle is formed by spike glycoprotein (s), the envelope (e), the membrane (m), and the nucleocapsid (n) (vilas boas et al., ) . it should be noted that their infectivity requires viral spike (s) protein. fusion inhibitor peptides combine with the s protein to interfere with its folding and prevent infection. besides, the s domain of the sars-cov s protein contains heptad repeat hr and hr sequences. peptide hr (hr : sltqinttlldltyemlslqqvvkalnesyidlkel) and its lipid-binding peptide is highly similar or even identical to the near-membrane portion of s protein ferredoxin, which interferes with refolding into post-fusion fusion-catalyzing domains (fds) (du et al., ; park and gallagher, ) . according to recent research, the lipopeptide ek c , derived from ek (sldqinvtfldleyemkkleeaikkleesyidlkel), is the most effective fusion inhibitor against covid- s proteinmediated membrane fusion . homology modeling and protein-peptide docking showed that temporin has potential therapeutic applications against mers-cov (marimuthu et al., ) . two amps from the non-structural protein nsp of sars-cov, k , and k , can inhibit sars-cov replication (ke et al., ) . furthermore, rhesus thetadefensin (rtd- ) treated animals have a marked reduction in mortality in the presence of sars-cov while the peptide alone shows airway inflammation and the one possible mechanism of action for rtd- is immunomodulatory (wohlford-lenane et al., ) . in general, amps against coronavirus can be roughly classified as i) peptides derived from hr , hr and rbd subunits of the spike protein, ii) peptides derived from other amps, iii) peptides derived from non-structural protein (mustafa et al., ) . furthermore, molecular docking analysis indicated that peptides were employed to disrupt the interaction between covid- and ace (angiotensin-converting enzyme ) to inhibit covid- entrance in cells ( figure b ) (souza et al., ) . finally, it should be noted that this therapy lacks clinical trials and the main method of animal experiments is an intranasal administration. this reminds us that nasal drug delivery (ndd) is a potential therapy for amps as anti-coronavirus drugs. besides, the antiviral database avpdb includes numerous antiviral peptides. parasitic protozoa can cause diseases in human and animals through a variety of routes, including animal-to-person or person-to-person contact, water, soil, and food (chalmers et al., ) . and with the increase in parasite drug resistance, the need for new treatments has increased. antiparasitic peptides show their killing effect on parasites which cause diseases such as malaria and leishmaniasis (mangoni et al., ; rhaiem and houimel, ) and amps like cathelicidin, temporins-shd show high inhibition activity against parasites (abbassi et al., ) . in recent research, epi- , a marine synthetic amp, can remarkably inhibit trichomonas vaginalis by destroying its membrane (neshani et al., ) . the peptide jellein derived from bee royal jelly which has introduced above and -amino acid amp kdel (lysine, aspartic acid, glutamic acid, and leucine) has shown a significant effect on the leishmania parasite (cao et al., ; zahedifard et al., ) . however, it should be noted that their mechanisms are not the same. cyanobacterial peptides differ from higher-eukaryote amps because their antiparasitic action depends on specific protein targets. thus, these target parasites can be distinguished accurately even though they belong to the same family or genus (rivas and rojas, ) . the acps show anticancer mechanisms by ( ) recruiting immune cells (such as dendritic cells) to kill tumor cells, ( ) inducing the necrosis or apoptosis of cancer cells, ( ) inhibiting angiogenesis to eliminate tumor nutrition and prevent metastasis, and ( ) activating certain regulatory functional proteins to interfere with the gene transcription and translation of tumor cells (wu d. et al., ; ma et al., ) . tritrpticin and its analogs induce considerable toxicity toward jurkat cells in vitro, whereas indolicidin and puroindoline a can also act as acps (arias et al., ) . it should be noted that both net charge and hydrophobicity play important roles in optimizing the anticancer activity of acps and they can constrain and influence each other. thus, achieving a balance between net charge and hydrophobicity is important for better anticancer activity. besides the peptide mentioned above, anti-inflammatory, anti-diabetic peptides, spermicidal peptides etc. have been noticed, but they are not the same as antimicrobial peptides. simply put, anti-inflammatory peptides decrease the release of inflammatory mediators and inflammatory cytokines (nitric oxide, interleukin- , and interleukin- β) and some of them also inhibit inflammatory signals like nf-κb, mapk, and jak-stat pathways (meram and wu, ; gao et al., ) . anti-diabetic peptides play their function by modulating the g proteincoupled receptor kinase (grk / ) or activating glucagonlike peptide- (glp- ), glucagon receptors (marya et al., ; graham et al., ) . however, it is not accurate to classify these types of peptides as amps and bioactive peptides may be more convincing. proline is a typical non-polar amino acid. pramps behave differently from other amps, that is, they enter bacterial cytoplasm by the inner membrane transporter sbma instead of killing bacteria through membrane destruction (mattiuzzo et al., ) . once in the cytoplasm, pramps target ribosomes and block the binding of aminoacyl-trna to peptidyltransferase center or trap decoding release factors on the ribosome during the termination of translation to interfere with protein synthesis (seefeldt et al., ) . for instance, tur a, which is an orthologous amp of bovine pramp bac discovered from tursiops truncatus, interferes with the transition from the initial phase to the extension phase of protein synthesis by binding to ribosomes. in addition, different pramps lack a high sequence similarity but have short motifs containing repeating proline and arginine (arg) residues (e.g., -ppxr-in bac and -prpxin bac ) (mardirossian et al., (mardirossian et al., , . although pramps mainly kill gram-positive bacteria, ppr-amp , a proline-rich amp identified from crab (scylla paramamosain), exhibits antimicrobial activity against gram-positive and gram-negative bacteria (imjongjirak et al., ) . besides, pieces of research have shown that pramps have immunostimulation activity (li w. et al., ) . tryptophan (trp), as a non-polar amino acid, has a remarkable effect on the interface region of the lipid bilayer, whereas arg, as a basic amino acid, confers peptide charge and hydrogen bond interactions, which are essential properties to combine with the bacterial membrane's abundant anionic component. and it seems that trp residues play the role of natural aromatic activators of arg-rich amps by ion-pair-π interactions (walrant et al., ) , thereby promoting enhanced peptide-membrane interactions (chan et al., ) . in addition to indolicidin and triptrpticin which both are famous amps that rich in arg and trp residues. octa (rrwwrwwr) is also a typical trp-and arg-rich amp that inhibits gram-negative e. coli and pseudomonas aeruginosa and gram-positive s. aureus. and short trp-and arg-rich amps designed based on bovine and murine lactoferricin have also shown strong inhibitory action against bacteria (strøm et al., ; bacalum et al., ) . histidine is a common basic amino acid, and histidine-rich amps show good membrane permeation activity. hv is a histidinerich amp designed based on rr(xh) xdpgx(yh) rr-nh (where x represents i, w, v, and f). this peptide increases the permeability of bacterial cell membranes to cause cell membrane rupture and death. in addition, hv inhibits bacterial movement in a concentration-dependent manner and shows a strong anti-inflammatory effect by inhibiting the production of tumor necrosis factor α (tnf-α) ). an amp designed based on octa has shown good therapeutic potential by replacing its arg residues with histidine (bacalum et al., ) . furthermore, l h , which is designed based on the linear cationic amphiphilic peptide magainin, also shows good antibacterial activity and cell penetration properties by inserting four histidine sequences in leucine and alanine (lointier et al., ) . the r group of glycine is generally classified as a non-polar amino acid in biology. glycine-rich amps, such as attacins and diptericins, widely exist in nature (lee et al., ; kwon et al., ) . these peptides contain % to % glycine residues, which have an important effect on the tertiary structure of the peptide chain. a glycine-rich amp derived from salmonid cathelicidins activates phagocyte-mediated microbicidal mechanisms, which differ from the mechanism of conventional amps (d'este et al., ) . furthermore, the glycine-rich central-symmetrical gg is an ideal commercial drug candidate against clinical gramnegative bacteria . antimicrobial peptides can be divided into four categories based on their structures including linear α-helical peptides, β-sheet peptides, linear extension structure, and both α-helix and β-sheet peptides ( figure ) (lei et al., ) . moreover, progressively cyclic peptides and amps with more complex topologies (including lasso peptides and thioether bridged structures) are reported (koehbach and craik, ) . the membrane-targeting mechanisms of amps can be described through models, including the pole and carpet models and the pole model can be further divided into the toroidal pore and barrel-stave models (figure ). the toroidal pore model is also known as the wormhole model. in this model, amps vertically embedded in the cell membrane accumulate and then bend to form a ring hole with a diameter of - nm (matsuzaki et al., (matsuzaki et al., , . the typical examples of this model are magainin , lacticin q, and arenicin. furthermore, cationic peptides, including tc , tc , and bp , compromise the membrane barrier by creating fluid domains (omardien et al., ) . antimicrobial peptides aggregate with each other, penetrate the bilayer of the cell membrane in the form of multimers, and form channels that result in the cytoplasmic outflow. in severe cases, amps can induce cell membrane collapse and lead to cell death (lohner and prossnigg, ). for instance, alamethicin performs its pore-forming activity by using this model. besides, hairpin amp protegrin- can form stable octameric β-barrels and tetrameric arcs (half barrels) in implicit and explicit membranes by simulations (lipkin and lazaridis, ) . antimicrobial peptides are arranged parallel to the cell membrane. their hydrophilic end faces the solution, and their hydrophobic end faces the phospholipid bilayer. amps will cover the membrane surface that similar to a carpet and destroy the cell membrane in a 'detergent'-like manner (oren and shai, ) . however, this pore-forming mechanism requires a certain concentration threshold and the required concentration of amps is high. human cathelicidin ll- exhibits its activity through this mechanism, and amps with β-sheet structure also play a role in this model (shenkarev et al., ; corrêa et al., ) . polarized light-attenuated total reflection fourier transform infrared spectroscopy (atr-ftir) was used to study the effect of amp cecropin p on the bacterial cell membrane and found that it was an applied flat on the surface of the pathogen's cell membrane to destabilize and eventually destroy the cell membrane . membrane targeting mechanisms (the cell membrane composition differences of bacteria and fungi shown in figure ) can be further refined to address the large differences in the lipid composition of the cell membranes of bacteria, fungi, and mammals. the main lipids in cell membranes include glycerophospholipids (gpls), lysolipids, sphingolipids, and sterols. phosphatidylethanolamine (pe), phosphatidylglycerol (pg), and cardiolipin (cl) are the most common anionic lipids in bacteria, whereas phosphatidylcholine (pc), phosphatidylinositol (pi), pe, and phosphatidic acid (pa) are the main gpls in fungal cell membranes (ejsing et al., ; singh and prasad, ; li et al., ) . furthermore, fungal cell membranes are more anionic than mammalian cell membranes and have higher pc content. meanwhile, ergosterol is the sterol found in the plasma membrane of lower eukaryotes, such as fungi, whereas that of animals contains cholesterol (faruck et al., ) . many amps take advantage of differences in membrane components to exert their effects. antimicrobial peptides are promising to be anti-biofilm agents but it should be noticed that they are different from the cell penetrating peptides (cpps) which typically comprise - amino acids and can translocate across the cell membrane. cpps could be categorized according to physicochemical properties into three classes: cationic, amphipathic, and hydrophobic, but anti-biofilm peptides have stricter requirements for these physicochemical properties. anti-biofilm peptides target the biofilms by different mechanisms including ( ) degradation of signals within biofilms; ( ) permeabilize within cytoplasmic membrane/eps; ( ) modulating eps production etc. and then can address chronic multi-resistant bacterial infections (pletzer et al., ; ribeiro et al., ; guidotti et al., ; derakhshankhah and jafari, ; rajput and kumar, ) . for instance, saap- , synthesized based on ll- , showed activity to prevent biofilm formation by s. aureus and a. baumannii (crunkhorn, ) . the way of amps entering cells is direct penetration or endocytosis. after entering the cytoplasm, amps will identify and act on the target. depending on the target, amps can be divided into the following categories. antimicrobial peptides affect transcription, translation, and assembly into functional peptides through molecular chaperone folding by interfering with related enzymes and effector molecules. for example, bac - targets ribosomes to inhibit protein translation (mardirossian et al., ) , whereas tur a inhibits protein synthesis in e. coli and thermus thermophilus by inhibiting the transition from the initial phase to the extension phase. however, the differences between tur a and bac also lead to various ways of binding to ribosomes and interacting with the ribosomal peptide exit tunnel (mardirossian et al., ) . but some amps' have different targets. for instance, genome-wide transcription shows that the amp dm can affect many important intracellular pathways of protein biosynthesis . chaperones are key proteins for correctly folding and assembling newly synthesized proteins and make them have stereoisomerism, which makes amps have cell selectivity and can prevent cytotoxicity. according to a previous review: both pyrhocoricin and drosocin can prevent dnak from refolding misfolded proteins by inducing a permanent closure of the dnak peptide-binding cavity (kragol et al., ; le et al., ; wrońska and boguś, ) . antimicrobial peptides can affect key enzymes or induce the degradation of nucleic acid molecules to inhibit nucleic acid biosynthesis. indolicidin, a c-terminal-amidated cationic trprich amp with amino acids, specifically targets the abasic site of dna to crosslink single-or double-stranded dna and it can also inhibit dna topoisomerase i (subbalakshmi and sitaram, ) . tfp (tissue factor pathway inhibitor) - tc , which is an amp from tongues, enters the cytoplasm of target cells after the rupture of cell membrane and then degrades dna and rna (he et al., ) . many amps can inhibit various metabolic activities by inhibiting protease activity. for example, histatin has a strong inhibitory effect on the proteases secreted by the host and bacteria. amps enap- and indolicidin inhibit microbial serine proteases, elastase, and chymotrypsin (le et al., ) . cathelicidin-bf is a peptide isolated from the venom of bungarus fasciatus, it can effectively inhibit thrombin-induced platelet aggregation and further block protease-activated receptor (shu et al., ) . antimicrobial peptides inhibit cell division by inhibiting dna replication and dna damage response (sos response), blocking the cell cycle or causing the failure of chromosome separation (lutkenhaus, ) . for instance, app (glaraltrllrqltrqltra), which is an amp with amino acid residues, can efficiently kill c. albicans because of its cell-penetrating efficiency, strong dna-binding affinity, and ability to induce s-phase arrest in intracellular environment . mciz, which has amino acid residues, is an effective inhibitor of bacterial cell division, z-ring formation, and localization (cruz et al., ) . moreover, it has been reported that several afps have damaging effects on the organelles of fungi. for example, histintin can interact with mitochondria, causing the production of ros, and inducing cell death (helmerhorst et al., ) . in addition to intracellular targets, differences in cell wall composition, such as lipopolysaccharide (lps), lipid a and mannoproteins, are potential targets for amps. specifically, gram-positive and gram-negative bacteria are classified based on their bacterial cell wall structure. gram-positive bacteria have a layer of cross-linked peptidoglycan, whereas gram-negative bacteria have an additional outer membrane with an inner leaflet containing only phosphatidic acid and an outer leaflet made of lps. lps has numerous negatively charged phosphate groups, which combine with a salt bridge with a divalent cation (e.g., ca + and mg + ) to form an electrostatic network (nikaido, ) . this electrostatic zone is the main barrier against hydrophobic antibiotics and causes the low permeability of gram-negative bacteria. the main components of the fungal cell wall are mannoprotein, β-glucans and chitin (polymers of , -β-n-acetylglucosamine) and the mutations in the relevant genes of the lps pathway and phospholipid trafficking provide resistance to the amps (cabib, ; spohn et al., ) . mannoproteins in fungal cell walls include a variety of proteins, including structural proteins, cell adhesion proteins (floccrin and lectin) and enzymes involved in cell wall synthesis and remodeling (hydrolytic enzymes and transglycosylase). these proteins differ from human cell membrane proteins and are potential targets of afps (rautenbach et al., ) . furthermore, teichoic acid and lipoteichoic acid in the cell wall are also potential targets of amps and these theories could support the design of amps with low cytotoxicity. antimicrobial peptides have good application prospects. however, amps have the following problems. ( ) amps damage the cell membrane of eukaryotes and cause hemolytic side effects; ( ) rising production costs and technical problems limit their manufacture; ( ) their stability is limited at certain ph; ( ) amps have reduced activity under the presence of iron and certain serum; ( ) amps are easily hydrolyzed by proteases. therefore, the ideal amp should meet the following characteristics: (i) high antimicrobial activity; (ii) low toxicity to mammalian membranes; (iii) high protease and environment stability; (iv) low serum binding capacity and (v) ease of access and low cost production . therefore, designing amps to achieve the desired effect has attracted increasing attention. the rational design of antibacterial peptides should focus on the following five aspects: chain length, secondary structure, net charge, hydrophobicity, and amphiphilicity and these have been mentioned in many studies and this review will focus more on several specific methods of antimicrobial peptide design. site-directed mutation refers to the redesign of natural antimicrobial peptides by adding, deleting or replacing one, or several amino acid residues (torres et al., ) . the de novo design of peptides attaches importance to the design of amphiphilic amps (guha et al., ) . for example, gala is a well-known de novo-designed amp. amphipathic α-helical peptide gala is created by placing protonatable glutamic acid residues in most positions with the spacing of i to i + (goormaghtigh et al., ) . the repeated sequence (xxyy)n, where x and x are hydrophobic amino acids, y and y are cationic amino acids, and n is the number of repeat units, is designed based on the hydrophobicity cycle that mimics natural α-helical amps and successfully designs broadspectrum α-helical amps. sequences (lkkl) and (wkkw) . have the highest selectivity (khara et al., ) . moreover, l l k m w model peptides are also de novo-designed peptides. amphipathic helical properties were conferred by using leucines and lysines, and two tryptophan residues were positioned at the amphipathic interface between the hydrophilic ending side and the hydrophobic starting side. among the model peptides, l k w has good anti-mrsa activity (lee et al., ) . sequence templates can be obtained by comparing a large number of structurally homologous fragments of natural amps (such as hdps) and extracting conservative patterns based on the type of residue (such as charged, polar, hydrophobic, etc.) (zelezetsky and tossi, ) . based on the modification, the parameters, such as helix formation tendency, cationic, amphiphilicity and overall hydrophobicity, can be systematically changed. for instance, cecropin, magainin, protegrin, and lactoferrin have all been used as amp templates (fjell et al., ) . peptides can form nanostructures, such as micelles, vesicles, nanotubes, nanoparticle nanobelt, and nanofibre nanotube, and can increase or impart antibacterial activity to amps during the self-assembly of peptides. for example, kld- (kld) is a self-assembling peptide with amino acid residues that can adopt nanostructures and are known for their tissue engineering properties. the addition of arg residues in kld shows no remarkable change in its β-sheet secondary structure and the self-assembly characteristics of the forming nanostructures (tripathi et al., ) . dimer structure can also be used to enhance the antimicrobial activity of amps and reduce toxicity, but membrane-destabilizing effects are reduced after dimer formation (malekkhaiat häffner and malmsten, ). various chemical modifications of amps, including residue phosphorylation, the addition of d-amino acids or unnatural amino acids (homoarginine), cyclization, halogenation, acetylation, and peptidomimetics, have been used to improve the stability of peptides against proteases. given that the enzyme is stereospecific, the incorporation of unnatural d-amino acids into the amp sequence can reverse the stereochemistry and prevent protease degradation (zhong et al., ) . the so-called peptidomimetics, whose main elements mimic the structure of peptides, are usually produced by modifications, such as chain extension or heteroatom incorporation of existing peptides (patch and barron, ) . ornine, which is an unnatural residue with a positive charge and has a high resistance to protease activity, is also used in non-chemical modification. replacing trp residues with family residues, such as β-dihydrophenylalanine, can stabilize secondary structures and improve antibacterial properties (maurya et al., ) . halogenation is highly related to the activity, specificity, and stability of amps. in the latest report, halogen is introduced into jelleine-i which is a short peptide isolated from the royal jelly of honeybees (apis mellifera) by replacing phenylalanine with a halogenated phenylalanine analog, increasing the antibacterial activity in vitro and anti-biofilm activity. in addition, the proteolytic stability of jelleine- is increased by - times by halogenation (jia et al., ) . the halogenated peptidomimetic α,α-disubstituted β-amino amides are also promising bacteriostatic drugs that have inhibitory effects on more than multi-resistant clinical isolates of gram-positive and gram-negative bacteria (paulsen et al., ) . halogenation is also related to the specificity of amps. the o-fluorine substitution in phenylalanine residues maintains the activity of temporin l on e. coli but leads to the loss of activity on s. aureus and p. aeruginosa (setty et al., ) . three modes of cyclisation, including cyclisation via disulfide bonds, head-to-tail cyclisation and internal bonding between side chains, have been found in natural amps. the synthesis of disulfide bonds often complicates the development of synthetic peptides. the circularisation of the main chain of arenicin- molecule resulted in increased activity against drug-resistant clinical isolates but caused no substantial effect on cytotoxicity (orlov et al., ) . the hdps tachyplesins i, ii, and iii and their cyclic analogs cti, ctii, and ctiii, respectively, have similar structures and activities and can resist bacterial and cancer cells. the cyclisation of the backbone reduces the hemolytic activity and improves the stability of the peptides whilst maintaining effective anticancer and antibacterial activities (vernen et al., ) . capping refers to the addition of specific motifs or modifications, such as amidation at the c-terminus and acetylation at the n-terminus, rendering amps with more natural peptide characteristics. post-translational modifications play an important role in the function of amps and are the most commonly used in peptide design. the c-terminal rana box (consisting of a c-terminal cyclic heptapeptide with a conservative disulfide bond) and amide group are important c-terminal capping methods. for example, the c-terminal amide group of maximin h can enhance antibacterial efficacy without increasing lytic ability (dennison et al., ) . the n-terminal lipidated analog c vg krkp shows enhanced antibacterial activity against various gram-negative bacteria. the functions of n-terminal lipidation include (i) increasing lps neutralization, (ii) increasing stability to proteases and peptidases, and (iii) reducing cytotoxicity (datta et al., ) . furthermore, hydrophobic end labeling is a commonly used method to increase the activity of antimicrobial peptides. acyl lipid peptides have a linear or cyclic structure in which one or more hydrocarbon tails are connected to the n-terminus of a short oligopeptide (chu-kung et al., ) . lipopeptides have covalently attached hydrophobic moieties, such as sterols or fatty acids. aromatic amino acid terminal labeling is also the main hydrophobic terminal labeling method. tryptophan (w) and phenylalanine (f) are the commonly used aromatic amino acids. their large and polarisable residues have an affinity for the interface, and the w/f tag is also sensitive to the differences between ergosterol and cholesterol and can prevent self-assembly. this condition results in low aggregation numbers and high critical aggregation concentrations (schmidtchen et al., ) . peptide conjugation has been the goal of most research in recent years to produce active and stable amps with high selectivity. different side chains or amp fragments can be used aside from the repetition of the same amino acid motifs. for example, conjugating fatty acids with a length of - carbon atoms to the th or th side chain of the d-amino acids of ano-d , improves antibacterial selectivity and anti-biofilm activity. in addition, the new peptide exhibits high stability against trypsin, serum, salt, and different ph environments (zhong et al., ) . the conjugation of different amps can also be performed. for example, the hybrid peptide (pa -gnu ) constructed by the addition of pa to gnu has a high activity and specificity to p. aeruginosa . smamps include a broad family of molecular entities based on the structure and function of amps. however, their backbones are not entirely based on α-amino acids, including β-amino acid oligomers, arylamide oligomers, and phenylene ethynylenes (michael henderson and lee, ) . for instance, smamp , which is a potential drug for intravenous treatment, causes no drug resistance and has a strong inhibitory effect on mrsa and vancomycin-resistant enterococcus faecium (tew et al., ) . peptoids are peptide isomers, in which the side chain is bonded to the main chain nitrogen instead of α-carbon or poly-nsubstituted glycine in which the side chain is connected to amide nitrogen instead of the α-carbon on the main chain (andreev et al., ) . for example, the cationic peptide sa (iowagolfolfo-nh ) and its poly-n-substituted glycine homolog spo (ninonwnangnonlnfnonlnfno-nh ) inhibit the planktonic and biofilm formation of a. baumannii strains, which are susceptible to multi-drug resistance (sharma et al., ) . motifs with specific functions have been reported increasingly. these motifs can be repeated units for combining into new antimicrobial peptides, or specific amino acid combination units appearing at the end (such as capping) of or even in the peptide chain. this motif includes two tripeptide structures, including gly-gly-his or val-ile-his, which are added at the end of the peptide chain. atcun-containing amps in the presence of hydrogen peroxide and ascorbic acid combine with cu + to induce the valence of copper ions between + and + oxidation states and form an atcun-cu (ii) complex, generating ros by fenton-like reactions. extracellular polymeric substances (eps) are important for biofilms and can enhance the resistance of cells to antibacterial agents (flemming, ) . atcun-amps have been used to degrade environmental dna, which is one of the major components of eps. several related practical applications have been reported. for example, the biological activity against carbapenem-resistant enterobacteriaceae is increased by adding this motif to the n-terminus of an alpha-helical amp (such as cm ). besides, the cu-atcun derivative of ov- containing a c-terminal ggc sequence showed high levels of membrane permeation and lipid peroxidation. the concept of catalytic metal drugs has attracted widespread attention although the concept is still in its infancy because of the role of metal ions (alexander et al., ; agbale et al., ) . rana box: rana box is a heptapeptide motif (cglxglc) from the nigrocin family. rana box consists of two cysteine residues that are separated by four or five other residues on the side and can form a cyclic disulfide bond. rana box peptide has shown structural analogies with polymyxin (colistin), and the primary structure of the rana box motif is important in determining bacteriostatic activity (kozić et al., ) . the deletion of the 'rana box' motif will cause the amp antibacterial effect to disappear, but replacing the natural 'rana box' sequence of amps with amidated phenylalanine can expand its efficacy against antibiotic-resistant microorganisms, including mrsa and p. aeruginosa, and reduce cytotoxicity. this phenomenon also shows that the effect of the motif on amps needs to be determined based on the specific situation and is not completely beneficial (bao et al., ) . the lps binding motif (g-wkrkrf-g) can produce a broad spectrum of antibacterial activity when introduced into the c-terminus of temporin- ta and temporin- tb (close isoforms of temporin) (mohanram and bhattacharjya, ) . antifungal peptides have a conserved gxc(x − ) c γ-core motif (residues - , gkcykkdnic; d-isomer) at its n-terminus, which is a cation part of the ring. this conserved motif interferes with the integrity of the plasma membrane of the cell (yount and yeaman, ) . conserved γ-core motifs are directly involved in protein-membrane interactions and strongly contribute to membrane binding (utesch et al., ) . if replace d-phe -pro sequence in peptide chain with d-phe- -abz turn motif ( -abz is an abbreviation of -aminobenzoic acid d-amino acid) in amp tyrc a, and nuclear magnetic resonance shows that this change retains the β-hairpin structure. unlike the traditional β-turn motif, the d-phe- -abz motif can be used as a tool for β-hairpin libraries. the hydrophobic peptide can be formed into the nucleated β-hairpin formation by adding the d-phe- -abz motif. moreover, the inclusion of this part in two designed cationic amphiphilic peptides can produce broadspectrum antibacterial activity and low hemolysis rate (cameron et al., ; cameron et al., ) . the ngr motif is composed of asn-gly-arg, and amps with this structure have strong cytotoxicity ( table ). the data indicate that the new amps containing ngr may bind to cd + or αvβ + tumor cells by binding to cd or αvβ , respectively, to exert anti-tumor activity, especially on cd + tumor cells . the central gxxxg motif can induce strong self-assembly and have been already used in the design of amps (brosig and langosch, ; krauson et al., ) . bovine lactoferrin b is an amp composed of amino acid residues and has antibacterial, antifungal, and antiparasitic activities. the multivalent molecules lfcinb ( - ) and lfcinb ( - ) contain the lfcinb ( - ) motif (rrwqwr) and show inhibition activity against e. coli, p. aeruginosa, and s. aureus. chimeric peptide chimera containing two motifs, namely, the rrwqwr of lfcinb ( - ) and the rllr of bfii computer design includes simple statistical modeling, structureactivity relationships study (abdel monaim et al., ) , neural networks (müller et al., ) , deep learning (veltri et al., ) , word embedding (hamid and friedberg, ) and machine learning. for example, a machine learning method by matlab is proposed based on the concept of scoring the contribution of each amino acid's antibacterial activity (wu x. et al., ) . the genetic algorithm was used to design the amphiphilic α-helical peptide guavalin , which has an uncommon amino acid composition (three tyrosine and three glutamine residues) and interestingly causes membrane hyperpolarization, which is a different mechanism from those of other amps (porto et al., ) . two research methods have been developed based on the research background of quantitative structure-activity relationships: prediction method based on amp therapeutic index and the identification of novel potential amps from the expressed sequence tag database based on the principles of the highly conserved signal peptide subclasses related to amps (juretić et al., ) . in this way, a variety of amp variants can be obtained. if combined with high-throughput screening, it can effectively obtain the desired amp. for instance, some new amps are designed by the combinatorial peptide library of melittin and show higher activity and lower cytotoxicity (krauson et al., ) . cations, such as na + and mg + , may affect amp activity . however, the different valences of metal ions have varied effects on amps. for example, divalent cations show stronger antagonism to bacteria than monovalent cations with thanatin and s-thanatin, which are insect amps (wu et al., ) . in the presence of nacl, the signal response during the association phase remarkably decreased in single-cycle and multi-cycle kinetic experiments, resulting in a decreased association rate. this occurrence may be caused by the shielding effect of nacl between the cationic peptide and the zwitterionic membrane. another possible reason is that na + can bind to the phospholipid bilayer, where the ions interact with the phosphate and the carbonyl oxygen of lipid head groups (sabapathy et al., ) . the reduced activity of synthetic peptide [rllr] under high salt concentration is possibly caused by the destruction of its α-helix structure. table shows that several amps, including histatin, myxinidin, and hepcidin, contain atcun motifs (amino terminal copper and nickel with xxh sequence). iron is the most abundant metal ion in human saliva, but the combination with this metal ion results in the loss of the α-helix of histatin and greatly reduces its antifungal activity (puri et al., ) . however, the coordination of copper (ii) and nickel (ii) ions can induce the formation of ros, which is essential for bactericidal activity (jeżowska-bojczuk and stokowa-sołtys, ). anionic amps have a large number of negatively charged aspartic and glutamic acid residues (lakshmaiah narayana and chen, ) . they require zinc as a functional cofactor and the zinc complex shows stronger antibacterial activity (jiang et al., ) . several of these amps use metal ions to form cationic salt bridges with the negatively charged components of the microbial membrane to penetrate the membrane. anionic amps may attach to ribosomes or inhibit ribonuclease activity when in the cytoplasm (jeżowska-bojczuk and stokowa-sołtys, ). metal ions also affect the self-assembly of peptides. these ions can recognize specific amino acids, such as lysine and glutamic acid, and may form salt bridges between peptide molecules to induce peptide self-assembly. for example, zn + can stabilize the aggregation of peptides on the cell membrane, which results in the enhanced antibacterial effect of dcd- l in the presence of zn + (tian et al., ) . ph many amps are stable and retain their antimicrobial activity in a wide ph range. amps have enhanced activity at low ph because of their basic properties. this condition is related to the protonation of histidine at acidic ph, which promotes electrostatic interactions with anionic surfaces, including lps and the anions of phospholipids, and subsequently enhances antibacterial properties. the effect of ph on the antibacterial activity of amps varies. for example, thanatin's activity at neutral ph is slightly higher than that under acidic conditions. by contrast, the activity of xylan on e. coli, listeria, and c. albicans is remarkably higher at ph . than at ph . (holdbrook et al., ) . the inactivation of the histidinecontaining amp c g-his under low ph conditions involves ph-dependent changes in the state of the aggregates in the solution, because the aggregates, which are sensitive to ph and lipid composition, may be affected by binding and conformation. peptides can also enhance bacterial membrane permeability at low ph (hitchner et al., ) . thrombin-derived c-terminal peptides (tcps) will also change the mode of cd (a protein that is abundant in human plasma) from anti-inflammatory mode to bacterial elimination mode from ph . to ph . (holdbrook et al., ) . a dimer (e.g., p- ) can be created to provide amps with resistance to a higher ph range. the sensitivity of this ph-sensitive amp can be used to achieve a certain targeting effect in practical applications. in addition, charge interaction is one of the most important factors in peptide self-assembly. ph affects the charge state of amino acid and substituent functional groups. therefore, adjusting the ph is the most common method for controlling peptide assembly and disassembly (tian et al., ) . proteases have a strong destructive effect on amps. for instance, ll- , which has the strongest inhibitory effect on chlamydial infection, is inhibited by the protease chlamydial protease-like activity factor (cpaf) secreted by chlamydia (tang et al., ) . studies have been focused on the design of amp carriers to solve this problem (lewies et al., ; nordström et al., ) . the presence of chitosan-silica solid support of kr- peptide can protect it be hydrolyzed by α-trypsin, and the degree of protection is increased by % compared with the free kr- (diosa et al., ) . however, several enzymes, such as protease , esterase and phosphatase , cut the blocking group of the peptide and trigger the self-assembly of the peptide, which positively affects amps (tian et al., ) . antimicrobial peptides can regulate pro-inflammatory reactions, recruit cells, stimulate the proliferation of cells, promote wound healing, modify gene expression and kill cancer cells to participate in the immune regulation of human skin, respiratory infections, and inflammatory diseases (de la fuente-núñez et al., ) . for example, α-defensins hnp- , hnp- , and hnp- showed effective antibacterial activity against adenovirus, human papilloma virus, herpes virus, influenza virus and cytomegalovirus. pulmonary diseases, such as idiopathic pulmonary fibrosis, alveolar proteinosis, and acute respiratory distress syndrome, show elevated levels of amps (guaní-guerra et al., ) . likewise, amps secreted by the paneth cells in the mammalian gut are important to shape the gut microbiota (bevins and salzman, ) . the application of amps in medicine, such as dental, surgical infection, wound healing and ophthalmology is developing now. but there are only three amps that have been approved by fda including gramicidin, daptomycin, and colistin. dental caries, endodontic infections, candidiasis, and periodontal disease are common diseases in the human oral cavity. dental caries is a prevalent oral disease and some acidogenic bacteria like streptococcus sp. are the main cariesassociated pathogens (izadi et al., ) . several amps have good application potential. for instance, peptide zxr- (fkiggfikklwrslla) has shown potent activities against pathogenic bacteria of dental caries, streptococcus mutans, streptococcus sobrinus, and porphyromonas gingivalis and peptide pac- (clinical trial identifier: nct ) that has been sold over the counter in taiwan for treating oral candidiasis (chen l. et al., ) . in surgical infection and wound healing: surgical infection occurs after surgery, burns, accidental injury, skin disease, and chronic wound infections have a serious hazard to human life (thapa et al., ) . several amps have shown the therapeutic potential of these diseases. for example, amp pxl shows pronounced efficacy as an anti-infective agent in burn wounds in mice and amp d a has been in the third phase of clinical trials for treating burn wound infections (björn et al., ) . in ophthalmology: human eyes are prone to be infected by several organisms including bacteria and fungi in which s. aureus, streptococcus pneumoniae, p. aeruginosa, aspergillus spp., and c. albicans are the most relevant pathogens (silva et al., ) . although amps such as lactoferricin b, protegrin- exhibited antimicrobial activity against these pathogenic bacteria, their application in the field of ophthalmology is only at the theoretical stage. with the popularity of contact lenses and the increase in cases of related eye infections, antimicrobial peptides have shown good application prospects in ophthalmology (khan and lee, ) . additional methods need to be performed for the application of amps as drugs in medicine. the main strategies include ( ) constructing precursors to reduce cytotoxicity and improve protease stability, ( ) using amps in combination with existing antibacterial agents, ( ) inducing the correct expression of amps with appropriate drugs and using engineering probiotics as vectors to express amps. for example, in the field of wound repair, different formulation strategies, such as loading amps in nanoparticles, hydrogels, creams, gels, ointments, or glutinous rice paper capsules, have been developed to effectively deliver amps to the wound (borro et al., ; thapa et al., ) . in recent research, the sponges developed from modified starch and hs-peg-sh are covalently immobilized with amp showed effective antibacterial activity (yang et al., ) . more technical means, including pheromone-labeled amps, local environment-triggered amps (enzyme precursor drug release system, ph-activated amps, etc.), have been developed to improve the targeting mechanism of amps. furthermore, nanotubes, quantum dots, graphene, and metal nanoparticles have been proposed to be a potential method to enhance drug delivery of amps (magana et al., ) . hybrid peptides have also been used to build targeting peptides. for example, pa , which is a p. aeruginosa-targeting peptide, was combined with gnu (a broad-spectrum amp) to construct a hybrid peptide (pa -gnu ) that targets oprf protein and has good bactericidal activity and specificity . furthermore, some antibiotics, for instance, daptomycin (a lipopeptide), lugdunin which is a -membered cyclic peptide consists of amino acid residues plus a thiazolidine moiety and telavancin (a glycopeptide) have been widely used for the clinic (durand et al., ; lampejo, ) . although they are antibiotics, they have provided broader ideas for the design of amps. food preservatives have potential harm to the human body. therefore, natural preservatives are being advocated by more people. amps have a good inhibitory effect on common bacteria and fungi in food, and many amps are resistant to acids, alkalis, and high temperatures are easily hydrolyzed by proteases in the human body. thus, amps are a promising alternative to preservatives. nisin is a bacteriocin produced by l. lactis subspecies. lactic acid bacteria have been widely used as food preservatives. nisin is categorized as generally recognized as safe (gras) by the us food and drug administration (fda) and is used as a food preservative in other countries (khan and oh, ) . however, only nisin and polylysine are currently approved by the fda as food additives (santos et al., ) . pedocin pa- , a bacteriocin consisting of amino acids produced by a diplococcus, is also used as a food preservative and is sold on the market under the trade name alta . pedocin pa- is used as a food additive to inhibit the growth of l. monocytogenes, which can cause meat deterioration (settanni and corsetti, ) . enterocin as- is an amp used to preserve cider, fruit and vegetable juices, and enterocin ccm is used to preserve soy milk (rai et al., ; santos et al., ) . encapsulating bacteriocins into liposomes is a new method used to overcome the problems of amps in food applications (such as proteolytic degradation or interaction with food ingredients) (da silva malheiros et al., ) . moreover, active packaging by adding amps is a novel packaging method that has great potential in the food industry. for instance, ε-poly-l-lysine is used in conjunction with starch biofilms to show good inhibitory effects on aspergillus parasiticus (aflatoxin producer) and penicillium expansum and nisin have the potential to be dairy preservative because it is a highly surface-active molecule (luz et al., ) . the european union banned the use of animal growth promoters in animal feed in . thus, a new antibacterial strategy is needed. many amps are the potential to be used in poultry, swine, and ruminants breeding and aquaculture because they can improve production performance (liu et al., ; bao et al., ) , immunity and promote intestinal health and some of them have a stronger inhibitory effect on bacterial inflammation if used with antibiotics cote et al., ) . for example, siamp has a good effect on the treatment of ibv in chicken (sun et al., ) . by adding swine gut intestinal antimicrobial peptides (sgamp), broilers showed higher average daily gain and feed efficiency under chronic heat stress conditions (hu et al., ) . frog caerin . , european sea bass dicentracin and nk-lysine peptides (nklps) have good inhibitory effects on nodavirus, septicaemia haemorrhagic virus, infectious pancreatic necrosis virus and spring viremia carp virus, which are devastating to fish farming (león et al., ) . the amp in soybean meal fermented by b. subtilis e effectively inhibits v. parahaemolyticus and vibrio alginolyticus and enhances the resistance level of litopenaeus vannamei against v. parahaemolyticus when added to feeds (cheng et al., ) . for agriculture, the plant pathogenic infection of bacteria and fungi causes the loss of economy, for instance, aspergillus flavus infection of corn and peanuts, citrus green mold caused by penicillium digitatum, gray mold disease caused by botrytis cinerea on strawberries and geotrichum citriaurantii infection of citrus fruit all cause great harm to the growth and post-harvest of agricultural products (liu et al., ; liu et al., ) . several afps have shown prospect to control these problems. however, the practical application of antimicrobial peptides in the transportation and preservation of agricultural products is still lacking, because the use of antimicrobial peptides will greatly increase the cost in the transportation of fruits and vegetables (application examples of amps in these four fields are shown in table ). antimicrobial peptides constitute a global research hotspot, but many key issues in design and application need to be solved urgently. several restrictive factors hinder the application of amps. the interaction of multidisciplinary subjects, such as biology, materials science, chemistry, bioinformatics, molecular informatics and pharmacy can further develop prospective amps. computer molecular dynamics simulation, cell membrane simulation, and more methods are being applied to study the mechanism of amps. how to further understand the correlation between amps and various targets instead of conducting one-sided experimental research might improve experimental designs to obtain stronger systemic and scientific demonstrations. on this basis, further animal experiments are required instead of simple cell-level experiments to test the effect of amps under complex physiological conditions. several complicated methods, such as the chemical method of peptidomimetics and non-natural amino acid modifications, have been applied in designing amps to solve the problem of protease hydrolysis. most methods use chemical substrates, but the cost of these methods cannot be ignored in practice. in addition, chemical synthesis and the use of engineered bacteria are currently the mainstream for such procedures. finding a better biological preparation method, reducing the cost and increasing the yield is important problems in practical application. furthermore, studying the amp expression of the organism itself and finding a better expression vector are necessary for mass production in the future as more amps in nature are discovered. further research is needed on the reported amps to solve the problem on structure-function relationship. as a branch of peptide drugs, amps need to progress with the advancement of medical science against the background of the current low success rate of the clinical application of amps. more attention can be 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promises antimicrobial potency against multidrug-resistant bacteria isolation and structure of corticostatin peptides from rabbit fetal and adult lung characterization of antimicrobial activity and mechanisms of low amphipathic peptides with different α-helical propensity we thank the national key r&d program of china ( yfd ). key: cord- -qb nxl authors: donno, d.; cerutti, a.k.; mellano, m.g.; prgomet, z.; beccaro, g.l. title: serviceberry, a berry fruit with growing interest of industry: physicochemical and quali-quantitative health-related compound characterisation date: - - journal: j funct foods doi: . /j.jff. . . sha: doc_id: cord_uid: qb nxl amelanchier canadensis (l.) medik., commonly called serviceberry, is a potential functional food that is also used for its medicinal purposes. this work evaluated the potential of a cultivated serviceberry species as a functional food by characterising its physicochemical characteristics, antioxidant capacity, vitamin c, phenolics and other phytochemicals selected as health-promoting biomarkers, using high-performance liquid chromatography. the most important compound class identified was polyphenols ( . %), followed by organic acids ( . %), monoterpenes ( . %), and vitamins ( . %). results showed that serviceberry fruits could be good sources of phenolic constituents, as catechins ( . ± . mg/ g(fw)), anthocyanins ( . ± . mg/ g(fw)), and tannins ( . ± . mg/ g(fw)) (fw = fresh weight). these results highlight the potential role of a. canadensis fruits as a functional food. further studies are needed to identify several genotypes for breeding to get suitable cultivars for fresh consumption and processing. new demands are constantly directed to food producers by consumers. rising consumer incomes, changes in lifestyle and demographics, and shifting preferences due to advanced knowledge about the relationship between food and health contribute to new demands for foods (dolgopolova, teuber, & bruschi, ) . today, foods are intended to not only satisfy hunger and provide necessary nutrients, but also to prevent nutritionrelated diseases and improve physical and mental well-being (annunziata & vecchio, ) : functional foods could play an important role in human health. underutilised fruits, as serviceberry, could be an important source of health promoting phytonutrients with medicinal properties. furthermore, these fruits have often high pigment contents, which could represent an alternative to synthetic dyes (rymbai et al., ) . the genus amelanchier (family rosaceae) is represented by approximately species widespread in north america and in parts of eurasia (michalczyk & macura, ) . serviceberry is native to north america from alaska, across western canada, and in the western and north-central usa (lim, ) . it is less known and rarely grown in europe (except scandinavian countries) in spite of its high frost resistance, decorative value as a shrub, and edible fruits (bakowska- barczak, marianchuk, & kolodziejczyk, ) . in its native environment, serviceberry is found in thickets, woodland margins, banks of streams, canyons, and hillsides, from sea level to m altitude. it prefers a rich, well-drained loamy soil but it can also grow in any sandy or clayey soil that is not waterlogged or too dry. it is relatively drought and salt tolerant and thrives in a sunny or semishade position (lim, ) . a. alnifolia, a. arborea and a. canadensis are the best known species. a. alnifolia (saskatoon serviceberry or alder-leaved serviceberry) is a western north american species. it has a variable habitus but it is usually found as a multi-stemmed shrub ranging from - m in height: some genotypes are cultivated for commercial fruit production in western usa and canada. its fruits are approximately cm in diameter, blue-purple and ripen in july (hu, kwok, & kitts, ) . a. arborea (downy serviceberry) is an eastern north american large shrub or small tree - m high and is crossed with a. laevis to produce cultivars for the landscape industry in the usa. the fruits of a. arborea are purple-black, slightly sweet and ripen in late june (adhikari, francis, schutzki, chandra, & nair, ) . a. canadensis (shadblow serviceberry) is another eastern north american species. it ripens in early june and it is used in the landscape trade. it is a stoloniferous shrub or small tree reaching m in height with a fastigiate crown and smooth ash-grey bark. twigs are slender, reddish-brown becoming glabrous during flowering, while leaves are alternate, simple, oval-obovate to nearly round. the maroon-purple fruits are similar to a. arborea ones (pome, - mm across, glabrous, wax-coated) (lim, ) . north american indigenous people uses different parts of the serviceberry plant for several medicinal purposes: in canada, the fruits are used as juice for treating stomach ailments and as a laxative. eye-and ear-drops are also prepared from ripe serviceberries (kershaw, ) . the boiled bark is used as a disinfectant, while the root infusion is used to prevent miscarriage after an injury (lim, ) . native american communities prepare a tea from the twigs and stem and administer it to women just after childbirth. moreover, a tonic from the bark is given to women after delivery to hasten discharge of the placenta (turner, ) . amelanchier spp. can also be used as a windbreak plant. the wood can be used for tool handles, canes, canoe crossbars, and small implements, because it is hard, strong, and fine-grained, while the young stems are used to make basket rims, handles, arrows, combs, digging sticks, salmon spreaders, and pipes. fruits provide a purple dye (lim, ) . the ripe fruit of amelanchier spp. is sweet with a hint of apple, and there is growing interest in using it in the food industry (fresh, pies, pastries, preserves, jams, jellies, spreads cereals, and snack food). the fruits are also been added into cider, wine, beer, or tea (adhikari et al., ; bakowska-barczak & kolodziejczyk, ) . the native people and early settlers of the north american prairies used serviceberry as one of their main food sources, but its use was limited because of its natural distribution area in the wild. in the last two decades, however, there has been growing interest in the industrial cultivation and utilisation of this fruit in canada and usa (michigan) (adhikari et al., ; kershaw, ) . fruit processing has an important role in exploiting the raw material because serviceberry has a relatively short harvest period (michalczyk & macura, ) . innovative methods of processing, freezing, and packaging have greatly increased the uses of this fruit. moreover, growers are promoting it as a potential functional food (jamin, ), alongside other fruit species, as ribes nigrum (donno et al., b) , morus nigra (donno, cerutti, prgomet, mellano, & beccaro, c) and lycium spp. (donno, beccaro, mellano, cerutti, & bounous, a) . the composition of fruits considerably varies depending on genotype, ripening stage at harvest and growing conditions (michalczyk & macura, ) . there are few reports concerning the chemical composition of amelanchier spp.: phytochemical studies on a. canadensis are rarely found in the literature. however, the available literature usually emphasises its important health benefits: serviceberry appears to be an excellent source of manganese, magnesium, and iron, and a relatively good source of calcium, potassium, copper, and carotenoids (e.g. lutein). moreover, the fruit is rich in nutraceuticals, particularly phenolic compounds, as anthocyanins, chlorogenic acid, catechins and rutin (bakowska-barczak & kolodziejczyk, ; bakowska-barczak et al., ) . in addition, amelanchier spp. seed oil may serve as a potential dietary source of tocopherols, sterols, and unsaturated fatty acids (lim, ) . several cultivars of amelanchier spp. were found to possess free radical scavenging activity in a concentration-dependent manner related to their relatively high anthocyanin content (hu et al., ) , and antiviral activity against enteric coronavirus. moreover, serviceberry fruits show antidiabetic properties (as aldose reductase inhibitor activity), and exhibit the ability to regulate lipid metabolism and energy expenditure in a manner consistent with improving metabolic syndrome (burns kraft et al., ) . the identification and quantification of bioactive compounds in fruits and the evaluation of their biological activities are important to gauge their efficacy as dietary interventions (donno et al., ; fu et al., ) . chromatographic fingerprinting could be considered an easy and reliable technique to characterise and differentiate amelanchier spp. checking the fruit quality and safety (donno et al., c) . the technique shows a relatively complete picture of fruit extracts and provides insight into the synergistic and additive biological effects of the bioactive constituents to total phytocomplex (donno, beccaro, mellano, cerutti, & bounous, ) . this work aimed to evaluate the potential of a cultivated serviceberry species (a. canadensis (l.) medik.) as a functional food by characterising its physicochemical characteristics, and antioxidant capacity. furthermore, the characterisation and quantification of several phytochemicals, selected as healthpromoting biomarkers, was performed, using high-performance liquid chromatography-diode array detection (hplc-dad). fully ripened fruits ( . kg for each replication) were collected from a cultivated genotype of a. canadensis (l.) medik in the middle of june , in chieri ( ° ' "n, ° ' "e, at m a.s.l.), piedmont (north-western italy), in the fruit tree germplasm collection of the department of agricultural, forest and food sciences, university of turin. the climate of the area is temperate, with rains in spring and autumn, and a rainfall of approximately mm/year; the soil is loam-clay. immediately after harvest, samples were protected from light to avoid loss of antioxidant components, as reported by cazares-franco et al. ( ) , and sent directly to the laboratory where they were divided into two equal portions. one portion was used to determine their physicochemical parameters, on the same day of harvest. the second portion was stored at °c and % relative humidity (rh), until extraction and nutraceutical analysis. sodium carbonate, folin-ciocalteu phenol reagent, sodium acetate, citric acid, potassium chloride, hydrochloric acid, iron(iii) chloride hexahydrate, , , -tripyridyl-s-triazine, , phenylenediamine dihydrochloride (opda), all polyphenolic and terpenic standards, potassium dihydrogen phosphate, phosphoric acid and hplc-grade methanol and acetonitrile were purchased from sigma-aldrich (st. louis, mo, usa). acetic acid, ethanol, organic acids and hplc-grade formic acid were purchased from fluka biochemika, buchs, switzerland. ethylenediaminetetraacetic acid disodium salt was purchased from amresco (solon, oh, usa). sodium fluoride was purchased from riedel-de haen (seelze, germany). cetyltrimethylammonium bromide (cetrimide), ascorbic acid (aa) and dehydroascorbic acid (dhaa) were purchased from extrasynthése (genay, france). milli-q ultrapure water was produced by sartorius stedim biotech mod. arium (sartorius, göettingen, germany). approximately % of the ripe fruits were washed, drained, and dried with paper towels. their width and length were measured using a . mm sensitive digital calliper (traceable digital caliper- ″, vwr international, milano, italy) and their weight determined to the nearest . g (mettler, greifensee, switzerland). the fruit was then homogenised in a blender and centrifuged ( rpm, min), and ph, total soluble solids (tss), and titratable acidity (ta) were evaluated. the remaining fresh fruits were immediately stored at °c and % rh, until further analysis. the ta (meq · l − ) was determined in a mixture of ml serviceberry juice diluted in ml milli-q water, by titration with . m naoh using an automatic titrator (crison, alella, spain) to an end-point of ph . . the ph of the fruit juice was measured directly. the tss was measured directly in serviceberry juice with a digital refractometer (tsingtao unicom-optics instruments, laixi, china), and the results were expressed as°brix. the total polyphenol content (tpc) was determined following the folin-ciocalteu colorimetric method (slinkard & singleton, ) , and the results were expressed as mg of gallic acid equivalents (gae) per g of fresh weight (fw). gallic acid standard solutions were prepared at . - . mg · ml − . the total anthocyanin content (tac) in the extracts was determined using the ph-differential method (giusti & wrolstad, ; lee, durst, & wrolstad, ) , and expressed as milligrams of cyanidin- -o-glucoside (c g) per grams of fresh weight (mgc g/ gfw). the antioxidant activity was evaluated by the ferric reducing antioxidant power (frap) assay (benzie & strain, ) , and the results were expressed as millimoles of ferrous iron (fe + ) equivalents per kilogram (solid food) of fw. the standard curve was obtained using feso · h o at - µmol · l − . samples were filtered with circular pre-injection filters ( . µm, polytetrafluoroethylene membrane) prior to hplc-dad analysis. in the case of vitamin c analysis, a c cartridge for solid phase extraction (sep-pak ® c- , waters, milford, ma, usa) was used to absorb the polyphenolic fraction. then, µl of opda solution ( . mmol · l − ) was added to µl of each sample for dhaa derivatisation into the fluorophore -( , dihydroxyethyl)furo( , -b)quinoxaline- -one. after min in the dark, these samples were analysed using hplc-dad (gonzalez-molina, moreno, & garcia-viguera, ). external standard calibration method was used for quantitative determinations. three manual injections of each standard ( µl) at the concentrations listed in table were performed. the calibration curves were obtained by plotting the peak area (y) of the compound at each concentration level versus the sample concentration (x). an agilent high-performance liquid chromatograph coupled to an agilent uv-vis diode array detector (agilent technologies, santa clara, ca, usa) was used for the chromatographic analysis. five chromatographic methods were used to separate the biomolecules on a kinetex c column ( . × mm, µm, phenomenex, torrance, ca, usa), as listed in table . several mobile phases were used for biomarker identification and uv spectra were recorded at different wavelengths, based on hplc methods previously tested and validated for herbal medicines (donno et al., b) . all the samples were analysed in triplicate, and standard deviations are given in order to assess the repeatability of the used methods. total bioactive compound content (tbcc) was determined as the sum of selected biomarkers having a positive role in human health ("multi-marker approach") (mok & chau, ) . five polyphenolic classes were considered: benzoic acids, catechins, cinnamic acids, flavonols, and tannins. monoterpenes, organic acids, and vitamin c (as the sum of ascorbic and dehydroascorbic acids) were also considered to obtain a complete analytical fingerprint. all the results were expressed as mg/ g of fw. all samples were prepared and analysed in triplicate. results were subjected to analysis of variance (anova) for mean comparison (spss . software) followed by hsd tukey multiple range test (p < . ). the present study characterises the morphological properties, quality parameters and phytochemical composition of a. canadensis fruits. the results are discussed in relation to the potential of serviceberry as a functional food. table presents the fruit weight and size, tss, ph, and ta of the analysed serviceberry genotype. results showed that the fresh fruit are small and spheroidal (length: . ± . mm; width: . ± . mm), with a mean weight of . ± . g. the cultivated serviceberry fruit size is on average greater than the size of the fruit harvested in the wild, as reported in the previous studies (bakowska- barczak & kolodziejczyk, ; cazares-franco et al., ) . the results (table ) corroborated with stushnoff ( ) , who reported that cultivated serviceberry showed up to % larger fruit size compared with wild fruit one. quality analysis reported a mean value of tss . ± . °brix, ta . ± . meq · l − and ph . ± . (table ). the tss in various serviceberry genotypes ranged from % to % fw, with - % sucrose and - % reducing sugars (mazza, ) . wolfe and wood ( ) found that the sugar content slowly increased as the fruit matured and then considerably accelerated before ripening: fructose content greatly decreased (− %) after the fruit ripened while the glucose content remained unaltered. the ph of wild fruits, as reported in previous studies (cazares-franco et al., ; lim, ) , is often lower than the ph of cultivated fruits: the higher ph indicates that the cultivated fruit is a low-acid food, a positive sensory feature, as confirmed by ta values reported in this study. moreover, a lower tss/ta ratio than other studies by hosseinian and beta ( ) showed that serviceberry had high potential value for the fresh market and foodderived manufacturers, because of their high polyphenolic content, in particular, anthocyanins.thetpc ( . ± . mggae/ gfw) and tac ( . ± . mgc g/ gfw) values obtained from the analysed extracts are reported in table . anthocyanins and total phenolics in serviceberries increase with fruit ripening (lim, ) . the phenolic compounds found in fruits and vegetables have attracted much interest due to their beneficial properties (faller & fialho, ) . based on the tpc, serviceberry fruits are excellent polyphenolic sources compared with commonly consumed fruits. for example,tpc reported for some berries (e.g. raspberry, blueberry, strawberry, and blackcurrant) (donno et al., b (donno et al., , c were markedly lower than tpc reported in this study for serviceberry. moreover, a high positive correlation between tpc and antioxidant capacity has been established (donno et al., ; faller & fialho, ) : therefore, a. canadensis is a very attractive fruit species from a functional point of view. in other studies (lim, ) , anthocyanin content was correlated with tpc,ta, ph, and sugar : acid ratio.the results in tables and suggested that high content of anthocyanins in serviceberries could be associated with high tpc and low ph and sugar : acid ratio. hellström, sinkkonen, karonen, and mattila ( ) found that fruits of different serviceberry genotypes contained proanthocyanidins from dimers to heptamers and higher polymers. in serviceberry, proanthocyanidins were generally of the procyanidin type, mainly consisting of epicatechin units linked by b-type bonds. bakowska- barczak et al. ( ) found serviceberries contained the following anthocyanidins: cyanidin, delphinidin, pelargonidin, petunidin, peonidin, and malvidin. in the current research, only tac was considered. the frap assay was used to evaluate antioxidant capacity of a. canadensis fruits. the frap assay is based on the ability of antioxidants to reduce ferric(iii) ions to ferrous(ii) ions: it is a simple and widely used method to evaluate antioxidant capacity (donno et al., a; fu et al., ) . in this study, fresh fruits showed a mean frap value of . ± . mmol fe + · kg − (table ). thus, serviceberry presented a higher frap value compared to some common fruit, as black mulberry, goji, raspberry, and apple (donno et al., c) . antioxidant activity values of the above mentioned common fruits are reported in other studies (cazares-franco et al., ; michalczyk & macura, ) . on the basis of tac and tpc measurements, it would appear that the antioxidant properties of serviceberry fruits are mainly affected by anthocyanins (pearson correlation index for tac/ antioxidant activity presented a value of . ). the chemical fingerprint of a. canadensis (l.) medik is reported in tables and (single compound composition). the tbcc was calculated as the sum of the main biomarkers selected for their healthy properties and detected in the extracts. results showed a mean tbcc value of . ± . mg/ gfw. these biomarkers were selected for the fingerprint because they have been described as important healtheffective molecules in humans (de cassia da silveira e sa, andrade, & de sousa, ). the phytochemical fingerprint of serviceberry fruit has been described: bioactive compounds were identified by hplc-dad (mazza, ; ozga, saeed, wismer, & reinecke, ) . in this study, a. canadensis fruits showed the following bioactive compound composition: four cinnamic acids (caffeic, chlorogenic, coumaric, and ferulic acids), five flavonols (hyperoside, isoquercitrin, quercetin, quercitrin, rutin), one benzoic acid (ellagic acid), one catechin (epicatechin), two tannins (castalagin, vescalagin), two monoterpenes (limonene, γ-terpinene), three organic acids (oxalic, succinic, and tartaric acids), and vitamin c (expressed as the sum of ascorbic acid and dehydroascorbic acid). catechin, phellandrene, sabinene, terpinolene, and citric, malic, gallic and quinic acids were not detected, in agreement with serviceberry fruit phytochemical fingerprints reported in similar studies (juríková et al., ; michalczyk & macura, ) . other peaks were detected in the obtained chromatograms, but they did not match any of the biomarkers used in the present work. further studies, adding other biomarkers with demonstrated biological activity, are needed for a complete identification of the chromatogram. the identified health-promoting agents were grouped into classes to evaluate the single contribution of each class to total fruit phytocomplex composition (fig. ) . the phytochemical fingerprint showed the prevalence of polyphenols (as the sum of anthocyanins, cinnamic acids, flavonols, benzoic acids, catechins, and tannins) and organic acids in all the analysed samples (mean values were considered). indeed, the most important class was polyphenols ( . ± . %), followed by organic acids ( . ± . %), monoterpenes ( . ± . %), and vitamins ( . ± . %). results showed that serviceberry fruits could be a good source of phenolic constituents and deserved special attention focused on studying their phytochemical profile: the main phenolic groups were catechins ( . ± . mg/ gfw) > anthocyanins ( . ± . mg/ gfw) > tannins ( . ± . mg/ gfw) > cinnamic acids ( . ± . mg/ gfw) > flavonols ( . ± . mg/ gfw) > benzoic acids ( . ± . mg/ gfw).the phenolic compounds detected in the present work were similar to those reported in other studies on different serviceberry genotypes (bakowska-barczak & kolodziejczyk, ; ozga et al., ) . the most important polyphenolic classes, in relation to the tpc, were catechins ( . ± . %) > anthocyanins ( . ± . %) > tannins ( . ± . %) > cinnamic acids ( . ± . %) > flavonols ( . ± . %) > benzoic acids ( . ± . %) (fig. ) . results showed few differences in phenolic constituents compared to other serviceberry genotypes: the variation of phenolic compounds in berries, as well as in other fruits, depends on many factors, as ripening stage, genetic differences, and environmental conditions during fruit development (sánchez-salcedo, mena, garcía-viguera, martínez, & hernández, ) . these results highlight the potential of a. canadensis fruits as a functional food due to their biological and pharmacological effects associated with the detected phytochemicals. plants containing flavonoids are often used to treat diabetes in several natural medicines; these compounds are reported to have glucose-lowering effects and repress hepatic glucose production in animals (wolfram et al., ) . the presence of ellagic acid ( . ± . mg/ gfw) in serviceberry makes this fruit an excellent candidate to evaluate several effects in vivo. the concentration of cinnamic acids, in particular chlorogenic acid ( . ± . mg/ gfw), may be important when fruits are processed, as these compounds are considered a preferential substrate for the catecholase activity of polyphenol oxidase (wojdyło, oszmiań ski, & bielicki, ) . the relative concentration of cinnamic acids could also influence the oxidation and colour development processes occurring during technological processing (sánchez-salcedo et al., ) . moreover, chlorogenic acid has been recognised for its extensive biological properties (radojkovic, zekovic, vidovic, kocar, & maskovic, ) . anthocyanin pigments are of prominent importance in a. canadensis fruits because of their dual value. first, they constitute an integral part of the sensory attributes, as their levels and various forms directly pertain to the colouration of the final product. on the other hand, they have been claimed to possess several biological properties and therefore they are considered as secondary metabolites with potential nutraceutical value (chen, xin, yuan, su, & liu, ) . the high content of tannins in serviceberry fruits is interesting, thanks to their wellknown association with human health, as anticarcinogenic and antimutagenic agents important in protecting cells by oxidative damages (chung, wong, wei, huang, & lin, ) . although flavonols are not the main phenolic components of serviceberry fruits, they should be considered in the phytochemical profile of amelanchier spp. fruits, since they may exert critical beneficial features related to human health (del rio et al. ). organic acids constituted an important component of the a. canadensis fruits ( . ± . mg/ gfw). these compounds affect fruit quality properties, as stability, colour, and flavour. they are mainly used to determine fruit maturity stage, identify adulteration in fruit juices, indicate the spoilage of fruit products, and function as food acidifiers (soyer, koca, & karadeniz, ) . in addition, organic acids are antioxidants with multi-purpose uses in pharmacology (eyduran et al., ) . although monoterpenes only represent a small fraction of the total phytocomplex of serviceberry fruits ( . ± . mg/ gfw), they could be an important bioactive class: monoterpenes are increasingly being referred to as a potential pharmacological group that act as anti-inflammatory drugs (de cassia da silveira e sa et al., ). dehydroascorbic acid shows biological activity and can easily be converted to ascorbic acid by humans (corral-aguayo, yahia, carrillo-lopez, & gonzalez-aguilar, ).thus, the sum of ascorbic and dehydroascorbic acids was considered as vitamin c, according to previous similar studies (cazares-franco et al., ; donno et al., a) . if compared on a fresh weight basis, the vitamin c content of serviceberry ( . ± . mg/ gfw) was higher than that found in most common fruits, as kiwifruit ( . ± . mg/ gfw), orange ( . ± . mg/ gfw), strawberry ( . ± . mg/ gfw), blackberry ( . ± . mg/ gfw), blueberry ( . ± . mg/ gfw), apple ( . ± . mg/ gfw), and black mulberry ( . ± . mg/ gfw) as previously reported (donno et al., a (donno et al., , c . the recommended daily intake of vitamin c for adults is - mg (monsen, ) and, therefore, a portion of g of ripe serviceberry fruits could contribute more than % of the recommended daily intake of vitamin c. evidence continues to emerge suggesting that many commonly consumed fruits and vegetables are beneficial to health in relation to the prevention of several diseases. for this reason, there is a growing interest in novel or less well-known foods that offer an opportunity for health maintenance. this study demonstrated that serviceberry fruits present a plethora of phytochemicals with the capacity to promote health and protect against chronic diseases. the results of this study provided a detailed assessment of the quality, chemical characteristics, and potential of a cultivated edible serviceberry as a functional fresh fruit. substantial amounts of simple phenolics, as chlorogenic, coumaric and ferulic acids, and flavonols are present in this fruit. some of these simple phenolics, together with complex polyphenols, monoterpenes and other phytochemicals (organic acids and vitamin c) make serviceberry an alternative fruit that could contribute to the prevention of chronic diseases resulting from oxidative stress. finally, as the opportunity for medium-scale production on marginal lands, further studies are needed to investigate this genotype by environment interactions and identify several genotypes suitable for fresh consumption, processing, or breeding research. quantification and characterisation of cyclooxygenase and lipid peroxidation inhibitory anthocyanins in fruits of amelanchier functional foods development in the european market: a consumer perspective evaluation of saskatoon berry (amelanchier alnifolia nutt.) cultivars for their polyphenol content, antioxidant properties, and storage 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assessment of their beneficial properties total phenol analysis: automation and comparison with manual methods organic acid profile of turkish white grapes and grape juices amelanchier species. genetic resources of temperate fruit and nut crops food plants of interior first peoples polyphenolic composition, antioxidant activity, and polyphenol oxidase (ppo) activity of quince (cydonia oblonga miller) varieties non-volatile organic acid and sugar composition of saskatoon berries (alnifolia spp.) during ripening epigallocatechin gallate supplementation alleviates diabetes in rodents supplementary data to this article can be found online at doi: . /j.jff. . . . key: cord- -mdes jg authors: bauvois, brigitte; dauzonne, daniel title: aminopeptidase‐n/cd (ec . . . ) inhibitors: chemistry, biological evaluations, and therapeutic prospects date: - - journal: med res rev doi: . /med. sha: doc_id: cord_uid: mdes jg aminopeptidase n (apn)/cd (ec . . . ) is a transmembrane protease present in a wide variety of human tissues and cell types (endothelial, epithelial, fibroblast, leukocyte). apn/cd expression is dysregulated in inflammatory diseases and in cancers (solid and hematologic tumors). apn/cd serves as a receptor for coronaviruses. natural and synthetic inhibitors of apn activity have been characterized. these inhibitors have revealed that apn is able to modulate bioactive peptide responses (pain management, vasopressin release) and to influence immune functions and major biological events (cell proliferation, secretion, invasion, angiogenesis). therefore, inhibition of apn/cd may lead to the development of anti‐cancer and anti‐inflammatory drugs. this review provides an update on the biological and pharmacological profiles of known natural and synthetic apn inhibitors. current status on their potential use as therapeutic agents is discussed with regard to toxicity and specificity. © wiley periodicals, inc. med res rev aminopeptidase n (ec . . . , apn) is a metallo-dependent integral membrane protease. the enzyme belongs to the m family of the ma clan of peptidases also called gluzincins. aminopeptidase n consists of amino acids with a short n-terminal cytoplasmic domain, a single transmembrane part, and a large cellular ectodomain containing the active site. this enzyme was first isolated in by pfleiderer and celliers from pig kidney and is known under several different names (alanine aminopeptidase; microsomal aminopeptidase; microsomal leucine aminopeptidase aminopeptidase m; amino oligopeptidase; gp ). in the last few years, certain surface molecules identified as cluster differentiation (cd) antigens were found to be identical to some membrane proteins. thus, cd is identical to apn. , soluble apn is detectable in plasma/serum and urine [ ] [ ] [ ] [ ] but the mechanism of release of membrane apn remains unknown. membrane-bound apn/cd is widely distributed outside the hematopoietic system (epithelial-, endothelial-, fibroblast-cell types) with main sources being brush border membranes of kidney proximal tubule cells and enterocytes, and in the hematopoietic compartment is not confined to a particular lineage. , , apn/cd is predominantly expressed on stem cells and on cells of the granulocytic and monocytic lineages at distinct stages of differentiation and is therefore considered as a marker of differentiation. , dysregulated expression of membrane and/or soluble forms of apn/cd is observed in many diseases. compiled observations indicate enhanced apn levels in tumor cells such as melanoma, , renal, pancreas, colon, prostate, gastric, and thyroid cancers. tumor-infiltrating t cells in renal and lung cancers are cd -positive. , apn activity is elevated in plasma and effusions of cancer patients. apn activity on neutrophils from patients affected by a rare adrenal gland tumor, adrenal pheochromocytoma, is significantly increased as compared with healthy controls. cd is overexpressed in acute and chronic myeloid leukemias , , [ ] [ ] [ ] and in anaplastic large cell lymphomas. , overexpression of apn/cd in t lymphocytes or neutrophils occurs in several inflammatory diseases (chronic pain, various forms of joint effusions, rheumatoid arthritis, multiple sclerosis, systemic sclerosis, systemic lupus erythematosus, polymyositis/dermatomyosytis, pulmonary sarcoidosis). [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] apn/cd may be therefore considered as a useful clinical marker. whether this protease critically contributes to the pathological behavior remains however unknown. in this review, we briefly summarise knowledge on the structure and the mechanisms of cleavage of apn/cd to integrate current knowledge in natural and synthetic apn inhibitors. the reader is referred to excellent reviews for the characteristics of apn/cd and substrate specificity. , [ ] [ ] [ ] [ ] [ ] [ ] various aspects on the roles of apn/cd are reviewed here in the context of the in vitro and in vivo use of certain apn inhibitors. apn is anchored to the plasma membrane, via an uncleaved signal sequence, by the c-terminus (type ii) facing extracellularly. membrane apn/cd is found as a dimer of two non covalently associated subunits with a relative molecular mass of kda (fig. a) . , , the human cd gene, cloned in and subsequently mapped to chromosome q - , possesses two promoters (fig. b) . [ ] [ ] [ ] [ ] [ ] [ ] the cdna sequence reveals the presence of the amino acid sequence his-glu-xaa-xaa-his which is a zn þþ binding motif found in one class of metallo-peptidases. site-directed mutagenesis indicates that extracellular cysteines in the molecule confer correct structure and consequently enzymatic activity and surface expression of apn. mutation of glutamic acid in an aminopeptidase conserved region (the gamen motif) leads to an inactive enzyme indicating that this glutamic acid belongs to the anionic binding site in apn and interacts with the n-terminal aamino group of the substrate. apn/cd cleaves preferentially neutral amino acids (with the exception of proline) (fig. c) from the unsubstituted n-terminus of oligopeptides. , biologically active peptide substrates cleaved by apn/cd are neuropeptides (met-and leu-enkephalins, neurokinin a, met-lys-bradykinin, and endorphins such as spinorphin), , - vasoactive peptides (kallidin, somatostatin, and angiotensins) - and chemotactic peptides (monocyte chemotactic protein//mcp- and n-formyl methionine leucine phenylalanine/f-mlp). , apart from its hydrolytic ability, apn serves as a receptor for coronaviruses. [ ] [ ] [ ] [ ] in humans, the e corona virus uses apn to enter alveolar cells and establish an upper respiratory tract infection. bradykinin (arg-pro-pro-gly-phe-ser-pro-phe-arg) and substance p (arg-pro-lys-pro-gln-gln-phe-phe-gly-leu-met-nh ) are natural peptides capable of inhibiting apn in micromolar concentrations. similarly, elevated concentrations of leucine, proline, l-alanine, l-arginine, l-glutamine, l-methionine, as well as divalent cations (co þ , zn þ , mn þ , ca þ , ni þ ) inhibit apn activity. (for review) moreover, molecules with a broad spectrum of action such as kcn, nan , ammonium oxalate, n-ethyl-maleimide, and -hydroxyquinoline inhibit apn/cd . (for review) apn activity is also inhibited by puromycin ( ), , lapstatin ( ), some n-phenylphthalimide derivatives such as compound , - several n-phenylhomophthalimide derivatives like piq- ( ) , which has later been described as a rather puromycin-sensitive aminopeptidase (psa) inhibitor by the same group, [ ] [ ] [ ] [ ] phosphinate dipeptide analogues illustrated by hphep[ch ]tyr ( ), pseudoglutamyl aminophosphinic peptides such as gluc(po ch )leu-ala ( ), several variously substituted -amino- -oxobutyramide exemplified by compound , a-aminoboronic derivatives such as the benzyl derivative , or a-aminobenzaldehydes illustrated by (s) -amino- methylpentanal ( ) . an eclectic set of compounds has been described and used for the biochemical characterization or/and inhibition of other proteases-e.g.: urokinase-type plasminogen activator, dipeptidylpeptidase iv (dppiv/cd ), or other different aminopeptidases including human enkephalin degrading aminopeptidase (heda), cytosolic leucine aminopeptidase (lapc), glutamyl aminopeptidase (apa), and arginyl aminopeptidase (ap-b). in this context, it is also worth mentioning two systematic studies devoted to hydroxylated naturally occurring flavonoids such as baicalein ( ), apigenin ( ) , or myricetin ( ) and related compounds which, aside their activity on neutral endopeptidase (nep/cd ) or angiotensin-converting enzyme (ace/cd ), exhibited a significant in vitro inhibitory effect toward apn. , formulas, ki, ic or inhibition percentages of enzymes for compounds - are depicted in figure . two recent publications describing either the irreversible inhibition of both apn/cd and dpp iv/cd enzymatic activities by aqueous extracts of a cistus incanus l. or ace, nep, and apn inhibition by extracts of epilobium angustifolium deserve also quotation. although the borderline is not easy to position, leaving out the above-mentioned studies dealing with non-specific compounds targeting other enzymes and, incidentally, revealing an inhibitory activity on apn, we have chosen to focus the present review on the data tightly dedicated to natural and synthetic inhibitors of apn/cd itself. the most widely used among the naturally occuring apn/cd inhibitors are microorganismproduced and have been purified from microbial culture filtrates. a large part of them are generated by bacteria belonging to the order actinomycetales, especially of the genera streptomyces: ( ) was first isolated by r. green and r. bhagwan singh from a malayan strain of actynomycetes. this compound was then listed as streptomyces cutter c/ (n.c.i.b. ). about years later, actinonin was also obtained from another strain referenced mg -hf and its inhibition against apn was found to be competitive with the substrate. the structural study and the chemical synthesis of and some analogues have aroused numerous works [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] completed by a structure-activity relationship investigation dealing with anti bacterial properties observed in this actinonin series. ( s, r)- -amino- -hydroxy- -phenylbutanoyl-l-valine) ( ) ) and two closely related derivatives: ahpa-val-pro-hyp ( s, r)- -amino- -hydroxy- -phenylbutanoyl-l-valyl-l-prolyl-(trans- hydroxy-l-proline) (mr a) ( ) and ahpa-val-pro-pro ( s, r)- -amino- -hydroxy- -phenylbutanoyl-l-valyl-l-prolyl-l-proline) (mr b) ( ) were obtained from the culture broth of streptomyces neyagawaensis sl- . [ ] [ ] [ ] the preparation of several novel synthetic ahpa derivatives (exemplified by ) bearing, for most of them, heterocyclic moieties and exhibiting interesting in vivo antitumor potencies ( - % inhibitory rate on s sarcoma) has been recently reported. ( s, r)- -amino- -hydroxy- -methylhexanoyl-l-valine-l-valine-l-aspartic acid) ( ) has been reported to be a slow-binding competitive inhibitor of apn. it was first isolated from the culture filtrate of streptomyces sp. me -m . and its structure has been unambiguously determined. several enantioselective syntheses of this tetrapeptide have been reported, , and some of its analogues have also been prepared in the context of a sar study. is an inhibitor of various leucine and arginine aminopeptidases, and an efficient inhibitor of lta hydrolase. [ ] [ ] [ ] [ ] however, in spite of its marked toxicity and of its relative lack of selectivity toward exopeptidases, it is one of the most used compound for its apn/cd inhibitory effects. bestatin has been described as a slow-binding competitive inhibitor of apn, and a schematic representation of within the active site of apn , is depicted in figure . bestatin was first isolated from a culture filtrate of streptomyces olivoreticuli (md -c ) and its chemical structure has been subsequently ascertained. several stereoselective total syntheses of have been reported, - the preparation of its stereoisomers has been performed and some ubenimex derivatives or analogues such as the para-hydroxybestatin ( ), the -thiolbestatin ( ), , the bestatin thioamide ( ), , or the reduced bestatin have also been prepared. ( s, r)- -amino- -hydroxy- -phenylbutanoyl-l-valyl-l-phenylalanine) ( ) is a tripeptide produced by streptomyces sp. mj -m . some stereoselective syntheses of have been recently reported. , , ( s, r)- -amino- -hydroxy- -phenylbutanoyl-l-valyl-l-prolyl-l-proline) ( ) is a tetrapeptide isolated from the culture of streptomyces azureus (mh - f ) and its structure has been unambiguously established. probestin has been described as a competitive inhibitor of apn and, here also, some total syntheses have been lately described. , , an overview of the formulas of compounds - reveals that, except the synthetic analogue prepared in its racemic form, they all possess the absolute configuration ( s, r) which appears crucial for activity. a comparable chiral framework is also existent in the side chain of the pharmacologically important series constituted by taxoids and, in this context, it is worth pointing out that numerous and various synthetic approaches to building blocks liable to lead to enantiomerically pure ( s, r)- -amino- -hydroxyalkanoic structures and/or their diastereomers have attracted considerable attention. . leuhistin ( r, s)- -amino- -hydroxy- - h-(imidazol- -ylmethyl)- -methylhexanoic acid ( ) has been isolated in by takeuchi and co-workers from the culture broth of a bacteria belonging to the phylum firmicutes: bacillus laterosporus bm - f . , this compound inhibits apn in a competitive manner with the substrate. the structure of and its absolute configuration have been thereafter ascertained by the same group. several naturally occuring apn inhibitors are of vegetal origin: benzo[c]phenantridines such as , -dimethoxy- -methyl , dioxolo[ , : , ]benzo[ , -c]phenanthridin- -ium chloride or chelerythrine ( ) and some closely related alkaloids have recently been isolated from extracts of the papaveraceae macleaya cordata (wild.) r. br. some of these compounds showed an efficacy against apn similar to that of amastatin ( ) or bestatin ( ) . a weaker inhibitory effect on dpp-iv has also been reported. (e,e- , -bis-( -hydroxy- -methoxyphenyl)- , -heptadiene- , -dione) ( ) is a yellow natural phenolic compound isolated from the rhizomes of asian perennial herbs extensively cultivated in tropical areas and belonging to the zingiberaceae family. all these plants are of the genera curcuma. the most exploited representative is curcuma longa l., whose dried rhizome is the source of the spice turmeric which is widely employed in food and has a long tradition of use in folk medicine. in addition to its irreversible apn/cd inhibition potencies, curcumin is now considered by oncologists as a potential cancer chemopreventive agent, , and clinical trials in this context are carried out in several laboratories. furthermore, curcumin possesses anti-inflammatory activity and is a potent inhibitor of reactive oxygen-generating enzymes (e.g. lipooxygenase/cyclooxygenase- , xanthine dehydrogenase/oxidase and inducible nitric oxide synthase). curcumin hinders also the initiation of carcinogenesis by inhibiting the cytochrome p- enzyme activity and increasing the levels of glutathione-s-transferase. its anti-tumor effect in the promotion and progression stages has been attributed, in part, to the arrest of cancer cells in s, g /m cycle phase, and induction of apoptosis. it has also been proposed that curcumin may suppress tumor promotion by blocking signal transduction pathways in the target cells. curcumin is a potent inhibitor of protein kinase c, egf-receptor tyrosine kinase and i-kb kinase. in addition, curcumin inhibits the activation of nf-kb and the expression of c-jun, c-fos, c-myc. , last, curcumin has been proposed as a hiv- or hiv- protease inhibitor, as a hiv- integrase inhibitor, and proved to be radioprotectant. , several chemical synthesis of , involving , -pentanedione and vanillin, have been reported [ ] [ ] [ ] [ ] [ ] as well as the preparations of some of its analogues designed as angiogenesis inhibitors through their ability to inhibit endothelial cell proliferation. ( b-hydroxylup- ( )-en- -oic acid) ( ) is a pentacyclic compound widely present in the plant kingdom. this oxidized derivative of betulin owes its trivial name to the fact that this class of lupane type triterpenes was first isolated from betula ssp. (birch trees). afterwards, betulinic acid has been obtained from various other vegetal species including ancistrocladus ssp.,arbutus ssp., diospyros ssp., paeonia ssp., picramnia ssp., syzygium ssp., tetracera ssp., tryphillum spp., zizyphus ssp. one of the main current sources of betulinic acid from natural origin is the bark of plane trees (e.g. platanus acerifolia) by employing a patented procedure. in addition to its apn inhibitory activity in a dose-dependent manner, and possibly as a partial consequence of this inhibitory potency, betulinic acid has been shown to modulate the immune response, to exhibit anti-inflammatory properties and to block hiv- entry into cells. it has also been reported to be a selective inhibitor of dna polymerase b and to induce apoptosis in tumor cells. the wide range of biological properties linked to betulinic acid have recently been recapitulated and analyzed in three excellent revues. [ ] [ ] [ ] several hemisynthesis of starting from betulin via betulonic acid [ ] [ ] [ ] [ ] or from various naturally occuring betulinic acid derivatives such as glycosides, [ ] [ ] [ ] [ ] sulfates, or dihydroxycinnamic esters have been reported. to our knowledge, only one naturally occuring apn inhibitor originates from animal kingdom: is a symmetrical disulfide compound bearing two hydroxyimino functional groups. this bis-bromotyrosine derivative was, almost simultaneously, first isolated in by three groups: from an unidentified marine sponge (probably of the verongidae family) collected in guam, from a psammaplysilla sp., and from thorectopsamma xana. its structure has been unambiguously and independently established by these different authors. thereafter, psammalin a has also been extracted from other sponges: psammaplysilla purpurea, , dysidea spp. (in this case, the authors have erroneously named «bisprasin»-the misspelled name of the psammalin a dimer-a compound which is obviously the psammalin a itself as judged by the reported formula) aplysinella rhax, [ ] [ ] [ ] pseudoceratina purpurea, or from a two-sponge association: poecillastra wondoensis and jaspis wondoensis. , a biosynthetic pathway has been proposed for the formation of involving modified cysteine and bromotyrosine , and, to our knowledge, only one laboratory preparation of psammalin a has been carried out starting from l-tyrosine through its its n,n -bis-(tetrahydropyran- -yl)oxime derivative. it is also worth pointing out that a library comprising about two hundred psammalin a type derivatives has recently been prepared by nicolaou and his co-workers by using solution phase combinatorial synthesis with the aim to evaluate their antibacterial activity. , in addition to its very recently reported ability to inhibit apn in a non-competitive manner thus inducing a suppression of in vitro angiogenesis, has been shown to induce a variety of biological effects: (i) a significant in vitro antibacterial activity against staphylococcus aureus and methicillin-resistant staphylococcus aureus , , which is assumed to be due to its ability to inhibit dna gyrase, (ii) a cytotoxicity against various human tumor cell lines, , [ ] [ ] [ ] (iii) an increase in ca þ release from the heavy fraction of skeletal muscle sarcoplasmic reticulum, (iv) an inhibition of topoisomerase ii, leucine aminopeptidase and farnesyl protein transferase, , mycothiol-s-conjugate amidase, chitinase, histone deacetylase and dna methyltransferase, and dna replication by targeting polymerase a-primase. some antifungal and insecticidal activities have been further reported. chemical structures of apn inhibitors - , and enzyme inhibition values are depicted in figure . several synthetic small molecules belonging to various chemical families have been reported to inhibit apn activity. a-aminomethylketones such as (s)- -amino- -methylpentan- -one hydrochloride (valine methyl ketone hydrochloride) ( ) alkyl d-cysteinates display also efficient competitive apn inhibitions. among the five esters tested, an optimal inhibitory activity has been observed with the n-butyl derivative ( ). -amino- -tetralone derivatives such as the -amino- , -dihydro- h-phenanthren- -one hydrochloride ( ) have been reported to be efficient and selective competitive inhibitors of apn. these compounds do not affect ap-a or ap-b and poorly inhibit lapc. -amino- -hydroxypropionaldehyde and -amino- -hydroxypropan- -one derivatives such as and , respectively. these competitive inhibitors of apn are very moderately active on lapc or apb. flavone- -acetic acid derivatives constitute a class of products whose the parent compound showed antiangiogenic properties. in this series, products bearing a nitro group in the -position such as the , -dinitroflavone- acetic acid ( ) proved the most potent apn inhibitors and act by reversibly binding to the catalytic site of the enzyme. these compounds present the advantage to exhibit no toxicity towards cultured human cells, to induce no apoptosis, and to be inactive on other proteases such as mmp- , ace, nep, g-glutamyl transpeptidase, cathepsin g, or dppiv. . n-hydroxy- -(naphthalene- -ylsulfanyl)acetamide n-hydroxy- -(naphthalene- -ylsulfanyl)acetamide ( ) has recently been identified as a potent apn inhibitor. it acts in a dose-dependent manner and is inactive on metalloenzymes mmp- , mmp- , mmp- , or a-lap. the design of synthetic apn inhibitors has often been relied to structure-activity studies based on active site models derived from structural data obtained on the zinc-dependent protease thermolysin crystallized with a variety of inhibitors. molecules capable of interacting with at least the s subsite of apn and which have a strong zinc-chelating group , were designed. according to these criteria, some a-aminophosphinic acids and derivatives such as or have been prepared and proved to be very potent apn inhibitors. according to the patterns of these models, synthesis of analogs such as the iodo derivative (rb ) have next been performed to give rise to the radiolabelled ( i)rb which represents a useful probe to investigate the physiological role of apn. , , in the same context, several b-aminothiols exemplified by or have been conceived and synthesized. the research in this field has then been extended to more elaborated series by roques and co-workers, and novel sulfur-containing molecules capable of inhibiting apn such as , , , or were prepared. from these works on b-aminothiols, two products emerged: pc (s)( -amino- -methylthiobutanethiol) ( ) the similarities between the active sites of apn and the membrane-bound protease neutral endopeptidase . (ec . . . , cd , nep) led to the idea that mixed inhibitors could be developed by selecting frameworks bearing a strong zinc-chelating group and a residue able to interact with at least one subsite (s , s , and s ) of each peptidase. , , - the first dual e . /apn inhibitors developed were hydroxamate-containing molecules such as kelatorphan ( ) or rb a ( ) , , whose several analogs have been synthesized and found to be also potent inhibitors of leukotriene a hydrolase. however, the important water solubility of these compounds is an impediment for crossing the blood-brain barrier and, consequently, for obtaining a good bioavailability. another strategy, involving more lipophilic derivatives, led to the synthesis of rb (n-((r,s)- -benzyl- ((s)( -amino- -methylthio)butyldithio)- -oxopropyl)-l-phenylalanine benzyl ester ( ) and rb (n-((s)- -benzyl- ((s)( -amino- -methylthio)butyldithio)- oxopropyl)-l-alanine benzyl ester ( ), two dual inhibitors in which a disulfide bridge links the apn inhibitor pc with analogs (the phenylalanine analog (st ) in the case of , or the alanine analogue in the case of ) of the benzyl ester of thiorphan, a specific nep inhibitor (fig. ) . , , such mixed inhibitors present the advantage to possess the above-mentioned disulfide bond which is relatively stable in plasma, in contrast to its rapid cleavage in brain, thus allowing the delivery of the nep and apn inhibitors in their active form toward their respective target. the development of such mixed inhibitors has constituted an important advance in the research of new antihypertensives and novel antinociceptive drugs devoid of opioid side effects. , (for reviews) more recently, a new generation of phosphinic acid derivatives have been prepared as nep/apn dual inhibitors, and compounds such as have been successfully tested in this context. , chemical structures of apn inhibitors - , and enzyme inhibition values are outlined in figure . the effects of some of these above described inhibitors on cell behavior have been assayed in in vitro approaches. table i provides a summary of most relevant studies in the human system. actinonin, bestatin, probestin, and psammaplin a (at - mm concentrations) were shown to reduce the growth of human t/b lymphocytes, dendritic and cord blood cd þ cells [ ] [ ] [ ] [ ] and human myeloid and lymphoid cell lines, , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] as well as the proliferation of , and various tumor and endothelial cell lines. , [ ] [ ] [ ] a question central to apn inhibition studies is how cell growth can be turned off by apn inhibitors. apn inhibitors may alter the processing of (unknown) growth factors directly involved in the regulation of growth. in addition, several studies indicate that inhibitors like actinonin and probestin may transmit intracellular-transduction signals by interfering with the map kinase signaling pathway. , , , a second cell signaling pathway involving the wnt- a proto-oncogene appears also affected by inhibition of apn by actinonin. it has to be pointed out that actinonin (at a mm concentration) inhibited the growth of both cd -positive myeloid and cd -negative lymphoma cell lines suggesting that the effects induced by actinonin are not likely to be mediated by cd . moreover, amastatin at a concentration which inhibits apn activity was found without any effect on the growth of human myeloid cell lines , . bestatin-mediated cell growth arrest is associated with an induction of cell maturation of clonogenic gm-cfu (granulocyte-macrophage colony forming unit) cells from human immature derived-bone marrow cells. , similarly, treatment of human myeloid u and nb cell lines with bestatin induced phenotypic changes characteristic of macrophage (u ) or neutrophil (nb ) maturation. , , cell growth arrest induced by apn inhibitors correlates with alternated secretion of proinflammatory and immunosuppressive cytokines involved in pathophysiological processes. bestatin ( . mm) increased the levels of il- secreted by endothelial cells, and of il- release from mouse peritoneal macrophages and il- release from concanavalin-stimulated t cells. probestin induces the synthesis and release of tgf-b . , recent observations point to the involvement of apn in the process of apoptosis (programmed cell death). bestatin and actinonin (starting mm) induce apoptosis in a large variety of cell lines, i.e. myeloid (p /tsu, hl- , u , nb ) and lymphoid (jurkat, bjab, nalm , boe) cells, and carcinoma (fibrosarcoma, cervical, and lung carcinoma). , , , , , betulinic acid induces apoptosis in the ht colon cancer cell line ( mm) and in acute leukemia cells ( mm). in a general way, cell motility (migration and invasion) may be influenced by the processing of chemokines and/or degradation of the extracellular matrix (ecm). the two small proteins with chemotactic activity, mcp- and f-mlp, are in vitro hydrolyzed by apn/cd . with regard to mcp- , there is no current data reporting the potential action of apn inhibitors on the mcp- -mediated migration. actinonin and amastatin were able to enhance the chemotactic response of human neutrophils toward f-mlp. one explanation of the effects of actinonin or amastatin would be that both inhibitors prevent the inactivation of f-mlp by apn, to further enhance the f-mlp-mediated chemotactic response. it has however to underline that both inhibitors weakly inhibited apn enzymatic activity over the range from À to À m, concentrations that are effective on neutrophil migration. apn inhibition by actinonin or bestatin significantly enhanced the in vitro migration of eosinophils across huvec monolayers. moreover, actinonin, bestatin as well as leuhistin ( - mm) significantly blocked the invasion of various human metastatic tumor cells into reconstituted basement membranes , or into matrigel. , [ ] [ ] [ ] these latter data suggested that apn could be indirectly involved in type iv collagen degradation by activating type iv procollagenase/prommp- . , , recent studies demonstrated that soluble apn/cd induces in vitro chemotactic migration of t lymphocytes, and that bestatin at high concentration ( mm) abolishes this process, suggesting that the enzymatic activity of apn was responsible for the chemotactic activity. , , , the demonstration of the participation of apn in angiogenesis has come from recent studies in which blocking apn activity by apn inhibitors resulted in the perturbation of ''angiogenic'' assays (table i) . apn/cd is expressed on the human umbilical vein endothelial cells (huvecs) of angiogenic, but not normal, vasculature. bestatin, betulinic acid, amastatin, curcumin, and psammaplin a ( - mm) abrogate the ability of the huvecs cultured on matrigel to organize a capillary network , , , [ ] [ ] [ ] without altering their proliferation rates. in contrast, one study underlines the proangiogenic effect of bestatin ( - mm) which instead causes matrix degradation and stimulates the invasion of microvascular endothelial cells into a fibrin matrix. in the chorioallantoic membrane (cam) assay, the angiogenic response is determined by measuring the number of avian extraembryonic capillary vessels that grow within a matrix polymer (containing an angiogenic molecule such as fibroblast growth factor- /fgf- ) placed on the yolk sac membrane of a day embryo in culture. the chick vasculature expresses a phenotype apn/cd , and subsequent treatment with bestatin or actinonin ( mg) inhibited fgf- -induced angiogenesis. in the mouse retinal neovascularization model, bestatin ( mg/mouse) leads to the blockade of hypoxia-induced retinal neovascularization in mice. the intraperitoneal administration of bestatin ( - mg/kg/day) after the orthotopic implantation of b -bl melanoma cells into mice reduces the number of vessels oriented toward the established primary tumor mass on the dorsal side of mice. compiled data documenting the involvement of apn/cd in pathophysiological events (cancer, inflammation, infection, pain suppression) have come from studies which blocked apn activity in rodent models (table i) . studies in rats indicate that administration of bestatin leads to the inhibition of fetal growth and the induction of placental apoptosis. , the in vivo anti-cancer activities of bestatin and betulinic acid have been reported through their capacities to inhibit the growth of syngeneic tumor (leukemia/ melanoma/ovarian/hepatoma/gastric carcinoma) cells implanted in mice , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and rats. , , doses as low as . mg/kg for bestatin and mg/kg for betulinic acid were used in these studies. moreover, high doses (up to mg/kg) did not lead to any cytotoxic effect in mice. bestatin, leuhistin, and betulinic acid have been investigated for anti-inflammatory properties. betulinic acid possessed moderate ant-inflammatory abilities at relatively high concentrations ( mg/kg/mouse, i.v.). in contrast, bestatin and leuhistin inhibit acute inflammation associated the accumulation of polymorphonuclear neutrophils in a mouse model ( mg/kg, i.v.). , moreover, oral administration of bestatin ( mg/kg) in carcinoma-bearing mice induces generation of cytotoxic t cells and nk (natural killer) cells. angiotensins ii and iii are two peptide effectors of the brain rennin-angiotensin system that participate in the control of blood pressure, increase water consumption and vasopressin release. in hypertensive rats, infusion of amastatin ( nmol/min i.v.) prevents degradation of angiotensins associated with blood pressure decrease. , . in the mouse brain, apn inhibition by pc or ec ( - mg injected intracerebroventricularly) increases the half life of angiotensin iii, resulting in enhanced vasopressin release. , , several studies report that bestatin exerts anti-infectious properties by augmenting host resistance to bacterial, viral or fungal experimental infections in mice by inducing neutrophil and macrophage activation , and enhancing antibody production. [ ] [ ] [ ] [ ] [ ] [ ] finally, in the central nervous system, enkephalins which modulate responses to painful stimuli, are inactivated by apn and the membrane-bound protease neutral endopeptidase . (ec . . . , cd ). this led to the idea that inhibition of these enzymes (alone or in combination) could achieve clinically efficient analgesia. actinonin as well as the dual inhibitors rb and rb ( mg/kg, i.v.; mg/kg, i.p.) exhibited analgesic properties against chronic pain in rats and mice. , , , , in first clinical trials, bestatin ( mg/daily) has been used to treat patients with acute and chronic myeloid leukemias (aml, cml) and lymphomas. [ ] [ ] [ ] [ ] [ ] therapeutic efficacy was demonstrated by a prolongation of survival in patients with aml , and lymphomas, , , and in promoting graft versus leukemia effects in patients following allogeneic bone marrow transplant. in a phase ib trial, activation of blood monocytes and increase in the cd /cd lymphocyte ratio were observed in hodgkin's and non-hodgkin's lymphoma patients treated orally with high doses of bestatin ( - mg/daily/ days) following autologous bone marrow transplantation. , , in phase iii trials in resected stage i squamous cell lung carcinoma, survival was statistically better for patients who were treated with bestatin ( mg/daily/ years) as a post-operative adjuvant therapy than those who received a placebo. , apn/cd , is useful in defining clinical subgroups of patients with various malignancies or inflammatory diseases. the use of natural and synthetic apn inhibitors has revealed that apn/cd participates to the control of major biological processes such as proliferation, secretion and apoptosis. dysregulation of apn/cd in tumors is often linked to tumor invasion and angiogenesis. studies on non-hematopoietic cells suggest that apn/cd may influence cell migration and invasion. apn/ cd inhibitors have been shown to alter angiogenesis in in vitro and in vivo assays. documented evidence underlines both the antiangiogenic and proangiogenic effects of bestatin. , , figure summarizes our current understanding of the involvement of apn inhibitors in the modulation of these events. the detailed molecular mechanisms underlying these effects are however yet unclear. importantly, the requirement for apn in these processes has been mostly confirmed with studies in which apn/cd expression was blocked by neutralizing cd antibodies , , , , or antisense cd oligonucleotides, , , or enhanced by the use of cd transfectants. it has however to be pointed out that most of apn inhibitors lack tight specificity by inhibiting other membrane-bound metalloproteases or secreted matrix metalloproteinases (mmps) ( table i) . for example, bestatin interacts with leucyl-aminopeptidase (ec . . . , oxytocinase, leu-ap), aminopeptidase b (ec . . . , ap-b) and aminopeptidase w (ec . . . , ap-w) , - thus suggesting that some of the observed chemotherapeutic actions of bestatin may be due to inhibition of other cell surface peptidases. actinonin was recently shown to interact with human peptide deformylase, , meprin a (ec . . . , endopeptidase . ), and mmp- . amastatin and probestin in the low micromolar range ( . - mm) inhibit aminopeptidase a (ec . . . , ap-a) and ap-w. , , leuhistin inhibits ap-a and ap-b to the same degree than apn. curcumin and betulinic acid block mmp- expression and collagenase activity through inhibition of nf-kb activation. [ ] [ ] [ ] [ ] [ ] in addition, the use of available apn inhibitors in some experimental situations has revealed complex effects on cell behavior. as mentioned in paragraph .a, cd -positive and cd negative cell lines are equally sensitive to the growth-inhibitory effect of actinonin ( - mm) thus emphasizing that actinonin may induce unspecific cytotoxic side-effects. moreover, betulinic acid inhibits tube formation of bovine aortic endothelial cells at a concentration which had no effect on the cell viability and in vivo apn activity of endothelial cells, thus indicating an apn-independent mode of action of betulinic acid. together, these observations emphasize the need for more specific and targeted apn inhibitors to (re)evaluate the actions of apn/cd in pathophysiological processes. future consideration has to be given to the obtention of the three-dimensional structure of apn determined by nmr spectroscopy to help apn inhibitor design strategy. further in vitro and in vivo studies with promising non cytototoxic apn inhibitors (such as psammaplin a, phosphonic derivatives, flavone- -acetic acid derivatives) are also required before clinically prescribing an apn inhibitor as an anti-cancer or anti-inflammatory agent. ectopeptidases in pathophysiology merops: the peptidase database families of zinc metalloproteases complete amino acid sequence of human intestinal aminopeptidase n as deduced from cloned cdna isolation of an aminopeptidase from kidney particles human myeloid plasma membrane glycoprotein cd (gp ) is identical to aminopeptidase n hematopoietic differentiation antigens that are membrane-associated enzymes: cutting is the key! characterization of particulate and soluble variants of the brush-border enzymes alanine aminopeptidase, alkaline phosphatase and gamma-glutamyltransferase in human urine cd (gp ; aminopeptidase-n): predominant functional activity in blood is localized to plasma and is not cell-surface associated high-molecular-mass isoform of aminopeptidase n/cd in serum from cholestatic patients soluble aminopeptidase n/cd in malignant and nonmalignant effusions and intratumoral fluid transmembrane proteases as disease markers and targets for therapy ontogenic and adult whole body distribution of aminopeptidase n in rat investigated by in vitro autoradiography the significance of aminopeptidases and haematopoietic cell differentiation induction of cd expression on fresh myeloid leukaemia: correlation of cd expression with aminopeptidase-n activity biochemical and functional characterization of aminopeptidase n expressed by human melanoma cells human melanoma invasion and metastasis enhancement by high expression of aminopeptidase n/cd urinary excretion of glycine.prolile dipeptidile aminopeptidase, n-acetyl-beta-d-glucosaminidase, alanine aminopeptidase, and low molecular protein in patients with renal cell carcinoma clinical significance of aminopeptidase n/cd expression in human pancreatic carcinoma aminopeptidase n is involved in cell motility and angiogenesis: its clinical significance in human colon cancer inhibition of aminopeptidase n (ap-n) and urokinase-type plasminogen activator (upa) by zinc suppresses the invasion activity in human urological cancer cells determinants essential for the transmissible gastroenteritis virus-receptor interaction reside within a domain of aminopeptidase-n that is distinct from the enzymatic site aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev further characterization of aminopeptidase-n as a receptor for coronaviruses human aminopeptidase n is a receptor for human coronavirus e substance p and bradykinin are natural inhibitors of cd / aminopeptidase n alanyl aminopeptidase from human seminal plasma: purification, characterization, and immunohistochemical localization in the male genital tract puromycin-sensitive alanyl aminopeptidase from human liver cytosol: purification and characterization lapstatin, a new aminopeptidase inhibitor produced by streptomyces rimosus, inhibits autogenous aminopeptidases novel potent nonpeptide aminopeptidase n inhibitors with a cyclic imide skeleton novel small molecule nonpeptide aminopeptidase n inhibitors with a cyclic imide skeleton nonpeptide small-molecular inhibitors of dipeptidyl peptidase iv: n-phenylphthalimide analogs preparation of novel specific aminopeptidase inhibitors with a cyclic imide skeleton potent homophthalimide-type inhibitors of b f /l mouse melanoma cell invasion specific inhibitor of puromycin-sensitive aminopeptidase with a homophthalimide skeleton: identification of the target molecule and a structure-activity relationship study specific nonpeptide inhibitors of puromycin-sensitive aminopeptidase with a , ( h, h)-quinazolinedione skeleton the most potent organophosphorus inhibitors of leucine aminopeptidase. structure-based design, chemistry, and activity potent and selective inhibition of zinc aminopeptidase a (ec . . . , apa) by glutamyl aminophosphinic peptides: importance of glutamyl aminophosphinic residue in the p position alpha-keto amide inhibitors of aminopeptidases alpha-aminoboronic acid derivatives: effective inhibitors of aminopeptidases alpha-aminoaldehydes: transition state analogue inhibitors of leucine aminopeptidase inhibition of zinc metallopeptidases by flavonoids and related phenolic compounds: structure-activity relationships inhibition of metallopeptidases by flavonoids and related compounds inhibition of human leukocyte function, alanyl aminopeptidase (apn, cd ) and dipeptidylpeptidase iv (dp iv, cd ) enzymatic activities by aqueous extracts of cistus incanus l. ssp. incanus compounds from epilobium angustifolium inhibit the specific metallopeptidases ace, nep, and apn actinonin: an antibiotic substance produced by an actinomycete production of actinonin, an inhibitor of aminopeptidase m, by actinomycetes studies concerning the antibiotic actinonin. part i. the constitution of actinonin. a natural hydroxamic acid with antibiotic activity studies concerning the antibiotic actinonin. part ii. total synthesis of actinonin and some structural analogues by the isomaleimide method studies concerning the antibiotic actinonin. part iii. synthesis of structural analogues of actinonin by the anhydride-imide method studies concerning the antibiotic actinonin. part iv. synthesis of structural analogues of actinonin by the mixed anhydride method studies concerning the antibiotic actinonin. part v. synthesis of structural analogues of actinonin by the anhydride-ester method studies concerning the antibiotic actinonin. part vi. synthesis of structural analogues of actinonin by dicyclohexylcarbodiimide coupling reactions studies concerning the antibiotic actinonin. part vii. mass spectra of actinonin and related compounds asymmetric synthesis of (À)-actinonin and (À)-epi-actinonin studies concerning the antibiotic actinonin. part viii. structure-activity relationships in the actinonin series bestatin analogue from streptomyces neyagawaensis sl- mr- a and b, new aminopeptidase n inhibitors, produced by streptomyces neyagawaensis sl- biosynthesis of peptide inhibitor mr- by streptomyces neyagawaensis synthesis and anti-cancer activity of ahpa derivatives inhibition of aminopeptidases by amastatin and bestatin derivatives amastatin, an inhibitor of aminopeptidase a, produced by actinomycetes structure and chemical synthesis of amastatin synthesis of ( s, r)- -amino- -hydroxy- -methylhexanoic acid derivatives. application to the synthesis of amastatin, an inhibitor of aminopeptidase synthesis and structure-activity relationships of amastatin analogues, inhibitors of aminopeptidase a leucine aminopeptidase: bestatin inhibition and a model for enzyme-catalyzed peptide hydrolysis leukotriene a hydrolase. inhibition by bestatin and intrinsic aminopeptidase activity establish its functional resemblance to metallohydrolase enzymes bestatin inhibits covalent coupling of [ h]lta to human leukocyte lta hydrolase effects of metalloproteinase inhibitors on leukotriene a hydrolase in human airway epithelial cells leukotriene a hydrolase: a critical role of glutamic acid- for the binding of bestatin bestatin as an experimental tool in mammals bestatin, an inhibitor of aminopeptidase b, produced by actinomycetes the structure of bestatin the chemical synthesis of bestatin a stereocontrolled synthesis of (À)-bestatin from an acyclic allylamine by iodocyclocarbamation synthesis of [beta]-amino-[alplha]-hydroxy acids via aldol condensation of a chiral glycolate enolate. synthesis of (À)-bestatin a stereospecific synthesis of (À)l-bestatin from -malic acid synthesis of the peptidic [alpha]-hydroxy amides phebestin, probestin, and bestatin from [alpha]-keto amide precursors acylnitrene route to vicinal amino alcohols. application to the synthesis of (À)-bestatin and analogues a new one-pot method for the synthesis of [alpha]-siloxyamides from aldehydes or ketones and its application to the synthesis of (À)-bestatin new stereoselective synthesis of the peptidic aminopeptidase inhibitors bestatin, phebestin and probestin application of acyl cyanophosphorane methodology to the synthesis of protease inhibitors: poststatin, eurystatin, phebestin, probestin, and bestatin chirospecific synthesis of the( s, r)-and( s, s)- -amino- -hydroxy- -phenylbutanoic acids from sugar: application to (À)-bestatin synthesis and structure-activity relationships of bestatin analogues, inhibitors of aminopeptidase b synthesis of p-hydroxyubenimex synthesis of sulfur-containing analogues of bestatin. inhibition of aminopeptidases by alpha-thiolbestatin analogues design of novel inhibitors of aminopeptidases. synthesis of peptide-derived diamino thiols and sulfur replacement analogues of bestatin development of selective tight-binding inhibitors of leukotriene a hydrolase inhibition of arginine aminopeptidase by bestatin and arphamenine analogues phebestin, a new inhibitor of aminopeptidase n, produced by streptomyces sp. mj -m probestin, a new inhibitor of aminopeptidase m, produced by streptomyces azureus mh - f . i. taxonomy, production, isolation, physico-chemical properties, and biological activities probestin, a new inhibitor of aminopeptidase m, produced by streptomyces azureus mh - f . ii. structure determination of probestin x-ray structure determination of ( s, r)- -amino- -hydroxy- -phenylbutanoic acid, a new amino acid component of bestatin regio-and stereo-specific synthesis of threo- -amino- -hydroxy-acids, novelamino-acids contained in aminopeptidase inhibitors of microbial origin diastereo-and enantioselective synthesis of , -amino alcohols from glycol aldehyde hydrazones; asymmetric synthesis of (r,r)-statin versatile synthetic routes to threo-[beta]-amino hydroxy carboxylic acids, statine and its analogues synthesis of taxol and taxotere side chains by -(trimethylsilyl)thiazole based homologation of l-phenylglycine a catalytic asymmetric synthesis of cyclohexylnorstatine catalytic asymmetric aminohydroxylation (aa) of olefins n-halocarbamate salts lead to more efficient catalytic assymetric aminohydroxylation the enantioselective synthesis of [beta]-aminoacids, their [alpha]-hydroxy derivatives, and the n-terminal components of bestatin and microginin an enantioselective, stereodivergent approach to anti-and syn-[alpha]-hydroxy-[beta]-amino acids from anti- -amino- , -diols. synthesis of the ready for coupling taxotere(r) side chain stereoselective synthesis of (À)-n-boc-statine and (À)-n-boc-norstatine metal halide-mediated opening of three membered rings: enantioselective synthesis of ( s, r)- -amino- -hydroxydecanoic acid and ( r)- -aminodecanoic acid enantiospecific and diastereoselective synthesis of syn-[beta]-amino-[alpha]-hydroxy acids practical synthesis of ( s, s)- -amino- -hydroxy- -phenylbutyric acid, a key component of hiv protease inhibitors a convenient preparation of ( sr, s)- amino- -hydroxy- -phenylbutanoic acid; an important peptide bond isostere a novel synthesis of allophenylnorstatine from (r)-aspartic acid asymmetric synthesis of -amino- -hydroxy- -phenylbutanoate diastereoselective synthesis of n-protected [beta]-amino-[alpha]-hydroxyacids (norstatines) from urethane n-carboxyanhydrides (uncas) process for producing erythro- -amino- -hydroxybutyric acid derivatives the s-thioester enolate/imine condensation: a shortcut to [beta]-lactams new synthetic technology for efficient construction of [alpha]-hydroxy-[beta]-amino amides via the passerini reaction analysis of amide bond formation with an [alpha]-hydroxy-[beta]-amino acid derivative, -amino- -hydroxy- -phenylbutanoic acid, as an acyl component: byproduction of homobislactone a short and enantioselective synthesis of n-terminal components of bestatin, amastatin, and microginin stereoselective synthesis of [alpha]-hydroxy-[beta]-amino acids: the chiral pool approach leuhistin, a new inhibitor of aminopeptidase m, produced by bacillus laterosporus bmi - f . i. taxonomy, production, isolation, physico-chemical properties, and biological activities biosynthetic study of leuhistin, a new inhibitor of aminopeptidase m leuhistin, a new inhibitor of aminopeptidase m, produced by bacillus laterosporus bmi - f . ii. structure determination of leuhistin quaternary benzo[c]phenanthridine alkaloids as inhibitors of aminopeptidase n and dipeptidyl peptidase iv irreversible inhibition of cd /aminopeptidase n by the antiangiogenic agent curcumin molecular mechanisms of chemopreventive effects of selected dietary and medicinal phenolic substances role of chemopreventive agents in cancer therapy anticancer potential of curcumin: preclinical and clinical studies molecular mechanisms underlying chemopreventive activities of anti-inflammatory phytochemicals: down-regulation of cox- and inos through suppression of nf-kappa b activation chemotherapeutic potential of curcumin for colorectal cancer cell signaling pathways altered by natural chemopreventive agents the molecular mechanisms for the antitumorigenic effect of curcumin inhibition of the hiv- and hiv- proteases by curcumin and curcumin boron complexes current lead natural products for the chemotherapy of human immunodeficiency virus (hiv) infection effect of curcumin on radiation-impaired healing of excisional wounds in mice role of curcumin, a naturally occurring phenolic compound of turmeric in accelerating the repair of excision wound, in mice whole-body exposed to various doses of gammaradiation synthesis of curcumin and related compounds synthesis of naturally occuring curcuminoids and related compounds simplified condition for synthesis of curcumin i and other curcuminoids curcumin analogs with altered potencies against hiv- integrase as probes for biochemical mechanisms of drug action electron ionization mass spectrometry of curcumin analogues: an olefin metathesis reaction in the fragmentation of radical cations design, synthesis, and biological evaluation of angiogenesis inhibitors: aromatic enone and dienone analogues of curcumin hydrazinocurcumin, a novel synthetic curcumin derivative, is a potent inhibitor of endothelial cell proliferation method of producing betulinic acid betulinic acid inhibits aminopeptidase n activity a review of natural and modified betulinic, ursolic and echinocystic acid derivatives as potential antitumor and anti-hiv agents betulinic acid: a promising anticancer candidate chemistry, biological activity, and chemotherapeutic potential of betulinic acid for the prevention and treatment of cancer and hiv infection a concise semi-synthetic approach to betulinic acid from betulin the synthesis of betulinic acid from betulin and its solubilization with liposomes synthesis of betulinic acid derivatives with activity against human melanoma lupane triterpenes and derivatives with antiviral activity pavophylline, a new saponin from the stem of pavonia zeylanica ( )-en- -oic acid from the stem of dillenia pentagyna a saponin from asparagus gonocladus a betulinic acid glycoside from schefflera venulosa two new triterpenoid sulfates from the leaves of schefflera octophylla studies on anti-inflammatory agents. v. a new anti-inflammatory constituent of pyracantha crenulata roem brominated tyrosine metabolites from an unidentified sponge phenolic constituents of psammaplysilla two bromotyrosine-cysteine derived metabolites from a sponge novel marine sponge derived amino acids . additional psammaplin derivatives from psammaplysilla purpurea bisprasin, a novel ca( þ) releaser with caffeine-like properties from a marine sponge, dysidea spp., acts on ca( þ)-induced ca( þ) release channels of skeletal muscle sarcoplasmic reticulum new bromotyrosine metabolites from the sponge aplysinella rhax isolation of psammaplin a ¢-sulfate and bisaprasin ¢-sulfate from the marine sponge aplysinella rhax psammaplin a, a chitinase inhibitor isolated from the fijian marine sponge aplysinella rhax psammaplins from the sponge pseudoceratina purpurea: inhibition of both histone deacetylase and dna methyltransferase cytotoxic compounds from a two-sponge association new bromotyrosine derivatives from an association of two sponges, jaspis wondoensis and poecillastra wondoensis combinatorial synthesis through disulfide exchange: discovery of potent psammaplin a type antibacterial agents active against methicillinresistant staphylococcus aureus (mrsa) optimization and mechanistic studies of psammaplin a type antibacterial agents active against methicillin-resistant staphylococcus aureus (mrsa) psammaplin a, a marine natural product, inhibits aminopeptidase n and suppresses angiogenesis in vitro psammaplin a, a natural bromotyrosine derivative from a sponge, possesses the antibacterial activity against methicillin-resistant staphylococcus aureus and the dna gyrase-inhibitory activity psammaplin a, a natural phenolic compound, has inhibitory effect on human topoisomerase ii and is cytotoxic to cancer cells bromotyrosine-derived natural and synthetic products as inhibitors of mycothiol-s-conjugate amidase cytotoxicity of psammaplin a from a two-sponge association may correlate with the inhibition of dna replication [alpha]-aminoketone-ein beitrag zur synthese optish aktiver derivate von aminosäuren und peptiden inhibition of aminopeptidase m by alkyl d-cysteinates -amino- -tetralone derivatives: novel potent and selective inhibitors of aminopeptidase-m (ec . . . ) -amino- -hydroxy-propionaldehyde and -amino- -hydroxypropan- -one derivatives: new classes of aminopeptidase inhibitors effect of flavone acetic acid on endothelial cell proliferation: evidence for antiangiogenic properties synthesis and biological evaluation of novel flavone- -acetic acid derivatives as reversible inhibitors of aminopeptidase n/cd n-hydroxy- -(naphthalene- -ylsulfanyl)-acetamide, a novel hydroxamic acid-based inhibitor of aminopeptidase n and its anti-angiogenic activity the binding of l-valyl-l-tryptophan to crystalline thermolysin illustrates the mode of interaction of a product of peptide hydrolysis potent and systemically active aminopeptidase n inhibitors designed from active-site investigation phosphinic derivatives as new dual enkephalin-degrading enzyme inhibitors: synthesis, biological properties, and antinociceptive activities design of the first highly potent and selective aminopeptidase n (ec . . . ) inhibitor synthesis of (s)-benzyl- -[hydroxy( ¢(r)-aminoethyl)phosphinyl]propanoyl-l- -[ i]-iodotyrosine: a radiolabelled inhibitor of aminopeptidase n binding properties of a highly potent and selective iodinated aminopeptidase n inhibitor appropriate for radioautography first discrete autoradiographic distribution of aminopeptidase n in various structures of rat brain and spinal cord using the selective iodinated inhibitor [ i]rb potent inhibition of cerebral aminopeptidases by carbaphethiol, a parenterally active compound investigation of the active site of aminopeptidase a using a series of new thiol-containing inhibitors differential inhibition of aminopeptidase a and aminopeptidase n by new beta-amino thiols pc , a specific aminopeptidase n inhibitor, induces vasopressin release by increasing the half-life of brain angiotensin iii aminopeptidase a, which generates one of the main effector peptides of the brain rennin-angiotensin system, angiotensin iii, has a key role in central control of arterial blood pressure analgesic effects of kelatorphan, a new highly potent inhibitor of multiple enkephalin degrading enzymes mixed inhibitorprodrug as a new approach toward systemically active inhibitors of enkephalin-degrading enzymes dual inhibitors of enkephalin-degrading enzymes (neutral endopeptidase . and aminopeptidase n) as potential new medications in the management of pain and opioid addiction inhibitors of neprilysin: design, pharmacological and clinical applications analgesic responses elicited by endogenous enkephalins (protected by mixed peptidase inhibitors) in a variety of morphine-sensitive noxious tests inhibition of the enkephalin-metabolizing enzymes by the first systemically active mixed inhibitor prodrug rb induces potent analgesic responses in mice and rats kelatorphan and related analogs: potent and selective inhibitors of leukotriene a hydrolase molecular pharmacology of endothelin converting enzymes zinc metallopeptidases: active site structure and design of selective and mixed inhibitors: new approaches in the search for analgesics and anti-hypertensives peptidomimetics as receptors agonists or peptidase inhibitors: a structural approach in the field of enkephalins, anp, and cck aminophosphinic inhibitors as transition state analogues of enkephalin-degrading enzymes: a class of central analgesics inhibitory effect of bestatin on the growth of human leukemic cells induction of apoptosis by bestatin (ubenimex) in human leukemic cell lines cd /n-aminopeptidase is involved in the development of dendritic cells and macrophages from cord blood cd (þ) cells inhibitory effect of bestatin on the growth of human lymphocytes bestatin, an inhibitor of aminopeptidase b, suppresses the proliferation and differentiation of human b-cells in vitro inhibition of alanylaminopeptidase suppresses the activation-dependent induction of glycogen synthase kinase- beta (gsk- beta) in human t cells inhibition of alanyl aminopeptidase induces map-kinase p /erk in the human t cell line karpas- effect of ubenimex on the proliferation and differentiation of u human histiocytic lymphoma cells cell cycle retardation in monocytoid cells induced by aminopeptidase n (cd ) aminopeptidase inhibitors inhibit proliferation and induce apoptosis of k and sti -resistant k cell lines through the mapk and gsk- beta pathways expression of cd /aminopeptidase n and cd /neutral endopeptidase on cultured human keratinocytes identification of extra-and intracellular alanyl aminopeptidases as new targets to modulate keratinocyte growth and differentiation expression of aminopeptidase n on human choriocarcinoma cells and cell growth suppression by the inhibition of aminopeptidase n activity growth inhibitory effect of bestatin on choriocarcinoma cell lines in vitro antitumor activity of actinonin in vitro and in vivo aminopeptidase n/cd is directly linked to signal transduction pathways in monocytes aminopeptidase n-mediated signal transduction and inhibition of proliferation of human myeloid cells modulation of wnt- a expression by actinonin: linkage of apn to the wnt-pathway? augmentation of death ligand-induced apoptosis by aminopeptidase inhibitors in human solid tumor cell lines enhancing effect of ubenimex (bestatin) on proliferation and differentiation of hematopoietic progenitor cells, and the suppressive effect on proliferation of leukemic cell lines via peptidase regulation modulation of bone marrow cell functions in vitro by bestatin (ubenimex) enhancement of sensitivity by bestatin of acute promyelocytic leukemia nb cells to all-trans retinoic acid leukemic cell-surface cd /aminopeptidase n and resistance to apoptosis mediated by endothelial cells enhancement of interleukin and interleukin releases by ubenimex synergistic action of dpivand apn in the regulation of t cell function aminopeptidase inhibitor bestatin induces hl- cell apoptosis through activating caspase induction of apoptosis by ubenimex (bestatin) in human non-small-cell lung cancer cell lines betulinic acid-induced apoptosis in leukemia cells effects of aminopeptidase inhibitors actinonin and amastatin on chemotactic and phagocytic responses of human neutrophils differential regulation of aminopeptidase n (cd ) by transendothelial migration and cytokines on human eosinophils role of aminopeptidase n (cd ) in tumor-cell invasion and extracellular matrix degradation inhibition of tumor invasion and extracellular matrix degradation by ubenimex (bestatin) aminopeptidase n regulated by zinc in human prostate participates in tumor cell invasion inhibition of tumor cell invasion and matrix degradation by aminopeptidase inhibitors possible contribution of aminopeptidase n (apn/cd ) to invasive potential enhanced by interleukin- and soluble interleukin- receptor in human osteosarcoma cell lines role of cd / aminopeptidase n in rat lymphocytic alveolitis caused by thoracic irradiation aminopeptidase n is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis cd /apn is activated by angiogenic signals and is essential for capillary tube formation anti-tumor angiogenesis effect of aminopeptidase inhibitor bestatin against b -bl melanoma cells orthotopically implanted into syngeneic mice betulinic acid inhibits growth factorinduced in vitro angiogenesis via the modulation of mitochondrial function in endothelial cells aminopeptidase inhibitor bestatin stimulates microvascular endothelial cell invasion in a fibrin matrix the chick embryo chorioallantoic membrane as a model for in vivo research on anti-angiogenesis bestatin results in pathophysiological changes similar to preeclampsia in rats via induction of placental apoptosis effects of bestatin on intrauterine growth of rat fetuses antitumor cells found in tumor-bearing mice given ubenimex effect of bestatin on syngeneic tumors in mice antitumor effect of bestatin combined with bleomycin against hepatoma ah subcutaneously transplanted in rats preclinical approaches to the development of effective immunotherapeutic protocols for the treatment of metastasis inhibition of lymph node metastasis of p leukemia by bestatin in mice discovery of betulinic acid as a selective inhibitor of human melanoma that functions by induction of apoptosis selective cytotoxicity of betulinic acid on tumor cell lines, but not on normal cells sterol and triterpene derivatives from plants inhibit the effects of a tumor promoter, and sitosterol and betulinic acid inhibit tumor formation in mouse skin two-stage carcinogenesis inhibitory effect of rikkunshi-to, a traditional chinese herbal prescription, on tumor promotion in two-stage carcinogenesis in mouse skin enhancement of antitumor effect of cytotoxic agents by bestatin the effect of ubenimex on n-methyl-n¢-nitro-n-nitrosoguanidine-induced stomach tumor in rats spinorphin as an endogenous inhibitor of enkephalin-degrading enzymes: roles in pain and inflammation use of aminopeptidase m as a hypotensive agent in spontaneously hypertensive rats studies on the mechanisms of action of the immunomodulator bestatin in various screening test systems enhancement by ubenimex (bestatin) of host resistance to candida albicans infection enhancement of antibody formation against herpes simplex virus in mice by the t-cell mitogen bestatin stimulation of cell-mediated immunity by bestatin correlates with reduction of bacterial persistence in experimental chronic salmonella typhimurium infection study of the prophylactic effect of ubenimex on experimental pyelonephritis induced by pseudomonas in neutropenic mice the mode of immunopotentiating action of bestatin: enhanced resistance to listeria monocytogenes infection analgesic effect of actinonin, a new potent inhibitor of multiple enkephalin degrading enzymes repeated systemic administration of the mixed inhibitor of enkephalin-degrading enzymes, rb , does not induce either antinociceptive tolerance or cross-tolerance with morphine pain-suppressive effects on various nociceptive stimuli (thermal, chemical, electrical and inflammatory) of the first orally active enkephalin-metabolizing enzyme inhibitor rb long lasting antinociceptive properties of enkephalin degrading enzyme (nep and apn) inhibitor prodrugs depressant effect on a c-fibre reflex in the rat, of rb , a dual inhibitor of enkephalin-degrading enzymes facilitation of enkephalins catabolism inhibitor-induced antinociception by drugs classically used in pain management immunotherapy with bestatin for acute nonlymphocytic leukemia in adults results of follow-up studies on prognosis after immunotherapy with bestatin in acute nonlymphocytic leukemia monocyte activation by an oral immunomodulator (bestatin) in lymphoma patients following autologous bone marrow transplantation the effect of bestatin on patients with acute and chronic leukemia and malignant lymphoma bestatin treatment of myelodysplastic syndromes and chronic myelogenous leukemia immunopotentiation with ubenimex for prevention of leukemia relapse after allogeneic bmt. the study group of ubenimex for bmt partial review of immunotherapeutic pharmacology in stem cell transplantation randomized double-blind placebo-controlled trial of bestatin in patients with resected stage i squamous-cell lung carcinoma bestatin in resected lung cancer. a randomized clinical trial antisense-mediated inhibition of aminopeptidase n (cd ) markedly decreases growth rates of hematopoietic tumour cells inhibition of aminopeptidase b and leucine aminopeptidase by bestatin and its stereoisomer action of ubenimex on aminopeptidase activities in spleen cells and peritoneal macrophages from mice inhibition of aminopeptidases n, a and w. a re-evaluation of the actions of bestatin and inhibitors of angiotensin converting enzyme enhancement of delayed-type hypersensitivity by bestatin, an inhibitor of aminopeptidase b and leucine aminopeptidase the slow, tight binding of bestatin and amastatin to aminopeptidases a new human peptide deformylase inhibitable by actinonin human mitochondrial peptide deformylase, a new anticancer target of actinonin-based antibiotics human meprin alpha and beta homo-oligomers: cleavage of basement membrane proteins and sensitivity to metalloprotease inhibitors endostatin binds to the catalytic domain of matrix metalloproteinase- identification and properties of the cell membrane bound leucine aminopeptidase interacting with the potential immunostimulant and chemotherapeutic agent bestatin curcumin exhibits antimetastatic properties by modulating integrin receptors, collagenase activity, and expression of nm and e-cadherin curcumin (diferuloylmethane) down-regulates cigarette smoke-induced nf-kappab activation through inhibition of ikappabalpha kinase in human lung epithelial cells: correlation with suppression of cox- , mmp- , and cyclin d betulinic acid suppresses carcinogen-induced nf-kappa b activation through inhibition of i kappa b alpha kinase and p phosphorylation: abrogation of cyclooxygenase- and matrix metalloprotease- inhibition of growth and survival of human head and neck squamous cell carcinoma cells by curcumin via modulation of nuclear factor-kappab signaling curcuminoids inhibit the angiogenic response stimulated by fibroblast growth factor- , including expression of matrix metalloproteinase gelatinase b characterization of aminopeptidase n from the brush border membrane of the larvae midgut of silkworm, bombyx mori as a zinc enzyme metabolism of aspartame by human and pig intestinal microvillar peptidases actinonin, a naturally occurring antibacterial agent, is a potent deformylase inhibitor the metabolism of neuropeptides. phase separation of synaptic membrane preparations with triton x- reveals the presence of aminopeptidase n the aminopeptidase activity in the human t-cell lymphoma line (jurkat) is not at the cell surface and is not aminopeptidase n (cd- ) metabolism of vasoactive peptides by plasma and purified renal aminopeptidase m purification by affinity chromatography using amastatin and properties of aminopeptidase a from pig kidney enzymic and molecular properties of aminopeptidase w insights into peptide and protein function: a convergent approach after a post-doctoral stay with professor a. g. m. barrett at the imperial college in london, he joined the cnrs (centre national de la recherche scientifique) at institut curie (paris), being appointed «attaché de recherche», then «directeur de recherche directeur de recherche'' in . she is appointed to her current position in the inserm laboratory of prof key: cord- -i reanan authors: nan title: ecb : th european congess on biotechnology date: - - journal: j biotechnol doi: . /j.jbiotec. . . sha: doc_id: cord_uid: i reanan nan in the last years biotechnology has made tremendous progress in its different application fields: red biotechnology, the use of biological methods for medical purposes, is firmly established in the development of new drugs. the use of plant or green biotechnology is under controversial discussion in politics and public. nevertheless, genetically modified herbicide and insect resistant crops are cultivated to a large extent. industrial biotechnology, now often named white biotechnology, seems widely underestimated in the public perception. it includes all industrial processes for the production of chemical products and enzymes, which fully or partly rely on the biological toolbox of nature. white biotechnology processes are carried out in a contained environment, typically in a bioreactor in a dedicated industrial plant. well-known examples are the fermentative productions of antibiotics, amino acids, vitamins and enzymes, products related to medical, food and feed applications. many products like the amino acids glutamic acid, lysine, threonine and tryptophane are exclusively produced using microbes in large scale industrial processes. in other cases, like the water soluble vitamin b , biotechnological processes successfully replaced chemical productions, due to lower costs and improved ecoefficiency. in contrast to this, most industrial chemicals and polymers are produced by chemical synthesis based on oil and gas. however, there are some examples for bioproducts among industrial chemicals. the solvents acetone and butanol, for instance, were manufactured by fermentation for several decades in the last century. since the s these fermentations have been replaced by more efficient and cheaper chemical synthesis. recently, new pilot and production processes for biopolymers like pha or biomonomers like , -propanediol or lactic acid were announced by different companies. currently, ethanol is by far the largest white biotech product by volume. in brazil, where bioethanol is used as liquid transportation fuel, the annual production is in the range of mio m . bioethanol is of growing importance also in the united states. business consultants predict a tremendous growth of biotechnological products within the chemical industry. high prices for crude oil, dropping prices for renewable resources, and scientific progresses nourish the expectation that industrial biotechnology will replace many bulk chemicals. is this realistic? will we switch from a petrochemical industry to a biobased chemistry within the next years? based on economic considerations it can be stated that this is a long term goal. to achieve this it remains a scientific challenge to make renewable raw materials available for competitive bioproduction of bulk chemicals at low costs. conversion of lignocellulosic material to fermentation sugar may be a solution. also green biotechnology can contribute to the supply of cheap fermentation raw materials. innovative ideas for downstream processing or further chemical conversion of fermentation products are required to enter the chemical value chains. furthermore, the identification of new higher value bioproducts is a chance for short term successes in white biotechnology. enzyme and protein engineering has the potential to create new biomolecules, metabolic engineering can contribute to develop new metabolic pathways, may be even for unnatural compounds. by continuously increasing the efficiency and throughput of dna sequencing we, together with colleagues, have sequenced the human genome and the genomes of all the major model organisms. the challenge now centers on understanding these vast instruction sets. our ability to read these instructions must be enhanced through collection of key additional data sets. one productive path for delineating the functional sequences and inferring their function is comparative sequence. the mouse genome sequence, for example, led to estimates that only % of the human genome is functional. sequencing of an extensive set of additional mammalian genomes promises to define these functional sequences with a resolution of less than base pairs. on a different course, we have sequenced the chimpanzee genome to learn what has changed in the evolution of humans. beyond providing for the first time a catalog of the differences between the two genomes, the comparison of the chimpanzee and human genomes reveals the patterns of neutral mutation and regions that deviate from that. the talk will summarize these and related findings. the sequence of additional primate genomes will help delineate what has changed specifically in humans and add power to the analysis. ultimately, capturing human sequence variation and correlating with phenotypic variation will be required to understand function. but learning what these functional elements do requires new sets of experimental data. for this, we have turned to the nematode c. elegans. in this simple system most of the ∼ k genes have been defined and experimentally confirmed. beyond the hundreds of genes with already known mutants, two centers are systematically producing gene knockouts or rnai can be used to inhibit any gene temporarily. sequences of three caenorhabditis species are already available, and two more are underway. expression data has been collected for all the genes under many conditions and time points through development. to enhance the resolution of expression data and to simplify phenotypic analysis of embryonic mutants, we are developing a system that will automatically trace the cell lineage and assign gene expression to precise cells with high temporal resolution. the latest results with the system will be described. in the longer term, this and similar datasets should provide an understanding of how the genome specifies the form and behavior of the worm. uhlen department of biotechnlogy, albanova university center, royal institute of technology (kth), stockholm, sweden here, we present a new protein atlas database (www.proteinatlas.org) showing the expression and localization of human protein in normal and cancer tissues. the atlas is based on the use of antibodies (agaton et al., ) to generate high-resolution immunohistochemistry images representing normal tissues and different cancer types (uhlen and ponten, ) . each antibody is used to generate more than individual images and each image has been annotated by a pathologist (kampf et al., in press) . the database has been created by the swedish human proteome resource (hpr) and the program has been set-up to allow the exploration of the human proteome with antibody-based proteomics (nilsson et al., in press) . the basic concept is to generate, in a systematic and high-throughput manner (uhlen and ponten, ) , specific antibodies to all human proteins, and subsequently used these for functional analysis of the corresponding proteins in a wide range of assay platforms, including (i) a protein atlas for tissue profiles (kampf et al., in press) , (ii) specific probes to evaluate the functional role of individual proteins, and (iii) affinity reagents for purification of the specific proteins and their associated complexes for structural and biochemical analyses. ments, most effective source of variation was perturbation in growth medium, followed by perturbation in growth rate. effect of gene deletion on data variation was found to be less apparent when compared to other perturbations. a significant similarity in variation of metabolome and mrna data was observed, which may be used as the key point for integration of these two sets of data in functional analysis of genes. projection to latent structures (partial least squares, pls) is used for integration of transcriptome and metabolome data. comparison of pca and pls shows that linear model constructed via pls to predict the metabolome data does not make use of all the variation in transcriptome data. thus, pls allows the discrimination between the portion of gene expression change that affects the metabolome profile and the portion that is not directly effective on metabolome. both pca and pls can be used to detect the open reading frames (orfs) which are the main sources of variation in transcriptome data and/or effective on metabolome profile. extracellular metabolomics to accelerate the discovery of key genes involved in fibre degradation silas g. villas-bôas, geoffrey lane, graeme attwood, adrian cookson agresearch limited, grasslands research centre, tennent drive, private bag , palmerston north, new zealand. e-mail: silas.villas-boas@agresearch.co.nz (s.g. villas-bôas) the genome of the hemicellulose-degrading microbe clostridium proteoclasticum is been sequenced and an array of candidate genes with diverse activity relevant to fibre degradation have been identified by automated gene annotation methods. c. proteoclasticum falls within the butyrivibrio-pseudobutyrivibrio assemblage of rumen bacteria which are though to play an important role in the degradation of plant hemicellulose-lignin complexes which limit fibre degradation in the rumen. for new zealand it makes strategic sense to invest in microbial genomics efforts applied to agriculture where the country holds a strong competitive advantage and where ruminants constitute the vast majority of farmed animals. in conjunction with dna sequencing, proteomics and transcriptomics (micro-array analysis) we are using metabolomics as an additional functional genomics tool for gene discovery. we have established a footprinting approach for microbial metabolome analysis focused mainly on metabolic intermediates of polysaccharide degradation to provide quantitative information on end products of fibre-degrading enzymes. a gc-ms method has been developed that is able to resolve complex biological mixtures containing mono-, di, and oligosaccharides, in addition to a series of organic acids. we are currently phenotyping a series of c. proteoclasticum mutants to validate our analytical methodology and we are going to fully characterize the fibrolytic ability of c. proteoclasticum to be compared with other fibre-degrading microbes. we believe that our metabolomics data will complement current proteomic analysis of fibre-degrading enzymes and micro-array analysis of gene expression from a series of mutants by providing direct evidence of the metabolic function of key genes involved in fibre-degradation processes. many gram-negative bacteria utilize cell-to-cell communication systems that rely on diffusible n-acyl homoserine lactone (ahl) signal molecules to monitor the size of the population in a process known as quorum sensing (qs). in human pathogens this form of gene regulation ensures that the cells remain invisible to the immune system of the host until the pathogen has reached a critical population density sufficient to overwhelm host defenses and to establish the infection. the qs regulon of pseudomonas aeruginosa and burkholderia cepacia, two important pathogens for patients suffering from cystic fibrosis, has been studied by proteome analyses. comparative twodimensional gelelectrophoresis of pre-fractionated protein mixtures (extra-, surface-, and intracellular proteins) coupled to mass spectrometry analysis or n-terminal sequencing has been employed to recognize and identify qs-controlled proteins. our findings strongly support the importance of ahl-mediated cell-cell-communication as a global regulatory system and suggest that qs control also operates via post-translational mechanisms. as qs has been proven to be a central regulator for the expression of pathogenic traits and biofilm formation in opportunistic human pathogens it represents a highly attractive target for the development of novel anti-infective compounds. functional genomics technologies (transcriptomics and proteomics) have been exploited to validate the target specificity of natural and synthetic qs inhibitors, thus having a great potential as alternative therapeutics for the treatment of bacterial infections. modeling cell cycle complex formation from high-throughput data sets lars juhl jensen european molecular biology laboratory, meyerhofstrasse , heidelberg, germany. e-mail: jensen@embl. de to analyze the dynamics of protein complexes during the mitotic cell cycle, we integrated data on protein interactions and gene expression. the resulting time-dependent interaction network for the first time places both periodically and constitutively expressed proteins in a temporal cell cycle context, thereby revealing novel components and modules. we discover that most complexes consist of both periodically and constitutively expressed subunits, suggesting that the former control complex activity by a mechanism of just-in-time assembly. consistent with this, we show that additional regulation through targeted degradation and phosphorylation by cdk (cdc p) specifically affects the periodically expressed proteins. alessandra luchini, andrea callegaro, silvio bicciato department of chemical engineering processes, university of padova, padova, italy. e-mail: alessandra.luchini@unipd.it (a. luchini) since transcriptional control is the result of complex networks, analyzing dynamical states of gene expression is of paramount importance to detect the multivariate nature of biological mechanisms. although hundreds of studies fully demonstrated the relevancy of microarrays in describing different physiological conditions, to reconstruct complex interaction pathways it is necessary to analyze the temporal evolution of transcriptional states. however, a robust experimental design for identifying differentially expressed genes over a temporal window would require large amounts of microarrays. unfortunately, replicates for each time point and experimental condition are not always available, because of cost limitations and/or biological samples scarcity. in addition, common data analysis tools, like anova, require replicates and disregard correlation structure among times. we present a method for the identification of differentially expressed genes in un-replicated time-course experiments. the procedure does not assume any model or distribution function, takes into account the correlation of data, and does not require sample replicates at the various time points, other than the presence of an initial time point for all analyzed conditions. the identification of differentially expressed genes as the result of a system perturbation is formally stated as a hypothesis testing problem in which a defined statistic is used to rank transcripts in order of evidence against the null hypothesis. specifically, (i) data are structured so that measurements are correlated in time, within the same biological condition; (ii) the null hypothesis is formulated so that changes in expression levels at different time points are equivalent; (iii) time point t represents the system before the perturbation. therefore, modulated genes are detected testing the statistical significance of expression differences between physiological states at each time point, once corrected by the variability at t , and given an empirical null distribution constructed using permutations. statistical significance is assessed by the q-value. the method has been tested on time-course microarray experiments aimed at studying the temporal changes of gene expression in: (i) skeletal muscle cells treated with a histone deacetylase inhibitor (iezzi et al., ) and (ii) immature mouse dendritic cells (dc) exposed to larval and egg stages of s. mansoni (trottein et al., ) . differentially expressed genes, identified using the proposed algorithm, have been compared with results obtained from anova model and sam paired test. the biological significance and soundness of selected transcripts was also verified using global functional profiling by means of ontotools. results demonstrate that this novel procedure allows the identification of biologically relevant genes using half of the replicates required by standard model-based approaches. carbon sources. interesting data on the expression profile of the sty and paa genes in pseudomonas sp. y have been obtained, and have raised new questions on styrene and paa degradation by this bacterium. the calcium-dependent antibiotic (cda) is a lipopeptide synthesised non-ribosomally and produced by streptomyces coelicolor a ( ). cda contains several non-proteinogenic amino acid residues. hydroxyphenylglycine ( -hpg) is one of the unusual amino acids in the structure of the cda and vancomycin groups of antibiotics. for the members of the vancomycin group of antibiotics, the -hpg residue plays crucial roles in the structure and function of the final glycopeptide antibiotic. to reveal the putative biosynthetic pathway of this amino acid in cda, a standard "double crossover replacement strategy" was used to delete -hydroxymandelic acid synthase ( -hmas, encoded by hpd) from s. coelicolor mt and , using the delivery plasmid pzmh . there was no cda production in the disrupted strains. plates containing a gradient of hydroxymandelic acid were used to restore cda production in both s. coelicolor mt hpd and hpd. exogenous supply of -hydroxyl phenylglyoxylate and -hydroxyphenylglycine reestablished cda production by the hpd mutant. feeding analogs of these precursors to the mutant resulted in the directed biosynthesis of novel lipopeptides with modified arylglycine residues (mutasynthesis). a cxcl tandem repeat promoter polymorphism is associated with susceptibility to severe sepsis in the spanish population n. maca-meyer , c. flores , l. pérez-méndez , r. sangüesa , e. espinosa , j. villar : research institute, hospital universitario n.s. de candelaria, s/c tenerife , spain; department of anesthesiology, hospital universitario n. s. de candelaria, s/c tenerife , spain. e-mail: nmacame@ull.es (n. maca-meyer) sepsis describes a complex clinical syndrome resulting from a systemic inflammatory response to bacteria, and remains an important cause of mortality in the intensive care unit. cxcl chemokine (or mip- ) exhibits a pivotal role in the immune response, and several functional studies in animal models of sepsis have catalogued cxcl as a candidate gene for the development of sepsis. we have performed a case-control association study of cxcl gene variants and susceptibility to severe sepsis in hospitalised patients and healthy individuals. after the examination of linkage disequilibrium in the region, we analysed whether two promoter polymorphisms (snp rs and a newly described polymorphic short tandem repeat d s ) were associated with the syndrome. we found a significant association of common variants at d s with the development of severe sepsis (heterozygote carriers or . ; % ci . - . , and homozygote carriers or . ; % ci . - . ; mantel-haenszel χ test for linear trend p = . ). the risks remained significant even after a genomic control adjustment, based on additional genotyped polymorphisms not linked to the candidate gene. these preliminary results suggest that cxcl gene variants may contribute to the development of severe sepsis. kasper møller , ana paula oliveira , jens nielsen , mark johnston : center for microbial biotechnology, biocentrum, technical university of denmark, denmark; department of genetics, school of medicine, washington university, st. louis, usa glucose is the preferred carbon and energy source for most cells. in saccharomyces cerevisiae, a complex regulatory network ensures that s. cerevisiae ferments glucose to ethanol even in the presence of oxygen. to obtain a better understanding of this crabtree effect and the logic of the glucose signalling network in s. cerevisiae, we are analyzing glucose sensing and signalling in the related species saccharomyces kluyveri, which exhibits much less of a crabtree effect (it prefers not to ferment glucose when oxygen is available). we show that there are only two major glucose transporters in s. kluyveri, and that these are regulated in response to the availability of glucose via a glucose sensor and a signalling pathway similar to the glucose induction (rgt /snf -rgt ) pathway in s. cerevisiae. we have used dna-microarrays for s. kluyveri to find targets of the s. kluyveri glucose induction pathway, as well as to evaluate the global response to a change in environment from growth on ethanol to growth on glucose. this study identifies a number of differences in the regulation of glucose uptake and global responses to glucose between s. kluyveri and s. cerevisiae, which may contribute to their different glucose metabolism. detection and analysis of microrna using lna probes nana jacobsen, christian lomholt, peter mouritzen, peter stein nielsen, mikkel noerholm exiqon a/s, bygstubben , dk- vedbaek, denmark. e-mail: mouritzen@exiqon.com (p. mouritzen) micrornas are a class of short endogenous rnas that act as post-transcriptional modulators of gene expression. growing evidence suggest that micrornas exhibit a wide variety of regulatory functions and exert significant effects on cell growth, development, and differentiation. recent studies have shown that human microrna genes are frequently located in cancer associated genomic regions and perturbed microrna expression patterns have been observed in many malignant tumors. we have exploited the significantly improved hybridization properties of lna oligonucleotides against rna targets to design lna-modified dna probes for detection of different micrornas in animal and plants by northern blot analysis, microarray hybridization and in situ hybridization. we will describe the results obtained from detection and analysis of different micrornas in c. elegans, zebrafish, mouse, and plants. in addition, we will describe a novel lna-based method for expression profiling of mature micrornas by quantitative rt-pcr. expression profile of the sty and paa genes in pseudomonas sp. y by means of dna microarrays david bartolomé-martín , david juck , m a teresa del peso-santos , charles w. greer , julián perera : departamento de bioquímica y biología molecular i, facultad de ciencias biológicas, universidad complutense de madrid, madrid, spain; environmental microbiology group, biotechnology research institute, national research council canada, montréal, que., canada h p r . e-mail: perera@bio.ucm.es (j. perera) dna microarrays are a new and powerful tool to study gene expression in very diverse systems. environmental biotechnology and biodegradation are some of the fields of research where this technology may be very promising. pseudomonas sp. y is a bacterium able to grow in minimal medium plus either styrene (sty) or phenylacetic acid (paa) as the sole carbon and energy sources. this bacterium is the only organism where the genes that code for both the upper (sty genes) and the lower (paa genes) catabolic pathways for the styrene degradation have been described till now. it is unique in having two active copies of the genes encoding the lower pathway (paa and paa gene clusters). we have designed a dna microarray with the sty and paa genes in order to analyse their expression in the wild type pseudomonas sp. y , in p. sp. y t (a paa deletion mutant) and in p. sp. y c (a crc gene mutant). this analysis has been performed on bacterial cultures grown in media with different carbon sources. interesting data on the expression profile of the sty and paa genes in pseudomonas sp. y have been obtained, and have raised new questions on styrene and paa degradation by this bacterium. dynamics in induced repression of phosphomannose isomerase pmi gene of saccharomyces cerevisiae anssi törmä , , juha-pekka pitkänen , , laura huopaniemi , risto renkonen : medicel ltd., haartmaninkatu , helsinki, finland; rational drug design program, department of bacteriology and immunology, haartman institute and biomedicum, university of helsinki, p.o. box , helsinki, finland. e-mail: juhapekka.pitkanen@medicel.com (j.-p. pitkänen) gdp-mannose is the precursor of cell wall biosynthesis in s. cerevisiae. to understand the system level role of gdp-mannose, we studied a conditional knock-out strain of the key enzyme in its synthesis; pmi . the experimental procedure allowed us to study the order of mechanisms the cells launch in order to adjust to a sudden malfunction in the metabolic machinery. we collected samples from continuous cultivations over h and measured genome-wide gene expression levels, enzyme activities, and concentrations of intracellular metabolites. for sampling we have built a sample robot, which automatically takes and preserves the samples. in order to carry out this magnitude of experimentations and generated data, we have constructed a proprietary software platform to handle all the phases from project management in wet-lab to workflow and pathway management in in silico. after normalization and clustering, significantly changed genes and metabolites were searched for enrichment in biological processes and molecular complexes. further, gene expression levels, metabolite concentrations, and enzyme activities were searched against each other for causality over time. overall, we focused on thorough analysis of our own data and known database data in order to reward our efforts with knowledge. at the transcriptome level, repression of pmi led to two major types of activation profiles, one peaking at the time when pmi p activity and gdp-mannose were depleted and the other later during recovery from the perturbation. the primary response was most enriched with genes known to play roles in mating and filamentous growth and associated with the transcription factors ste p, tec p, dig p, and mcm p, whereas the secondary response consisted of genes involved in carbon metabolism and associated with the general stress response regulators msn p and msn p. skn p, a high-level transcription factor was associated with both the primary and the secondary response, consistent with its suggested role of coordinating environmental responses and developmental processes. transcriptome of pig ovarian cells: discriminant genes involved in follicular development bonnet a., le cao k.a., low-so g., san cristobal m., tosser-klopp g., hatey f. laboratoire de génétique cellulaire, centre inra de toulouse, castanet-tolosan , france in order to identify genes and gene networks involved in pig ovarian follicular development, we built subtractive suppressive hybridization libraries (ssh) from granulosa cells of healthy follicles (small, medium or large). the rna isolated from these cells was used to hybridize cdna nylon micro-arrays. data analysis using a gaussian linear mixed model showed that % of the variability is due to the genes. two hundred fifty one regulated genes (from the expressed) were identified and clustered into three groups according to the follicle size. moreover, we found previously identified genes such as aromatase, igfbp which supported the validity of our experimental model. ramdom forest analysis put forward the most discriminant genes between the three follicle classes. this study put forward gene sets such as those involved in cell modeling, regulation of transcription, apoptosis during follicle growth. the next step will be to describe more precisely the spatio-temporal expression patterns at the mrna levels of the genes identified by these experiments. microalgae constitute a significant source of valuable natural products, e.g. sulfated polysaccharides, polyunsaturated fatty acids, and phycobiliproteins that find applications in wide range of industries, including food, pharmaceutical, agricultural and cosmetics. however, genomic and molecular genetic studies of microalgae lag far behind those of higher plants. in order to accelerate red microalgal genomic studies by taking advantage of current genomics and post-genomic technologies, we have generated expressed sequence tag (est) databases of two red microalgae porphyridium sp. and dixoniella grisea grown under various physiological conditions. to date we have sequenced and ests of porphyridium sp. and d. grisea, respectively. the sequence assembly resulted into, ca. non-redundant unigenes for each microalga, only % of which were identified by similarity to sequences in the public databases. porphyridium sp. and d. grisea unigenes were compared with the whole-genome predicted proteomes of three microalgae and those of representative eukaryotic and prokaryotic organisms. both microalgae have highest similarity to the red microalga cyanidioschyzon merolae. the order of sequence similarity to other organisms examined was arabidopsis thaliana, oryza sativa, chlamydomonas reinhardtii, thalassiosira pseudonana (diatom), saccharomyces cerevisiae, caenorhabditis elegans, archaea and cyanobacteria. although red microalgae are considered as phylogenetic bridge between prokaryotes and eukaryotes, our data show that the red microalgae have strong similarity to eukaryotes and only distant similarity to prokaryotes. gene expression profiles were studied by analyzing cdna and subtraction libraries constructed from algae grown under various physiological conditions. we observed that top three most abundant ests in the stationary phase of porphyridium sp. were adp ribosylation factor like- , flavohemoglobin and adp ribosylation factor- . in addition, we have identified several genes which were specific to nitrate-and sulfate starvation. the sarco(endo)plasmic reticulum ca + -atpase (serca, "the calcium pump"), is responsible for pumping the ca + released into the cytoplasm during muscle contraction back into the sarcoplasmic reticulum store while proton are pumped the opposite way as counter-cations. these transport processes go against the concentration gradients and are therefore energy consuming. the energy is derived from atp hydrolysis via formation and break-down of a phospho-enzyme intermediate. over the last year a number of new crystal structures have been published which have added to our understanding of how this task is accomplished, which provides an impressive insight to the mechanism of a molecular pump. rhomboids are a family of intramembrane serine proteases that are widely conserved throughout evolution. among diverse functions discovered so far, rhomboids participate in intercellular signalling, parasite invasion, membrane dynamics and bacterial quorum sensing, making them potentially valuable therapeutic targets. the identification of physiological substrates and of selective inhibitors will be key towards their evaluation as drug targets. we have developed an in vitro cleavage assay to monitor rhomboid activity in the detergent solubilised state, enabling the first isolation of a highly pure rhomboid with catalytic activity. analysis of purified mutant proteins suggests that rhomboids use a serine protease catalytic dyad instead of the previously proposed triad, and gives insights into subsidiary functions like ligand binding and water supply. this work was supported principally by embo and the mrc. structure and target-specificity of thioredoxin h kenji maeda , anette henriksen , per hägglund , christine finnie , birte svensson : biochemistry and nutrition group, biocentrum-dtu, technical university of denmark, dk- kgs. lyngby, denmark; biostructure group, carlsberg laboratory, dk- valby, denmark. e-mail: kenji@biocentrum.dtu.dk (k. maeda) thioredoxins are ubiquitous small proteins with protein disulphide reductase activity. thioredoxins can alter the structures and the activities of various target proteins by reducing their disulphide bonds. seeds of several plants are abundant in cytosolic thioredoxins referred as h-type. in barley, two thioredoxin h isoforms, hvtrxh and hvtrxh that share % sequence identity but differ in temporal and spatial distributions were previously identified and characterised. in the present study, the relationship between structures and targetspecificities of h-type thioredoxins are analysed. the d-structures of hvtrxh and hvtrxh are determined by x-ray crystallography as the first crystal structures of thioredoxin h. comparison of the structures shows that the majority of solvent exposed residues near the active sites are conserved between the two isoforms. this is in agreement with previously observed similarity in target-specificity of the two isoforms. thioredoxins from organisms distantly related to barley, such as e. coli, have highly similar folds but different surface charge distributions compared to barley thioredoxins. a comparison of the target-specificities of hvtrxh , hvtrxh , e. coli thioredoxin and several thioredoxin mutants will be attempted to reveal the structural features that influence specificity of barley thioredoxin h isoforms. enbrel is a dimeric fusion protein consisting of the extracellular ligand binding portion of the human kda (p ) tumor necrosis factor receptor (tnfr) linked to the fc portion of human igg . the cho-expressed molecule contains both n-and o-linked oligosaccharides with a total carbohydrate content of % by mass. the o-linked oligosaccharides were released by hydrazinolysis and their structure determined by exoglycosidase sequencing and maldi-tof mass spectrometry. to locate precisely the o-linked sites, the glycosylation heterogeneity of tnfr:fc was simplified by treatment with n-acetyl neuraminidase and n-glycanase. the remaining molecule, which only carries core o-linked glycan structures, was cleaved by trypsin and analyzed by lc-ms. precise localization of o-glycosylation sites was determined based on the concept of a specific modification of the o-glycosylated serine into aminopropenoic acid and o-glycosylated threonine into -amino- butenoic acid. the deficit in mass resulting from this transformation was the marker used to localize the modified residues on the peptides by tandem mass spectrometry sequencing (ms-ms). ms-ms spectrum of enbrel glycopeptides were interpreted based on the presence of -aminopropenoic acid and -amino- -butenoic acid, resulting in a complete map of o-linked glycans precisely located at different sites. anu mursula, beatrix fahnert, sari krapu, eija-riitta hämäläinen, ritva isomäki, peter neubauer bioprocess engineering laboratory and biocenter oulu, university of oulu, oulu, finland. e-mail: anu.mursula@oulu.fi (a. mursula) wnt proteins form a highly conserved family of secreted glycoproteins important in cell-cell signaling events during embryogenesis and adult tissue maintenance. impairments within this complex signaling pathway can lead for example to developmental defects in embryos, degenerative diseases and cancer. respectively, wnt proteins can be used as tools in basic research concerning wnt function, developmental biology, screening for interacting compounds, and for medical applications (e.g. therapeutics, stem cells). hence, recombinant wnts provide a valuable basis for these purposes. however, production of recombinant wnt proteins is challenging, because they contain multiple disulfide bonds making the folding very difficult. a process for production of murine wnt- in e. coli has been developed in our laboratory. the knowledge obtained from this research has also been applied to the expression of other wnts, namely wnt- and wnt- , and can be used to approach other cysteine-rich proteins as well. since the expression level of wnt proteins is rather low so far, tools for monitoring and optimizing the production process have been established. by means of this sandwich hybridization method the level of target (wnt) mrna can be measured. the technique has already been applied to analyzing wnt- mrna levels. probes also for wnt- and wnt- have been generated. thus, transcription of wnt genes in all kind of cells (e.g. tissue, recombinant hosts) in general as well as kinetics of transcription can be studied using these tools. in growth factor signaling, stimulation of cell-surface receptors first triggers activation of the receptor itself and then of a large number of intracellular effector molecules. the stimulus is integrated with a host of other cellular processes, leading to cytoskeletal changes, activating transcriptional programs in the nucleus and ultimately resulting in cell proliferation, differentiation or motility. classical signaling pathways and networks depict potential protein-protein interactions only in a static form. in the cell, these interactions are dynamic and occur in an ordered fashion. here, we apply a mass spectrometric method that converts temporal changes to differences in peptide isotopic abundance in order to study the global dynamics of signaling events. briefly, three cell populations are metabolically labeled with either normal arginine or a c substituted form, or a c n variant (stable isotope labeling by amino acids in cell culture, silac). each population was then stimulated with egf for a different time period and tyrosine phosphorylated proteins were affinity purified with anti-phosphotyrosine antibodies. the proteins from the precipitated complexes were quantitatively analyzed and identified using lc-ms/ms. arginine containing peptides occurred in three forms, directly indicating protein activation at the corre-sponding time point. combination of two experiments sharing one common time point of activation then generated five-point dynamic profiles. from the proteins quantified, we identified signaling proteins, including virtually all known egfr substrates and novel effectors, and the time course of their activation upon egf stimulation. discriminating proteins involved in the signaling network from unspecific binders was straightforward as these presented an activation profile. we have now further extended this study by directly measuring in vivo phosphorylation sites in response to growth factor stimulation and monitoring the time evolution of the phosphorylation events. finally, we determined and quantitatively compared the global egf and pdgf tyrosine phosphoproteomes in human mesenchymal stem cells and revealed a control point in their differentiation into bone-forming cells. such global activation profiles provide a novel perspective in cell signaling and will be crucial to model the highly dynamic signaling networks in a systems biology approach. klaus schneider , dave g smith , steven skaper , alastair d. reith : discovery research, glaxosmithkline, coldharbour road, harlow, essex, uk; neurology & gastrointestinal cedd, glaxo-smithkline, coldharbour road, harlow, essex, uk over the last years, progress in signal transduction research has revealed an astonishing degree of complexity in cell signalling which is manifested in positive and negative regulations and feedback loops within signalling pathways and by cross-talks between pathways, all of which are highly cell-type dependent. it has become evident that protein phosphorylation by protein kinases plays a major role in this complex regulation of cell signalling (hunter, ) . due to the importance of signal transduction in disease processes, many protein kinases may constitute key targets for disease intervention. yet, the lack of a full understanding of the regulation, the activation and, importantly, of downstream substrates of particular protein kinases requires often more detailed studies before initiation of resource-intensive efforts to find disease-modifying molecules. technologies for the study of protein kinase signalling include p labelling, mutational and knock-out studies and more recently rna interference. these tools are complemented by approaches that are based on proteomic technologies developed over the course of the last years. in this presentation, the scope of proteomics technologies to contribute to an understanding of kinase signalling will be discussed. an overview of available technologies will be given and results will be presented from a proteomic study of glycogen-synthase kinase (gsk ) signalling (coghlan et al., ) . novel findings will be presented on a study of gsk inhibition in a primary neuronal cell line by differential d gel electrophoresis, which resulted in the identification of more than proteins that were significantly regulated. proteome analysis is typically done by nanospray lc/ms in order to achieve higher sensitivity and thus a greater number of protein identifications. however, nano-scale lc systems can be more challenging to use and maintain. to obtain the best chromatographic performance, connections must be made carefully to minimize band broadening. improved chromatographic performance can enhance the mass spectrometric results by tandem ms as a greater number of peptides can be detected. a microfluidic chip-based system has been developed (yin et al., ) that minimizes the number of connections and the delay volumes. this work evaluates the performance of this device against the traditional nanospray approach. a yeast extract sample was separated by sds-page and bands were excised from the gel for further analysis. after in-gel digestion, the sample was analyzed by both traditional nanospray and the microfluidicbased chip device. after protein database searching, the identified proteins and the protein sequence coverage's were compared for the two approaches. the microfluidic device was demonstrated to be equivalent or better compared to the traditional approach. yin, h., killeen, k., brennen, r., et al., . anal. chem. , - . plant cytochromes p (p s) play key roles in the biosynthesis of most bioactive compounds with agronomic and therapeutic applications. a collection of about plant p s was expressed in yeast. the cdnas were isolated from the model plant with a sequenced genome arabidopsis thaliana, some others from wheat, helianthus tuberosus or vicia sativa. they were expressed under the control of a galactose-inducible promoter in an engineered strain of saccharomyces cerevisiae in which the gene of the native p reductase was replaced with the gene of a p reductase from a. thaliana under the control of the same galactose-inducible promoter, in order to provide an optimal context for plant p expression and activity (pompon et al., ) . an original procedure was designed for the high-throughput functional screening of this enzyme collection. it is based on the detection of oxygen consumed during the catalytic reaction by a fluorochrome embedded in the bottom of the microwell plates. this method was validated using several recombinant p s of known activity. it also allows for a very efficient screening for enzyme inhibitors. the advantages and limits of the method will be discussed. this work was carried out with the support of génoplante programme (no ) . reference pompon, d., louerat, b., bronine, a., urban, p., . methods enzymol , - . folding of a bacterial membrane protein studied by protein engineering daniel e.otzen, pankaj sehgal, peter a. christensen department of life sciences, aalborg university, sohngaardsholmsvej , dk - aalborg. e-mail: dao@bio.aau.dk (d.e. otzen) we have carried out a kinetic analysis of the folding of the -helix transmembrane protein dsbb in a mixed micelle system consisting of varying molar ratios of sodium dodecyl sulfate and dodecyl maltoside. this analysis incorporates both folding and unfolding rates, making it possible to determine both the stability of the native state and the process by which the protein folds. the analysis also takes into account the composition of the mixed micelles, which is different from the bulk detergent composition. refolding and unfolding are consistent with a three-state folding scheme involving the sdsdenatured state, the native state and an unfolding intermediate that accumulates only under unfolding conditions at high mole fractions of sds. the temperature-dependence of the folding reaction displays an unusual decrease in heat capacity accompanying unfolding, which probably reflects the amphiphilic environment of the membrane protein. destabilization of dsbb by different short-chain alcohols correlates very well with the alcohols' respective hydrophobicities. data from a series of ala-scanning mutants tentatively identify a nucleus for folding, which is relatively diffuse and involves all four helices. we are currently complementing this work with studies of the association of peptides corresponding to individual transmembrane segments of dsbb. the reca protein of e. coli plays a crucial role in homologous recombination and dna repair. the recombination process takes place in a filamentous complex, in which the protein monomers are arranged in a helical manner around a single-stranded dna (ss-dna). in the presence of atp the filament can accommodate a second, double-stranded dna (ds-dna) and the strand exchange reaction can occur. the three-dimensional structure of reca itself and its complex with adp have been determined by x-ray crystallography. the active nucleoprotein filament, however, has only been studied at low resolution. both electron microscopy (em) and small-angle neutron scattering (sans) indicate significant differences between the structures of the active nucleoprotein filament and the compressed, inactive filament of only reca. we have presented a structural model of the reca protein in its active filament with ss-dna, using data obtained by linear dichroism (ld) polarized-light spectroscopy, based on a technique, we call "site-specific linear dichroism", which allows the orientation of a set of amino acids to be determined from ld data by systematic modification of the protein. here, we show that ld data of the nucleoprotein filament with ds-dna is over all similar to the data of the complex with ss-dna, indicating that the orientation as well as internal structure of reca in the active filament is not significantly altered when the bound dna is changed from single-stranded to double-stranded. this result supports the idea that the strand exchange reaction occurs without large conformational change of the reca protein. the choline-binding modules: a powerful biotechnological tool jesús m. sanz instituto de biología molecular y celular, universidad miguel hernández, elche, spain choline-binding modules (chbms) are present in some virulence factors of streptococcus pneumoniae (pneumococcus). the most extensively studied chbm is c-lyta, the carboxy-terminal domain of the pneumococcal cell-wall amidase lyta. the three-dimensional structure of choline-ligated c-lyta is built up from six loop-hairpin structures ("choline binding repeats", chbrs) forming a left-handed ␤-solenoid with four choline binding sites. although the structure of the ligand-free form is not yet known, our folding studies suggest that it is more loosely packed, with a partially unfolded amino-terminal region and a stable carboxy-terminal moiety that is extremely resistant to chemical denaturation (maestro and sanz, ) . the affinity of c-lyta for choline and other structural analogues allows its use as an efficient affinity tag for overexpression, immobilization and single-step purification of proteins of biomedical interest (c-lytag fusion protein purification system). this system presents many advantages when compared to current commercial methods, namely simplicity, compatibility with buffers and robustness. the availability of multiple supports that specifically bind chbms (such as multiwell plates) has recently allowed the development of a new procedure for the immobilization of c-lyta-containing hybrid proteins that may be used in proteomics, diagnostics and peptide display. in this communication, we present our last results about the stability, folding and engineering of c-lyta, together with a compendium of the current biotechnological potential of this protein, and highlight the productive link between basic molecular studies and their application. many of the modern approaches for studying disease compare steady state functions, such as repair, growth, and regulated gene expression within the various biological compartments organised by specialized function, be it mitochondria or blood vessels. the assignment of protein identities, which are linked to key biological mechanisms, which are associated with disease processes and disease progressions are an important area of this work (marko-varga and fehniger, ) . today, the technology available for studying proteome expression and resolving exact protein and peptide identities in complex mixtures of biological samples allows global protein expression within cells, fluids, and tissue to be approached with confidence. this confidence is due in part to reproducible repetitive sampling and analysis technologies including robotics data acquisition and high level mass spectrometry including both laser-desorbtion and electro spray ionisation. the precision in defining differences between normal and diseased steady states is aided by the creation of compiled reference and master data sets and by new methods for multiplexing the analysis of samples in groups. the establishment of key representative reference proteome systems representing the dynamic changes in protein expression during disease will be vital to the interpretation of changes observed in specific samplings of disease states and specific cells obtained from these samples. the creation of reference databases of proteins linked to disease pathways will play an important role in furthering our understanding of the "proteome of disease". examples will be given where protein expression patterns have been generated from compartments within tissue sections. marko-varga, g., fehniger, t.e., . j. proteome res. , - . adaptation of the saccharomyces cerevisiae proteome to nutrient limitations studied by metabolic stable isotope labeling and mass spectrometry albert j.r. heck netherlands proteomics centre and utrecht university, the netherlands. e-mail: heck@npc.genomics.nl. url: www.netherlandsproteomicscentre.nl one of the major aims of proteomics is to provide quantitative data on differential protein expression levels. recently, mass spectrometry-based methods have been introduced that can provide quantitative data on differential protein expression, mostly using stable isotope labeling (goshe and smith, ) . we opted for metabolic labeling as this provides efficient means to quantify differential protein expression, and has the advantage that all proteins are labeled universally (romijn et al., ) . in their natural habitat microorganisms encounter non-optimal growth conditions and often growth is limited by one nutrient. microorganisms need to respond rapidly to changes in the environment in order to survive. in the present study, we investigate the proteome response of chemostat cultivated wildtype saccharomyces cerevisiae to two different nutrient limitations, namely carbon and nitrogen limitation. yeast was metabolically labeled in well-controlled chemostat cultures. n and n labeled proteins were separated using d gel electrophoresis followed by rp-lc-esi-ms on a lc-q. relative quantification was performed by using relex software (maccoss et al., ) . we quantified proteins, using on average peptide peak pairs per protein. this analysis revealed that proteins showed a significant increase/decrease in expression level. the functional annotation of these proteins revealed that the yeast cells change expression levels of enzymes involved in metabolism of the growth-limiting compound. the protein expression ratios were compared with corresponding transcript levels. moreover, we compared the accuracy of quantifica-profiles mainly reflected differences in cellular origins in addition to different functional roles. mass spectrometric analysis identified proteins pertaining to several functional classes, i.e. acute phase proteins, antioxidant proteins and proteins involved in protein synthesis/maturation/degradation, cytoskeletal (re)organization and in lipid metabolism. several proteins not previously implicated in nerve regeneration were identified, e.g. translationally-controlled tumor protein, annexin a / , vitamin d-binding protein, ␣-crystallin b, ␣-synuclein, dimethylargininases and reticulocalbin. real-time pcr analysis of selected genes showed which were expressed in the nerve versus the dorsal root ganglion neurons. in conclusion, this study highlights the complexity and temporal aspect of the molecular process underlying nerve regeneration and points to the importance of glial and inflammatory determinants. yeasts plasma membrane macromolecular components involved in stress resistance paola branduardi, paola paganoni, danilo porro dipartimento di biotecnologie e bioscienze, università degli studi di milano-bicocca, piazza della scienza, - milano, italy. e-mail: paola.branduardi@unimib.it (p. branduardi) the plasma membrane is a universal structure of living cells constituting an essential barrier dividing and defining the intracellular from the extracellular environment. it is consequently easy to deduce the crucial role played by said structure for any cell of any living organism, and especially for unicellular organisms, since all the information deriving from the external environment as well as many of the consequent cellular responses have to pass through this barrier. unicellular organisms, thanks to easy manipulation and cultivation techniques, can represent a very useful model for studying the plasma membrane function and response. in addition microorganisms, and among them yeasts, can be considered advantageous cell factories for recombinant productions. in this contest, the implementation of any process of production has to take into account, among others, the response and the tolerance of the host to the external environment. from these considerations derives the interest of our group to analyse the main macromolecular components of yeasts plasma membranes (proteins, lipoproteins and lipids), isolated from cells grown under different stress conditions, with particular attention to acidic environments. here, we present our recent data about separation and identification (by sequencing analyses) of lipoproteins isolated from the conventional yeast saccharomyces cerevisiae as well as from the non-conventional and acid tolerant yeast zygosaccharomyces bailii cell cultures grown in different conditions. in parallel, the protein fraction is under evaluation through a differential d proteomic approach and consequent analyses. effect of fungal polysaccharides on the expression of pancreatic proteins in streptozotocin-induced diabetic rats sang woo kim , hye jin hwang , kwang bon koo , jang won choi , jong won yun * : department of biotechnology; department of bioindustry, daegu university, kyungsan, in an attempt to search novel biomarkers for monitoring diabetes prognosis, we examined the influence of the hypoglycemic fungal extracellular polysaccharides (eps) on the differential expression of pancreatic proteins in streptozotocin-induced diabetic rats. the results of diabetic study revealed that orally administrated eps exhibited excellent hypoglycemic effect, lowering the average plasma glucose level of the diabetic rats to . %. pancreatic proteome were analyzed by -de system, which separated more than individual spots. the -de analysis demonstrated that thirty-four proteins from a total of about matched spots were differentially expressed, of which spots were identified as the proteins whose expression has previously been associated with diabetes. twenty-two overexpressed and twelve underexpressed proteins were significant (p < . ) between the healthy and diabetic rats, and the altered proteins were restored (p < . ) upon eps treatment. it was first found that carbonyl reductase ( . -fold, p < . ) and mawdbp ( . -fold, p < . ) were surprisingly upregulated upon diabetes induction, and then those two protein concentrations were completely restored by eps treatment. moreover, we obtained eight unidentified proteins that have not been reported to be related with diabetes mellitus. these results evidenced the effect of fungal eps on searching potential markers for diagnosis and therapeutic manipulation of diabetes mellitus. although molecular basis of protein modulation after eps administration in diabetic rats was not verified in this study, the results of the proteomic analysis provide impetus for further studies of mining the biomarkers for diabetic therapy. the model established in our experiment is expected to mimic human diabetic status, which will help us to interpret the roles of biomarkers in diabetic state. the use of polyol-responsive monoclonal antibodies in immunoaffinity chromatography and as a probe for unfolding of wild-type and altered (t i) amidase from pseudomonas aeruginosa s. martins , j. andrade , a. karmali , a.i. custódio , m.l. since immunoaffinity chromatography is a powerful protein purification technique of interest in proteomics, monoclonal antibodies (mabs) against mutant (t i) amidase from p. aeruginosa were raised by hybridoma technology. in order to identify mabs that bind t i amidase tightly but release under gentle conditions, hybridoma clones secreting polyol-responsive mabs (pr-mabs) were previously screened. nearly % of elisa assay-positive hybridoma produced clones secreting pr-mabs with potential application as ligands for immunoaffinity chromatography. to select the optimal conditions for amidase elution, an elisa-elution assay was carried out, with two of these clones (f g ; e a ). the dissociation of ag-ab complex required % of propylene glycol and either . m (nh ) so or . m nacl. the binding of purified mab of igm class (e a ) to wild-type and mutant amidases was investigate by direct elisa, which revealed that it recognised specifically a common epitope on both amidases. conformational changes on antigen molecule were studied. mab e a showed a higher affinity for heat denatured forms than for native forms as revealed by affinity constants suggesting that the mab recognizes a cryptic epitope. the effect of mab e a on amidase activity was also investigated. the binding of mab to wild-type and mutant amidases exhibited an inhibition and activation of % as a function of time, respectively. this pr-mab is useful as a probe to detect conformational changes in native and denatured amidases as well as a ligand in immunoaffinity chromatography, which is of great interest in protein purification and proteomics. fragility and solubility of non-classical inclusion bodieš s. peternel , a. ristič , , v. gaberc-porekar , v. menart , : national institute of chemistry, ljubljana, si- ; lek pharmaceuticals d.d., ljubljana, si- . e-mail: spela.peternel@ki.si (Š. peternel) human granulocyte colony stimulating factor (g-csf) is a pharmaceutically important cytokine. when overexpressed in escherichia coli, it is usually accumulated in the form of inclusion bodies (ibs) . when produced at • c classical insoluble ibs are formed while at • c non-classical ibs containing a high amount of correctly folded g-csf are formed. as higher fragility and solubility of non-classical ibs were noticed, we decided to check whether bacterial cell disruption method has any influence on their mechanical stability and solubility. enzymatic lysis, sonication and homogenization, methods often used for disruption of bacterial cells during the isolation of ibs were compared. lysozyme treatment of bacterial cells appears to be mild enough disruption method not influencing the integrity of ibs. homogenization of bacterial cells at high pressure ( . - . kpa) shows no impact on classical ibs while some loss of target protein from the non-classical ibs is observed. sonication seems to be most harmful as even at rather low sonication altitudes, noticeable disassembling and solubilization of non-classical ibs occurs while no effect on classical ibs is perceived. our studies show that non-classical ibs are much more fragile and soluble than classical ones. therefore, one should extremely carefully choose the method for cell disruption to avoid undesirable loss of the target protein. the danish tick ixodes ricinus parasitize three different hosts both mammals and birds during the -year life cycle. the aim of this study was to identify the last blood host being the host, which the nymph had parasitized before molting to the adult instar. the reason for the study was to reveal the origin of the host contributing the most to the life cycle of the tick and thereby the maintenance of tick-borne diseases in denmark. the most common tick-borne diseases are lyme borreliosis and tick-borne encephalitis (tbe) causing illness in both animals and humans. we analyzed adult ticks, which were collected from known hosts. the analysis was performed at different heat stable proteins, which could be detected during the off host period by elisa. we found that heat stable proteins could be used as identification markers for host recognition. mushroom polysaccharides alter the expression of diabetesassociated proteins in the liver of streptozotocin-induced diabetic rats hye-jin hwang , sang-woo kim , kwang-bon koo , jang-won choi , jong-won yun * : department of biotechnology, daegu university, kyungsan, kyungbuk - , korea; department of bioindustry, daegu university, kyungsan, kyungbuk - , korea. e-mail: jwyun@daegu.ac.kr (j.-w. yun) in the present study, we investigated the influence of the hypoglycemic fungal extracellular polysaccharides (eps) on the differential expression of liver proteins in streptozotocin (stz)-induced diabetic rats. the results of diabetic study revealed that orally administrated eps exhibited an excellent hypoglycemic effect, lowering the average plasma glucose level in eps-fed rats to . %. in the next step, we analyzed the differential expression patterns of rat liver proteins from each group, to discover potent candidates for diabetesassociated proteins. a total of proteins of the -de gel were expressed differentially between diabetic and healthy rats. among them, proteins were upregulated and proteins were downregulated upon diabetes induction. many of these changes were in accordance with observations in previously published studies. surprisingly, the altered levels of most proteins in diabetic group were fully or partially restored to those of non-diabetic control group by eps treatment. moreover, we obtained unidentified proteins that have not been reported to be related with diabetes mellitus. although molecular basis of protein modulation after eps administration in diabetic rats was not verified in this study, the results of the proteomic analysis provide impetus for further studies of mining the biomarkers for diabetic therapy. potential is still limited by the differences observed in the structure of plant and mammalian n-glycans. indeed, theses differences and particularly the presence of ␤ , -xylose and ␣ , -fucose glycoepitopes are responsible for the immunogenicity of plant n-glycans. in order to reduce the structural differences between plant and mammalian n-glycans, current strategies are to knock out plant-specific glycosyltransferases or to humanize plant n-glycans by expression of mammalian glycosyltransferases in plants. in the present study, we have expressed a human ␤ , -galactosyltransferase in alfalfa. in order to further increase the efficiency of the human ␤ , -galactosyltransferase in the plant golgi apparatus, we have exchanged the endogenous targeting signal of this human glycosyltransferase for the ones from plant glycosyltransferases recently characterized in our laboratory. we will illustrate this approach of targeted expression with the results obtained by fusion of the catalytic domain of human ␤ , -galactosyltransferase with the n-terminal sequence of a plant glycosyltransferase that targets the fusion to the very early compartments of the golgi apparatus. the efficiency of natural versus targeted expression of human ␤ , galactosyltransferase in alfalfa will be compared in term of n-glycan humanization. altogether, our results clearly illustrate that we are now on the way to get perfect copy of mammalian glycoproteins in alfalfa plants. construction of recb-recd gene fusion and analysis of fusion enzyme activities oytun portakal , gerald r. smith , pakize dogan : department of biochemistry, hacettepe university medical school, , ankara, turkey; divisions of basic sciences, fred hutchinson cancer research center, seattle, wa - , usa. e-mail: oytun@hacettepe.edu.tr (o. portakal) protein folding is a fundamental process to gain protein function. in an oligomeric protein, the interaction between polypeptides affects the folding process and assembly to the holoenzyme. recbcd is a heterotrimeric and multifunctional enzyme that plays an essential role for major pathway of homologous recombination in e. coli. it is composed of recb, recc and recd gene products. recd is the fast motor unit of the recbcd enzyme. recd also plays a role for high affinity dsdna binding, nuclease activity and chidependent regulation. this study was designed to test the hypothesis that recd polypeptide regulates the essential reca loading activity. the approaching of the study was to fuse recd gene to subsequent recb gene and to observe the changes in enzyme activity and structure. for these purpose two genetic fusion mutations, two-nucleotide deletion and three-codon substitution were created at the overlap sites (ta) of recb and recd genes. fusion mutations were constructed by phage-mediated recombination system, which is called recombineering. this technology requires red function but not host reca protein function. here, we showed the recbd fusion polypeptides in crude extracts. genetic characterization tests were revealed that both fusion enzymes are recombination proficient and have wild-type phenotype. biochemical assays demonstrated that recbdc fusion heterotrimers have dsdna exonuclease, unwinding and chi cutting activities. they were also resistant to dna damaging agents. western blot analysis also detected a wild type length recd polypeptide together with recbd fusion polypeptides and a decreased heterotrimer compared to wild type. our findings suggest that recb-recd genetic fusions may affect recd assembling to the heterotrimer, but not affect it's native folding. sandwich immunoassay-a simple strategy for enhancement of the sensitivity and the specificity in prostate specific antigen detection based on surface plasmon resonance cuong cao, sang jun sim department of chemical engineering sungkyunkwan university, chunchun-dong, jangan-gu suwon, prostate cancer is a deadly disease in men. prostate specific antigen (psa) has been proved to be the most reliable and specific biomarker in preoperative diagnosis, monitoring and followup of patients with prostate cancer. in this study, a biochip based on surface plasmon resonance was fabricated to detect psa at concentrations ranging from to ng/ml. to reduce nonspecific binding, the chemical surface of sensor was constructed by using various ethyleneglycol mixtures of different molar ratios of hs(ch ) (och ch ) cooh and hs(ch ) (och ch ) oh. we also biotinylated the sams surface to enhance the orientation of protein immobilization. by using this surface, spr-based psa detection gave a positive ru value at the fist response in the whole range of psa concentrations. however, this ru value could get better and more reliable by simply applying a secondary interactant, the psa polyclonal antibody, in sandwich immunoassay. the results shown this approach could satisfy our purpose without modify the secondary interactant, which has usually been done by the other report. expression of epitopic domains of human coagulation factor viii in escherichia coli amir amiri yekta , , naser amirizadeh , alireza zomorodipour * , fariba ataei : department of mol genet. national institute for genet eng & biotechnol tehran-iran p.o. box: - , tehran, iran; islamic azad university of jahrom, jahrom, iran; department of hematol, faculty of med, tarbiat modarres university, tehran, iran. e-mails: amir amiriyekta@yahoo.com (a.a. yekta), * zomorodi@nrcgeb.ac.ir (a. zomorodipour) human factor viii (hfviii) plays major role in the intrinsic pathway of blood coagulation and is used to treat individuals with hemophilia a for bleeding episodes. many researches have been focused on the molecular aspects of this protein. in this regard, epitopes of hfviii as well as their corresponding antibodies have many important applications. bacterially produced fviii-epitopes are capable to neutralize the alloantibodies that inhibit hfviii activity. the purpose of present study was to over-express two epitope-containing fragments of fviii in e. coli under t promoter (novagen). two dna fragments from light-and heavy-chains of hfviii ( bp-c c and bp-a a , respectively) were subcloned in the expression vector. the use of his -tagged tail was also considered for detection and purification purposes. in each of the examined clones, a protein of expected size was detectable. in the c c -expressing clone the specificity of the over-expressed protein was confirmed by its reaction with the rabbit serum directed against native hfviii as well as anti-his-tag antibody. in the heavy chain-related-expressing clone, the expression level was low, but it was detectable by immunoblotting experiments. manipulations of the growth as well as induction may be required. the over-expression of the other epitopes reported in the heavy chain may be achievable by the expression of (a) sub-fragment(s) of this region. the over-expressed his-tagged c c -related protein was appeared to be trapped in the cell as non-soluble inclusion bodies. therefore, after homogenizing of the induced recombinant cells, the nonsoluble fraction was dissolved in a solution of denaturant (guanidine hydrochloride) and subjected for the purification, using a ni-nta resin (qiagen) followed by protein measurement. accordingly, an expression level of mg/l (of culture) of the purified c c -related peptide was obtained. the recombinant hfviii c c -derived peptide has provided useful mean for further experimental and medical applications. isotachophoresis has almost exclusively been applied for contracting and stacking samples ions before zone electrophoretic separation of proteins. this study attempts to apply microfluidic isotachophoresis (itp) as a high resolution analytical method for proteins. beta-lactoglobulin and other milk proteins with slightly different pi were labelled with fluorescent red and analysed by the micralyne tk system using microfluidic glass chips, either with simple cross (sc) or double cross (tt) injection or designed d-itp-cze chips with double tt injection and sc for transfer to the second dimension cze channel, efficiently non-covalently coated with . % w/v epoxy-polydimethylacrylamide to lower electroendoosmosis. capillary zone electrophoresis (cze) in borate or phosphate buffer was reproducibly perform for more than consecutive runs using upchurch tm reservoirs glued to the wells to enable larger buffer volumes and greater run-to-run stability. finally, isotachophoretic anionic separation of the proteins were done using phosphate (ph . ) or chloride (ph . ) as leading ion and -amino-caproic acid (ph . ) as terminating ion. the effect of narrow cut ampholytes as spacers needs further investigations. the perspective aim is to combine the migrating itp separated zones with second dimension capillary zone electrophoresis as a new microfluidic proteomic danalysis. development of strategies for heterologous expression of glucose dehydrogenase from the halophilic archaeon halobacterium sp. nrc- juan carlos cruz-jiménez , lorenzo saliceti-piazza , rafael montalvo : chemical engineering, university of puerto rico-mayagüez campus, mayagüez , puerto rico; biology, university of puerto rico-mayagüez campus, mayagüez , puerto rico. e-mail: juancruzj@hotmail.com (j.c. cruz-jiménez) halophilic archaea are excellent model organisms and valuable for biotechnology applications; they are easy to culture in the lab, genetically tractable, and exhibit a variety of interesting and useful characteristics. most halophilic archaea require . m nacl to sustain growth and structural integrity. among the . genes in halobacterium, we are studying the gene encoding a glucose dehydrogenase, gene id is , located between the and bases (halobacterium sp. nrc- genome project). this extremozyme is bioengineerable, and its use as a model for studying biocatalysis in aqueous/organic and nonaqueous media has not been explored to date. the utilization of enzymes in organic solvents has several potential advantages over aqueous systems. a major benefit is the increased solubility of many substrates, resulting in higher concentrations of reactant and products, hence reducing and purification costs and simplifying recovery protocols. cells were grown aerobically during seven days at • c in a complex medium, harvested by centrifugation and their genomic dna extracted. for cloning of the gene, primers were designed based on the sequence recently published by the halobacterium sp. nrc- genome project. the forward ( -ccgcatgcgcc cacagtccc- ) and reverse ( -ccggcctctagaacggcctgg- ) primers were designed to incorporate restriction sites for sph i and xba i, respectively (in bold). we are pcr amplifying the genomic dna and developing methods for the heterologous expression using the mesophilic escherichia coli, as well as purifying the enzyme. the purification procedure will be carried out using high resolution methods based on the protein's halophilicity. bioinformatics methods will be used to facilitate conforming of protein function and for comparison with a native enzyme. quantitative measurements by mass spectrometry of hundreds of proteins simultaneously using the new proteinchip systemseries p. iversen, e. fernvik ciphergen biosystems inc., symbion research park, fruebjergvej , dk- copenhagen, denmark. e-mail: piversen@ciphergen.com (p. iversen) most mass spectrometry methods used in proteomics allow for the identification of multiple proteins in a limited number of complex samples, but lack the ability to assess the quantity of the proteins and their modifications. however, mounting evidence shows specific cleavage of well-known proteins as being strong candidates for specific biomarkers, and in order to discover these biomarkers one has to be able to monitor the quantity and mass of hundreds of proteins from hundreds of complex samples reproducibly. the new series instrument in connection with proteinchip arrays ® from ciphergen biosystems enables this. the new series instrument is optimized for sensitivity, reproducibility and quantitation. new ion optics allows the use of higher acceleration voltages thus increasing the sensitivity, but without lowering the resolution. a new method of blanking the detector in connection with a non-linear gain of the detector also increases the sensitivity to the effect that igg can be detected down to . fmol. furthermore, the unique design of the instrument permits the detection of proteins with great variation in both mass and concentration and thus making it ideal for proteomics studies. a unique feature of the series instrument is the possibility to normalize the output by controlling the laser and detector so that results can be read with equal precision on different instruments, which is not often possible in mass spectrometry where individual instruments yield different results. this feature is vital in the validation of research results beyond individual laboratories. the coupling of liquid chromatography with mass spectrometry is now firmly established as a routine method for the identification of proteins that have been subjected to enzymatic digestion. in an on-line lc-ms experiment, the column eluent is coupled to the electrospray source via an emitter and any tryptic peptides present in the mixture are mass analyses as they elute from the hplc column. should there be any co-eluting species in the eluent, these will be separated in the mass analyser by their mass-to-charge ratio. it has become increasingly clear that relative quantification of protein expression changes is important in modern biology and medicine. several current approaches have been developed that utilise stable isotope labelling of samples in combination with separation and subsequent analysis by mass spectrometry. however, we have recently described an lc-ms strategy where quantification is achieved via normalisation of the ms datasets and comparison of the peptide intensities (of the observed tryptic peptides) across samples is performed. in this case, it is desirable to perform replicate injections and hence reduce statistical errors. this approach places a requirement upon good chromatography, especially in terms of retention time reproducibility. in addition exact mass measurement of the eluting ions is required as well as the ability to generate reproducible and reliable peak intensity, or area, calculations for the eluting tryptic peptides. the ability to measure the mass to charge ratios of ions accurately, across injections and across samples, increases confidence that the same ions have been matched from each sample injection. in this presentation our current strategy for the relative quantification of proteins will be discussed using, as examples, complex protein mixtures from salmonella enterica, eschericia coli and human serum. proteomic analysis for the production of rhctla ig in transgenic rice cell cultures using dige ji-suk cho, song-jae lee, inha university, difference in gel electrophoresis (dige) technology using fluorescent dyes is a novel method, which simplifies the process of detecting and matching proteins between multiple gels by allowing the separation of up to three separate protein samples in the same gel. it provides accurate quantitative and reproducible differential expression values for proteins in several samples. recombinant human cytotoxic t lymphocyte-associated antigen -immunoglobulin (rhctla ig) was produced in the transgenic rice suspension cell cultures using ␣-amylase promoter system. this system is efficient for the production of recombinant proteins, as it secretes target proteins into culture medium under sugar-depleting condition. in this study, the intracellular proteins expressed at both growth and induction stages of culture were separated and analyzed using -d dige. each sample from different conditions and internal standard were labeled with n-hydroxy succinimidyl ester-derivatives of cy , cy and cy dyes and run within a single dige gel. using decyder tm software, spots were detected with two-fold thresholds with % confidence and it was found that proteins underwent significant change during the production of rhctla ig with normalization method improving data distribution. a pooled sample mixture for the picking gel was prepared with deep purple staining and analyzed with mass spectrometry. in addition, the intracellular rhctla ig spots were identified with western blot analysis using goat anti-human igg (fc) antibody after dige gel was transferred to pvdf membrane. study of substrate specificity of rnr-exoribonucelases using hybrid proteins ana barbas, mónica amblar, cecília m. arraiano instituto de tecnologia química e biológica, ean, - oeiras, portugal. e-mail: ab@itqb.unl.pt (a. barbas) the ribonucleases are essential enzymes responsible for the regulation of gene expression and have shown to be important for biotechnology purposes. for instance, commercial mutants deficient in ribonucleases have been quite relevant for the over-production of recombinant proteins. escherichia coli rnase ii is a processive - exoribonuclease prototype of the rnr family that has homologues widespread in the majority of the sequenced genomes. by sequence alignment it has been proposed for the rnr type proteins the existence of three different domains: an n-terminal cold shock nucleotide binding domain (csd), a rnb catalytic domain, and a c-terminal s nucleotide binding domain. we have constructed several rnase ii deletion mutants to enable the characterization of each domain. these studies have allowed us to determine that both csd and s are involved in the binding of the enzyme to the rna substrate, being the s domain the most important. in rna-binding proteins it has been shown that the s domain's conformation is highly conserved. however, it is not known whether the substrate specificity is s -dependent. in order to characterize the s domain and verify if it is directly related to substrate specificity, we have constructed rnase ii hybrid proteins in which the s domain was substituted by the s of two other exoribonucleases, rnase r (rnii-rnr) and pnpase (rnii-pnp). preliminary results have demonstrated that both quimeric proteins are capable of binding and degrading various rna substrates. in addition, studies are currently being carried out to verify the possibility that s domain of pnp in the hybrid protein might be involved in multimerization and/or interaction with other proteins. the murine monoclonal antibody igg , anti-digoxin was produced in a rolling bottle fermentor. purification was performed on a protein g column. cd spectra were recorded on a jasco- spectropolarimeter. protein concentrations of - g/ml and path length of cm were used for measurements in a far uv region. all measurements were performed in a cell holder thermostand with an accuracy of ± . at • c. at this temperature the predominance of ␤-strands is indicated. large conformational changes occur at • c. at this temperature the spectra tense to irregular ␤-strands and unordered structures. these evidences confirming temperaturedependent conformational changes of protein and also high thermal stability of mentioned monoclonal antibody. generation of monoclonal antibodies for the assessment of protein purification by recombinant ribosomal coupling janni kristensen, kim kusk mortensen, hans peter sørensen laboratory of biodesign, department of molecular biology, aarhus university, gustav wieds vej c, dk- aarhus c, denmark. e-mail: hans.peter.sorensen@teknologisk.dk (h.p. sørensen) we recently described a conceptually novel method for the purification of recombinant proteins with a propensity to form inclusion bodies in the cytoplasm of escherichia coli. recombinant proteins were covalently coupled to the e. coli ribosome by fusing them to ribosomal protein (rpl ) followed by expression in an rpl deficient strain of e. coli. this allowed for the isolation of ribsomes with covalently coupled target proteins which could be efficiently purified by centrifugation after in vitro proteolysis at a specific site incorporated between rpl and the target protein. to assess the efficiency of separation of target protein from ribosomes, by site specific proteolysis, we required monoclonal antibodies directed against rpl and gfp. we therefore purified rpl -gfp-his, rpl -his and gfp from e. coli recombinants using affinity, ion-exchange and hydrophobic interaction chromatography. these proteins could be purified with yields of , and g per gram cellular wet weight, respectively. however, rpl -gfp-his could only be expressed in a soluble form and subsequently purified, when cells were cultivated at reduced temperatures. the purified rpl -gfp-his fusion protein was used to immunize balb/c mice and the hybridoma cell lines resulting from in vitro cell fusion were screened by elisa using rpl -his and gfp to select for monoclonal antibodies specific for each protein. this resulted in antibodies directed against rpl and antibodies directed against gfp. antibodies were screened for isotypes and their efficiency in western immunoblots. the most efficient antibody against rpl and gfp were purified by protein g sepharose affinity chromatography. the purified antibodies were used to evaluate the separation of ribosomes from gfp, streptavidin, murine interleukin- , a phagedisplay antibody and yeast elongation factor a by centrifugation, when ribosomes with covalently coupled target protein were cleaved at specific proteolytic cleavage sites. proteomic analysis for the production of rhctla ig in transgenic rice cell cultures using dige ji-suk cho, song-jae lee, inha university, difference in gel electrophoresis (dige) technology using fluorescent dyes is a novel method, which simplifies the process of detecting and matching proteins between multiple gels by allowing the separation of up to three separate protein samples in the same gel. it provides accurate quantitative and reproducible differential expression values for proteins in several samples. recombinant human cytotoxic t lymphocyte-associated antigen -immunoglobulin (rhctla ig) was produced in the transgenic rice suspension cell cultures using ␣-amylase promoter system. this system is efficient for the production of recombinant proteins, as it secretes target proteins into culture medium under sugar-depleting condition. in this study, the intracellular proteins expressed at both growth and induction stages of culture were separated and analyzed using -d dige. each sample from different conditions and internal standard were labeled with n-hydroxy succinimidyl ester-derivatives of cy , cy and cy dyes and run within a single dige gel. using decydertm software, spots were detected with two-fold thresholds with % confidence and it was found that proteins underwent significant change during the production of rhctla ig with normalization method improving data distribution. a pooled sample mixture for the picking gel was prepared with deep purple staining and analyzed with mass spectrometry. in addition, the intracellular rhctla ig spots were identified with western blot analysis using goat anti-human igg (fc) antibody after dige gel was transferred to pvdf membrane. similarity searches and multiple alignment of s and s protein of sars-cov for modeling d structure and its evolution (origin) mohammad soltany rezaee rad, iman tavassoly, negar mottaghi, banafsheh rezaee. e-mail: mohammad.soltany@gmail.com (m.s.r. rad) aims: the exact origin of the cause of severe acute syndrome (sars) is still an open question. nowadays recombinant origins for this virus have been found. s and s subunit of spike protein of this virus are the most important proteins responsible for severe acute respiratory syndrome. in fact they are glycoproteins of this virus exist on its surface. they are responsible for mediating fusion of viral and cellular membrane. the classification and modeling d structure of this virus can help us to suggest new ideas about its charististics and function, which may lead to new therapeutic and preventing modalities. methods: we used nucleotide sequence of s and s subunit of s (spike) protein for multiple alignments. we have done multiple alignments with different bioinformatics software (clusterx, entrez) for comparing the sequence with the other viruses and, we used weblab view software for modeling and identifying d structure of these proteins. findings: the similarity searches on nucleotide sequence of this protein with the single strands rna (ssrna) shows the virus belong to a known classification named coronaviridae. these d structures show the responsibility of s protein in this syndrome. another findings based on these alignments is an important similarity between these subunits and genome of hiv- showing they have familiar mechanism in pathogenesis. discussion: multiple alignments are powerful tool in classification of new recombinational virus and emerging infection. d structure model of this virus is an important guide to understand the mechanism of this virus. the shape of glycoprotein that modeled with bioinformatics software can help us in understanding mechanism of binding this virus to human cells. this fact can be used in designing drug and vaccine to cure and prevent the sars. blocking these origins and sites leads to inhibiting the virus attachment. also the similarity between this virus and hiv- shows us that both of them have similar proteins that cause pathogenesis of these viruses. the simulated moving bed (smb) technology is a continuous countercurrent chromatographic separation technique that has been applied successfully in the last years to a number of significant problems. an smb consists of a series of fixed bed chromatographic columns connected in a loop, and outperforms column chromatography in terms of productivity and solvent consumption. the use of smb instead of batch processes for bioseparations, i.e. separations involving large and rather complex molecules with multiple d configurations depending on parameters such as ph, temperature, etc., is becoming of greater and greater interest. examples of these are therapeutic proteins, antibodies and plasmid dna among others. for all chromatographic processes in this field, one of the most crucial issues is the cleaning of the chromatographic media with a special solvent system, an operation usually referred to as cleaning in place (cip). in single column chromatography this is easily done off-line, but this is not compatible with standard smb operation. in order to overcome this limitation, the standard smb configuration has been modified by adding a dedicated section plus an additional section for the re-equilibration of the freshly cleaned column with the working solvent before it is re-inserted into the smb loop. in such a way, cip according to gmp criteria can be incorporated into the smb unit and operation, which is then called cip-smb. this new smb configuration is also related to the three fraction separation unit called f-smb that has been recently introduced and applied to the separation of nucleosides. in this work we apply cip-smb using a size exclusion stationary phase to the separation of plasmid dna from the filtered cell lysate solution. plasmid purification has become a key issue in the last years as a result of the advances in gene therapy, whereas traditional laboratory methods are not always suitable for therapeutic purposes. we report about separation performances, which are then discussed in the light of smb design criteria and compared to column chromatography performance. computer guided optimization of adsorptive bioseparation processes bernt nilsson department of chemical engineering, lund university, lund, sweden. e-mail: bernt.nilsson@chemeng.lth.se separation processes like chromatography can be highly nonlinear and the behavior can sometimes be hard to predict. optimization of preparative chromatography is often done experimentally, which is both time consuming and expensive. a model-based approach to optimization is therefore an attractive and challenging way to overcome some drawbacks in the traditional working procedure in biotechnical industry. efficient model-based optimization for industrial needs requires three parts; models, methods and tools. model-based methodology requires a set of chromatography column model structures, which can capture the phenomenon of interest. for instance they have to capture column load variations, elution profile changes, operation condition disturbances, column configurations and stationary phase properties. to derive a reliable model for optimization it has to be calibrated and validated to experimental data, which requires an efficient calibration procedure. different calibration procedures are discussed and compared. after validation the model can be used in the design of a separation step. to do a robust design a set of requirements have to be fulfilled. the column size and operation conditions are used to optimize the performance of the step, which requires a constraint nonlinear optimization method. the choice of objective function for optimization and corresponding constraints are not obvious and the resulting operation conditions are often not robust. therefore there have to be additional methods available for analysis of the performance, like sensitivity and robustness analysis. optimization of the purification of antibodies is discussed and exemplified. the work with mathematical models and numerical methods has to be supported by a set of computer tools of different kinds in order to solve industrial problems effective. there is a need for different kinds of tools; customized tool to solve specific problems by a non skilled user and general toolbox for the expert. an example of a toolbox is presented. tina tarmann, alois jungbauer department of biotechnology, university of natural resources and applied life sciences, vienna, austria plasmids and viruses are the contemporary vehicles for genetherapy and genetic vaccination. extremely promising results have been reported from in-vitro, in-vivo and clinical studies. currently a lot of these compounds are manufactured with a technology which has been directly transferred from laboratory to pilot scale without further engineering. membrane based separations, chromatographic separations and precipitation have been employed for separation of plasmids and viruses. chromatographic separation have been designed with aim of protein separation. thus such processes suffer from either mass transfer limitations or low capacity. monoliths without intraskeleton mesopores and chromatography particles with giga pores are excellently suited for adsorption and separation of plasmids and viruses. low mass transfer resistance and high capacity compared to conventional beaded materials can be observed. adsorption kinetics were derived from infinite and finite bath methods and isotherms were constructed. these data also suggest that a conformational change of the plasmids takes place upon adsorption. discussion of the mass transfer properties and an example of scale up of a chromatographic separation process using these novel materials will be shown and discussed in respect to already existing processes. the recent developments in molecular therapies such as non-viral gene therapy and dna vaccination have fostered the development of efficient plasmid dna (pdna) purification processes. the separation of supercoiled (sc) and open circular (oc) isoforms is one of the key steps in the large scale purification of pdna vectors intended for a therapeutic use. although escherichia coli produces mainly the more compact sc pdna isoform, oc, linear and denatured pdna isoforms are usually present and are likely to be less efficient in transferring gene expression. for this reason, regulatory agencies specify that more than % of pdna in a therapeutic product is in the sc isoform. in this work histidine-base recognition is explored as a mean to separate pdna isoforms. the agarose gel used here combines the mild hydrophobic characteristics of an epoxy spacer arm with a pseudo-affinity histidine ligand. chromatographic profiles were obtained by injection of native plasmid (sc + oc) samples in the histidine-agarose support showing an efficient and baseline separation of both isoforms. the high resolution obtained with this support indicates that the method is potentially applicable to the separation of pdna at preparative and analytical scale. affinity ligand development with a novel encoded bead screening technology ib johannsen, versamatrix a/s, gamle carlsberg vej , dk- valby, denmark. e-mail: www.versamatrix.com the presentation describes a new invention for fast development of affinity ligands, where up to , ligands can be screened onbead and identified in a few hours. combinatorial synthesis by the split and mix procedure is a powerful technique for generating vast numbers of diverse chemical compounds on polymer beads with relatively little effort. traditionally, the technique is hampered by the laborious spectroscopic and chemical analysis, needed to determine the exact structures of the ligand on selected beads. in this way, - month analysis time could easily be spent just to analyze a tiny fraction of the library. in the versaffin tm technology each bead is encoded, individually tracked, and identified during the synthesis and screening. this decreases the whole ligand development time from months to weeks and increases the amount of information significantly. the bead code further enables evaluation of the ligandprotein binding under varying binding and elution conditions. the instrument for reading the encoded beads and for quantifying the amount of bound protein is presented. the encoded beads we use are based on functional cross-linked polyethylenglycol (peg), which is compatible with water as well as most organic solvents. thus, the combinatorial synthesis can be carried out in organic solvents and the resulting compounds can be evaluated, still bound to the parent beads, under aqueous conditions. a further advantage of using peg based beads for on-bead screening is the fact that peg is biologically inert and therefore does not interfere in a bioassay. in the biopharmaceutical industry, pressure is mounting to shorten development times and thereby time to market, e.g. in the field of monoclonal antibodies, generic processes have been established which allow for more rapid development from gene to production of pre-clinical and proof of concept/phase i material. for non-mab products from various expression systems, productspecific approaches still prevail. however, for most product types similar issues like clearance of process-and product-related impurities, overall purity and yield, or manufacturing issues have to be dealt with in downstream process development. integrated and timely approaches based on process science and developed orthogonal analytical tools are often hampered by tight time frames and limited resources available. on the other hand, thorough understanding and analytical characterization of product characteristics but also of (process-related) impurities are pre-requisites for fully exploiting separation power and for achieving final purities way above % in a robust and cost-effective manner. from primary separation to polishing steps, we have made several attempts to improve the efficiencies of process steps themselves but also of ways to develop them. the strategies applied comprise implementation of innovative processing tools, rational streamlining and optimization of a sequence of unit operations, tech transfer and scale up considerations. also in this context, the applicability of scale down and ultra scale down models for process development and optimization purposes, their potential for speeding up and their limitations will be discussed. chromatographic monoliths are rather new chromatographic stationary phases. they consist of a single piece of a highly porous material. the pores are interconnected forming a network of channels. since the transport mechanism is predominantly based on convection, mass transfer between mobile and stationary phase is significantly enhanced resulting in short separation times. because of that they seem to be an ideal support for separation and purification of extremely large molecules like proteins, dna or even viruses. in this talk, various features of the monoliths like high porosity, fast mass transfer, surface accessibility and dynamic binding capacity will be described. effect of each feature on the separation and purification efficiency will be discussed in terms of molecular size and properties. while chromatographic monoliths are already widely accepted in microchip fluid devices, capillary columns as well as analytical columns, very few reports about preparative monolithic columns can be found. reasons for lack of preparative chromatographic columns will be elucidated and preparation strategy for construction of several liter volume methacrylate based monoliths will be presented. finally, several examples of biomolecule purification like large proteins, plasmid and genomic dna and viruses on cim convective interaction media ® monolithic columns will be given. further, their application as bioreactors and supports for solid state synthesis will be demonstrated. hubbuch institute of biotechnology, forschungszentrum juelich, juelich, germany. e-mail: m.schroeder@fz-juelich. de (m. schroeder) the intraparticle diffusion coefficient is an important parameter for modeling of chromatographic separation processes. a new method based on dynamic measurements of intraparticle concentration profiles of proteins in process chromatographic media with a confocal laser scanning microscope is presented. the diffusion coefficient is determined by fitting experimental data to a spherical diffusion model. excellent agreement of experimental data with simulation results is obtained. the diffusion coefficient is measured for seven proteins in sepharose ff, spanning molecular weights from . to kda. in addition, multicomponent diffusion processes for combination of differently sized proteins are analyzed and the influence of adsorbed proteins on the diffusion coefficient is measured in sp or q sepharose ff. taken together the presented method allows measuring the diffusion coefficient of proteins in process chromatographic media in a packed column. use of automated docking for predicting chromatographic behavior of proteins in hydrophobic interaction chromatography andrea mahn, m. elena. lienqueo contreras university of chile, santiago, chile in the present work, we have extended and automated the methodology proposed by mahn et al., for predicting protein behavior in hydrophobic interaction chromatography (hic). this methodology is based on the good correlation level between the average surface hydrophobicity of the interfacial zone (local hydrophobicity lh) and protein retention time in hic, for only three different ribonucleases. for determining the lh it is necessary to select the most probable protein-ligand conformation. in this work, we have determined the most probable conformation, of more than proteins, using (i) first, the module insight ii affinity by accelrys for providing automated docking (grid method) of ligands (phenyl) to the proteins ( conformations); (ii) then, the different probable docked protein-ligand conformations were automatically scored using the module insight ii ludi by accelrys; (iii) after that, each conformation was clustered and each cluster was scored by using the average score of each cluster (iv) finally, the most probable conformation was selected using a function based on the number of cluster components, and the average score value. then, when the most probable conformation was selected, the local hydrophobicity (lh) was calculated using the graphical representation and analysis of structural properties (grasp) program. the results have shown an acceptable correlation level (r > . ) between lh and the experimental dimensionless retention time (drt). in view of these results, we consider that this methodology could be used to adequately represent the chromatographic behavior in hic for all kinds of proteins (with a heterogeneous and homogeneous surface hydrophobicity distribution) and without a large number of tedious experiments, but only using computational simulation and adequate score criterion. potato tuber proteins are nutritious and show potential as functional ingredient in food systems. however, the present bulk processing technology can only recover byproduct protein for animal feed use. an expanded bed adsorption (eba) process for isolating functional food-grade protein from crude potato starch effluent was previously developed. moderate capture efficiency ( - %) of the total crude protein was most likely caused by diffusion limitations and aggregated protein, inaccessible for adsorption. we employed the same adsorption ligand attached to agarose-tungsten carbide beads to create stable beds of . - . × expansion using flow rates at - cm h − . a pilot scale eba process was run in a commercial processing plant over a three month campaign of starch production from potatoes of mixed variety. fresh crude effluent ( - l/cycle) was applied to a column ( cm × m) containing l of resin. protein capture by eba was reliable in operation, producing a refined protein material, which after dewatering and gentle drying, showed improved functionality over heat-coagulated protein produced at the same plant. overall productivity increased. however, finding a robust operating window of predictable productivity is challenging since the potato fruit water is complex and deteriorates easily. from breakthrough curves, it is observed that the major bulk protein, patatin, displays non-langmuir adsorption behavior. this may indicate a range of interactions for different species of the same protein. chlorogenic acid (ca), the main polyphenolic substance in potato tuber, causes enzymatic browning and undesirable flavor changes, but polyphenols can also react with protein. assessing the effects of interacting cell components therefore applies to the bioprocessing of plant material. at present the acceptance of biochip technology for on site use, e.g. diagnosis or environmental control is hindered by rather expensive and complex instrumental systems. there is a need to provide reliable and cost-effective systems that can be operated with minimal training. the construction of electronic biochip microarrays using semiconductor technology enables the construction of compact systems with high integration at acceptable production costs. the key feature of the fully electrical biochip technology are micro arrays made in advanced si-technology and carrying several array positions with interdigitated nanometer gold electrodes on its surface. the chips are fabricated by standard silicon fabrication methods allow-ing high volume production and to minimise the cost per chip. the advantage of fully electronic microarrays is the intrinsic high spatial resolution and direct signal coupling of the biosensing element and the transducer. the function of fully electronic biochips is also based on the electrochemical transduction and quantification of the formation of affinity complexes on the chip surface. a portable device for field applications and point of care diagnosis have been designed and manufactured. the amperometric device enables the recognition of biomolecular interactions by measuring the redox recycling products of elisa enzyme labels. the highly sensitive signal transduction is achieved with a -channel interdigitated ultramicroelectrode array. one major advantage of fully electronic microarrays is the direct signal coupling of the biosensing element and the resulting robustness and opportunity for miniaturisation. those electrical biochip arrays have been adapted for the detection of all types of affinity complexes, such as for dna, rna, proteins and haptens. self assembling of capture oligonucleotides via thiol-gold coupling have been used to construct the array chip nucleic acid interface. thus e.g. pathogenic micro organisms have been identified and quantified via their genomic dna or ribosomal rna respectively. another application based on immobilized antibodies is shown to sense extreme low concentration of bioagent toxins. for processing the assay formats and the electrical read out of the detection of affinity complexes a modular fully automated measurement system has been developed. it is manufactured in industrial lines and available at market. dynamics and self-assembly of organic molecules on surfaces revealed by high-resolution, fast-scanning stm flemming besenbacher interdisciplinary nanoscience center (inano), university of aarhus, dk- aarhus c, denmark. e-mail: fbe@inano.dk in the interdisciplinary area of nanoscience and nanotechnology, the adsorption and self-assembly of organic molecules on singlecrystal surfaces have attracted much attention lately due to the potential applications in fields ranging from molecular electronics to biocompatible interfaces. the supramolecular structures formed upon deposition of molecular species on solid surfaces depend on the molecular architecture and the distribution of functional groups on one hand, which determines the thermodynamically stable molecular arrangement, and on the other hand, on kinetic factors like thermal diffusion, spontaneous rotations and conformational dynamics. i will show how the unique aspect of our aarhus stm and the time-resolved, high-resolution stm imaging can be used to obtain important new insight into the dynamics, and can provide very important new information on the atomic-scale realm and on the dynamics of molecular nanostructures. the time-resolved stm data are visualized in the form of stm movies (see www.inano.dk/spm) which can subsequently be analyzed in order to extract quantitative information on the activation energy, the prefactors and the adsorbate-promoted diffusion. i will specifically discuss: (i) the self-assembly of guanine quartets on au( ) and the influence of cooperative hydrogen bonds, and (ii) the molecular recognition in binary mixtures of dna bases. g molecules are found to self-assemble into a hydrogen-bonded network of g-quartets, whose structure corresponds perfectly with the quartet structure of telomeric dna determined by x-ray crystallography. the strong preference of g molecules to form quartets can be explained by a cooperative effect that strengthens the hydrogen bonds within the g-quartet network over the hydrogen bonds in isolated dimers. by means of a combination of stm experiments and dft calculations we compare the d molecular networks formed on deposition of the binary mixtures g-c (purine-pyrimidine pair of complementary bases) and a-c (purine-pyrimidine pair of non-complementary bases). we find that the non-complementary bases segregate into islands of pure a and a network of pure c, whereas the complementary bases g and c form a network that cannot be separated by annealing up to the desorption temperature for c. high-resolution stm images allow us to identify the structures for the enhanced thermal stability as structures that contain g-c bonds, possibly with the same structure as the watson-crick pairs in dna molecules. kühnle, r., et al., . nature , . otero, r., et al., . angewandte chemie int. ed. , . otero, r., et al., . nat. mater. , . otero, r., et al., . angewandte chemie int. ed. , . rosei, f., et al., . science , . schunack, m., et al., . biomedical and pharmaceutical companies are using an increasing number of carbohydrate polymers in the formulation of drugs. one such polymer with highly attractive features is chitosan that can be produced from crustacean shells. chitosan is non-toxic, biocompatible and biodegradable. chitosan can be formulated as nanoparticles or membranes and have enhanced several bioprocesses. among the well-documented features are enhanced drug uptake by tight junction relaxation and enhanced in vivo uptake and protection of nucleic acid formulations. chitosan research has increased throughout this decade. research programs are addressing the potential of chitosan applications but preparations with variable molecular size and charge are not easily available. specifically companies working with the development of new drugs and enhancement of drug functionality are in need of formulation technology that provides well characterized biocompatible material. in the chitosan innovation consortium the danish companies, coloplast, novozymes biopolymers, pipeline biotech, and zgene, together with the research center inano (aarhus university) and bioneer a/s have developed a series of chitosan preparations suitable for research of biopharmaceutical applications (www.chitosan.dk). the ability to obtain functional formulations is currently being tested in both in vitro and in vivo experiments. the consortium participants have established a platform from which chitosan processing, characterization and formulation technology can be extracted. by providing specified chitosan preparations the polymer feature can be adjusted to fit specialized biopharmaceutical applications. high throughput bioprocessing govind rao center for advanced sensor technology, umbc, baltimore, md , usa the post genome era holds a great deal of promise. an enormous number of new proteins await study. these will require sophisticated culture techniques, as cells will have to be grown under large numbers of environmental conditions to elucidate expression triggers. unfortunately, unlike molecular biology, bioreactor technology is little changed since its inception. the primary reason has been a lack of sensor technology that can be readily employed to monitor the cellular environment. we will take a look at the current status of the technology and report on promising optical sensor technology that permits low-cost high throughput cell culture and fermentation. noninvasive sensors that monitor ph, po and pco and high sensitivity solutions for glucose and glutamine measurements will be presented. in addition, the mixing characteristics that determine bioreactor performance will be examined at the small scale and their relevance to the large scale will be demonstrated. on-line monitoring and fed-batch operation in shake flask and micro titre plate cultures jochen büchs , frank kensy , markus jeude , tibor anderlei , barbara dittrich , doris klee : biochemical engineering, rwth aachen university, aachen, germany; ac biotec, jülich, germany; textile chemistry and macromolecular chemistry, rwth aachen university, aachen, germany although methods of molecular biology has led to rational design of micro-organisms to suit our requirements, screening of large numbers of strains and media is still one of the most important tasks in biotechnology. batch operation of shaken bioreactors and absence of on-line monitoring is still the general state of the art for that purpose. it is also a very common practice in screening projects that only the final product titre is measured at the end of the culture for the evaluation of the "best performers". in the recent years several approaches were introduced to follow microbial cultures also in shaken bioreactors like shake flasks or micro titre plates (mtp's). it became obvious that the cultures can behave quite unexpected and most relevant and essential information is lost, if only the final product titre at the end of the cultures is utilised for evaluation. as a result, the screening may be directed to an unknown and non-intended direction or may even fail. this is demonstrated with some examples in this contribution. new methods and techniques are introduced to measure the oxygen and carbon dioxide transfer rate and the respiratory quotient in shake flasks and the optical density, nadh fluorescence, ph and do in mtp's. if the desired product can be fused to a fluorescence protein, like gfp or yfp, also the product formation can be monitored on-line in mtp's. it is of utmost importance that the operating conditions of the applied shaking bioreactors are suitable and shaking motion is not stopped during the measurement. otherwise, e.g. power input, mixing and oxygen supply is interrupted and the micro-organisms will ongoingly rearrange their metabolism to cope with these disturbances of their environmental conditions. another problem of screening is the commonly applied batch operation mode. a lot of microbial systems display an overflow metabolism, substrate or osmotic inhibition or are characterised by a catabolite repressed product formation. in all these cases, batch operation is not the preferred operation mode and, therefore, these cultures are run in fed-batch in larger scales. in particular, it is nearly impossible to screen systems, which are catabolite repressed by the carbon source, in batch mode in defined mineral media. after initial growth has led to a nearly complete consumption of the carbon source, the product formation is derepressed. but then no more carbon source is available to continue with production. it is quite questionable whether in batch operation mode suitable strains can be selected for later fed-batch operation or not. this consideration has resulted in the development of a new technique which allows to run the screening in fed-batch operation mode. this technique is applicable in shake flasks as well as in mtp's. dramatic increases in product titre between -and -folds were observed under these conditions compared to conventional batch screenings. mikkel nordkvist, john villadsen center for microbial biotechnology, technical university of denmark, dk- lyngby. e-mail: mnq@biocentrum.dtu.dk (m. nordkvist) efficient mixing and mass transfer are highly important in the chemical industry and in the fermentation industry. poor mixing can result in low yield and variable product quality in a number of cultivation processes, and mass transfer can easily become the limiting step in aerobic cultivations, especially at high cell density. we have tested a new tank reactor system, where liquid is withdrawn from the bottom of a tank, rapidly circulated, and injected back into the bulk liquid through the nozzles of rotary jet heads. liquid feed as well as gas is added in the recirculation loop and thereby distributed via the rotary jet heads. solid feed in powder form can also be added in the loop with advantage, and heat is efficiently removed in a plate-type heat exchanger, which is part of the loop. the system has a very simple design with no internal baffles or heat exchange area, and between batches the rotary jet heads are used for cleaning in place. a number of applications ranging from dispersion of liquid and powder to mass transfer will be presented. mass transfer applications include baker's yeast cultivation and oxidation of lactose to lactobionic acid by a carbohydrate oxidase. fast and accurate analytical information that can be used to rapidly evaluate the interactions between biological systems and bioprocess operations is essential for optimization of biological production processes. we have researched and developed a multiplexed microbioreactor system for the parallel operation of multiple microbial fermentations. the microbioreactors have working volumes from to l, and are instrumented for real-time monitoring of dissolved oxygen, ph and optical density. the growth profiles obtained with escherichia coli compare favorably to results obtained from conventional ml batch bioreactors. we also demonstrate the use of our microbioreactors coupled to dna microarray analy-sis, as a tool for accelerated discovery and elucidation of metabolic pathways and gene expression profiles. the multiplexed system represents a significant step towards high-throughput data acquisition and has the potential to replace current instrumented bioreactors, which are bulky, expensive to run, and require many mechanical manipulations. design of a laboratory scale bioreactor to study solid-state tobacco fermentation m. di giacomo, l. nappi, d. silvestro, m. paolino, d. parente r&d biology department, british american tobacco italia, naples , italy italian toscano cigar production is based on the fermentation of dark fire-cured tobacco. the process starts with the rise of leaf moisture to levels of water activities assuring development of the wild phylloplane microflora in the absence of free water. the intense growth of microorganisms modifies leaf characteristics (ph rise from acidic to alkaline condition) contributing to define the toscano typical smoke profile. tobacco fermentation takes place in great bulks of kg which cause considerable amount of heat evolution as a function of the metabolic activities of the microorganisms. this heat accumulates and temperature can rise to as high as • c. a laboratory cylindrical packed-bed bioreactor was designed to work under isothermal conditions. the reactor was ideal to ferment small quantities of tobacco ( g) and was made up of a column aerated from the bottom with humidified air and placed in a thermoregulated room. experiments were conducted with constant temperature and air flow. moreover, bioenrichment experiments were conducted in the presence of different microbial starter cultures. fermentation courses were monitored by measuring microbial counts and chemical/physical modification of the substrate. with this laboratory scale system we obtained different kinds of information on the role and dynamics of the microorganisms involved in the fermentation process and on the influence of different environmental conditions. for the future, the design of an adiabatic device for tobacco fermentation is planned. on-line liquid chromatography as a process analytical technology for monitoring and control of biotech processes rick e. cooley , : process analytics center of excellence, dionex corporation, sunnyvale, ca, usa; eli lilly and company, indianapolis, in, usa (retired) biotech processes, used to produce an active pharmaceutical ingredient (api), generally differ from small molecule api manufacturing processes in that the starting materials tend to be more variable, more complex, and the product of interest in lower concentration due to the fact that they originate from a biological rather than a chemical process. this complexity and low starting concentration has generated a high level of interest in developing technologies that can be utilized to increase yield and reduce variability in the initial bioreactor phase, as well as, downstream isolation and purification operations. the use of process analytics, or on-line analytical measurements, is a technology approach that can contribute to increased process understanding and control. this presentation will provide examples of how various analytical measurement technologies have been utilized to monitor typical bioprocess unit operations leading to increased automation and control. examples of the use on-line liquid chromatography to monitor bioreactors, process scale chromatography columns, and enzymatic reactions will be presented in more detail. application of advanced monitoring strategies for recombinant protein production karl bayer department of biotechnology, university of natural resources and applied life sciences, vienna a- , austria high yield in combination with the required quality are the key objectives of large-scale production of recombinant proteins using different host/vector systems. however, in the past the efficient production of recombinant proteins was frequently limited due to inadequate exploitation of the cell factory and deficiencies in process design. to achieve optimal exploitation of microbial cell factories the key requirement is to enhance the monitoring capabilities to improve the insight into the host metabolism dynamics and to cope with limited understanding of the interaction of recombinant protein synthesis with host cell metabolism. since each protein exerts an individual influence on the host/vector system, the selection of appropriate analytical methods is even more important. in order to overcome these problems high throughput technology platforms, such as dna microarrays for transcriptome and differential -d electrophoresis (dige) for proteome analysis provide extensive data to screen for significant analytes and provide an appropriate basis for in silico modelling and reverse engineering of regulatory networks. taking advantage from such an iterative process experimental design will be improved and further aid to increase the performance of modelling. in addition, transcriptome data are used to screen fast stress responsive promoters to set up gfp based reporter gene fusions for in-situ monitoring of the metabolic load due to recombinant gene expression. moreover chemometric methods are frequently applied to model complex data from easily obtainable on-line data sets to overcome the limited monitoring capabilities due to the high complexity and nonlinearity of biological reactions and reaction networks. this strategy has been successfully applied to model bdm (bacterial dry matter), pcn (plasmid copy number) and the amount of recombinant protein using data sets acquired from off-gas analysis (o consumption, co evolution), alkaline consumption rate, in-situ capacitance and multi-wavelength fluorescence measurements. at-line monitoring of bioprocess-relevant marker genes using an electric dna-chip britta jürgen , daniel pioch , le thi hoi , jörg albers , rainer hintsche , stefan evers , karl-heinz-maurer , michael hecker , thomas schweder : institut für pharmazie, ernst-moritz-arndt-universität, greifswald, germany; ebiochipsystems, itzehoe, germany; vtb-enzymtechnologie, henkel kgaa, düsseldorf, germany; institut für mikrobiologie, ernst-moritz-arndt-universität, greifswald, germany. e-mail: britta.juergen@uni-greifswald.de (b. jürgen) the gram-positive bacteria bacillus licheniformis and bacillus subtilis represent important industrial hosts for the production of enzymes (e.g., proteases and amylases) or antibiotics (e.g., bacitracin). both organisms are attractive for this purpose because of their apathogenity and their classification as gras organisms (generally regarded as save). moreover, their easy cultivation and their high natural capacity to secrete proteins into the growth medium qualify them for the industrial overproduction of homologous or heterologous proteins (simonen and palva, ) . for the control of the physiological state and the productivity of the production cells efficient analysis tools are of great interest. a prerequisite for the evaluation of the physiological state of cells during industrial fermentation processes is the analysis of so-called process-relevant marker genes, the expression of which indicates unfavorable growth and production conditions (schweder and hecker, ) . by means of proteome and transcriptome analyses we have identified critical process-relevant genes of b. licheniformis and b. subtilis cells under different nutrient limitation conditions and during industrialclose bioprocesses. dna-chips with probes for such process-relevant marker genes could be valuable diagnostic tools for the monitoring of the cellular physiology during microbial bioprocesses. in order to provide reliable tools for the monitoring of the cell physiology during microbial bioprocesses, we have developed a fast mrna analytical approach, which allows an at-line monitoring of the transcriptional activity of selected marker genes during bioprocesses. this approach is based on an easy, fast and reliable rna isolation procedure and the measurement of specific mrnas by means of an electric dna-chip barken et al., a robust bioprocess is crucial to ensure the consistent process performance and provide the high quality of product for drug manufacturing in biopharmaceutical industries. existing methodologies for bioprocess design do not involve establishing mechanisms to achieve the desirable robust bioprocesses and have low capacity in handling uncertainty in the product manufacturing. also, the solutions are often obtained step wise and do not account for interactions between the steps. despite its importance, the robustness of a bioprocess has not been properly defined and studies carried out in statistic sense are often retrospective. in addition, the computational cost is expensive due to using a line search algorithm for finding an optimal operating solution. finally, the existing methodologies are difficult to apply to the whole bioprocess in biopharmaceutical industries. this paper attempts to define rigorously a measure for process robustness and presents a new methodology for evaluating the robustness of bioprocess operations and their performance. the methodology is based on the concept of 'windows of operation' which shows the whole range of possible operating regions. the methodology also establishes a lower bound for the largest variation of a design variable to ensure the performance. these bounds are achieved by min-max optimization techniques. a direct search algorithm has been developed and its computational cost is much lower than the line search algorithm. results include visualization of robust operating regions and a set of indices which compare the performance of different operating strategies. the capabilities and efficiency of this methodology are illustrated by applying it to the centrifuge selection for the clarification of high solids density cell broths. the research work will impact considerably upon robust bioprocess operation. when grown on methanol, pichia pastoris is able to synthesize proteins to high titres as well as secreting and glycosylation, thereby making this organism a very interesting host for the production of recombinant drugs at large scale. the methanol residual concentration has been reported to strongly influence the specific productivity, the optimum concentration being around g/l. a suitable monitoring and control technique is therefore necessary to study and improve the productivity of p. pastoris fermentations. the current research aims at showing how a mid-infrared spectrometer (atr-ftir) can be calibrated in-situ in order to monitor and control p. pastoris fermentations. this method is simple and fast, and eliminates the need of both standards preparation and off-line calibration. it is based on the observation that during fed-batch processes, only substrate and biomass concentrations vary significantly. the method therefore consists in adding a known amount of methanol at the beginning of the process, just after inoculation, and subsequently calibrating the instrument. financial support for the swiss national science foundation is gratefully acknowledged. implantable biomaterials are subjected to inflammatory responses mediated by adherent phagocytes such as monocytes and macrophages. these cellular responses and behavior have been shown to be dependent on the type of protein that adsorbs to the surface. surface modification is necessary to control and prevent protein adsorption, and thus modulate the inflammatory responses. hydrophilic surfaces that adsorb minimal amounts of protein are considered useful for minimizing the inflammatory reactions to biomaterials. in this study we have used two routes to modify polyethylene terephthalate (pet) films: ( ) a wet-chemical method for attachment of linear polyethylene glycol chains (mpeg); and ( ) gas-phase plasma polymerisation of diethylene glycol vinyl ether (degve) to generate peg-like surfaces. the surface chemistry was assessed by x-ray photoelctron spectroscopy (xps), fourier transform infrared spectroscopy (ftir) and time-flight secondary ion mass spectrometry (tof-sims). the two pegylated surfaces were compared for their ability to minimise both fibrinogen adsorption and the adhesion and activation of macrophage-like human leukocytes. adsorbed fibrinogen has been shown to be one of the key proteins in stimulating inflammatory responses to biomaterials. adsorption was investigated quantitatively using i-radiolabeled human fibrinogen. in addition, the conformation of the adsorbed protein was tested using an antifibrinogen monoclonal antibody in an enzyme-linked immunosorbent assay. the results showed that pegylated surfaces adsorbed up to % less fibrinogen, and that unfolding of adsorbed fibrinogen was more pronounced on the linear mpeg layers than on the peg-like plasma polymer surfaces. adhesion of in-vitro differentiated macrophage-like u cells was reduced on both the peg-like plasma polymer surfaces and the linear mpeg layers compared to the unmodified pet surface, but cells adhering to the peg-like plasma polymer surfaces secreted less tumor necrosis factor-␣ (tnf-␣) than cells adhering to the linear mpeg layers. thus, the linear mpeg surface is relatively efficient at reducing adhesion of macrophage-like cells, but those cells that do attach are in a more activated and proinflammatory state. analysis of ceramides from biological samples m. budvytiene , j. liesiene , b. niemeyer , n. ceramides in human skin play an important role in the regulation of cell growth, differentiation and apoptosis. moreover, they are involved in the numerous signaling pathways. the growing interest in the investigations of ceramides physiological functions requires efficient separation methods of ceramides from biological resources and sensitive analytical methods. in this work some sensitive and selective methods, involving thin layer chromatography (tlc), high performance liquid chromatography (hplc), mass spectrometry (ms) and nuclear magnetic resonance spectroscopy (nmr) were employed for the separation and characterization of ceramides from human foreskin. epidermal lipids were extracted from human foreskin for h at room temperature using three solvent mixtures (chloroform/ethanol/water : : . , v/v/v); (chloroform/ethanol : , v/v), and (chloroform/ethanol : , v/v). ceramides retention char-acteristics in tlc and hplc were compared with the retention of commercial standards. the best separation was obtained using normal phase column packed with hilic silica m. the elution was performed using mixture of chloroform and ethanol / (v/v) as an eluent. two commercial standards n-stearoyl-sphingosine cer(ns), r f = . , and n-palmitoyl-sphingosine cer(np), r f = . , were selectively separated with hplc system in above mentioned conditions. the retention time of cer(np) and cer(ns) was . and . min, respectively. the same lipids were detected by hplc in the human foreskin extracts. the structure of the lipids from collected fractions was confirmed by means mass spectrometry and nmr. the physiological functions of the separated ceramides are investigated. production recombinant human thymosin-␣ overexpressed as intein fusion protein in e. coli roman s. esipov, vasily n. stepanenko, anatoly i. miroshnikov, shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, ul. miklukho-maklaya / , moscow, russia. e-mail: esipov@ibch.ru (roman s. esipov) medicines based on polypeptides consisting of and more amino acid residues are widely spread in pharmaceutical market at the present time. practically all polypeptide medicines known are prepared by general chemical synthesis that caused high cost of their production. that's why biotechnological way of polypeptide medicines preparation using recombinant gene expression in bacteria seems to be promising. during our work we design hybrid construction allowing solving a problem of specific cleavage of target polypeptide from the hybrid protein using system of protein splicing from new england biolabs. in case of thymosin-␣ production system intein mediated purification with affinity chitin (impac system)-binding tag has been used, where the modified sce vma from s. cerevisiae has been applied as intein. in contrast to other systems, impact allows the preparation of target protein without using of serine protease and other factors that may cleave the hybrid protein. in the presence of thiol reagents, such as dithiothreitol, mercaptoethanol, or cystein the hybrid protein can be site-specifically cleaved to give intein, the target protein and small fragment of nextein. using of this system does not allow to obtain the target protein with ser, cys or thr on n-terminal of protein, because in those cases target protein and n-extein ligation product will be formed. ser is n-terminal amino-acid in thymosin-␣ . it was recently found that some metal ions essentially affect the splicing. we tried to use zncl and we have found that, in case of intein-thymosin-␣ , the maximal yield of target polypeptide and the minimal yield of splicing products are observed in the absence of dithithreitol and in the presence of . - mm zinc chloride in buffers on all stages of thymosin ␣ isolation. the structure of recombinant thymosin-␣ of human was confirmed by the determination of n-and c-terminal amino-acid sequences and by maldi tof mass-spectrometry. we present a microbioreactor with thermoelectric cooling to inactivate cellular metabolism by cell culture freezing. small-scale cultivation methods have gained increased importance and their development has been supported by advances in bioprocess monitoring methods. yet efficient sampling methods for off-line analysis remain important where in-situ real-time measurements are difficult, for example for intracellular metabolite concentration or enzyme activity measurements, and for all methods which are invasive by nature, such as protein purification. freeze-stop measurements of metabolite levels are ideal, because they are inert, i.e. do not require the addition of a chemical. in large systems, the chilling time is often the limiting factor, and alternative methods for cell metabolism inactivation are required, such as the spraying of the cell suspension in % methanol at a temperature of − • c, or the use of boiling buffered ethanol. due to the small thermal mass of microsystems, shorter chilling times can be expected. sample cooling to • c in a microbioreactor has been presented previously. in this contribution, we investigate sample freezing to completely inactivate the cell metabolism from microbioreactor working volumes of approximately l. the use of multi-parameter flow cytometry for characterisation and monitoring of insect cell-baculovirus fermentations in a mechanically-agitated bioreactor bojan isailovic , alvin w. nienow , ian w. taylor , ryan hicks , christopher j. bacteria and mammalian cells have been traditionally used as hosts for commercial recombinant protein production. however, in recent years, the insect cell-baculovirus system has emerged as a potentially attractive recombinant protein expression vehicle. although flow cytometry has been used widely for analysis of mammalian and microbial cells, there is very little information on applications of this powerful technique in insect cell culture. here we compared cell ratiometric counts and viability (propidium iodide and calcein am) of sf- cell cultures using flow cytometry to those determined by more traditional methods using a haemocytometer and the trypan-blue exclusion dye. flow cytometry has also been used to monitor various parameters during cultures of sf infected with the recombinant autographa californica nuclear polyhedrosis virus (acnpv) containing the inserted nucleic acid sequence amfp coding for am-cyan coral protein, which emits natural green fluorescence. the optimization of a fermentation process requires the organism to be cultivated under desirable conditions, which depends on how well the fermentation process is controlled. inadequate mixing and mass transfer are responsible for the heterogeneous environment at large scale in terms of nutrient concentration and ph profile, resulting in lower product yields. these have been associated with, inadequate control of ph and the production of acetate or formate in response to over-feeding of glucose and oxygen deficiency. rapid analyses of substrates and indicator metabolites in a fermentation process is critical for optimal control. this can be achieved in real time with nir spectroscopy. in this study, nir-spectroscopy has been applied to monitor the concentrations of glucose, acetate, formate, ammonium hydroxide and biomass in the cultivation of e. coli (w ). a comparison of partial least square models built using water standards, synthetic medium standards and fermentation samples has been made. the control of the brewing process is important for improving product quality, and lowering costs. infrared spectroscopy is a technique that can be used at-line and on-line to rapidly measure component concentrations of unprocessed whole broth samples in real time. in this study, both mid -infrared (mir) and near-infrared (nir) spectroscopy have been used and compared for the monitoring of ethanol, flavour components, wort sugars, biomass and specific gravity during the brewing. partial least-squares regression (pls) was used to model relationships between component concentrations and spectra. the performance of these models was evaluated in terms of the standard error of prediction (sep), number of pls factors and the correlation coefficient (r). calibration models were constructed using spectra acquired for multi-component mixtures, intended to simulate brewing fermentation conditions, and actual brewing fermentation samples. chemometric results indicated that nir is a powerful tool for accurately measuring sugar, ethanol and biomass concentrations. when choosing an expression host for production of a specific recombinant protein, one can essentially select from a multiplicity of different systems. while e. coli bacterium is usually the starting point for any cloning and expression effort, there is no universal expression host that would work optimally for all proteins. practical issues to consider include e.g. need for post-translational modifications and protease activity of the host. we have produced recombinant hiv- nef in different host cell systems: e. coli, p. pastoris yeast and stable drosophila s insect cells. using strain/cell line development, production and purification data from practical experiments, we were able to conduct a techno-economical comparison of the different host cell systems. the annual production goal was set at mg of high-purity nef. this was supposed to be produced campaign-wise in - batches using laboratory-scale bioreactors and other equipment. in this study, it was shown that although the production costs of the different systems were in the same range, the production in e. coli was most inexpensive, and the s cell system was the most expensive. regardless of the selected host system, the labour costs incurred the most expenses. when comparing different stages of the work (strain/cell line development, bioreactor production and down-stream processing), the strain development, the most man-hours demanding stage, involved approximately half of the costs of every production system. although e. coli was the most inexpensive host system for producing nef, it has some definite disadvantages: e.g. the production of endotoxins and the disability to perform post-translational modifications. if these disadvantages are of importance, the production must be done using more expensive system. modelling and control of industrial fermentation j.k. rasmussen, s.b. jørgensen capec, department of chemical engineering, technical university of denmark, dk- lyngby, denmark. e-mail: jkr@kt.dtu.dk (j.k. rasmussen) fed-batch processes play a very important role in chemical and biochemical industry. fermentations in biochemical industry are most often carried out as fed-batch processes. present control schemes do not utilize the full potential of the production facilities and may fail to achieve uniform product quality and optimal productivity. the introduction of model based control strategies is considered difficult because suitable models are not readily available. first principle engineering models can be used but the usually limited knowledge of the regulatory network in the micro-organism makes model development very time consuming. another strategy is to use a purely data-driven approach where only limited prior knowledge of the process is required. a framework for generation of such blackbox models is used in this project. this framework is called "grid of linear models" (golm), it uses a large number of linear models which each describes the behaviour of the process within a certain time interval. the combination of these models results in a model which covers the entire time span of the fermentation and approximates the nonlinear time varying behaviour. a procedure for deriving golm models from operational data has been developed and because they consist of a large set of linear models it makes them suitable for model predictive control implementation with iterative learning capabilities from batch to batch. iterative learning model predictive control (ilmpc) based on a golm model is being implemented on a fermentor at novozymes a/s. the results will be evaluated in terms of the controller's capability to ensure uniform product quality and reject both intra and inter batch process disturbances. the model based approach renders optimization of the process recipe possible by using the ilmpc capability. this opportunity provides a great potential for increase of productivity and reduction of cost. j.m. viader-salvadó, j.a. fuentes-garibay, l.j. galán-wong, m. guerrero-olazarán institute of biotechnology, biological science school, autonomous university of nuevo león, san nicolás de los garza, n.l., méxico, the production of recombinant trypsin and trypsinogen has been reported as difficult due to a probably toxicity on host or its instability. in an effort to attain high-level production of shrimp trypsin for aquaculture applications, we have evaluated shrimp trypsinogen production by recombinant pichia pastoris strains in -l bioreactors. a p. pastoris gs mut+ containing in its genome the litopenaeus vannamei trypsinogen cdna fused in frame to saccharomyces cerevisiae ␣-factor secretion signal, previously constructed in our laboratory, was used. four three-step fermentations (glycerol batch, glycerol and methanol fed-batch) were carried out. the glycerol batch step ( l of basal salts medium, g/l glycerol, . ml biotin . %, . ml ptm , l antifoam, ph adjusted to with % nh oh) was carried out until glycerol was completely exhausted ( h). the glycerol fed-batch step was carried out feeding with % glycerol ( ml/l biotin . % and ptm ) at . ml/min by h and min of posterior starvation. the methanol fed-batch step was carried out feeding with % methanol ( ml/l biotin . % and ptm ) by h using a methanol concentration on/off feedback control to maintain constant the methanol concentration in the culture medium to g/l. in all the fermentations the air flow rate and the agitation were set at l/min and - rpm, respectively. with the four fermentation assays, the influence of the ph and temperature in the production phase to the recombinant shrimp trypsinogen production were evaluated. in the four fermentations, at the end of the second step a biomass of g/l wet weight were obtained (od ). the methanol demand in the four fermentations surprisingly was not increasing rather initially it was . ml/min, after h decreased . times for h, increased to . ml/min for h and afterwards it was decreased manually to a constant value of . ml/min for that the dissolved oxygen will not decrease to values less than % (last h). the total protein amount in the culture medium supernatant increased during the production step until values of . g/l (assay at ph ), . times more than the worst assay (ph ) recombinant shrimp trypsinogen production was confirmed by sds-page (about mg/l) and trypsin enzymatic activity was detected using bapna as substrate after trypsinogen activation with shrimp hepatopancreas extract. large conformational change on giant dna induced by ascorbic acid: a nobel scheme on its antioxidative activity yuko yoshikawa, emi sakai, yoshiko oda department of food and nutrition, nagoya bunri college, nagoya - , japan ascorbic acid is often regarded as an antioxidant in vivo, where it protects against dna damage by scavenging reactive oxygen species. in the present, we will show another potent scenario on the protective effect of ascorbic acid through a significant structural change of giant dna. recently, we examined the effect of ascorbic acid on the higher order structure of dna through single molecular observation with fluorescence microscopy, and found that ascorbic acid generates a pearling structure in giant dna molecules, where elongated and compact parts coexist along a molecular chain. the results of observations with atomic force microscopy indicate that the compact parts assume a loosely packed conformation. as the extension, here we study the protective effect against double-strand breaks by reactive oxygen at different concentrations of ascorbic acid, in relation to the change of the higher order structure of giant dna. we have performed a real time observation on the doublestrand breaks on individual dna molecules by use of fluorescence microscopy. we have found that the double-strand break is markedly protected when ascorbic acid exists over millimolar concentrations. it is found that such a protective effect of ascorbic acid corresponds well to the above mentioned change on the higher order structure of dna. it has been reported that human circulating immune cells, such as neutrophils, monocytes and lymphocytes, accumulate ascorbic acid in millimolar concentrations. therefore, it is expected that the ascorbic acid concentration that induces the large conformational change on dna may be of physiological significance. , y., et al., . febs lett. , - . yoshikawa, y., et al., . plant proteins are increasingly being used as an alternative to proteins from animal sources and substantially contribute to the human diet in several developing countries. there are many process both industrial and food based which employ protein hydrolysis and hydrolytic products have a wide variety of applications from industrial fermentation media to food additives. traditionally, proteins are hydrolysed by chemical means. acid hydrolysates of protein are used to produce food ingredients and flavour compounds. however, hydrolysis by chemical reagents produce potentially hazardous byproducts and these non-selective hydrolysis products cannot easily be defined. the use of enzymes allows for selective hydrolysis of protein and thus produces a potentially safer and more defined material. the present investigation describes the effects of substrate, enzyme and hydrolysate concentration on the hydrolysis of corn gluten. the corn gluten was hydrolysed by a commercial protease preparation neutrase. the protein hydrolysis reactions were carried out in . l of aqueous solutions at the temperature of • c and ph and were monitored by using ph-stat method. the degree of hydrolysis (%) and soluble protein concentration depending on time were investigated by using , , , , and % (w/v) substrate concentrations; and . , . , . , . and . , % (v/v) enzyme concentrations; and , , and % (v/v) this paper describes medium optimization for urease production by aspergillus niger ptcc by one-factor-at-a-time and orthogonal array design methods. the one-factor-at-a-time method was used to study the effects of carbon and nitrogen sources on urease production. among various carbon and nitrogen sources used, sucrose and yeast extract were the most suitable for urease production, respectively. subsequently, the concentration of sucrose, yeast extract and mineral sources were optimized using the orthogonal array method in two stages. the effects of nutritional components for urease production by a. niger ptcc in the first and second stages were in order of urea > niso > sucrose >kh po > k hpo > cacl > yeast extract > mgso and yeast extract > sucrose > k hpo > kh po > urea > cacl > niso , respectively. the optimal concentrations of nutritional components for improved urease production were determined as g/l sucrose, . g/l urea, . g/l yeast extract, . g/l niso · h o, . g/l mgso · h o, . g/l cacl , . g/l kh po , and . g/l k hpo . these results showed that urea, niso , yeast extract and sucrose had significant effect on urease production by a. niger ptcc . tween and mgso had negligible effect on urease production by this strain. the subsequent confirmation experiments determined the validity of the models. maximum urease activity in optimized media by onefactor-at-a-time and orthogonal array methods were about . and . times greater than with the basal medium, respectively. carbon sources create fingerprint fermentation characteristics pınar Ç alık , güzide Ç alık , tunçer h.Özdamar : bre lab, department of chemical engineering, ankara university, ankara, turkey; ib lab, department of chemical engineering, metu, ankara, turkey. e-mail: calik@eng.ankara.edu.tr (g. Ç alık) this work reports on a systematic investigation of the interactions between the single-carbon sources, i.e. glucose and citric acid, and complex-medium components, i.e., carbon and nitrogen sources, in enzyme fermentation processes, i.e. serine alkaline protease (sap; ec . . . ),) and ␤-lactamase (ec . . . ), with the oxygentransfer and ph conditions to demonstrate the influences of carbon sources that create the fingerprint fermentation characteristics, moreover, their influences on the product and by-product formations and the intracellular reaction rates. the influence of the medium composition i.e. citric acid-, glucose-, molasses-and soybean-based media together with the oxygen transfer (ot)-and ph-conditions applied, on the product and by-product distributions and ot characteristics were investigated in batch bioreactors. for sap, in general, under uncontrolled-ph operations the variation of the medium ph in sap fermentation process has a tendency to increase in the sap production phase; however, depending on the carbon source used, its behaviour changes in the early stages of the fermentation as the consequences of the directed functioning of the intracellular bioreaction network. the loci of the dissolved oxygen (do) curves also strongly depend on the carbon source(s) utilised, in addition to the applied ot conditions. the complex media profiles are significantly different compared to the defined media as the ph and do profiles are interrelated owing to the bioreactor operation conditions affecting the metabolic reaction network. the highest volumetric oxygen uptake rates were obtained with soybean-based medium that was ca. three-fold higher than the values reported in citrate-based and glucose based media, and ca. . - -fold higher than the values reported in molasses-based medium. the significant changes, moreover, the drastic change observed with the use of soybean-based complex medium are due to the compositions of the fermentation media used, and its influence on the intracellular bioreaction network. thus, we conclude that the change in medium composition based on the carbon source changes the fermentation characteristics under the designed bioreactor operation conditions that appear as the fingerprints of the bioprocess. effects of oxygen transfer on ␣-amylase production by b. amyloliquefaciens nurhan güngör, güzide Ç alık bre lab, department of chemical engineering, ankara university, ankara, turkey ␣-amylase (e.c. . . . ) a commercially important enzyme used in food, textile, detergent, brewing, paper, and animal feed industries; hydrolyses ␣- , glucosydic bonds in amylose, amylopectin, and related polysaccharides. optimum medium composition and influence of bioreactor operation parameters on ␣-amylase production with high yield and selectivity were determined together with the metabolic flux distributions using b. amyloliquefaciens (nrrl b- ) which is found to be a good producer of the enzyme. systematic investigation of oxygen transfer in relation with the metabolic fluxes for ␣-amylase is not available in the literature. shake-flask experiments were conducted in . dm airfiltered bioreactors in orbital shakers with agitation and heating controls (b. braun, certomat-bs ). laboratory-scale bioreactors were composed of agitation, heating, foam, dissolved-oxygen and ph-controlled . and . dm systems (b. braun, biostat q; and chemap). after separation of the cells with a sorval rc s ultracentrifuge, ␣-amylase activity was measured by the dns method (bernfeld, ) . amino acid concentrations were determined with a hplc (waters), protein and organic acid concentrations were measured with a hpce (waters, quanta e) (Ç alık et al., ) . oxygen transfer characteristics in the bioreactor systems were calculated using the dynamic method (rainer, ) . in the mass flux balance-based analyses, a pseudo-steady state approximation for the intracellular metabolites and the accumulation rates of the extracellular metabolites measured throughout the fermentations in considerations of the biochemical feature of the system were used to acquire the flux distributions. in laboratory scale, the effects of different c sources i.e., glucose, fructose, maltose, lactose and soluble starch; n sources i.e., (nh ) hpo , (nh ) so , and nh cl and/or their concentrations; and the operation parameters, ph and temperature on cell growth, substrate utilization, ␣-amylase and byproduct concentrations, and ␣-amylase activity were investigated. in pilot scale, the fermentation and oxygen transfer characteristics of the bioreaction system together with the metabolic fluxes were determined. oxygen transfer regulates benzaldehyde lyase production in e. coli pınar Ç alık iblab, department of chemical engineering, metu, ankara, turkey. e-mail: pcalik@metu.edu.tr the effects of oxygen transfer rate on benzaldehyde lyase (bal) production by puc ::bal carrying recombinant e. coli on a defined medium with . kg/m glucose were investigated in order to finetune the bioreactor performance, in v = dm batch bioreactors at five different conditions with the parameters at, i.e. q /v r = . vvm and n = , , and min − and; q /v r = . vvm and n = min − . the concentrations of the product and by-products amino acids and organic acids were determined in addition to bal activities. medium oxygen transfer rate conditions and uncontrolled ph operation at ph . are optimum for maximum bal activity, i.e. u/cm at h, and productivity and selectivity. on the bases of the data, response of the intracellular bioreaction network of r-e. coli to oxygen transfer conditions were analysed using a mass-flux balance based stoichiometric model that contains metabolites and reaction fluxes. the results reveal that metabolic reactions are intimately coupled with the oxygen transfer conditions. oxygen transfer rate showed diverse effects on the product formation by influencing metabolic pathways and changing metabolic fluxes. metabolic flux analysis was helpful to describe the interactions between the cell and the bioreactor by predicting the changes in the fluxes and the rate controlling step(s) in the metabolic pathways. therefore, knowing the distribution of the metabolic fluxes during the growth, and bal and by-product formations provide new information for understanding physiological characteristics of the r-e.coli, and reveals important features of the regulation of the bioprocess and opens new avenues to successful application of metabolic engineering. the detection and diagnosis of pathogenic bacteria causing many diseases to the human body is an area of important research to public welfare. food is the most important energy source to humans, but it can give rise to disease caused by pathogenic bacteria not performing adequate detection tests. oligonucleotide-based microarrays are becoming increasingly useful for the analysis of expression profiles and polymorphisms among interested genes. here, we checked the possibility of development of oligonucleotide-based microarrays for detection and diagnosis of foodborne pathogenic bacteria. the oligonucleotide chip technology was applied to one control strain and seven foodborne pathogenic bacteria strains. it was designed repeated spots of eight hyperspecific and two highly conserved (control) capture probes from s rdna sequences. in order to validate experimental quality and to certificate specificities among specific spots at a glance by d and d views, quantitative visualization tool was developed. using the proposed oligonucleotide chip, we could classify and diagnose species and even subtypes of some pathogens. induction of in vitro neuro-muscular junctions using neuroblastoma and fibroblast cell lines for facilitating receptor-binding studies with botulinum toxin arindam chaudhury, bal ram singh department of chemistry and biochemistry, university of massachusetts dartmouth, old westport road, north dartmouth, ma - , usa. e-mail: g achaudhury@umassd.edu (a. chaudhury) botulinum toxin (bont), the most potent biological toxin known, is responsible for botulism, a fatal paralytic disease of the neuromuscular transmission. it blocks the release of acetylcholine at the neurotransmitter junction of the synapse. the objective of the current study was to induce in vitro neuro-muscular junctions through co-culturing of nerve and precursor-muscle cell lines. presently no known primary cultures or cell lines are available for nerve-muscle co-culture, thus validating the current work. j - t fibroblast cell line was first adapted to grow in media conducive for growth of sh-sy y neuroblastoma cell line. the two cell lines were then splitted and co-cultured and observed for junction formations. light and fluorescent microscopic studies revealed en plaque (twitch-type) and en grappe (bulbous nerve endings) nerve-muscle junctions. growth rate of j - t cells decreased substantially when the media was initially changed. structurally they were more spindle-shaped than the normal reticular shapes of j - t cells, when grown in a media tailor-made for them. the formation of nerve-muscle junctions were confirmed using markers specific for each cell type. future work is focusing on receptor identification for the botulinum toxin in the established in vitro neuro-muscular junctions and also the transcellular translocation of the toxin. fourier-transform infrared (ftir) spectrometers have recently enjoyed widespread popularity in bioprocess monitoring applications due to their non-invasiveness and in-situ sterilizability. their online applicability creates an interesting opportunity for process control and optimization. however, the precision and accuracy of the predicted analyte concentration values directly depend on the quality of the measured signal and the robustness of the calibration model. instability and time drift in the measured spectra are currently one of the main obstacles in ftir monitoring. the intensity of the detected signal is influenced both by random noise and structural drifts and offsets. as a result, it is often necessary to scale the measured spectrum with respect to a constant reference spectrum, a technique similar to the internal standard approach used in analytical assays, such as hplc. applying this technique has lead to a noticeable decrease in the standard error of prediction in the monitoring of an anaerobic s fermentation of the saccharomyces cerevisiae yeast. in order to test the robustness of the calibration model and to increase its resistance to signal instability, random spikes of known amounts of analytes were introduced into the measured medium. this approach can be used to fine-tune the calibration model on-line and is currently one of the aspects investigated in this laboratory. the effect of the stringent response induction on l-valine biosynthesis by corynebacterium glutamicum ilze denina , , longina paegle , liga zala , , maija ruklisha : faculty of biology, university of latvia, kronvalda blvd. , riga lv- , latvia; institute of microbiology and biotechnology, university of latvia, kronvalda blvd. , riga lv- , latvia. e-mail: ilzede@hotmail.com (i. denina) the present study was focused on methods of the stringent response induction and on investigation of its effect on valine overproduction by isoleucine auxotrophs of corynebacterium glutamicum. the intracellular level of guanosine -diphosphate diphosphate (ppgpp) increased and bacterial growth rate (µ) decreased during the short-term experiments when the exponentially growing cells were exposed to isoleucine limited conditions. the induction of the cellular stringent response resulted in a drastic increase in the activity of acetoxydroxy acid synthase (ahas), also by a significant increase in valine production. in contrast, an increase in ahas activity and valine synthesis by c. glutamicum was not achieved when bacterial growth was down-regulated in a ppgpp-independent manner. these results demonstrated that induction of the ppgpp-mediated stringent response might be significant in order to increase valine overproduction by c. glutamicum. infections with human cytomegalovirus (hcmv) continue to be an important health problem in certain patient populations, such as newborns, graft recipients of solid organs, or bone marrow and aids patients. in these groups, hcmv is a major cause of morbidity and mortality. the complex biology of hcmv necessarily begins with an initial interaction between the envelope of the infectious virus and the host cell. glycoprotein b (gb) is the major antigen, on the envelope of hcmv, for the induction of neutralizing antibodies. the region between aa and of hcmv gb (termed antigenic domain , ad- ) has been identified as the immunodominant target for the humoral immune response following natural infection. screening methods for detection of neutralizing antibodies have not been used because they are costly and labor intensive and thus far are not feasible for use on a large scale. for the development of reliable and inexpensive serodiagnostic tests the ad- of hcmv glycoprotein gp , which are known to bind neutralizing antibodies, was expressed in prokaryotic systems. in this work, one prokaryotically expressed fusion protein which codifies ad- with ␤-galactosidase was used. the influence of different process conditions, on the production of the fusion protein containing the ad- as well as sugars addition to the fermentation medium was investigated. in order to analyze the expression of fusion protein, the ␤-galactosidase activity was followed throughout the fermentation. lysis process was also optimized and some final confirmation tests about protein antigenicity were performed. polyenzymic systems for the preparation of drugs based on modified nucleosides d. chuvikovsky, r. esipov, t. muravyova, a. miroshnikov shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, ul. miklukho-maklaya / , moscow , russia. e-mail: esipov@ibch.ru (r. esipov) considerable progress in the preparation of nucleoside analogues was achieved by combination of chemical and biochemical transformations. enzyme-catalyzed chemical transformation is now widely recognized as practical alternative to traditional organic synthesis in pharmaceutical and chemical industries. pentofuranosyltransfer reaction catalyzed by nucleoside phosphorylases was successfully employed for the synthesis of a variety of base-and sugar-modified nucleosides. enzymes involved in the metabolism of ribose phosphate and deoxyribose phosphate, such as ribokinase and phosphopentomutase, were used for the preparation of sugar-modified nucleosides. nucleosides phosphorylases (thymidine phosphorylase (tp), uridine phosphorylase (up) and purine nucleoside phosphorylase (pnp)), ribokinase and phosphopentomutase from e. coli have been cloned and overexpressed in e. coli. fast and efficient methods for the purification of nucleosides phosphorylases have been developed. the amount of purified protein was about mg/l of cell culture, corresponding to , , and units, respectively, of the up, tp and pnp. synthesis of medicinal drugs ribavirin ( -(␤-d-ribofuranosyl)- , , -triazole- -carboxamide), cladribine ( -chloroadenine- -␤-d- -deoxyribofuranoside) and fludarabine ( -fluoroadenine- -␤-darabinofuranoside) with the use of recombinant enzymes were studied. several important factors affecting the modified nucleosides production (ph, temperature, enzyme concentration, donor/acceptor ratio) were investigated and optimized. under optimum conditions ribavirin, cladribine and fludarabine produced in the reaction mixture in yields of , and %, referred to , , -triazole- -carboxamide, -chloroadenosine and -fluoroadenosine, respectively. aggregation and adsorption of fibroin molecules in aqueous solution won hur school of biotechnology and bioengineering, kangwon national university, chunchon - , korea. e-mail: wonhur@kangwon.ac.kr fibroin, the structural protein from bombyx mori, is composed of heavy chain (generally called 'fibroin') and light chain polypeptides of about and kda, respectively. this study investigated the aggregation of fibroin and the adsorption between fibroin and surfaces. the variations of particle size and zeta potential were investigated by electrophoretic light scattering spectrophotometer (els). the adsorption of fibroin on surface was investigated in a continuous flow system by biacore applied surface plasmon resonance(spr) technique. the particle size and zeta potential range of aqueous fibroin were nm, ± mv, respectively. iso-electric point(ph iep )of fibroin was ph . . the amount of fibroin adsorbed on a gold surface was less than . g/ml even in the presence of high concentration of fibroin. the modification of gold surface was accomplished by applying chemicals known to form self-assembled monolayer, those are carrying nh + , coo − , benzene ring and peptide that similar structure with fibroin. the adsorbed amount of fibroin on the self-assembly monolayers (sams) increased in the following order: nh + > benzene ring > coo − > peptide surface. the deposition of fibroin in aqueous solution on non-waven fabric was affected by nacl and high temperature. ph influences metabolite profiling of ␤-lactamse producing b. licheniformis nazarİleri , pınar Ç alık , ali Şengül : ib lab, department of chemical engineering, metu, ankara, turkey; gülhane sch med, dept immunol, ankara, turkey. e-mail: e @metu.edu.tr (n.İleri) ph conditions in the bioreactor affect product and by-product formations by influencing metabolic pathways and changing metabolic fluxes, based on its influence on, i.e. dna transcription, protein synthesis, transport mechanism, atp generation and cellular energetics. whereupon, some fermentation processes favours uncontrolled-ph conditions while others favours controlled-ph conditions. on the bases of the interactions between the cell and the bioreactor through a process, carried out at either uncontrolled-or controlled-ph conditions, intracellular ph can be widely different and variable during the fermentation process. consequently, if one aims towards a quantitative understanding of the cell metabolism, one has to take into account the time variations of the intracellular ph and its effects on the in-vivo kinetics of the metabolic steps involved. moreover, since the presence of dormant or dead cells in the cultivation medium have negative effect on the synthesis of the production of desired product; the physiological state of the culture has great importance. in this context, the effects of ph on the regulation of intracellular ph, transport mechanism, and metabolic activity of b. licheniformis during production of ␤-lactamase (ec . . . ), an industrial enzyme catalyzing the hydrolysis of beta-lactam ring in beta-lactam antibiotics, was investigated. in addition, the physiological state of the organism and its effect on the production were observed. the optimal controlled-ph operation was found to be ph . with u/cm enzyme activity. the intracellular and extracellular na + , k + ion concentrations increased significantly throughout the process with increasing ph. on the other hand, the intracellular nh + ion concentration was relatively constant. isolation and characterization of angiotensin-i converting enzyme inhibitory peptides by use of anti-peptide antibody fida hasan , megumi kitagawa , yoichi kumada , naoya hashimoto , masami shiiba , shigeo katoh , masaaki terashima : graduate school of science and technology, kobe university, nada, kobe - , japan; department of human science, kobe college, okadayama, nishinomiya - , japan inhibitory peptides against angiotensin-i converting enzyme can be promising bio-functional peptides as natural alternatives for the non-peptide ace inhibitory drugs. these peptides are inactive within sequences of parent proteins and can be released during enzymatic digestion or food processing. immunointeraction is very effective for the purification of proteins and peptides with high purity. in this study, ace inhibitory peptides from hydrolysate of bonito meat were isolated by an anti-peptide antibody column and hplc, and kinetics of production of these ace inhibitory peptides was studies. an anti-peptide antibody against an ace inhibitory peptide, which was found by kohama et al. from tuna was obtained by immunization of the antigen peptide pc-iace (kkpthikwgd). water extract of bonito meat was digested at • c in a modified gastric juice, . mg/ml nacl containing g/ml pepsin (ph ). peptides in hydrolysates were purified by use of an affinity column coupled with the antipeptide antibody and separated by hplc equipped with a reverse phase column (cosmosil c -ms-ii, . cm × cm). amino acid sequences and ic values of the potent ace inhibitory peptides were determined. sds-page and western blotting experiments clarified that bonito protein contained peptides having similar sequence to the antigen peptide. a fraction of retention time - min in hplc purification samples showed high inhibitory activity, and several peptides in this fraction were separated. after h digestion, two major inhibitory peptides, herdpthikwgd and pthikwgd, were found to be relatively stable in the gastric juice. kluyveromyces marxianus physiology on several levels of carbon, nitrogen sources and oxygenation during inulinase production silva-santisteban yépez, o. bernardo, francisco maugeri department of food eng./unicamp, - , campinas, sp-brazil. email: maugeri@fea.unicamp.br (f. maugeri) inulinase produced by yeasts is an interesting alternative compared with the one produced by filamentous molds, as culture conditions can be better controlled. during the assays, it was observed that inulinase production levels varied with nutritional conditions in batch culture. kluyveromyces marxianus atcc culture is described by two main phases, the first one being the growth phase, where substrate consumption and basal inulinase production were performed, and the second one being the phase where some metabolites are uptaken and high inulinase production is observed. the metabolic fluxes analyses were used to describe the cell physiology in the first phase, in a variety of conditions of sucrose and ammonium sulfate concentration and aeration condition. the metabolic network included the main metabolic pathways such as glycolisis, pentose phosphate pathway, krebs cycle, oxidative phosphorylation and biomass biosynthesis. the physiology in this phase was correlated with high inulinase production in the second phase. it was also noticed that inulinase production diminished when sucrose was in high concentration, leading, additionally, to ethanol production. in these terms, it was unveiled a kind of crabtree effect performed by this strain. forward extraction of l-aspartic acid from fermentation broths by reverse micelles and backward extraction by gas hydrate methodÖ. aydogan , e. bayraktar ,Ü. mehmetoglu , m. parlaktuna , t. mehmetoglu : department of chemical engineering, faculty of engineering, ankara university, tandogan, ankara , turkey; department of petroleum and natural gas engineering, middle east technical university, ankara , turkey. e-mail: mehmet@eng.ankara.edu.tr (Ü. mehmetoglu) recently gas hydrate method has been applied as a technique for backward extraction of amino acids from reverse micelle systems. in this study, backward extraction of l-aspartic acid was investigated by gas hydrate method. at the first stage, production of l-aspartic acid was carried out using ml of . m ammonium fumarate (ph . ) as substrate at circc in an orbital shaker at rpm for h. e. coli (atcc ) was used as biocatalyst. at the end of reaction excess fumaric acid was extracted in reverse micelle phase. then forward extraction of l-aspartic acid was carried out with injection method in reverse micelle phase. for back extraction, co is used to form gas hydrates crystalline structure. back extraction experiments were carried out between - bar g pressure and at circc. at the end of the back extraction l-aspartic acid was obtained in crystalline form. the results indicate that recovery of l-aspartic acid from reverse micelles by forming gas hydrate can be achieved with a yield of . %. consequently, gas hydrate method can be used as a new technique for backward extraction of amino acids from reverse micelles. aerobic and anaerobic cultivations of aspergillus niger on different nitrogen sources susan meijer, gianni panagiotou, lisbeth olsson and jens nielsen center of microbial biotechnology, biocentrum-dtu, technical university of denmark, dk- lyngby, denmark in this study, we aim at creating a succinic acid producing strain of a. niger. a. niger is known to be a strictly aerobic organism, meaning it is not able to use the reductive part of the tca cycle to produce succinate. during aerobic growth a. niger uses oxygen as electron acceptor in the respiratory chain, thereby reoxidizing the produced nadh to nad + . however, under anaerobic conditions other compounds than oxygen, such as no − are required as electron acceptor (denitrification). this process consists of no − reduction to nh + coupled to substrate-level phosphorylation that supports growth under anaerobic conditions. in the present study, our aim was to investigate the effect of different nitrogen sources on the physiology of a. niger during growth under aerobic and anaerobic conditions. aerobic growth experiments on three different nitrogen sources; ammonium, nitrate and nitrite, showed that ammonium and nitrate could be consumed by the filamentous fungus. nitrite on the other hand could not facilitate growth, indicating the absence of a nitrite uptake system. however, under anaerobic conditions notable growth was only observed on nitrate. these data support the hypothesis of the existence of an alternative electron acceptor that might facilitate anaerobic growth of a. niger. among the therapeutic proteins derived from mammalian cells, recombinant antibodies received a great deal of attention as a prominent product through biotech pipelines toward the marketplace. they now occupy about % of the estimated medicines in clinical development and many more antibodies which lead the value of the market going forward are reported. there are various environmental factors affecting rcho cell cultures such as medium components, temperature, ph, and byproducts (ammonia, lactate, and, etc.) . because most of mammalian cells are very sensitive to their environmental change, appropriate control of environmental parameters is a very important consideration to enhance cell growth and production of target proteins. balanced addition of limiting medium components plays an essential role on improvement of cell density and product concentration. temperature and ph are easily adjustable process parameters, being reported to influence cell growth and recombinant protein production. ammonia and lactate are well-known byproducts which have an inhibitory effect on cell growth when their concentrations exceed a specific level. in this work, effects of various environmental factors including temperature, ph, amino acids, vitamins, hormones, and metabolic byproducts on cell growth and recombinant antibody production were investigated in the cultivation of recombinant chinese hamster ovary cells. the most suitable condition of each environmental condition was proposed for enhancement of the cell growth and the productivity of recombinant antibody. the present study was carried out in order to assess the protective effects of calycosin- -o-␤-d-glucopyranoside isolated from astragali radix (ar) on hyaluronidase (haase) and the recombinant human interleukin- ␤ (il- ␤) induced matrix degradation in human articular cartilage and chondrocytes. we isolated the active component from the n-butanol soluble fraction of ar as haase inhibitor and structurally identified as calycosin- -o-␤-d-glucopyranoside by lc-ms, ir, h nmr, and c nmr analyses. the protective effect of arbu on the matrix gene expression of immortalized chondrocyte cell line c- /i treated with haase was investigated using a reverse transcription polymerase chain reaction (rt-pcr). its effect on haase and il- ␤-induced matrix degradation in human articular cartilage was determined by using a staining method and calculating the amount of degraded glycosaminoglycan (gag) from the cultured media. pretreatment with calycosin- -o-␤-d-glucopyranoside effectively protected against matrix degradation of the human chondrocytes and articular cartilage. therefore, it would appear that calycosin- -o-␤-d-glucopyranoside from ar is a potential natural ant-inflammatory or anti-osteoarthritis agent and can be effectively used to protect against proteoglycan (pg) degradation. catechol-o-methyltransferase (comt) is an enzyme that catalyses a variety of endogenous and exogenous catechol substrates by transferring a methyl group from s-adenosylmethionine (sam) to either the meta-or the para-hydroxyl group of the catechol ring. the enzyme has a physiological role in the metabolism of the catechol estrogens, inactivation of the catecholamine neurotransmitters such as dopamine and epinephrine and detoxification of a variety of xenobiotic catechols. comt activity has been identified in various tissues; however with the developments in molecular biology and gene technology, the production and purification of large amounts of recombinant comt is a good option for biochemical, pharmacological and structural studies. in this work, cultures of recombinant e. coli harbouring a model plasmid were grown in a ml shake-flask containing ml of complex medium. the influence of medium composition and induction time on comt production, recovery and clarification by sonication, ammonium sulphate precipitation and purification by hydrophobic interaction chromatography onto a butyl-sepharose column will be presented and discussed. bioactive bacterial exopolysaccharides corinne sinquin , karim senni , jacqueline ratiskol , farida guéniche , jean guézennec , gaston godeau , sylvia colliec-jouault : ifremer, nantes cedex , france; ea université rené descartes, montrouge, france. e-mail: corinne.sinquin@ifremer.fr (c. sinquin) interest in mass culture of microorganisms from the marine environment has increased considerably, representing an innovative approach to the biotechnological use of under-exploited resources. marine bacteria associated with deep-sea hydrothermal conditions have demonstrated their ability to produce in an aerobic carbohydrate-based medium, unusual extracellular polymers (guezennec, ; colliec-jouault et al., ) . these exopolysaccharides (eps) present original structural features that can be modified to design innovative bioactive compounds and improve their specificity. with the aim of promoting biological activities, chemical modifications (depolymerization and substitution reactions) of one eps produced by vibrio diabolicus have been undertaken (raguenes et al., ) . the structure of the native eps has been described (rougeaux et al., ) the potential of the eps derivatives as therapeutical agents will be presented. physiological responses of e. coli to glucose and oxygen shifts in fed-batch fermentations jaakko soini , christina saarimaa , arne matzen , peter neubauer : bioprocess engineering laboratory, department of process and environmental engineering, university of oulu, oulu fi- , finland; sanofi-aventis, germany. e-mail: jaakko.soini@oulu.fi (j. soini) in high-cell density fermentations e. coli cells are often subjects of transient changes in microenvironment around them. these changes can be, for example, medium component gradients or differences in oxygen availability. we have studied the physiological response of e. coli w cells to simultaneous oxygen limitation and overfeeding of glucose. the aim is to obtain more information of physiological changes for better understanding of the bottlenecks in such processes. the response of the cells for glucose and oxygen shifts was studied by analyzing key metabolites and proteins and mrna transcript levels. the transcript levels were measured using a sandwich hybridization technique (rautio et al., ) proteomic analysis was carried out by d-electrophoresis and the metabolite analysis by hplc. the main focus of this study is to monitor the expression patterns of marker genes involved in mixed acid fermentation, glycolytic pathway and tricarbonic acid cycle. rautio et al., . sandwich hybridisation assay for quantitative detection of yeast rnas in crude cell lysates. microb. cell fact. influence of ner on genetic instability of the (ctg/cag) tracts in bacterial chromosome sylwia szwarocka , paweł parniewski : department of microbiology and immunology, university of Łódź, - Łódź, banacha / , poland; centre for medical biology, polish academy of sciences, - Łódź, lodowa , poland many human hereditary neurological diseases, including fragile x syndrome, myotonic dystrophy and friedreich's ataxia, are associated with expansions of triplet repeat sequences (trs) (cgg/ccg, ctg/cag and gaa/ttc) in or near specific genes. mechanisms that mediate the expansions and deletions of trs include dna replication, repair and recombination. many investigations suggest that the structural properties of the trs play a consequential role in their genetic instabilities. nucleotide excision repair (ner) is the major cellular system in both prokaryotes and eukaryotes and recognises damages due to distortion of the dna helix. involvement of ner in the hairpin loop repair that can form within ctg tracts has been reported. the participation of this repair systems in the trs instability was investigated in e. coli only on multicopy plasmids. the results showed that deficiency of some ner functions dramatically affects the stability of long (ctg/cag) inserts. in this work we present a chromosomal model to study the instability of the trs in e. coli. we introduced the (ctg/cag)n tracts into the chromosome of e. coli and used strains with some deficiency of the ner and investigated genetic stability of these tracts after multiple recultivations. in general, our results show that the (ctg/cag)n repeats are much more stable in the chromosome than in plasmids. these data may suggest that instability of trs in plasmids is associated with interaction between repetitive tracts on different plasmid molecules inside the cell. however, mutations of ner genes may increase (uvra and uvrb mutants) or decrease (uvrc and uvrd mutants) stability of the trs in the e. coli chromosome. this study was partially funded by the kbn grant p a . performance analyses of a multi-stage integrated fermentation process for lactic acid production hsun-tung lin, feng-sheng wang department of chemical engineering, national chung cheng university, chia-yi - , taiwan. e-mail: chmfsw@ccu.edu.tw (f.s. wang) in this work, we considered a multi-stage integrated continuous fermentation process for producing lactic acid. each stage consists of a mixing tank, a fermenter, a cell recycle unit and an extractor. the generalized kinetic model is first applied to formulate the integrated process. we have compared the overall productivity and conversion of the integrated process with those of two simplified processes. from the design equations, we obtain that three processes have the identical overall conversion. however, the proposed process has the greatest overall productivity. the specific kinetic model for lactic production (youssef et al., ) was applied to the integrated process in order to find the maximum overall productivity. two optimization problems are respectively considered to determine the optimal stages, operating conditions and design variables. the first problem supposes that the integrated process has the equal working volume ratio for each fermenter. such a process requires four stages to yield the maximum overall productivity and the nearly complete overall conversion. however, if the working volume ratio for each stage is considered as the decision variables in the second optimization problem, three stages is enough to achieve the identical overall productivity. youssef, c.b., guillou, v., olmos-dichara, a., . contr. eng. pract. , - . modelling of the binding of ligands to macromolecules jørgen m. mollerup department of chemical engineering, building , dtu, lyngby, denmark a variety of factors that govern the properties of proteins are utilized in the development of chromatographic processes for the recovery of biological products including the binding and release of protons, the non-covalent association with non-polar groups (often hydrophobic interactions), the association of small ions (ion exchange) and the highly specific antigen-antibody interaction (affinity interactions). the fulcrum point in the understanding and modelling a chromatographic separation is the adsorption isotherm that determines the peak shape at preparative load. to enable an efficient chromatographic process development strategy it is necessary to conduct theoretical and experimental investigations of the adsorptive behaviour of proteins. thermodynamically consistent models for ion exchange chromatography and hydrophobic interaction chromatography have been developed and can be utilised in the simulation of a chromatographic separation. besides, measurements on hic media can be utilised to determine the cohn salting-out coefficient. the lig-and binding process can frequently be coupled to associated structure changes in the protein, the ligand or both. this gives rise to nonlinear adsorptive behaviour known as cooperativity which cannot be modelled using conventional models which displays convex behaviour. examples of cooperative behaviour are the reversible binding of oxygen and carbon monoxide to haemoglobins and the binding of nad + to yeast glyceraldehydes -phosphate dehydrogenase. in the paper we discuss the modelling of reversible binding of mobile as well as immobilised ligands to macromolecules and compare modelling to experiment. comparative analysis of the temperature policy for processes with a deactivating native enzyme i. grubecki, m. wojcik department of chemical and biochemical engineering, university of technology and agriculture, - bydgoszcz, ul. seminaryjna , poland a comparative analysis of the temperature policy for an enzymatic reaction with michaelis-menten kinetics in a batch reactor has been carried out. both isothermal and optimal temperature policies for processes with deactivating native enzyme have been considered. in the model, the thermal deactivation was described by a first-order reaction, and the arrhenius-type dependence between rate parameters and temperature was assumed. as an indicator for a direct comparison between the isothermal and optimal temperature policies the quotient of conversions under identical initial and final condition was used. a method was presented to calculate this indicator, which is based on the analytical and numerical solutions. this method can be of great importance for the industrial practice. application of changeable temperature policy could result in significant increase in conversion when ratio of activation energy for deactivation and activation energy for reaction is high. studies on the impact of mixing during brewing using near and mid-infrared spectroscopy georgina mcleod , alvin w. nienow , graham poulter , reg wilson , henri tapp , christopher j. the control of the brewing process is important for improving product quality, and lowering costs. infrared spectroscopy is a technique that can be used at-line and on-line to rapidly measure component concentrations of unprocessed whole broth samples in real time. in this study, both mid -infrared (mir) and near-infrared (nir) spectroscopy have been used and compared for the monitoring of ethanol, flavour components, wort sugars, biomass and specific gravity during the brewing. partial least-squares regression (pls) was used to model relationships between component concentrations and spectra. the performance of these models was evaluated in terms of the standard error of prediction (sep), number of pls factors and the correlation coefficient (r). calibration models were constructed using spectra acquired for multi-component mixtures, intended to simulate brewing fermentation conditions, and actual brewing fermentation samples. chemometric results indicated that nir is a powerful tool for accurately measuring sugar, ethanol and biomass concentrations. the optimization of a fermentation process requires the organism to be cultivated under desirable conditions, which depends on how well the fermentation process is controlled. inadequate mixing and mass transfer are responsible for the heterogeneous environment at large scale in terms of nutrient concentration and ph profile, resulting in lower product yields. these have been associated with, inadequate control of ph and the production of acetate or formate in response to over-feeding of glucose and oxygen deficiency. rapid analyses of substrates and indicator metabolites in a fermentation process is critical for optimal control. this can be achieved in real time with nir spectroscopy. in this study, nir-spectroscopy has been applied to monitor the concentrations of glucose, acetate, formate, ammonium hydroxide and biomass in the cultivation of e. coli (w ). a comparison of partial least square models built using water standards, synthetic medium standards and fermentation samples has been made. template refolding utilizing biospecific interactions shigeo katoh , yoichi kumada , nanae maeshima , daisuke nohara : graduate school of science and technologykobe university, kobe - , japan; department of biomolecular sciencegifu university, gifu - , japan. e-mail: katoh@kobe-u.ac.jp (s. katoh) recombinant proteins over-expressed in e. coli are often accumulated as insoluble particles called inclusion bodies. proteins in inclusion bodies must be solubilized by a denaturing agent, such as urea and guanidine hydrochloride, and refolded to recover their native structures having biological activities. in bioprocesses it is important to obtain high refolding efficiencies and high throughputs at high protein concentrations. in refolding operation, a denatured protein solution is usually added batch-wise into a large volume of a refolding buffer in order to start refolding by reducing the concentration of a denaturant and to prevent aggregate formation of renaturing molecules. thus, a large volume of a stirred tank is required, and the concentration of protein after renaturation becomes low. biointeractions between a pair of biomolecules, such as enzyme-inhibitor, antigen-antibody and hormone-receptor, are highly specific and have been used for detection and separation of biomolecules. these interactions may be used as templates for refolding of target molecules, which can be captured with the templates and are prevented from aggregate formation and, in the case of proteases, from autoproteolysis. the specific interaction might promote refolding of the target molecules. these might improve the refolding efficiency. the biointeractions between antigen-antibody and enzyme-inhibitor were used for efficient refolding in packed columns, in which template ligands (antibody, inhibitor) were coupled on gel support. denatured solutions of target molecules (carbonic anhydrase and s. griseus trypsin) were mixed with refolding buffer and supplied to the affinity column coupled with the template ligands for refolding. with refolding in the column, higher refolding efficiencies were obtained than those by the batch dilution method with relatively low concentrations of denaturants. by increasing the adsorption capacity of the column, throughput of refolding can be increased without decrease in the refolding efficiency. the use of multi-parameter flow cytometry for characterisation and monitoring of insect cell-baculovirus fermentations in a mechanically-agitated bioreactor bojan isailovic , alvin w. nienow , ian w. taylor , ryan hicks , christopher j. bacteria and mammalian cells have been traditionally used as hosts for commercial recombinant protein production. however, in recent years, the insect cell-baculovirus system has emerged as a potentially attractive recombinant protein expression vehicle. although flow cytometry has been used widely for analysis of mammalian and microbial cells, there is very little information on applications of this powerful technique in insect cell culture. here we compared cell ratiometric counts and viability (propidium iodide and calcein am) of sf- cell cultures using flow cytometry to those determined by more traditional methods using a haemocytometer and the trypan-blue exclusion dye. flow cytometry has also been used to monitor various parameters during cultures of sf infected with the recombinant autographa californica nuclear polyhedrosis virus (acnpv) containing the inserted nucleic acid sequence amfp coding for am-cyan coral protein, which emits natural green fluorescence. carbon sources create fingerprint fermentation characteristics pınar Ç alık , güzide Ç alık , tunçer h.Özdamar bre lab, department of chemical engineering, ankara university, ankara, turkey; ib lab, department of chemical engineering, metu, ankara, turkey. e-mail: calik@eng.ankara.edu.tr (g. Ç alık) this work reports on a systematic investigation of the interactions between the single-carbon sources, i.e., glucose and citric acid, and complex-medium components, i.e., carbon and nitrogen sources, in enzyme fermentation processes, i.e., serine alkaline protease (sap; ec . . . ),) and ␤-lactamase (ec . . . ), with the oxygentransfer and ph conditions to demonstrate the influences of carbon sources that create the fingerprint fermentation characteristics, moreover, their influences on the product and by-product formations and the intracellular reaction rates. the influence of the medium composition i.e. citric acid-, glucose-, molasses-and soybean-based media together with the oxygen transfer (ot)-and ph-conditions applied, on the product and by-product distributions and ot characteristics were investigated in batch bioreactors. for sap, in general, under uncontrolled-ph operations the variation of the medium ph in sap fermentation process has a tendency to increase in the sap production phase; however, depending on the carbon source used, its behaviour changes in the early stages of the fermentation as the consequences of the directed functioning of the intracellular bioreaction network. the loci of the dissolved oxygen (do) curves also strongly depend on the carbon source(s) utilised, in addition to the applied ot conditions. the complex media profiles are significantly different compared to the defined media as the ph and do profiles are interrelated owing to the bioreactor operation conditions affecting the metabolic reaction network. the highest volumetric oxygen uptake rates were obtained with soybean-based medium that was ca. three-fold higher than the values reported in citrate-based and glucose based media, and ca. . - -fold higher than the values reported in molasses-based medium. the significant changes, moreover, the drastic change observed with the use of soybean-based complex medium are due to the compositions of the fermentation media used, and its influence on the intracellular bioreaction network. thus, we conclude that the change in medium composition based on the carbon source changes the fermentation characteristics under the designed bioreactor operation conditions that appear as the fingerprints of the bioprocess. kinetic resolution of racemic benzoin with different lyophilized microorganisms Ç . babaarslan ,Ü. mehmetoglu , a.s. demir : ankara university, faculty of engineering, department of chemical engineering, , ankara, turkey; middle east technical university, department of chemistry, ankara, turkey. e-mail: barslan@eng.ankara.edu.tr (Ç . babaarslan) the biocatalytic resolution of racemates is valuable tool for enantioselective synthesis and proved to be a convenient method for obtaining optically enriched compounds from their racemic form. in this work, enantiomerically pure benzoin which is one of the -hydroxy ketones was synthesized by kinetic resolution of racemic benzoin using different lyophilized microorganisms as lipase sources. the effect of lyophilized microorganism type, solvent type and acyl donor type on enantioselectivity were studied. in kinetic resolution experiments, lyophilized microorganism was resuspended in the media containing solvent, racemic benzoin and acyl donor at • c and rpm on orbital shaker. the reaction was followed by tlc during the experiment and the enantiomeric ratio of benzoin was determined by hplc analysis using chiral cell ob column. the best enantioselectivity value was obtained with lyophilized rhizopus orayzae cbs - as ee s = % and ee p = % (conversion = %) using thf as solvent and vinyl acetate as acyl donor. otimizing the fermentation broth for tanase production by a new isolated strain paecilomyces variotii vania battestin, gláucia pastore, gabriela macedo department of food science, unicamp, p.o. box , campinas, cep - , são paulo, brazil tannase is an inducible enzyme that catalyses the breakdown of ester linkages in hydrolysable tannins, resulting in gallic acid and glucose. the fermentation broth can use by-products as wheat bran, rice or oats, adding tannic acid. the use of by products or residues rich in carbon source for fermentation purposes an alternative to solve pollution problems that can be caused by an incorrect environmental disposal. in the present study we have optimized the production of an extracellular tannase by a new isolated paecilomyces variotii using response surface methodology. the first step was identify the variables having a significant effect on enzyme production. the variables evaluated were temperature, residues ratio (coffe: wheat bran), concentration of tannic acid, salt solution during , and days of fermentation time. results showed that temperature, residues ratio (coffe: wheat bran) and tannic acid had significant effects on tannase production. commercial wheat bran (cwb) and coffe rusk residues (cr) were used as solid substrate. for fermentation the mediun was composed by, cwb:cr were mixed with distilled water and transferred into ml capacity erlenmeyers flasks and autoclaved at • c for min. the medium was then inoculated with spores ( . × ) and the flaks were incubated at • c. tannase was assayed according to the methodology of mondal et al. ( ) . acording to the statist analyses, the optimum conditions to produce tannase was the range of temperature ( - • c); tannic acid ( . - %); residues % (coffe: wheat bran) ( : ) and days fermentation time. the enzyme production increased . times more enzyme production than that was obtained before this optimization. how to cope with fda's pat-initiative with respect to fermentation process monitoring and control marco jenzsch , andreas luebbert , rimvydas simutis : institute of bioengineering, martin-luther-university halle-wittenberg, halle (saale), germany; process control department, kaunas university of technology, kaunas, lithuania with its pat initiative, fda forces drug manufacturers to increase their activities in innovative manufacturing techniques, and, more than previously, to focus on quality assurance. the agency particularly places emphasis on making use of modern process supervision and control techniques such as up-to-date process analytics, multivariate data acquisition and analysis tools in order to improve process monitoring and control. in this contribution we show by means of practical examples how this guidance can be applied to cultivations of genetically modified microorganisms. a comparison of different multivariate state estimation techniques will be presented and compared with more knowledge-based techniques such as the extended kalman filter. the comparison was made for the model system gfp expressed from e. coli bacteria (bl /de /gfp) for which more than full data sets are available. all these techniques have already been used during real protein formation at productionscale fermenters, with the same success. hence, the requirements expressed in the pat initiative can immediately be put into practice. feedback control of the recombinant protein production processes based on such estimations is show for several cultivation systems. simple parameter adaptive controllers are compared with model supported controllers, for instance, generic model controllers and model predictive controllers. the results clearly show that we have at hand a rather extended arsenal of feedback control procedures that can be used successfully to tightly control the processes even along set-point profiles of physiological variables such as the specific growth rate (µ). again, fda's suggestion with respect to "control in the engineering sense" can be applied immediately to reduce batch-to-batch variances and thus to increase process quality. extending life by alternative respiration? alexander kern, franz hartner, anton glieder institute of biotechnology, graz university of technology, a- graz, austria. e-mail: a.kern@tugraz.at (a. kern) alternative oxidase transfers electrons directly from the ubiquinol pool in mitochondria to oxygen, allowing cell respiration in presence of complexs iii and iv inhibitors like antimycin a or cyanide. electron transfer by alternative oxidase is not coupled with proton transfer across the mitochondrial membrane, thereby uncoupling the supply of small metabolic intermediates by the central metabolic pathway from energy production in the cell. alternative oxidase is present in mitochondria of plants, many fungi and a few, mostly crabtree-negative yeasts, but not in p. angusta (hansenula polymorpha) and s. cerevisiae. alternative oxidase has multiple functions in different organisms. it is involved in stress answers, in programmed cell death, maintenance of the cellular redox balance, and also citric acid accumulation in a. niger. we isolated the alternative oxidase gene from the methylotrophic yeast p. pastoris in order to study its effects on the cellular energy content, respiratory activity, its protective role against oxidative stress. our results indicate the importance of an exact regulation of the alternative oxidase due to its impact on many cellular functions. new types of energy efficient fermenters with better mass transport, mixing and cooling properties than the current crop of rushton turbine derived tank bioreactors are likely to be required in the future. such fermenters will be needed in order to meet the increasing pressure on costs for low price commodity type products such as single cell protein or food and technical grade enzymes, and to meet the demands of the new wave of white biotech, in which bio-produced chemicals must be made at prices competitive with those of the traditional chemical industry. with this in mind, a prototype pilot scale ( l) u-loop fermenter has recently been commissioned at biocentrum-dtu. in this fermenter, liquid circulation is driven by a propeller pump through a vertical u-shaped pipe, which is connected at the top with a de-gassing tank. we present here a study of liquid mixing and dispersion in the prototype u-loop fermenter. sub-sequently we show that the results can be described with the tanks in series model. mixing was characterised using pulses of nacl tracer, which were detected with a conductivity probe in various parts of the fermenter. bodenstein numbers (bo) were determined for flow rates corresponding to a linear fluid velocity of . m/s in the 'legs' of the reactor and showed that the majority of the mixing occurred in the top degassing part (bo = ) rather than in the u-loop section (bo = ). it was also observed that the time for mixing to % homogeneity after tracer pulse addition was a function of the number of cycles through the reactor ( - . ) within the range of flow velocities (u) studied (u = . m/s to u = . m/s). the mixing time to % homogeneity was between . s (at u = . m/s) and s (at u = . m/s). today many biotechnological processes are operated at suboptimal conditions and according to best practice. however, the current industrial development is towards analyzing more parameters and in particular there is a large interest in analysis of biological/biochemical variables. the quality of products and also the possibility to optimize production in submerged cultivations would be greatly enhanced if more on-line/real-time information were at hand. the present investigation was undertaken with the aim of evaluating the potential in using multi-wavelength fluorescence for monitoring and control of filamentous fungi fed-batch cultivations. a recombinant a. oryzae expressing a heterologous lipase was applied as model system. spectra of multi-wavelength fluorescence were collected every five minutes with the bioview ® system (delta, denmark) and both explorative and predictive models, correlating the fluorescence data with the important biological parameters cell mass and lipase activity, were built. the models will be presented, furthermore, advantages and disadvantages of multiwavelength fluorescence for monitoring of cultivation processes will be discussed. moving from r&d to pharmaceutical development is a costly process. it is therefore of paramount importance to design a manufacturing process that combines robust and well-documented technological platforms. therapeutic recombinant proteins designed for human administration should be as close to the authentic product as possible. here, the use of a scalable process and an economically sound affinity tag can be a relevant choice. the tagzyme tm system has been designed to allow for the precise removal of amino terminal affinity tags. the system is based on the use of recombinant aminopeptidases including dipeptidyl peptidase i (dapase tm ). dapase tm is currently produced under cgmp providing a suitable strategy for its use in pharmaceutical production. the tagzyme tm system is superior to other methods since: ( ) it is based on exopeptidases, precluding, e.g., unspecific protein cleavage reported when using so-called site-specific endoproteases. ( ) it has been tested for production of more than recombinant proteins. ( ) it is easily scalable from lab scale to kg of processed protein. ( ) it allows the use of his-tags for commercial production without patent infringment, due to our ipr position. ( ) the commercial use of tagzyme tm does not require any licensing, only purchase of the enzyme(s). ( ) the use of aminopeptidases for pharmaceutical production has been extensively documented for approved drugs. ( ) a number of therapeutics is currently being developed using tagzyme tm . ( ) unizyme can assist in the optimization of the dsp to enable further cost reduction in the process. these aspects will be discussed and illustrated in the presented poster. website sphingolipids are biologically active molecules involved in the regulation of a large quantity of biological responses. they function in cell proliferation, survival and death (apoptosis) as messengers. dysregulation of apoptosis has significance in numerous pathological conditions including cancer. several anticancer agents act by increasing tumor cell ceramide (a kind of sphingolipid) content. so, a novel approach to cancer therapy would be the pharmacological manipulation of sphingolipid metabolism. in this study, sphingolipid metabolism in baker's yeast s. cerevisiae is used as a model system as many of its sphingolipid related genes and proteins have been characterized. gepasi-biochemical kinetics simulator was used for metabolic control analysis (mca) of the above-specified system. the concentration control coefficients (ccc), flux control coefficients (fcc) and elasticity coefficients were calculated, and their significance in identification of anticancer drug targets is determined. elementary flux modes were also identified and metabolic pathway analysis (mpa) was performed. quantitatively, control effective flux (cef) values were used for potential drug target identification. the results from mca and mpa indicate almost the same potential drug targets: serine palmitoyl transferase, ceramide synthase and ceramidase. drugs against these targets are in preclinical and clinical development. for the identification of new potential drug targets, the cccs, fccs, cefs and elasticity coefficients were examined with an objective function of maximizing the cell ceramide concentrations. it was found that manipulation of inositol- -phosphate synthase and phosphoinositide kinase activities have considerable effects on ceramide concentrations. if a drug targeting the two enzymes at the same time is designed, it might give a better outcome in terms of cancer therapy. in recent years, there is a growing interest in utilization of airlift reactors (alrs) to biotechnological processes. nevertheless, their industrial application still remains limited because of a lack of reliable studies on transfer phenomena and mixing enabling a suggestion of suitable scale-up procedure. the way to more widely utilization of alrs to biological processes lies in experimental research (on a model medium as well as on a real fermentation medium) followed by mathematical modelling and scaling-up of the processes. this paper deals with a modelling of a glucose-gluconic acid fermentation by a. niger in an internal loop airlift reactor. knowledge of the stoichiometric relationship in the key reaction provides a good opportunity for estimation of substrate and product concentration. the model is based on material balance equations and has been adjusted to experimental data obtained from three internal loop airlift reactors ( . , and l) . in the model, the alr is divided into ideal stirred tanks in series. in each zone (tank) of the alr the material balance is calculated in two phases (the gas and the liquid phase). this work was supported by the slovak scientific grand agency, grant number vega / / alkaline phosphatase (ap, e.c. . . . ) is a thermolabile enzyme which is indigenous to all dairy products. it has an inactivation temperature slightly above the value that is required to destroy the most resistant pathogenic microorganism likely to be found in milk. due to that feature, this enzyme is used as an indicator of proper pasteurization. the effect of temperature treatment on the activity of ap was investigated in raw cow's and goat's milk. the stability of alkaline phosphatase in raw milk was compared with the stability of this enzyme in a . m potassium phosphate buffer with ph . . the ph value of the buffer was approximately the same as that of raw milk. the inactivation curves were measured in the temperature range from to • c. ap in cow's milk was completely inactivated at • c during s but approximately % of activity remained at • c after min of treatment. the time required for a complete inactivation of the enzyme in the raw cow's milk was reduced from to min as the temperature increased by • c. heat treatment of goat's milk caused the decrease of activity of the enzyme in the same temperature range as in the case of cow's milk. the increase of temperature from to • c reduced the inactivation time from min to s. the study of thermal stability of the alkaline phosphatase in the buffer solution showed that the time required for inactivation of enzyme was significantly shorter than in milk. milk thus had a protective effect on the activity of alkaline phosphatase. the experi-mental curves were fitted simultaneously using kinetic models where the initial heating period was considered. this work was supported by a grant of th framework program of eu, project foodpro, no. sme- - - . during the process of separation, purification and concentration of monoclonal antibodies (mabs) at industrial scale, the chromatographic unit operations have an important role. three different protein-binding modes are employed: ion-exchange, hydrophobic and affinity binding. two adsorbent properties are of uppermost importance: a high selectivity and adsorption capacity. in the case of ion-exchange/hydrophobic chromatography, the binding of charged proteins can be affected by ph and ionic strength. in this work, the adsorption capacity of eight commercially available adsorbents designed for separation of mabs (mabselect, rprotein a sepharose ff, poros a, prosep-va, fractogel emd se hicap (m), sp sepharose ff, mep hypercel, s ceramic hyperd f) was measured as a function of ph. as a model mab and contaminant proteins, human immunoglobulin (igg), human serum albumin (hsa) and horse skeletal muscle myoglobin (myo) were used. the resin properties were investigated within the range of ph - . the experiments were conducted in a batch-mode, individually for each model protein. the results showed that ion-exchange and hydrophobic resins provided the best selectivity for igg at ph . the selectivity of affinity adsorbents was essentially unaffected by ph, however, the highest capacity for igg was at ph . another investigated aspect was the dynamics of protein binding. the solution of individual protein in contact with tested adsorbent was circulated through an uv spectrophotometer, what enabled the measurement of time-dependent decrease of protein concentration. the results indicated that affinity adsorbents with a rigid matrix needed approximately four times shorter time to reach the adsorption equilibrium with igg in comparison with a gel. the gels, however, provided higher adsorption capacity. at ion-exchange resins, the time necessary to adsorb % of total amount of igg was about . - h. the affinity adsorbents were highly selective and therefore they adsorb very small amount of tested contaminant proteins (hsa, myo). the adsorption capacity was saturated by % in less than min in all cases of dynamic adsorption measurements. this work was supported by a grant of th framework program of eu, project aims, no. nmp -ct- - . microtechnology has for several years been applied within chemical reaction engineering. the advantages of microtechnology are that it makes it possible to develop light weight and compact systems, and the systems enable large surface-to-volume ratio, which results in low mass-transfer distances. in addition, parameters like pressure, temperature, residence time, and flow rate are more easily controlled. the use of microtechnology is also beginning to find its ways into the field of biotechnology. what we are aiming at is the development of a microreactor that can be applied as a production tool in industry as an alternative to conventional enzymatic reactors. our strategy is to use a small plate of a suitable material with microchannels fabricated into its surface, and the approach is to covalently couple enzymes into the microchannels. substrate can then be pumped through the channels and the enzymatic conversion will take place within the channels. as model enzyme in the development of the microreactor we are applying celb, a thermostable ␤-glycosidase from pyrococcus furiosus. kinetic resolution of racemic benzoin with different lyophilized microorganisms Ç . babaarslan ,Ü. mehmetoglu , a.s. demir : ankara university, faculty of engineering, department of chemical engineering, , ankara, turkey; middle east technical university, department of chemistry, , ankara, turkey. email: barslan@eng.ankara.edu.tr (Ç . babaarslan) the biocatalytic resolution of racemates is valuable tool for enantioselective synthesis and proved to be a convenient method for obtaining optically enriched compounds from their racemic form. in this work, enantiomerically pure benzoin which is one of the -hydroxy ketones was synthesized by kinetic resolution of racemic benzoin using different lyophilized microorganisms as lipase sources. the effect of lyophilized microorganism type, solvent type and acyl donor type on enantioselectivity were studied. in kinetic resolution experiments, lyophilized microorganism was resuspended in the media containing solvent, racemic benzoin and acyl donor at • c and rpm on orbital shaker. the reaction was followed by tlc during the experiment and the enantiomeric ratio of benzoin was determined by hplc analysis using chiral cell ob column. the best enantioselectivity value was obtained with lyophilized rhizopus orayzae cbs - as ee s = % and ee p = % (conversion = %) using thf as solvent and vinyl acetate as acyl donor. chromatography is one of the most important unit operations at separation, purification and concentration of monoclonal antibodies (mabs) at industrial scale. since these proteins belong to the group of immunoglobulins, their molecular weight (about , g/mol) or hydrodynamic radius ( . nm), respectively, is relatively large. the adsorbents used in ion-exchange/affinity chromatography of these biomolecules should thus provide a high pore accessibility coupled with a high value of specific surface area in order to ensure a sufficient ligand density and a high binding capacity. in this study, the pore accessibility of eight commercially available adsorbents designed for separation of mabs (mabselect, rprotein a sepharose ff, poros a, prosep -va, fractogel emd se hicap (m), sp sepharose ff, mep hypercel, s ceramic hyperd f) was investigated via size exclusion of standard-sized molecules (glucose, sucrose and dextrans with molar weight range - × g/mol) at nonbinding conditions. the experiments were conducted in a batch and column-mode. the batch experiments provided absolute partition coefficients, which were calculated from a mass balance and represent the fraction of pore water accessible to a solute. it was found that several adsorbents contained a small fraction of very small pores (less than nm), whereas some adsorbents contained a significant fraction of pores larger than nm. the column measurements provided relative partition coefficients, which were calculated from the retention volumes of solutes and represented the relative accessibility of pores, scaled between the accessibility of the smallest and largest solute used. when the absolute partition coefficients were recalculated into the relative form, it was found that the coefficients obtained by both methods correlated very well. the relative partition coefficients of solutes with the hydrodynamic radius of nm (corresponding to mabs) was about . - . at ion-exchange and hydrophobic adsorbents and . - . at affinity adsorbents. the relation between hydrodynamic radius of the solutes and their partition coefficients was successfully described with the giddings random plane model. this work was supported by a grant of th framework program of eu, project aims, no. nmp -ct- - . the transfer of laboratory results to a larger scale is often a critical step in process development to industrial application. the objective of this study was to scale-up the bioreactor for the fructosyltransferase production based on the results obtained in a dm stirred bioreactor. the investigations made in this bioreactor provided a clear picture of the effect of medium composition on the obtained fructosyltransferase (ftase) activity but the influence of mixing intensity was unequivocal. the increase of agitation rate had a positive effect up to a certain level where both fructosyltransferase and biomass production increased. the final optimal yield factor of ftase per dry cell mass obtained in the laboratory bioreactor was , u g − . we studied the effect of oxygen transfer on the process of ftase production at a larger scale, in and dm mechanically stirred bioreactors and in air-lift bioreactors, and dm whilst the medium composition was kept constant. the yield factors of ftase were comparable in both mechanically stirred bioreactors and they were about u g − . this decrease compared to that in the laboratory bioreactor could be explained by a slower cell growth. this fact was also confirmed by that glucose was not depleted till the end of fermentation and free fructose concentration was also lower. the yield factor of ftase was u g − in the dm air-lift reactor and u g − in the dm air-lift bioreactor. the lower yield of ftase in dm bioreactor was caused by a better biomass growth. this work was supported by slovak scientific grand agency, grant numbers vega / / and / / and by science and technology assistance agency, grant number apvt- - . the poster gives an overview of the objectives and achieved results of an interdisciplinary project on direct product isolation from crude feedstocks using magnetic micro adsorbents in combination with suitable magnet technology. the project was funded by the deutsche bundesstiftung umwelt and was running between august and november . in the course of the project several milestones could be met, which can be looked at as critical key points on a route towards an industrial realization of the process. among these milestones are: (i) the production of magnetic micro adsorbents with high capacity and selectivity in batches up to - g; (ii) the proof that the micro adsorbents can be reused many times; (iii) generation of a variety of recombinant tagged, active enzymes; and (iv) the design, assembly and operation of a fully automated pilot plant capable of generating approx. g/h (≈ % purity) protein. the process was also simulated by help of the software tool superpro designer and simple mass balance and sorption equilibrium approaches were used to derive rules for estimating optimum process parameters and productivities. finally an environmental performance evaluation was conducted externally by the german dechema. in this study the effect of fed-batch cysteine addition to a culture of a high-gsh-accumulating yeast strain on the metabolism of glutathione was investigated. it is known that cysteine is the rate limiting amino acid in the biosynthesis of gsh. the influence of the consumption rate of cysteine on glutathione metabolism and growth of s. cerevisiae mt- was determined. the results show that for rates of consumption below a critical value the microorganism growth is similar to a culture without feed of cysteine, but glutathione production is increased two-fold. on the other hand, if cysteine consumption rate is above the critical value the changes of cell metabolism implies ethanol accumulation in the extracellular media which diminishes biomass synthesis. the maximum specific glutathione production in this case is maintained at two-fold; however, gamma-glutamylcysteine accumulation is increased. cysteine present in culture media directs cell metabolism to a greater synthesis of ammonia and amino acids. hydrophobic interaction chromatography (hic) exploits the hydrophobic properties of protein surfaces for separation and purification by performing interactions with chromatographic sorbents of hydrophobic nature. in contrast to reversed phase chromatography this methodology is less detrimental to the protein and is therefore more commonly used in industrial scale as well as in bench scale when the conformational integrity of the protein is important. hydrophobic interactions are promoted by salt and thus proteins are retained in presence of a cosmotropic salt. when proteins are injected on hic columns with increasing salt concentrations under isocratic conditions only, a fraction of the applied amount is eluted. the higher the salt concentration the lower is the amount eluted protein. the rest can be desorbed with a buffer of low salt concentration or water. it has been proposed that the stronger retained protein fraction has partially changed the conformation upon adsorption. this has been also corroborated by physicochemical measurements. the retention data of five different model proteins and different stationary phases were evaluated. partial unfolding of proteins upon adsorption on surfaces of hic-media were assumed and a model describing the adsorption of native and partial unfolded fraction was developed. furthermore we hypothesize that the surface acts as catalyst for partial unfolding, since the fraction of partial unfolded protein is increasing with length of the alkyl chain. stationary phases for bioseparation of glycoproteins j. aniulyte , j. liesiene , b. niemeyer : department of chemical technology, kaunas university of technology (ktu), radvilenu pl. , kaunas, lithuania; institute of thermodynamic, helmut-schmidt-university/university of the federal armed forces hamburg, holstenhofweg , hamburg, germany d- nowadays glycomics raise new challenges for affinity chromatography related with an abundance of glycoconjugates in living organisms and with scaling-up of the preparative processes. economics, efficiency and practicality dictate the search of novel chromatographic biospecific adsorbents that could contribute to enhancing the productivity of the affinity separation process. the purpose of the work was to prepare cellulose-and silica-based biospecific adsorbents with immobilized lectins and to evaluate them for the affinity chromatography of glycoproteins. cellulose-based matrix granocel and silica coated with hydrophilic polymers were used as a support. the effect of support characteristics, such as pore size, chemistry of active groups and their density on the support' surface on lectin immobilization and on the efficiency of adsorbents obtained were evaluated.three different methods were used for the activation of the support: oxidation with sodium periodate, modification with pentaethylenhexamine (spacer arm of atoms) and carbonyldiimidazole activation.cona and wga two lectins of different molecular weight and shape were selected to notice differences resulting from the size and diffusion behaviour. chromatographic performances of the adsorbents were studied applying two different glycoproteins (god and fetuin) carrying specific terminal glycomoieties of mannose (god), and n-acetylglucosamine (fetuin) for specific interaction with cona and wga, respectively. the adsorbents demonstrated high affinity to glycoproteins with a sorption capacity in the column up to . mg per ml support and a high recovery (up to %). it was shown that spacer arm affected ligand coupling kinetic as well as the chromatographic behavior of the adsorbents obtained. the adsorption isotherms of god onto cona adsorbents reveal an adsorption behavior with high and low affinity binding sites. the dissociation constant k d of the ligand-sorbate complex is approximately × − m, and . × − m, respectively. it was supposed that the second step is related to the sorption of solvated god onto already adsorbed god forming sorbate dimers. cell disruption and chromatography are key unit operations in the downstream processing of an intracellular product. the cost involved in the extraction and purification of intracellular products can be reduced by selective release of proteins and reduction in the number of steps involved in the purification. the extent of disruption can be varied to provide a selective release, limiting the release of the contaminant proteins. the particle size distribution of the cell debris in the resulting suspension depends on the extent of disruption. expanded bed adsorption chromatography allows for the direct capture of the proteins from an unclarified suspension. this technique allows for the integration of solid-liquid separation, concentration and preliminary purification in one unit operation. a perfectly stable expanded bed can be obtained by choosing the appropriate flow conditions and a suitable adsorbent. the difference in the density between the adsorbent and the cell debris in the suspension, permits the cell debris to flow through the column without blocking, whilst the protein molecules in the suspension are adsorbed onto the adsorbent. after sample application, the bed is washed with buffer and the proteins eluted from the column in the packed bed mode. the presence of the cell debris in the feedstock influences the expansion of the bed and the adsorption of protein molecules. the physical properties of the suspension obtained after cell disruption depends on the extent of disruption. the particle size distribution of the cell debris, the viscosity and the release of soluble proteins and other intracellular components are influenced by the extent of disruption. the influence of the extent of disruption of e. coli on the expansion of the bed and the adsorption of ß-galactosidase is presented in the current study. e.coli cells were disrupted at different operating pressure using a high pressure homogenizer. the resulting crude homogenate is subjected to expanded bed adsorption chromatography using streamline deae as adsorbent. the disrupted suspension was characterised in terms of viscosity, density, particle size distribution of the cell debris and the extent of protein and ß-galactosidase released. the interaction between cell debris and adsorbent was quantified as the cell transmission index (ratio of the amount of cells present in the sample before and after passing through the bed). the expansion of the bed at a constant settled bed height and flow rate was measured. the influence of the cell debris on the extent of adsorption of ß-galactosidase has been quantified in terms of dynamic binding capacity (dbc) at % of the inlet concentration. the dbc of ß-galactosidase that was released by disruption at psi ( %, w/v, w/w, pass) was found to be u/ml of adsorbent while dbc of samples disrupted at psi ( % w/v, w/w, pass) was u/ml of adsorbent. the extent of disruption of e. coli over a wide range and its effect on the expansion and adsorption will be presented. study of dna binding during expanded bed adsorption and factors affecting adsorbent aggregation ayyoob arpanaei , niels mathiasen , timothy hobley , owen rt thomas , : center for microbial biotechnology, building , biocentrum-dtu, technical university of denmark, , lyngby, denmark; department of chemical engineering, university of birmingham, edgbaston, b tt, uk. e-mail: aa@biocentrum.dtu.dk (a. arpanaei) the adsorption of sonicated calf thymus dna (as a model dna molecule) to biosepra q hyper z adsorbents was evaluated in batch and expanded bed modes. stability of the expanded bed during feedstock loading was also studied. two batches of prototype q hyper z (batch and ) were examined, which had ionic capacities measured to be and mmol cl − /ml support respectively. in all adsorption experiments a mm tris-hcl ph buffer was used. maximum static binding capacities of adsorbent batches and were determined to be . and . mg dna/ml particle, respectively. dynamic binding capacity at % breakthrough (dbc % ) was measured in a -cm diameter eba column containing . ± . cm settled bed with a feed of g/ml dna. dbc % of the adsorbents were . and . mg dna/ml support for batches and , respectively in buffer containing no salt. however, the maximum dbc % for batch ( . mg dna/ml support) and ( . mg dna/ml support) were obtained in buffers containing . and . m nacl, respectively. further increases in salt concentration led to a decrease in dbc % for both adsorbent batches. the bed compression during loading that was observed in experiments at high conductivities (achieved by adding salt) was less than that seen with low conductivity ( ms/cm) solutions. aggregation of adsorbent particles and channeling of flow were not observed in the presence of salt concentrations more than . m. the effect of different concentrations of dna during loading in the presence of . m nacl was studied. it was found that increasing dna concentrations in the feed from to g/ml, to g/ml resulted in a decrease of dbc % by , and %, respectively. the bed compressed slower during loading of feedstock with low dna concentrations compared to that for higher concentrations. the expanded bed showed a partly reversible compression behavior during feedstock loading. this is attributed to the electrostatic interaction between dna adsorbed on the particles surface and rearrangements of dna strands as the number of free ligands on the adsorbent surfaces decrease during loading. large-scale production of plasmid dna for gene therapy and dna vaccination applications has become necessary as a result of the increasing number of approved protocols using non-viral vectors for gene delivery. a major challenge of large-scale plasmid production is to establish a robust cgmp manufacturing capable of producing hundreds of milligrams or grams of a pharmaceutical grade product. alcohol and salt precipitation are operations largely used in the early steps of plasmid downstream processes. however, there are few systematic studies on the influence of these precipitation agents in the final plasmid recovery and purity. in this work, alcohol and salt precipitation steps used in a plasmid purification process developed by our group have been optimized aiming at large-scale production. the optimization of alcohol precipitation indicated that almost % of the pdna precipitated when . vol. of isopropanol were used. the studies also indicated that the precipitation profile was strongly influenced by pdna initial concentration. finally, the final plasmid recovery and purity after a sequential alcohol and salt precipitation were strongly dependent on the concentrations of these precipitation agents. thus, a commitment between high recovery and purity level should be made during the development of the downstream processes. comparison of novel and conventional processes for protein refolding and initial purification h. ferré , , u. jørgensen , l. scale down of downstream processing unit operations is convenient for assessing process alternatives, particularly if feedstock is scarce. in this study it was imperative to use the smallest possible scale for comparison of a new system for continuous protein refolding and direct expanded bed adsorption (eba) capture with a traditional process composed of discrete operations of batch renaturation, centrifugation, microfiltration and packed bed chromatography (pbc). minimisation of the scale was restricted by the eba step: the smallest practical scale being a cm diameter column with - cm of settled bed, expanded two fold. in order to permit a fair comparison a similar column diameter and adsorbent volume was used in the packed bed process. in both alternatives, chelating media charged with cu + was used and a feedstock of denatured hat-tagged human beta- microglobulin (hat-h␤ m). following batch refolding and clarification, the performance of the packed column was severely hampered due to fouling of the top adapter. reducing the protein loaded to the packed bed to % of dbc working lead to a recovery of . % at a purity of % and . -fold concentration. the eba-based process performed unimpeded and productivity was calculated to be % higher than for that employing a packed bed. however, due to the severe scale restrictions placed on the eba process, which limited optimisation, significant productivity improvements of eba over packed bed are expected at larger scale. high gradient magnetic filtration has the potential for rapid processing of large volumes of crude bioprocess liquors when magnetic adsorbents are employed. the binding of a protein to a superparamagnetic solid support provides a unique selective 'handle'. typically the focus is placed on using the magnetic handle for direct capture of a protein from a fermentation broth. however, magnetic adsorbents may provide solutions to a range of downstream processing problems and in this presentation we illustrate this with a number of case studies. using whey as a model system, we show that the extent of the tryptic hydrolysis (ca. . mg/ml added enzyme) of proteins could be controlled by adding benzamidine-linked magnetic adsorbents after a given period ( - min), followed by removal of the loaded adsorbents using a magnetic filter. hydrolysis was stopped effectively and approx. % of the added trypsin could be recovered. a coupled process was devised for the refolding and purification of inclusion body proteins. solubilised (in m urea) inclusion bodies of recombinant histidine affinity tagged human beta microglobulin (hat-h-beta m), were refolded by dilution in a pipe reactor ( s), then captured directly on cu(ii) charged magnetic immobilised metal affinity adsorbents in a second pipe reactor ( s residence time). loaded adsorbents were retained in a magnetic filter, then washed and the protein eluted. a generic framework for the prediction of scale-up when using compressible chromatographic packings r. tran , j. joseph , a. sinclair , y. zhou , n. packed bed chromatography is the pre-eminent technique in the downstream purification of many biological products. the aspect ratio of a packed bed has a significant effect on the column pressure drop by virtue of wall support which is reduced at low aspect ratios. this can result in unexpectedly high pressures during manufacturing caused by the compression of the matrix via drag forces due to fluid flow through the bed. the need for an accurate model to predict flow conditions at increasing scale is essential for the scaling-up of chromatographic processes and for avoiding bed compression during operation so that maximum throughput can be achieved. several studies have generated correlations which allow for the prediction of column pressure drops but have either been mathematically complex, which makes their practical use unfeasible, or they have used highly specific empirical constants and hence require a large amount of experiments to be performed before they can be used. in this study, we have established relationships to link the critical velocity of operation, to bed height (l), column diameter (d c ), feed viscosity (µ) and also to the matrix rigidity through the level of agarose cross-linking (a%). the correlation is straight forward to use and involves very few system-specific constants thus significantly reducing the need for any preceding laboratory-scale experimentation. this paper describes the series of experiments that were performed to establish the correlation, using a range of cross-linked agarose matrices ( - %), at various aspect ratios, fluid flow rates and varying viscosities ( . - . mpas). a mathematical model was developed where parameter estimation for multi-variables was achieved by least squares optimisation. the model can be used to predict the extent of compression in industrial chromatography applications and will be useful in the development of chromatographic operations and for column sizing. institute of process engineering, swiss federal institute of technology, zurich. e-mail: makart@ipe.mavt.ethz.ch (s. makart) simulated moving bed (smb) technology receives increasing attention in biotechnology and in the biopharmaceutical industry as it enables an increase in productivity per unit mass of stationary phase, reduced solvent consumption, and fast and reliable scale-up. combining continuous chromatographic separation unit and reactor should enable the production of biopharmaceuticals and fine chemicals with high purity and yield at the same time. due to the increasing demand of enantiopure intermediates in the pharmaceutical industry, biocatalytic processes gain more and more importance because of their excellent enantioselectivity. yet the application of biocatalytic carbon-carbon bond formation on process scale is often hampered by an unfavourable equilibrium position and difficult downstream processing due to substrate/product mixtures. coupling a continuous separation unit to such a process would improve the feasibility by driving the reaction to completion and thus increasing the overall yield. we will discuss the design of such an integrated biocatalytic/smb process, taking the formation of l-allo-threonine from glycine and acetaldehyde, catalysed by the glycine-dependent aldolase glya from e. coli, as a model reaction. the enzyme exhibits absolute stereoselctivity at the c-alpha atom, whereas selectivity is less strict at c-beta. in situ product removal, by the integration of an smb unit, would aid to maintain a high diastereomeric excess as it shortens the residence time of the products in the reactor, in addition to shifting the reaction to the product side. the in-line coupling of the chromatographic unit to the enzyme reactor requires the use of the same solvents for reaction and separation, so the choice is limited to aqueous solutions close to physiological ph, limiting in turn the possible stationary phase materials. in a screening of different cation exchangers, amberlite cg- ii gave promising results: threonine is more retained than glycine, acetaldehyde is poorly and the cofactor plp is not retained. adsorption isotherms were determined by the retention time method and a smb under process conditions was simulated. by improving the packing of the column, i.e. achieving a more even particle size distribution, we tried to further increase the efficiency of the separation step. the application of enzymes for the synthesis of optically active substances is nowadays of growing importance in the pharmaceutical industry. this requires a proper cultivation of the microorganism as well as a posterior isolation process yielding a constant catalyst quality at high purity. goal of this project is the development of an integrated process for the production and isolation of a lipase from trichosporon beigilie and its posterior application for the enantioselective synthesis of pharmaceutical products. the cultivation of the microorganism is optimised in a laboratory and pilot-scale fermenter in a fed-batch mode. parameters like media composition, temperature, ph and aeration rate are set up. taking advantage of the localisation of the enzyme (covalently attached to the cell membrane) the first step of product isolation consists of a continuous cell disruption. optimal results are achieved with the continuous bead mill disruption process ( % enzyme release with a specific activity of . u/mg of protein). the non disrupted cells are recycled as inoculums for a new cultivation, increasing the yield of the overall process (by -times in the pilot-scale fermenter). in order to isolate the product two different process sequences are considered. the first one consists of an extraction (peg and phosphate buffer) coupled to an ion-exchange chromatography (q-sepharose ff). the second one applies a precipitation step with ammonia sulphate followed by a hydrophobic interaction chromatography (sepharose-hic) provid-ing a lipase yield of % ( -times higher than the one provided by combining extraction-chromatography). an ultrafiltration process is used in order to concentrate the lipase and its final properties (molecular weight, isoelectric point, activity, stability and kinetic data) are studied using p-nitrophenylacetate as model substrate. the relevance of the obtained product for its application in the pharmaceutical industry is proven by transforming (r,s)-naproxen-methylester into (s)-naproxen acid with an enantiomeric excess of > % (after h). biotensides (sugar fatty acid esters, sfaes) find nowadays a wide range of applications in pharmaceutical, personal care and food industry because of their biocompatibility, biodegradability and special surfactant properties. goal of this project is the development and optimisation of an integrated process for the enzymatic synthesis of sfaes from renewable sources to be used in cosmetic formulations. the following figure shows the scheme of the overall process. commercial and also new screened lipases are applied in the reaction between sugar and fatty acid. the mixture grade of the initial reaction system is increased by ultrasounds taking into account the influence on the catalyst characteristics and also the necessity of an organic solvent as adjuvant. the reaction takes place in an enzymatic membrane reactor (emr) equipped with an ultrafiltration membrane which retains the catalyst. the separation of the by-product (water) from the rest of the components can be achieved by means of a pervaporation unit which coupling to the emr allows the semi-batch process. in order to separate the esters from the fatty acid a stepwise elution chromatography method is developed using silica as adsorbent and ethyl acetate and methanol as eluents. with this system % of the dimer is isolated with purity (hplc) of %. the application of a dialysis membrane technique allows the separation of % of the fatty acid by building ester micelles changing the polarity of the organic solvent used as eluent. solubility and crystallisation properties of recombinant bacillus halmapalus ␣-amylase cornelius faber centre for microbial biotechnology, biocentrum dtu, building , lyngby, denmark a comprehensive knowledge of solubility properties is a prerequisite for the efficient design and operation of bulk enzyme recovery processes, however, complete phase diagrams are only available for very few proteins, in particular lysozyme of high purity. here, we present the results of detailed solubility studies in aqueous solutions of an industrially relevant ␣-amylase of technical grade. experiments were conducted in small scale batch mode (working volume of ml). the influence of selected cations and anions from the hofmeister series on the stability of the ␣-amylase was examined. the hofmeister series for anions was followed in the correct order at all salt concentrations studied, i.e. from to m, whereas the series was reversed for monovalent cations at concentrations up to . m, with the exception of lithium. to further investigate why the position for lithium was different to the hofmeister series established for lysozyme, the zeta potential of protein solutions at low concentrations of selected salts was determined. the results of these measurements indicate a pronounced effect of lithium on the zeta potential, as compared to other salts. in particular, the ph of zero zeta potential (i.e. the pi) was shifted approximately . ph units towards alkaline conditions in the presence of lithium, whereas the pi stayed almost constant for sodium and potassium. since the solubility exhibits a minimum at ph-values at or near the protein's pi, shifts in ph caused by salt addition are important to identify and quantify to avoid uncontrolled phase separation. the measurement of the zeta potential of proteins in solution holds significant promise as an attractive tool for understanding and controlling processes that are operated close to the solubility limit and which are often plagued by uncontrolled precipitation or crystallisation and thus rely on carefully chosen operating conditions. polyphenolic interactions with potato proteins during industrial expanded bed adsorption processing sissel løkra , knut olav straetkvern , bjørg egelandsdal , gerd vegarud : department of natural science & technology, hedmark university college, n- hamar, norway; norwegian university of life sciences, as, norway. e-mail: sissel.lokra@lnb.hihm.no (s. løkra) in plant extracts it has been shown that polyphenols have a tendency to react with proteins, either by covalent or non-covalent interactions. these reactions can induce changes in the surface properties of the proteins, and, e.g. cause proteins to be insoluble and precipitate at ph-values below their isoelectric point. potato proteins have a high nutritional quality and show interesting functional properties in food systems. moreover, chlorogenic acid (ca) and caffeic acid constitute about % of the total polyphenol content of potato tuber. we have experienced expanded bed adsorption (eba) chromatography to be a method well suited for recovering industrial proteins from potato starch effluent. the process separates proteins from polyphenolic pigments, fiber and minerals. during the adsorption step, patatin, the major potato tuber protein shows complex binding kinetics demonstrated by breakthrough curves. in addition to diffusion limitations in the eba resin, changes in protein structure and surface properties probably are likely to affect this adsorption behavior. reactions between ca and patatin might result in a range of interactions for different species of the same protein. this project therefore aims to assess the interactions between ca, patatin and other major tuber protein fractions and how these changes affect the protein capture in eba. changes in size and charge are screened in -d electrophoresis and analyzed further. samples of different protein fractions are taken from breakthrough curves and dynamic binding capacity experiments in model systems with real feedstock. sandwich hybridisation assay for analysis of brewery contaminants s. huhtamella , m. leinonen , t. nieminen , a. breitenstein , p. neubauer : bioprocess engineering laboratory, university of oulu, finland; scanbec gmbh, halle, germany. email: peter.neubauer@oulu.fi (p. neubauer) here we describe the development of a sensitive, cultivationindependent analytical method for the analysis of brewery contaminants which can be performed within three hours in crude sample extracts. the method is based on s rrna detection by a paramagnetic bead based sandwich hybridization assay (sha) with two oligonucleotide probes designed to either detect the species or a group of contaminants. the signals were read out either by a fluorimeter (rautio et al., ; leskelä et al., ) or potentiometrically with an electric biochip instrument (ebiochip systems) . this assay is advantageous over rt-pcr becasue it only detects viable cells and the method can be directly applied to crude cell extracts without prior purification. we describe the principle of designing and evaluating a series of groupspecific lactobacillus probes and the optimisation towards effective cell lysis and high assay sensitivity. the applicability of the sha was evaluated with real brewery samples and the results were compared to routine tests. in all steps of the evaluation the reliability and usability of the method was prioritised. the optimised method combined with a h pre-enrichment period gave reliable results, had a detection limits of about - cells per assay and was easily applicable in a brewery environment. biodesulfurization is one of the possibilities studied by the researchers to attain the maximum sulfur levels imposed for a near future by governments (european directive, ) . rhodococcus erythropolis igts is a natural and strictly aerobic microorganism able to remove the sulfur atom from dibenzothiophene (dbt) in a selective way ( s route (oldfield et al., )), obtaining hydroxibifenyl (hbp) and sulfate. growth is carried out using the experimental procedure performed in previous works dealing with the inoculum built up, media composition and operational conditions (del olmo et al., a (del olmo et al., , b . this work is focused to determine the oxygen uptake rate during the production of the biocatalyst. four experiments were carried out at a biostat b fermentor (braun biotech.) using as only variable the constant stirrer speed used: , , and rpm. oxygen uptake rate have been determined by means of two methods: dynamic technique at different times during growth for few seconds to ovoid influences and from oxygen profile when the term dealing with oxygen transfer rate is known (predicted by the model proposed in a previous work (garcía-ochoa and gómez, )). our values obtained from the techniques used present the same tendency in all the runs carried out: our values from dynamic technique is always lower than our values obtained from the oxygen profile. these values are modeled and the difference observed is explained due to the cellular economy principle: during the seconds employed in the dynamic technique determinations microorganism do not produce s route enzymes. it was studied different methods to recover a p. salmonis antigenic protein from recombinant e. coli cells. this protein has shown be highly effective in vivo vaccine. it has the ability to stimulate salmon immune system protecting them against of aggressive disease salmonid rickettsial syndrome resolving by this way a great problem of salmonid aquaculture. biomass obtained from iptg induced e. coli bl (de ) codon plus culture was used for soluble and insoluble antigenic protein recovery. it was evaluated recuperation by glass bead mill, freezing and thawing, osmotic shock and lisozyme/edta treatments, all of them applied in single or combined way. biomass was measured by dry weight of cells, soluble protein concentration was quantified according to bradford method, and antigenic protein was identified by sds-page and western-blot analysis. cells treated with lisozyme/osmotic shock and then glass bead mill the soluble protein was a . % of the dry weight cell mass whereas using lisozyme/edta and glass bead mill as a single treatment only a . and . % were obtained respectively. the freezing and thawing disruption treatment released less than % of soluble protein, as well as the osmotic shock procedure too. the sds-page and west-ernblot analysis revealed that the antigenic protein must be purified from the insoluble cell fraction when physical or mechanical disruption methods were employed and from the soluble cell fraction when chemical or enzymatic treatments were used. we propose investigate in further studies the inclusion bodies formation to design an efficient purification procedure for the target protein. the iso-peroxidase pox from garlic bulb allium sativum that represented the major peroxidase activity was purified to homogeneity. the enzyme is monomeric and has a molecular mass of kda, and a pi around . the optimum temperature ranged between and • c, while optimum ph was around . pox appeared remarkably thermostable since it retained % of its activity at • c for at least h. in addition, the enzyme was stable at a ph range from , to . kinetic constants were calculated, the apparent k m values were and m for gaïacol and h o , respectively. the high thermostability of pox may represent clear advantages in a number of processes including immobilizing peroxidase and use it as a biosensor to detect oxidant component as h o and other peroxides. immobilization of pox was achieved by binding covalently the enzyme to a sepharose matrix (bead and membrane va epoxy). the immobilized peroxidase showed great stability at heat and storage than the soluble enzyme. the native enzyme retained % of its activity at • c for mn while the immobilized pox retained full activity for mn at the same temperature. in other side, the free enzyme retained full activity for at least one month and a half during storage at • c, and lost m of its activity after months. the immobilized form of pox retained complete activity for months at the same temperature. the immobilized enzyme was used to detect h o in some food components such as milk and fruit juices. in a second study, same experiments were performed in order to detect the smaller quantity of added h o to the farming milk. purpose: a new research field has been created to begin to address protein function at level of regulation of enzyme activity. this new area has been given the name chemical proteomics, or activity-based proteomics (abps), and makes use of small molecules that can covalently attach to catalytic residues in an enzyme active site. the selectivity of the chemically reactive group allows specific proteins or protein subset to be tagged, purified and analyzed. methods: this molecule (abps) has three subsets: tag, linker, and warhead. warhead is a nucleophile and attach to active site. linker is a polypeptide that makes a simple connection between warhead and tag. tag is fluorcent or radioactive material that facilitates the detection of drugs. findings: several diseases such as cancer, rheumatoid arthritis and osteoporosis are associated with elevated levels of protease activity. serine hydrolyses abps have been used to profile enzyme activity in a diverse range of cancer cell lines. in studies comparing metastatic and non-metastatic human breast cancer models, it was shown that the former exhibited a higher activity of a ␥-glutathione-s-transferase, an enzyme that has not previously been associated with breast cancer. discussion: additionally, abps can be used to develop robust screens for small molecule inhibitors of a specific enzyme target within a large family of related enzymes. this method of inhibitor screening allows compounds to be assayed for both potency and selectivity against a set of related in complex biological samples. this technique is able to identify novel enzymatic proteins and drugs and has the potential to accelerate the discovery of new drug target. a cyclodextrin glycosyltransferase (cgtase) from a new isolated strain from bacillus clausii e , was purified through q-sepharose, gel filtration chromatography and deae-sephadex a- . the mw of the pure enzyme was kda with sds-page. the enzyme displayed optimum ph value and ph stability at ph . and in range of . - . , respectively. the optimum temperature and thermostability were at • c and up to • c by h, respectively. the k m and v max were . mg/ml and . mol/min mg and . mg/ml . mol/min mg using maltodextrin and soluble starch, respectively. the isoeletric point was . and the n-terminal region of the pure enzyme was sequencing by maldi-tof-ms. the ratio of ␣-, ␤and ␥-cd was . : . : . and : : with maltodextrin and soluble starch at . %. application of magnetic separation technology for recovery of immobilised lipases nadja schultz , anke neumann , george metreveli , matthias franzreb , christoph syldatk chair of technical biology, university of karlsruhe, engler bunte ring , d- karlsruhe; forschungszentrum karlsruhe, institute of technical chemistry, water-and geotechnology; inst. für wasserchemie, engler bunte ring , ka. e-mail: nadja.schultz@ciw.uni-karlsruhe.de (n. schultz). url: www.fzk.de/itc-wgt (m. franzreb) first results on the development of the magnetic separation technology for the recovery of immobilised lipase from a -phase-system, which should be suitable for a large scale use in future, known as high gradient magnetic separation (hgms) are presented. the application of immobilised lipases makes the reuse of the enzyme in a process possible and is therefore interesting for industrial applications. in this study immobilised lipase is used in a -phase-system. here the new approach to recycle and reuse the lipase, immobilised on magnetic particles, from a -phase-system with the help of the new high gradient magnetic separator (hgms) is examined in ml scale. as model enzyme for the immobilisation on magnetic microparticles (polyvinyl alcohol (pva), - m) the commercially available (novonordisc) lipase a (cala) from candida antarctica was used. necessary for screening of immobilisation methods and characterisation of the immobilised lipase (candida antarctica) was the development of a robust, simple and rapid chromophoric activity assay. therefore the pnpp-lipase assay was optimised for direct application on immobilised lipases (in preparation schultz et al., ) . further more a ph-stat-assay for measuring the activity of free and immobilised cala in a -phase-system of tributyrate and buffer was optimized for this system. another important basis for the realisation of the recovery of immobilised lipases was to optimise the immobilization technique of the lipase. furthermore approaches for the explication of generally empirical based immobilization techniques on insoluble support were made. hereby we successfully applied the zeta potential measurement on the immobilization behaviour of the lipase cala. for to determine the operating temperature for the biomagnetic separation procedure we studied stability analysis of free and immobilised lipase cala at different temperatures ( , , , − • c) and at different ph values (ph , and ). the optimal temperature and ph value for the free and immobilised lipase was determined. presently and constructively on the so far developed methods and techniques we intensively work on the demonstration and feasibility of the recovery of immobilized lipase from a -phase-system. challenging approaches and first results on the recovery of immobilized lipase from a -phase-system in ml scale were shown already. nowadays glycomics raise new challenges for affinity chromatography related with an abundance of glycoconjugates in living organisms and with scaling-up of the preparative processes. economics, efficiency and practicality dictate the search of novel chromatographic biospecific adsorbents that could contribute to enhancing the productivity of the affinity separation process. the purpose of the work was to prepare cellulose-and silica-based biospecific adsorbents with immobilized lectins and to evaluate them for the affinity chromatography of glycoproteins. cellulose-based matrix granocel and silica coated with hydrophilic polymers were used as a support. the effect of support characteristics, such as pore size, chemistry of active groups and their density on the support' surface on lectin immobilization and on the efficiency of adsorbents obtained were evaluated. three different methods were used for the activation of the support: oxidation with sodium periodate, modification with pentaethylenhexamine (spacer arm of atoms) and carbonyldiimidazole activation. cona and wga two lectins of different molecular weight and shape were selected to notice differences resulting from the size and diffusion behaviour. chromatographic performances of the adsorbents were studied applying two different glycoproteins (god and fetuin) carrying specific terminal glycomoieties of mannose (god), and n-acetylglucosamine (fetuin) for specific interaction with cona and wga, respectively. the adsorbents demonstrated high affinity to glycoproteins with a sorption capacity in the column up to . mg per ml support and a high recovery (up to %). it was shown that spacer arm affected ligand coupling kinetic as well as the chromatographic behavior of the adsorbents obtained. the adsorption isotherms of god onto cona adsorbents reveal an adsorption behavior with high and low affin-ity binding sites. the dissociation constant k d of the ligand-sorbate complex is approximately × − , and . × − m, respectively. it was supposed that the second step is related to the sorption of solvated god onto already adsorbed god forming sorbate dimers. quantitative methods in high throughput screening of aqueous two phase systems matthias bensch, björn selbach, jürgen hubbuch institute of biotechnologie , forschungszentrum jülich, germany purification of biopharmaceuticals is one of the most expensive and at the same time least understood steps in bioprocesses. during the process development for protein production, short time to market and the demand for cheap processes dominate today's process development. one way of reducing process costs is to implement integrative processes. aqueous two phase systems (atps) combine the advantages of removing cell debris and simultaneously purifying and concentrating the target protein, however, to the cost of highly complex systems which are difficult to predict and optimize. using high throughput screening techniques in the development of atps processes thus seems to be an ideal candidate for achieving both a reduced development time and an economical process without the need for preliminarily well characterized systems. in this study, we use the robotic system tecan freedom evo tm as an automation platform for the evaluation of aqueous two phase systems. central to this workstation are the integrated hardware as liquid handler, gripper, reader and centrifuge. we have created high throughput methods for rapid parameter estimations. as a first step, pipetting and mixing had to be calibrated for the use of highly viscous polymer solutions which are common in atps. the focus of the current work lies on the integration of the automatic preparation and analysis of two phase systems in microtiter plate scale. the robotic platform can now automatically create aqueous two phase systems and measure characteristic values such as binodal curves, protein concentrations and protein distributions between the two phases. the major bottleneck of hts processes, namely the rapid analysis of impure systems, is tackled by using automated elisa tools. depending on the intended use, the high number of measured partition coefficients and yields can be used for modelling or rapid process design. today, most optimisations of chromatography separations are based on experimental work and rule of thumb. the pat initiative has opened up for a model-based approach for downstream processing of pharmaceutical substances. this work uses a nonlinear chromatography model to optimize an ion exchange separation step. the general rate model with langmuir mpm kinetics described the behaviour of the components in the column. the optimal operating points using both productivity and yield as objective functions were found. the optimizations were run with both igg and bsa as target proteins respectively to compare their optimal operating points. the requirement on the optimal operating point was a purity of at least %. this requirement was added to the optimization problem as a nonlinear inequality constraint. flow rate, loading volume, start salt concentration in elution, elution gradient and cut points were used as decision variables in the optimization. the more retained component, bsa, was much easier to separate from igg with a gradient elution than igg from bsa while still retaining a high productivity and yield. the higher load volume at the optimal operating point, with bsa as target protein, causes a displacement of igg and thereby improving the separation. a high productivity at the yield optimum was still possible with bsa as target protein. both a lower productivity and yield was obtained with igg as target protein. optimisation and robustness analysis of a hydrophobic interaction chromatography step niklas jakobsson, marcus degerman, bernt nilsson department of chemical engineering, lund university, p.o. box , se- lund, sweden process development, optimisation and robustness analysis for chromatography separations are often entirely based on experimental work and generic knowledge. the present study proposes a method of gaining process knowledge and assisting in the robustness analysis and optimisation of a hydrophobic interaction chromatography step using a model-based approach. factorial experimental design is common practice in industry today for robustness analysis. the method presented in this study can be used to find the critical parameter variations and serve as a basis for reducing the experimental work. in addition, the calibrated model obtained with this approach is used to find the optimal operating conditions for the chromatography column. the methodology consists of threes consecutive steps. firstly, screening experiments are performed using a factorial design. secondly a kinetic-dispersive model is calibrated using gradient elution and column load experiments. finally the model is used to find optimal operating conditions and a robustness analysis is conducted at the optimal point. the process studied in this work is the separation of polyclonal igg from bsa using hydrophobic interaction chromatography. department of biochemistry and microbiology, ict prague, technicka , prague cz- , czech republic the display of novel metal binding sites on the surface of the biosorbent represents potent tool to increase its binding capacity and improve selectivity. in this study, the . kda transcriptional regulator merr of mercury-inducible mer operon of tn exhibiting high affinity and selectivity towards hg + , was displayed on the surface of s. cerevisiae. to achieve this, merr was genetically fused with gene encoding c-terminal domain of ␣-agglutinin which resulted in covalent attachment of the of the fusion protein on the cell wall glucan via glycosylphosphatidylinositol anchor. to evaluate the performance of such modified whole-cell biosorbent with specific regard to hg + , we constructed a new biosensor e. coli strain, which utilizes kanamycine resistance gene as a reporter under the control of mer promoter. it allowed determination of hg + in a range of - nm by simply monitoring the growth in the hg + /kanamycine-containing media. the effect of genetic engineering of s. cerevisiae surface by merr became significantly pronounced in biosorption experiments with solutions containing m hg + when modified cells accumulated . -fold more hg + than the control strain expressing mere anchoring domain. sensitivity analysis of amino acids in simulated moving bed chromatography ju weon lee, chong ho lee, yoon mo koo center for advanced bioseparation technology, inha university, inchon - , korea. e-mail: ymkoo@inha.ac.kr (y.m. koo) the difficulty of simulated moving bed (smb) design is that the optimization of the operation conditions relies on the determination of accurate adsorption isotherms. most smb chromatograph is carried out under nonlinear conditions, and the nonlinear behavior should be considered properly in the equilibrium isotherms. the other difficulty is the smb operation which has the characteristics of continuous process, all flow rates and switching time of valves should be maintained during the operation of smb. if the disturbances of operating conditions and isotherm parameters are occurred, it affects the zone flow rates and the migration velocity of the solutes, and these effects change the internal profiles of the solutes. therefore, it is the reason of decreasing the purity and the yield of products the objective of this work is to consider the sensitivity of isotherm parameters and operating parameters in smb chromatography process. two amino acids, phenylalanine and tryptophan, separation by smb process is selected as control system. application of ph and po probes during bacillus caldolyticus fermentation: an additional approach in improving a feeding strategy johannes bader , boris neumann , karima schwab , milan popovic , rakesh bajpai : studiengang biotechnologie, fachbereich v, tfh-berlin, seestr., berlin, germany proteome factory ag, dorotheenstr. , berlin, germany; department of chemical engineering, university of missouri-columbia, w ebe, columbia, mo, usa. e-mail: popovic@tfh-berlin.de (m. popovic) bacillus caldolyticus,a thermophilic microorganism, is a good producer of thermostable liquefying ␣-amylase. during optimisation of initial and feeding media for fed-batch fermentation a two component feeding containing starch and casitone was found advantageous. to approach the optimal feeding rate the method published by akesson et al., was extended to two component feeding. the key idea, discussed in this presentation, was using the po and ph probing signals to determine if the feeding of one or the other component should be increased or decreased. each of the probes offers information of different areas of feeding condition. to prevent excessive feeding of starch the ph probe is preferable. in case of excessive casitone feeding the po probe responds in very authentic way enabling together with the ph signal reliable and reproducible evaluation of feeding strategy. however a congruent response of po and ph probes means the approaching of the optimum feeding rate for both components. akesson, m., hagander, p., axelsson, j.p., . probing control of fed-batch cultivations: analysis and tuning. contr. eng. pract. , - . antibody immobilization by using the plasma polymerized acrylic acid r. jafari, m.tatoulian, f. arefi-khonsari laboratoire de génie des procédés plasmas et traitement de surface, enscp, upmc, rue pierre et marie curie, paris, france the objective of this work is therefore to produce a surface containing a high density of cooh functions on the polymer beads (ps) for the covalent immobilization of antibodies. we have investigated the plasma polymerization of acrylic acid in a fluidized bed reactor the polystyrene (ps) beads. for such application, there is a strong need to obtain stable plasma polymerized acrylic acid (ppaa) coating, resistant to washing with water. different physico-chemical analyses have been used (water contact angle measurements (wca), xps and sem analysis) to characterize the ppaa coating deposited on ps beads under different experimental conditions. the xps results showed that the pretreatment of surface of the beads before deposition of acrylic acid plays an important role on the stability of ppaa layer. the instability of the coating is partially due the fact that under certain conditions the coatings are soluble in water and secondly due to the bad adhesion of the polymer beads which are hydrophobic to the growing ppaa coatings. xps as well as tof-sims gives evidence of the immobilization of the antibody. xps results as well as static sims allows to detect nitrogen on the surface of the treated beads which proves the presence of the immobilized antibodies. under optimum condition the ppaa coatings provides the possibility to show a nitrogen uptake which varies between . and % of the apparent stoicheiometry of the surface. holst department of medical physiology, the panum institute, university of copenhagen, dk- copenhagen, denmark glp- (glucagon-like peptide- ), a peptide of amino acids secreted by endocrine cells in the gut in response to meal ingestion, was discovered during a systematic search for gut factors capable of enhancing insulin secretion. it turned out to be the most efficacious insulin releaser known, and unlike other factors, was shown to retain its insulinotropic activity also in patients with type diabetes. subsequent research has documented that the peptide not only releases insulin from the beta cells, but also enhance all steps of insulin biosynthesis, up-regulates beta cell gene transcription, and has trophic effects on the beta cells. the latter includes both proliferation of existing cells, neogenesis from ductal precursor cells, and inhibition of apoptosis. the peptide also inhibits glucagon secretion, reduces gastric emptying and reduces appetite and food intake. because of these actions, glp- administered to patients with type diabetes dramatically lowers blood glucose as well as glycated hemoglobin levels, and reduces body weight. however, natural glp- is extremely rapidly metabolized in the body, and the problem has been how to convert the unstable peptide into a clinically useful agent. the two main problems are its susceptibility to enzymatic degradation by ubiquitous dipeptidylpeptide peptidase iv (dpp-iv) and its rapid renal elimination. a related peptide, isolated from the saliva of a lizard, exendin- , was found to be a full agonist of the glp- receptor, to be resistant to dpp-iv and to be cleared more slowly by the kidneys. this peptide was highly effective in clinical studies and has now ( / ) been approved for diabetes treatment by the fda. other approaches include acylation of glp- whereby it attaches to albumin in the body and acquires resistance to dpp-iv as well as a slow renal elimination. also this analogue (liraglutide) has favourable clinical effects. fusion proteins of glp- and larger, slowly eliminated proteins in the body are currently being evaluated. small molecule, orally available inhibitors of dpp-iv have been demonstrated to protect endogenous glp- from degradation and to be efficacious in both experimental and clinical diabetes, and numerous inhibitors are currently in clinical development. the incretin hormones are released from gut endocrine cells upon meal ingestion. they enhance glucose-induced insulin secretion and nay be responsible for up to % of postprandial insulin secretion. the incretin hormones are glucagon-like peptide- (glp- ) and glucosedependent insulinotropic polypeptide (gip). in patients with type diabetes ( dm) the incretin effect is severely reduced or absent. in dm patients the secretion of gip is normal, but its effect on insulin secretion is almost completely lost. glp- secretion, on the other hand, may be impaired, but its insulinotropic actions are preserved and it may restore insulin secretion to near normal levels. substitution therapy with glp- might therefore be possible. glp- is a product of the glucagon gene and its actions include: ( ) potentiation of glucose-induced insulin secretion; ( ) stimulation of the expression of ␤-cell genes essential for insulin secretion, including the insulin gene; ( ) stimulation of ␤-cell proliferation and neogenesis (by enhancing endocrine differentiation of duct cells) and inhibition of ␤-cell apoptosis; ( ) inhibition of glucagon secretion; ( ) inhibition of gastrointestinal secretion and motility, notably gastric emptying; and ( ) inhibition of appetite and food intake. these actions make glp- particularly attractive as a therapeutic agent for dm. thus, continuous subcutaneous administration of glp- for weeks resulted in a mmol/l reduction in mean plasma glucose and a reduction in hgba c of . %; a weight loss of kg; improved insulin sensitivity; improved ␤-cell function; and the treatment was associated with no significant side effects. unfortunately, glp- is rapidly destroyed in the body by the ubiquitous enzyme, dipeptidylpeptidase iv (dpp-iv). clinical strategies therefore include: ( ) the development of metabolically stable analogues of glp- viz. activators of the glp- receptor; and ( ) inhibition of dpp-iv. orally active dpp-iv inhibitors have proven successful in experimental diabetes and several companies are now trying to develop clinically suitable inhibitors. so far the clinical experience is limited, but recent clinical studies have provided proof of concept. metabolically stable analogues/activators include the structurally related lizard peptide, exendin- or analogues thereof, as well as glp- derived molecules that bind to albumin and thereby assume the pharmacokinetics of albumin. these molecules are effective in animal experimental models of type diabetes, and have been employed in clinical studies of up to weeks' duration. on the basis of these studies it can be concluded that a therapy of type diabetes mellitus based on stimulation of glp- receptors is likely to be effective and to become a clinical reality within the not too distant future( - ). recombinant activated coagulation factor vii (rfviia) was developed to treat bleedings in hemophilia patients, who have developed inhibitors against fviii or fix, and has been demonstrated to have an efficacy rate of - % in major surgery as well as in serious bleedings in such patients. to use rfviia as a hemostatic agent in severe hemophilia is a new concept of treatment, not being a substitution therapy, but using a pharmacological dose of exogeneous rfviia to compensate for the lack of fviii or fix. the administration of extra rfviia has been found not only to bind to tissue factor (tf), but also to the negatively charged phospholipids surface of thrombin activated platelets. hemostasis occurs on surfaces being initiated on the tf-expressing cells as a result of exposure of tf, not normally exposed to the circulating blood, following an injury to the vessel wall. tf is a true receptor protein with an intramembraneous part and an intracellular tail. its ligand is fvii/fviia. as soon as tf is being exposed to the blood, it forms complexes with fviia already present in the circulation. these complexes activates fx and provide the initial limited amount of thrombin molecules activating the co-factors, fviii and fv, as well as fxi and platelets. following the thrombin activation of platelets, negatively charged phospholipids are being exposed on the outer surface of the platelets. on this surface most coagulation proteins bind tightly, facilitating the conversion of fx into fxa and the full thrombin burst, necessary for the formation of a tight fibrin hemostatic plug resistant against premature lysis. in hemophilia patients the initiation of hemostasis is essentially normal, but, since they lack fviii or fix, they do not form the fviiia-fixa complex necessary for full thrombin generation on the activated platelet surface. as a consequence the fibrin plug formed in hemophilia is loose, fagile and easily dissolved resulting in continuous bleeding. pharmacological doses of rfviia have been demonstrated to mediate direct binding of rfviia to the negatively charged thrombin activated platelet surface, thereby generating thrombin formation in the absence of fviii/fix. through this mechanism hemostasis is generated in hemophilia patients independent of fviii/fix. furthermore, by generating more thrombin at an increased rate the formation of stable, tight fibrin hemostatic plugs are facilitated. such fibrin plugs are more resistant against premature lysis and help not only to initiate but also to maintain hemostasis. based on its capacity of enhancing thrombin generation locally on the activated platelet surface, rfviia has been used to ensure hemostasis also in other situations than hemophilia, such as platelet defects including thrombocytopenia. recently, rfviia was shown to be hemostatically effective in patients with profuse bleedings as a result of vast trauma and tissue damage. in these patients with a complex hemostasis pattern including a host of changes leading to an impaired hemostatic function, extra rfviia seems to help generate a burst of thrombin resulting in the formation of a stable hemostatic plug more resistant against the ongoing lysis. in patients with intracerebral haemorrhage, one single dose of rfviia recently was found to limit the expansion of the haemorrhage and thereby leading to improved functional outcome. institute for medical microbiology and immunology, panum . . , blegdamsvej , dk- copenhagen n, denmark. email: s.buus@immi.ku.dk complete genomes from several species including many pathogenic microorganisms are rapidly becoming available along with the corresponding "proteomes". even at the peptide level, the diversity of proteome is enormous and easily represents a unique imprint of the originating organism. it is perhaps not surprising that the immune system considers peptides as key targets. recent immunological advances have shown that mhc molecules act as peptide selectors for immune recognition. we have proposed to generate accurate predictions of peptide binding to mhc and used these to identify immunogenic epitopes directly from genomic data. we have developed an iterative data-driven immunobioinformatics approach where data is used to generate predictors, and predictors are used to select new and complementary data for the next iteration. we have demonstrated the superior performance of this approach compared to a random data selection approach. we have also developed an efficient approach to select the most informative mhc molecules to investigate. the resulting, immunobioinformatics resource represents an immediate and powerful application and interpretation of genomic data, and will enable a rational approach to immunotherapy in the future. allergen specific immunotherapy is a causal treatment for igemediated allergic diseases such as hay-fever, and it has relied traditionally on preparations derived from aqueous extracts of various natural allergenic source materials. the cloning and production of an increasing number of allergens through the use of dna technology has not only facilitated the characterisation and analysis of the allergenic proteins, but also provided the opportunity to use these recombinant proteins instead of natural allergen extracts for the diagnosis and therapy of allergic disease. detailed physicochemical, biochemical and immunological characterisation are essential for the comparison of natural and recombinant proteins, and also provide a basis for developing derivatives. chemically modified allergens with attenuated ige-reactivity are currently used for immunotherapy in order to enable high doses to be achieved with a minimized risk of inducing allergic side reactions. dna technology provides the opportunity to develop and produce hypoallergenic allergen variants using strategies including gene mutation. the design of such variants must ensure that t cell reactivity and immunogenic activity are retained in order to preserve therapeutic potential. the recombinant allergens and their derivatives have several advantages over natural allergen extracts. they are relatively easy to produce in consistent pharmaceutical quality; the problems of natural extract standardisation can be avoided completely; the relative concentrations of the individual allergens can be controlled to obtain optimal dosages; nonallergenic proteins are excluded; the possible risks of contamination are avoided. the first clinical trials with grass pollen allergens and birch pollen hypoallergenic variants have yielded very encouraging results. the use of recombinant polyclonal antibodies (pabs) may improve the treatment of disease caused by complex targets such as infectious agents, when compared to monoclonal antibody therapy. symphogen has developed a method for reproducible production of target-specific fully human pab compositions, so-called symphobodies. the antibody genes are first isolated from donors with an immune response against the target and antibodies are screened for specificity. subsequently, the pabs are expressed in mammalian cells using the sympress technology, which is based on site-specific integration. this procedure ensures that each of the expression constructs encoding the antibody genes are stably integrated at the same site in each of the host cells, thereby eliminating genomic position effects and differential growth and production rates. further, the sympress technology comprises the generation of a polyclonal working cell bank (pwcb) which is used as inoculation material for the manufacturing. these cells display sufficient genetic stability to enable a controlled gmp production of recombinant polyclonal antibodies. symphogen's first product, sym , is a recombinant human polyclonal rhesus d-specific symphobody preparation consisting of different anti-rhesus d antibodies. this product is intended to be used for treatment of idiopathic thrombocytopenia purpura and prevention of hemolytic disease in newborns. recombinant anti-rhesus d symphobodies were produced and shown to be biologically active against rhesus d. the expression technology provided a compositional reproducibility between batches which is sufficient for manufacturing of such a polyclonal product for clinical use. scaledup production for clinical trials is currently ongoing. stem cells play an important role in renewing tissues such as skin and cornea. they are responsible for the continuous generation of the differentiated epithelium. we have characterized stem cells of the skin and cornea in situ and their fate in vitro in human skin reconstructed by tissue engineering using keratin (k) . in the outer root sheath of the hair follicle, stem cells (label-retaining cells) present in the basal layer of the bulge area express k and present a loosely arranged keratin filament network and low levels of k protein in their cytoplasm. in addition, another stem cell population (also labelretaining) is present in the first suprabasal layers. these cells exhibit a very dense keratin network and express k . three-dimensional tissue constructs (dermis and epidermis) obtained by the self-assembly approach of tissue engineering allow the preservation of k positive stem cells in the basal layer of the epithelium. in the eye, the stem cells are located in the limbal part but not in central cornea and they express k . the epithelium of reconstructed cornea is thinner compared to reconstructed skin and more transparent. the characterization of stem cells in reconstructed tissue is essential to evaluate the long-term survival of these tissues in vitro but also after grafting. these human reconstructed tissues are developed for fundamental (physiological, toxicological studies) and clinical applications such as transplantation for the permanent replacement of damaged organs. lg is holder of the canadian research chair (cihr) on stem cells and tissue engineering. alessandra gliozzi physical department, university of genoa, genoa, italy hollow nanometer-sized capsules can be prepared by means of different techniques. first "nanocapsules" were liposomes, however they are too unstable for many medical or pharmaceutical applications. in contrast, recently developed polyelectrolyte capsules prepared by means of the layer-by-layer technique are much more stable and seem to be a very promising way for coating living cells or tissues in order to prevent or reduce their immune rejection after implantation. several observations on single living cells encapsulated by the alternative adsorption of oppositely charged polyelectrolytes will be presented. the most relevant result is that cell preserve their metabolic activity, are still capable of dividing and performing specific functions. moreover, a technique to immobilize in defined arrays coated cells expressing green fluorescent protein by using a microcontact printing of polyelectrolytes will be presented. finally, tests performed to study the induction of fibrosis and vascularization by nanocapsules implanted in rat kidney and liver will be presented. over the past decade we have developed methods to generate spontaneously and synchronously beating tissue equivalents from neonatal rat heart cells in the culture dish. these tissue equivalents display the key morphological and functional features of intact myocardium and have been termed engineered heart tissue (eht). to generate ehts, heart cells are mixed with freshly neutralized, liquid collagen i, matrigel and growth supplements and grown in a circular casting mold around a central cylinder, which subjects the cells to a continuous mechanical load. this process is enforced by cyclic mechanical stretch. we use eht mainly for two purposes, as a test bed for the effects of pharmacological or genetic manipulations and for cardiac repair. as a cell culture model, ehts compare with standard d monolayer cultures of neonatal rat cardiac myocytes and freshly isolated adult cardiac myocytes. advantages of ehts are their functional similarities with intact heart muscles, the ability to easily measure force of contraction under mechanical load, the pos-sibility to transfect cardiac myocytes inside ehts with adenovirus at high efficiency and the reproducibility in large series. a disadvantage is that contractile function as measured at the end of the culture period also integrates influences on tissue development, cell-cellconnections, extracellular matrix production and on non-myocytes. at present we are working on downscaling the eht method to a well format for screening purposes. to use ehts for cardiac repair we created multi-looped ehts from five circular ehts large enough to cover the infarct scar days after coronary artery ligation in rats. ehts survived and formed a layer of muscle tissue on top of the infarct scar. ehts restored undelayed anterograde impulse propagation over the scar, prevented further ventricular dilatation, normalized enddiastolic pressure and relaxation, and partly restored contraction of the scar. thus, the study provides evidence that implanting ehts onto infarcted hearts can improve cardiac contractile function after myocardial infarction. the goal of tissue engineering is the development of skin, bones and even organs to restore, maintain and improve tissue function within the body. the current paper focuses on the investigation of invitro growth of osteoblast cells in different types of scaffolds. three of the scaffolds were made of pcl (polycaprolactone) % glycerol and % hca(hydroxylapetite). two of the scaffolds were made by compression molding, and one was made by fused deposition modeling utilizing the stratasys. the fourth cerabio was a commercially available product totally ceramic. the pores in compression molding were obtained by putting in % volume of sugar either and m which was later removed by leaching. the fused deposition scaffold was made by placing the filaments in a predetermined arrangement. the stratasys system was a computer designed model. the scaffolds were seeded with hfob . human fetal osteoblast cell line with vigorous shaking overnight and incubating at • c and % co . observation of the seeded scaffolds was made after days and days of incubation. the seeded cells were stained with bcip/nbp at • c overnight. the cell proliferation in the and m scaffolds appeared approximately the same with a possible advantage of m. the cerabio sample demonstrated the greatest proliferation among the four scaffolds studied and the stratasys sample exhibited a different type of cell adhesion with the cells were clustered in the interstices of the structure. denise freimark, ruth freitag, valérie jérôme chair of process biotechnology, university of bayreuth, d- bayreuth, germany. e-mail: denise.freimark@uni-bayreuth. de (d. freimark) tissue engineering is emerging as an alternative to bone grafts for the regeneration of defects that do not heal spontaneously. ultimately, the development of an optimum carrier and the identification of ideal inductive factors and cells may enable tissue engineering to provide an improvement over bone grafts in the future. bone formation and repair require a complex cascade involving growth factors, cytokines and angiogenesis. at present the complexity of the molecular mechanisms that control gene expression in bone forming cells in embryo as well as in adult is not fully understood. several factors like bone morphogenetic proteins (bmps), transforming growth factor beta (tgf-␤), vascular endothelial growth factor (vegf) and insuline-like growth factor (igf) have been identified and their ability to stimulate bone formation in vitro and in vivo has been investigated. while much is known about these factors per se, less is known about genetic regulation of artificially stimulated osteogenesis. interestingly, some in vitro investigations showed that only optimal growth factors concentrations lead to effective bone formation whereas higher concentrations had deleterious effects which suggest some variation in the activated regulation pathways. therefore, one of our goals is to analyze kinetics, dose-dependence and synergistic effects of growth factors and cytokines on regulation pathways of bone formation. moreover, the optimal vascularization of the scaffold is a major hurdle in the development of engineered bone. it is well known that: (i) vascular invasion precedes bone growth and (ii) osteogenesis takes place in the vicinity of newly formed vessels. thus, inadequate bone vascularization is associated with decreased bone formation. further analysis of the intercommunication between endothelial cells and osteoprogenitors in co-culture systems could provide key information that could be thereafter used to solve this problem. we propose to add some new knowledge to this complicated puzzle. a first step in our investigation is the production of some of the growth factors mentioned above in recombinant form. these factors are expressed in a novel vector (ptriex tm ; novagen) which allows recombinant protein production in prokaryotic or in eukaryotic systems with a single plasmid. afterwards, we analyze potential synergistic effects of these factors as well as kinetic and dose-depend parameters on the genetic regulation of downstream pathways in osteoblasts culture. in parallel, we develop an in vitro system allowing us to investigate the intercommunication between endothelial cells and osteoprogenitors. there has been significant interest in the therapeutic and scientific potential of human embryonic stem (es) cells since they were first isolated in . if human es cells could be differentiated into suitable cell types, stem cells might be used in cell replacement therapies for degenerative diseases such as type i diabetes and parkinson's disease, or to repopulate the heart following myocardial damage. however, there is a significant shortage of high quality human es cell lines and few research groups have experience in the propagation and manipulation of these cells. we are addressing this important issue using the combined expertise of the stem cell biology laboratory and the assisted conception unit at king's college, london. with local ethical approval and under licence from the uk human fertilisation and embryology authority, we have been establishing high quality human es cell lines from a novel source of human embryos. to date, we have derived three human es cell lines and are now focused on the generation of therapeutically important cell populations, including cells that may have clinical application in degenerative and traumatic injury to the brain and spinal cord, heart, retina and other target organs. wallenberg neuroscience center, department of physiological sciences, lund university, bmc a , s- lund, sweden cell replacement therapy for parkinson's disease is based on the idea that implanted dopamine neurons may be able to substitute for the lost nigrostriatal neurons. in rodent and primate models of parkinson's disease it has been shown that transplanted dopamine neuroblasts can re-establish a functional innervation and restore dopaminergic neurotransmission in the area of the striatum reached by the outgrowing axons; that the grafted neurons are spontaneously active and release dopamine in an impulse-dependent manner, at both synaptic and non-synaptic sites; and that they can reverse or ameliorate some of the parkinson-like motor impairments induced by damage to the nigrostriatal system. clinical trials in patients with advanced parkinson's disease have shown that dopamine neuroblasts obtained from fetal human mesencephalic tissue can survive and function also in the brains of pd patients, restore striatal dopamine release, and ameliorate impairments in motor behavior. the principal limitation of this approach is the problems associated with the use of tissue derived from aborted human fetuses, and the large numbers of donors needed to obtain good therapeutic effects. until now, transplantation of dopamine neurons has focused primarily on differentiated neuroblasts and young postmitotic neurons, at the stage of neuronal development that is optimal for survival and growth of the grafted cells. however, progenitors taken at earlier stages of development might prove more effective. efforts are now made to expand multipotent neural stem-or progenitor cells in vitro, and control their phenotypic differentiation into a dopaminergic neuronal fate. initial results suggest that in vitro expanded cells can survive and function after transplantation to the striatum in the rat pd model, but the overall yield of surviving dopamine neurons has been very low. with further development, expanded progenitors or dopamine neuron precursors, possibly in combination with cell engineering techniques, may offer new sources of cells for replacement therapy in pd. stem cell therapy has been very much in vogue for several years now. like gene therapy before it, it has raised unrealistic hopes of cures being available imminently. unlike gene therapy, it can cite proof of principle in the well established practice of bone marrow transplantation which is actually a good example of stem cell therapy. however, most of the uses that are now touted as targets for stem cell therapy, envisage the conversion of the stem cells into lineage restricted progenitor cells or more commonly, the final differentiated cell type. such conversions are extremely difficult to initiate and control. the procedures involve the manipulation, differentiation, and expansion of cell cultures in the laboratory, with unknown long term effects on the genetics and physiology of the cells. these issues are compounded when one considers as source material, human embryonic stem cells, where not only the final cell product requires significant scrutiny, but also there are safety issues surrounding the persistence of undifferentiated cells. on top of all these challenges are commercial (for stem cell companies), clinical, and regulatory pressures which will impact heavily on the pace of progress. nonetheless, despite all these hurdles, various academic groups and companies are making significant progress and examples of such developments in diabetes and cardiovascular repair will be given stem cells have the unique ability to perpetuate themselves while continually replenishing tissues throughout the life of an organism. the era of cellular and tissue regeneration for the treatment of disease and the effects of aging has indeed begun. it has been known that mechanical factors play an important role in the regulation of cell physiology. it is therefore reasonable to believe that mechanical factors also play a significant role in the metabolic activity and differentiation of mscs. in this study, we investigated the viscoelasticity of individual bone marrow-derived adult human mesenchymal stem cells (hmscs), and the role of specific cytoskeletal component -f-actin microfilaments on the mechanical properties of individual hmscs. the mechanical properties of hmscs were determined using the micropipette aspiration technique coupled with a viscoelastic solid model of the cell. for the hmscs under control conditions the instantaneous young"s modulus e was found to be ± (pa), the equilibrium young"s modulus e ∞ ± (pa), and the apparent viscosity ± (pas). after exposed to m of chemical agent-cytochalasin d that disrupt the f-actin microfilaments, the young's moduli of hmscs decreased by up to % and the apparent viscosity increased by %. these findings suggest that microfilaments are crucial in providing the viscoelastic properties of the hmscs, and changes in the structure and properties of them may influence the mechanical properties of hmscs significantly. pharmacologic transcription control of desired transgenes is essential for gene-function analysis, drug discovery, biopharmaceutical manufacturing, design of complex artificial regulatory networks, precise and timely reprogramming of key cell characteristics for gene therapy and engineering of preferred cell phenotypes for tissue engineering. capitalizing on our recent advances in the design of small molecule-responsive transcription control modalities we have used conditional molecular interventions for (i) improvement of specific productivity in biopharmaceutical manufacturing, (ii) transdifferentiation of therapeutically relevant cell phenotypes and (iii) design of artificial microtissues. we will also report on a completely new dimension of transgene control as well as engineering of hysteretic and epigenetic gene networks in mammalian cells. chronic diseases are a growing burden for the individual and society alike. one key factor driving this increase is an ageing population. currently, there are million persons aged years or over and this number is predicted to triple by the middle of the st century. effective prevention of irreversible damage to major organ systems requires early diagnosis and treatment yielding significant quality of life to the individual and sparing valuable health care resources. even when safe and effective medicines are available, a remaining problem to successful therapy are issues of patient compliance. historically, vaccines have been one of the major advances towards the longevity we enjoy today, with compliance rates close to %. hence, vaccines for early treatment of chronic diseases are ideally positioned for long-term therapy, and will take away the burden of self-medication associated with orally active drugs. here we will discuss a new generation of therapeutic vaccines based on virus-like particles (vlps). by displaying target molecules in a highly repetitive manner on vlps, it is possible to break b cell unresponsiveness in experimental animals as well as in humans. using such vaccines in animals, chronic diseases such as hypertension, alzheimer's disease, obesity and rheumatoid arthritis could be treated. furthermore, vaccination against nicotine resulted in high nictotine-specific antibody titers in humans, greatly facilitating smoking cessation in immunized individuals. interdependence of the impact of methanol and oxygen supply on protein production with recombinant pichia pastoris n.k. khatri, f. hoffmann martin-luther-university halle-wittenberg, institute for biotechnology, halle d- , germany. e-mail: f.hoffmann@biochemtech.uni-halle.de (f. hoofmann) the methylotrophic yeast pichia pastoris is a potent expression system for secretion of recombinant proteins. methanol as inductor of the foreign gene expression is also a substrate with high oxygen demand, which can lead to sudden oxygen depletion upon induction. thus, supply rates of methanol and oxygen are major process parameter during protein production with recombinant pichia pastoris. limiting dosage of methanol allowed maintenance of oxygen sufficient conditions during production of a single chain antibody fragment, but the product was degraded from the c-terminal end. in contrast, full-length product accumulated with controlled methanol concentrations despite oxygen limitation. the volumetric methanol uptake rate are limited by the oxygen transfer capacity of the reactor. higher methanol concentrations decreased the biomass yield and thereby increased the specific methanol uptake rate. this enabled prolonged production and yielded fivefold higher product concentrations. at the same time, the accumulation of small molecular weight contaminants was reduced. dosage of pure oxygen accelerated methanol uptake, grow and production. switching to dostat mode upon oxygen depletion led to an arrest of product accumulation, in contrast to persistent methanol feeding. the productivity was tenfold higher than without oxygen. combined with high methanol concentrations, however, fast methanol uptake led to toxication of the cells and early stop of production. flow cytometry revealed that perturbation of oxygen metabolism was followed by partial lysis of the culture. recombinant production of therapeutic proteins poses severe challenges due to their complexity (cystines, subunits, size). formation of the correct disulphide bridges is a prerequisite for activity but difficult to achieve in a prokaryotic host. there proteins mainly fold post-translationally as opposed to eukaryotic co-translational folding. thus the recombinant products are often not soluble and/or active. that is why different production parameters (e.g. host, induction conditions, temperature, compartment, proteinaceous fusion partners, co-expression of chaperones, foldases) are applied in order to gain functional recombinant proteins. the impact of all these strategies cannot be predicted and every problem of the production (expression, solubility, activity) might need to be solved for every target protein separately. nevertheless, much effort has been put into this for almost decades, because they are important targets for the pharmaceutical industry. human growth factors influencing cellular proliferation and/or differentiation are one example. this case study gives an overview of strategies tested within the development of two processes leading to an optimised yield of active protein. murine wnt- (wnt family) possesses conserved cysteines (most likely all involved in disulphide bridge formation and one in posttranslational modification) and could only be successfully produced in e. coli (fahnert, ) recently despite various attempts for many years. the other target protein is human collagen prolyl- -hydroxylase being a heterotetramer consisting of two ␣-subunits and ␤-subunits each. the ␣-subunit strongly aggregates if produced separately whereas the ␤-subunit is pdi and is suggested to have a chaperone function. therefore a sequential induction strategy was proposed for this protein . the moss physcomitrella patens has been recently recognized as an ideal producer of recombinant proteins with respect to glycosylation. due to the elaborated post-translational capabilities of moss cells, the glycosylation patterns have been manipulated to obtain similar proteins to those found in animal cells. the protein expression using moss in suspension offers important advantages in comparison to other systems e.g. cho cells. the recombinant proteins can be targeted into the mineral medium, simplifying the down stream processing. moreover, there are neither known moss viruses nor plant viruses that are pathogenic for humans. the moss are cultivated axenically in a filamentous stage, the so called protonema. a pilot l tubular photoreactor is used to characterize the response of p. patens to variations on the culture conditions. the phototrophic culture in bioreactors is systematically investigated, where light quantity and quality, stress, concentration of phytohormones, and moss morphology influence the differentiation, growth, and protein expression. a tight control of the moss morphology in suspension, quantified by image analysis, has shown to be advantageous in order to delay the cell differentiation and maintain the carbon dioxide uptake in long bioreactor runs. the introduced perfused culture system by means of cross flow filtration allowed for a continuous product separation and concentration, and feed back of the productive cells. the characterization of this highly controlled culture system is presented and the potential of p. patens as an alternative tool for molecular farming is discussed. (rnai) is an evolutionarily conserved, endogenous mechanism for sequence-specific gene silencing that uses small double-stranded rnas (called short interfering rnas or sirnas) to direct cleavage or prevent translation of homologous mrnas. harnessing rnai for therapy presents an opportunity for potentially treating a wide variety of diseases. the main obstacle is delivering sirnas into the cytosol of target cells in vivo. although we were able to protect mice from autoimmune hepatitis by hydrodynamic tail vein injection of sirnas targeting fas, this delivery method is unlikely to be adaptable for human use. alternate strategies to deliver sirnas in vivo as small molecule drugs using currently available, clinically acceptable injection methods that have shown promise in mouse models will be discussed. these include local delivery to mucosal surfaces and delivery into specific cells via cell surface receptors using an antibody fragment fused to protamine. these sirna complexes silence gene expression in vivo only in cells bearing the targeted receptor. experiments showing efficient, effective and cell-specific delivery in a mouse tumor model will be discussed. in addition, encouraging preliminary data using rnai for a microbicide to prevent sexually transmitted infection will be presented. rna interference (rnai) holds significant progress as a therapeutic approach to siolence disease-causing genes, particularly those that encode "non-druggable" targets. the key hurdle for rnai therapeutics is in vivo delivery. a critical requirement for achieving systemic rnai in vivo is the introduction of "drug-like" properties, such as stability, cellular delivery and tissue biodistribution, into synthetic sirnas. our progress in achieving in vivo silencing of endogenous genes with chemically modified sirnas will be discussed. hiv- replication in human t cells can be inhibited by stable expression of a short hairpin rna targeting the viral nef gene (shrna-nef). however, hiv- escape variants emerge after prolonged culturing, and all but one escape mutant acquire a mutation in the shrna-nef target sequence. we observed single and multiple nucleotide substitutions, but also partial or complete deletion of the target sequence. these results demonstrate the sequencespecificity of this antiviral approach. we observed an inverse correlation between the level of resistance and the stability of the shrna/target-rna duplex for most of the escape mutants. however, two escape variants did not follow this pattern, including an escape mutant with a single point mutation at position − upstream of the target sequence. these mutants provide a much higher level of resistance than expected based on duplex stability, which is obviously not affected in the − mutant. we demonstrate that these mutants adopt an alternative rna secondary structure that occludes the target sequence. this results in reduced shrna-nef binding and provides a novel mechanism for rnai-resistance. to avoid viral escape, one should ideally target hiv- with multiple effective shrnas against conserved genome sequences. we performed a large scale screening to identify such targets, and we have identified at least nine genome segments that can be targeted effectively with shrnas. these potent antivirals are currently being assembled in a lentiviral vector for gene therapy applications in hiv-infected individuals. furthermore, we will describe approaches to forecast viral escape routes and to effectively block such evolutionary paths with additional rnai measures. in this study we analyzed the effect of antibodies against electronegative ldl on the development of atherosclerotic lesions in low-density receptor-deficient (ldlr −/− ) mice. two groups of (ldlr −/− ) mice (eight females) fed . % cholesterol-enriched chow were used. the first group received a monoclonal antibody against electronegative ldl ( g) and the second one received pbs (controls). additionally, other two groups (eight males) of (ldlr −/− ) mice were treated with a polyclonal antibody against electronegative ldl ( g) or pbs (controls). antibodies were administered by intravenous route one week before starting the hypercholesterolemic diet and then every week over an experimental time of days. afterwards, quantification of atherosclerotic plaque area of heart and aortic arch was done by analysis of the slices stained with oil red/hematoxolin/light green with the image propus software. the passive immunization with either monoclonal or polyclonal antibodies against electronegative ldl significantly reduced the atherosclerotic plaque areas in atherosclerosis-prone ldlr −/− mice. in conclusion, antibodies against electronegative ldl administered by intravenous route may play a protective role in atherosclerosis. supported by fundação de amparoà pesquisa do estado de são paulo (fapesp, scholarships to d.m.g., l.s. and a.b. and grants to m.h.k. and d.s.p.a.). the enzyme asparaginase from the procaryote escherichia coli or erwinia crysanthemi is used for the treatment of lymphoblastic leukaemia. the drug causes immunological reactions in despite of the treatment efficiency. asparaginase may also be obtained from saccharomyces cerevisiae and this enzyme could provide an alternative to its bacterial counterparts. in this study, the periplasmic nitrogen regulated asparaginase ii from s. cerevisiae, that is coded by the asp gene, was cloned and expressed in the methylotrophic yeast pichia pastoris under the control of the aox gene promoter. the recombinant p. pastoris strain was cultured in shake flasks and in a l instrumented bioreactor. in both cases it was observed specific enzyme yields seven fold higher in comparison to that using a nitrogen derepressed strain of s. cerevisiae, reaching u/g dry cell mass. high cell density cultures carried out in the l bioreactor, in which it was attained g dry cell mass/l, resulted in a dramatic improvement in asparaginase fermentation. as such, it was measured enzyme yields of , u/l and productivities of u/l h. department of biochemistry and food chemistry, biotechnology, university of turku, tykistokatu , biocity th floor, turku, finland. e-mails: lorenzo.galluzzi@utu.fi; deadoc@libero.it; deadoc@aliceposta.it (l. galluzzi) the bacterial luciferase operon from the bacterium photorhabdus luminescens has been used, since its first description, for exceptionally different applications. these ranged from the environmental monitoring to the cell tagging, from the analysis of cellular metabolism to the high-throughput screening of novel compounds. the wild-type luxcdabe operon has been engineered in countless ways (for instance by changing the order of the constituent genes, by optimizing the codon usage and by coupling it to many promoters) and has been expressed in prokaryotic and eukaryotic organisms in order to meet precise research and commercial needs. upon the operon expression light is emitted as the side product of a chemical reaction catalyzed by the luciferase enzyme, an ␣␤ heterodimer encoded in luxa and luxb genes. the reaction involves the oxidation of a long-chain aliphatic aldehyde and reduced flavin mononucleotide (fmnh ) with the liberation of excess free energy in the form of a blue-green light at nm. the luxcde genes code for the polypeptides (transferase, synthetase, and reductase) forming the fatty acid reductase complex that catalyzes the conversion of fatty acids into the long-chain aldehyde required for the luminescent reaction. noteworthy is that for the production of the substrates for the luciferase both atp and nadph are required, while neither is involved in the actual light emitting reaction (wilson and hastings, ) . recently, the coupling of the luciferase operon to regulated promoters lead to the construction of genetically modified bacteria able to sense the presence of chemicals and to respond, in a dosespecific manner, with light emission. this approach has been applied to the detection of antibiotics in samples from the food industry as well as to the detection of heavy metal ions in environmental samples (kurittu et al., ; bechor et al., ) . in addition, it opened the possibility of screening wide libraries of new compounds looking for molecules with pre-determined features, able to induce the bioluminescent response by de-repressing the lux operon transcription when incubated with the appropriate bacterial sensor. the high throughput and low costs are the main advantages of this system, which shows as well a certain degree of specificity (galluzzi and karp, ; galluzzi et al., ) . nevertheless, since the in vivo bioluminescence relies upon a complex network of biochemical reactions, under certain circumstances the light emission is not a direct consequence of the lux operon transcriptional induction but it more likely originates at a posttranslational stage. as a matter of fact, one can suppose that a change in the light emission will be observed whenever the concentration of one or more substrates for the lux␣␤ reaction occurs. consequently, all the molecules sharing the ability to impair the delicate chemical equilibrium regarding the compounds involved in bioluminescence will be sensed by the bacteria as inducing compounds. this, in turn, will result in a loss of specificity of the assay. we investigated this aspect of the whole-cell assays based upon the bacterial luciferase operon for drug discovery by means of a reporter plasmid in which the luxabcde genes, rearranged and optimized for the after min (black downward arrow) of incubation at • c under vigorous shaking the following concentrations of trimethoprim were added to the growing cells: g/ml (triangles), g/ml (circles) and g/ml (rumbles). water was administered to control cultures (squares). light emission was quantified every min by means of the wallac victor multilabel counter (perkin-elmer, turku, finland) and normalized to the absorbance, measured at nm with the same device. multi- white-walled transparent-bottomed plates were used for the assays (nalge nunc, usa). between the measurements the plates were kept at + • c under vigorous shaking. filled symbols refers to the absorbance values (left side y-axis); open symbols to the normalized count per seconds (right side y-axis). expression in gram + organisms, are under the control of the qacr regulatory region from staphylococcus aureus . non-pathogenic s. aureus rn cells bearing the pqaclux plasmid (cultivated in lb broth supplemented with , % d-glucose and g/ml chloramphenicol) emit light upon the specific transcriptional induction with quaternary ammonium compounds, widely used as surface disinfectants and in many over-the-counter drugs . however, the incubation of the same cells with an inhibitor of the dihydrofolate reductase enzyme, trimethoprim (sigma-aldrich chemie, steinheim, germany), enhanced in a dosedependant manner the light emission observed upon the induction with the optimal concentration ( g/ml) of benzalkonium chloride (bc). the extent of this increase in luminescence varied from - % to more than %, according to the trimethoprim concentration and to the measurement time. interestingly, when the same plasmid is carried by escherichia coli xl cells, the lux operon is expressed constitutively (since the qacr regulatory region is not functional in xl cells) and at much higher levels than in induced rn cells. also in this experimental system the incubation with trimethoprim results in a dramatic increase of the luminescent signal from the cultures. the explanation for these observations can be found in the molecular mode of action of trimethoprim. the inhibition of dihydrofolate reductase, indeed, directly leads to the accumulation of its substrates, among which is nadph, deeply entangled in the biochemical network of reactions centred on the light emission from the lux operon. nadph provides the reducing power to restore the reduced flavin mononucleotide pool and it is as well involved in fig. . xl /pqaclux growing cells were incubated with the following concentration of trimethoprim: g/ml (triangles), g/ml (circles) and g/ml (rumbles). water was administered to control cultures (squares). light emission and absorbance measurements were performed as previously described for rn cells. filled symbols refers to the absorbance values (left side y-axis); open symbols to the normalized count per seconds (right side y-axis). the diverse level of growth observed for rn and xl cells can be accounted by the different antimicrobial activity exerted by trimethoprim towards gram− and gram+ cells and by the lower activity of the promoter which in pqaclux plasmid drives the transcription of the selection marker (chloramphenicol acetyl transferase). the production of the long-chain aldehyde, both substrates of the lux␣␤ heterodimer (wilson and hastings, ) . in conclusion, here we demonstrate that the use of light emission from the bacterial luciferase as a transcriptional reporter has to be very carefully controlled, since some molecules (here trimethoprim) could mimic to some extent a specific promoter activation by impairing the delicate intracellular biochemical equilibrium. on the reverse side of the coin, fig. . simplified scheme depicting the bacterial folate metabolic pathway. only part of the reactions and compounds are reported. trimethoprim inhibits the nadph-dependant reduction of dihydrofolic acid into tetrahydrofolic acid catalyzed by dihydrofolate reductase. this results in the accumulation of both substrates, which become available for other reactions, and in the depletion of tetrahydrofolate, the major c carrier in the synthesis of purines, thymidine, glycine, methionine, and pantothenate in bacteria. for antimicrobial purposes trimethoprim is often associated with sulfonamides, with which it displays a synergistic activity (since they act on the same pathway but at an earlier reaction) (scholar and pratt, ) . however, it has to be noted that the lux reporter system could be used in many different in vivo experimental setups, where the change in the intracellular concentration of nadph, atp or other metabolites would be sensibly detected. we have carried out the construction of five pichia pastoris strains harboring in their respective genome three different growth hormones cdna's ( and kda human growth hormones and bovine growth hormone), a shrimp (litopenaeus vannamei) trypsinogen cdna and a bacterial (bacillus subtilis) phytase gene. in all the cases, the same kind of expression vector and the same transformation technique were used. each dna was fused in frame to saccharomyces cerevisiae alpha-factor secretion signal to lead the secretion of the foreign protein into the culture medium. the induction of each recombinant strain, all with his + and mut + phenotype, was carried out in shake flaks using methanol buffered minimal medium (bmm) and growth rates on methanol determined. production level of secreted proteins was evaluated by protein analysis by sds-page. furthermore, the expression with three of the constructed strains was performed in a -l bioreactor and the level of secreted protein determined in each case. both human growth hormones (hghs) were secreted into the culture medium with a high degree of purity obtaining up to % of hghs of total proteins in crude fermentation medium and - mg/l of hghs. neither bovine growth hormone, shrimp trypsinogen or bacterial phytase were detected in methanol induced cultures of the respective strains, in spite of the same culture conditions were used for all p. pastoris strains. the growth rates on methanol were different between some strains. in fermentor cultures the hghs production were increased and shrimp trypsinogen was produced and secreted into the culture medium after improving the culture conditions. bovine growth hormone was detected only when the culture conditions were modified. the protein production level for each strain was affected by the proprieties of each recombinant protein produced. competitive advantages of the diagnostic method for invasive amoebiasis using preserved antigenic extracts without using enzymatic inhibitors m.s. flores , * , e. tamez we have patented a method to diagnose invasive amoebiasis using a novel assay that preserves antigenicity of extracts with high protease content without using enzymatic inhibitors (ic:mc). the available tests for serologic diagnosis of invasive amoebiasis like elisa and indirect haemaglutination (iha) do not have consistent results in endemic zones. here we show the advantages of the assay and the validation of this diagnostic test for invasive amoebiasis. we demonstrated the reduction of proteolytic activity of ic:mc compared with the proteolytic activity of crude extract and crude extract with enzymatic inhibitors using assays. we displayed the ic:mc sds-page pattern and the western blot (wb) pattern useful for diagnosis. to search the clinical utility of this test we examined the wb obtained with sera from patients with different liver diseases; patients had invasive amoebiasis and patients had other liver diseases. the results were compared with those of iha test. also we have tested the accuracy of wb using sera from people with multiple intestinal parasites, like giardia lamblia, hymenolepis nana, blastocystis hominis, entamoeba coli, etc. the sensibility of the wb using the preserved amoebic antigens was %, specificity was %, positive predictive value was %, negative predictive value was % and accuracy was %. the wb did not exhibit cross reactions with sera from persons with intestinal parasites. our test was better than the (iha) test commonly used in endemic zones. these results show the improvement of using the preserved amoebic antigens in diagnostic tests. also they prove the diagnostic accuracy of our new wb test. antibacterial and antifungal activity of heracleum sphondylium subsp. artvinense yasemin kaçar , sema tan , aysun ergene , perihan güler , semra mirici , ergin hamzaoglu , ahmet duran , sinem yildirim : mersin university, faculty of science and literature, department of biology, mersin, turkey; kırıkkale university, faculty of science and literature, department of biology, yahsihan-kırıkkale, turkey; akdeniz university, faculty of education, department of biology education, antalya, turkey; selcuk university, faculty of education, department of biology education, konya, turkey turkey is covered yearly with a huge number of plant species. about species are condenced on the region that between asia and europe. many plant species have been used in folkloric medicine to treat various ailments. heracleum l (apiaceae) is include over than species on the world. this variety is represent with species in turkey that seven species are endemic. heracleum sphondylium subsp. artvinense is endemic species for turkey. ethanolic and aquous extract of heracleum sphondylium subsp. artvinense were investigated for their antimicrobial activities against six bacterial species (e. feacalis, e. coli, s. aureus, p. aeruginosa, l. monocytogenes, shigella) and two yeast (c. albicans, c. krusei). both ethanolic and aqueous extract of heracleum sphondylium subsp. artvinense showed antimicrobial activity against the gram negative bacterium (shigella) and gave the best activity against c.albicans. to develop artificial vectors allowing nucleic acid to transfect into mammalian cells are crucial for extending gene therapy. synthetic vectors based on lipid molecules are particularly attractive because of their potential safety. however, the low encapsulation efficiency of nucleic acid is one of the problem to be solved. recent advances in dna-lipid complex have improved this drawback, and now some lipid molecules are used as transfection agents. in particular, cationic lipids interact with negatively-charged cell surfaces and nucleic acids. the former interaction results in delivery of the nucleic acids directly across the cell membrane. on the other hand, the latter interaction improves the efficiency of nucleic acids entrapment. in this study, we developed a preparation method of "nanovesicle" containing nucleic acids by using reverse micellar solubilization. since dna interacts spontaneously with cationic amphiphiles, complete extraction of the dna molecules into an organic phase using dimethyl distearyl ammonium bromide ( c ab), an oil-soluble cationic surfactant, is achieved. the re-encapsulation of the reverse micellar droplets solubilizing dna by water-soluble surfactants facilitates the formation of nanovesicles. a high salt concentration at the re-encapsulation step promotes the production of nanovesicles containing dna molecules. eventually, more than % of dna was encapsulated in this nanovesicles under the condition of m nacl and % (w/v) cetyltridecyl ammonium bromide (ctab). in addition, the nanovesicles prepared at • c was smaller than that prepared at room temperature. the resultant c ab/ctab nanovesicle was ca. nm. asymmetric and chemically-modifiable vesicle surface are effective to design gene delivery system. moreover, such a small dna (or rna) carrier has a potential for novel gene therapy application from skin. the key players in clinically important inflammatory diseases, endothelial cells and leukocytes, communicate through membranebound cell adhesion molecules (cam's). obvious strategies for therapeutic intervention include specific means to affect the expression of cam's on the cells involved. rna-interference (rnai) is a well known means to achieve specific gene-inhibition. for this study, two cam's were chosen, namely vascular cell adhesion molecule- (vcam- ) as a target for inhibition, and intercellular adhesion molecule- (icam- ) as a non-target reference. we designed short interfering rna (sirna) oligos for vcam- in order to selectively inhibit the expression of this cam in cultured human vascular endothelial cells (huvec). real-time rt-pcr showed an % down-regulation of vcam- mrna while the expression of icam- remained unaffected. neither cam was affected by non-specific sirna. in order to further substantiate the potential use of sirna for therapeutic purposes, we have set out to investigate two things: ( ) does vcam- -specific sirna affect the amount of vcam- on the surface of huvec? ( ) is adhesion between leukocytes and endothelial cells affected by vcam- -specific sirna? the manufacturing of plasmid dna (pdna) is crucial to obtain a consistent product for gene therapy applications. although flowsheets for pdna production are established on the basis of experience, simulation tools provide a valuable help for evaluating alternatives. a process designed to produce kg pdna/year is analysed with the superpro designer tool. the target pdna is amplified in escherichia coli. after harvest, alkaline lysis is used to disrupt cells and release pdna and impurities. precipitations with isopropanol and ammonium sulphate are performed to concentrate/pre-purify pdna prior to hydrophobic interaction chromatography. experimental data is used as input for simulation. inventory analysis identified water, yeast extract, tryptone, isopropanol and ammonium sulphate as the major raw materials. major raw material costs ( %) are related to fermentation components. economic analysis indicates a unit production cost of $ /g pdna. for a selling price of $ /g, the payback time was . years and the roi was . %. an environmental analysis highlighted the replacement of the isopropanol precipitation for a microfiltration step as a benefit which would: (i) reduce the cost of raw materials ( . %), (ii) reduce the environmental impact associated with isopropanol ( %) and (iii) reduce costs associated with the treatment/disposal of liquid waste ( . %). preparation of chitosan microspheres for controlled release of somatotropin s. simsek , j. introduction: proteins and peptides have received extensive interest for their therapeutic applications in clinical applications. in order to achieve high administration efficacy of proteins, polymeric particulate carriers have been developed as an effective way to control the drug release profile and to protect the protein molecules from degradation. somatotropin also known growth hormone is a protein hormone of about amino acids. growth hormone is of considerable interest as a drug used in both humans and animals. chitosan a natural linear biopolyaminosaccharide is obtained by alkaline deacetylation of chitin. properties such as biodegradability, low toxicity and good biocompatibility make it suitable for use in biomedical and pharmaceutical formulations. the aim of this study was to prepare chitosan microspheres containing somatotropin and to investigate these microsphere formulations in-vitro release properties. methods: somatotropin-chitosan microspheres were prepared as follows: chitosan was dissolved in acidic solution ( %) containing polysorbate . sodium sulphate solution ( % w/v) containing somatotropin was added into the chitosan solution and mixed rpm for a hour. resulting suspension was centrifuged , rpm min at • c. the formed microspheres were freeze-dried and sieved. in-vitro release studies were performed in ph . phosphate buffer and time interval samples were removed and analysed by bradford protein assay method. results: microspheres were obtained by using chitosan. protein encapsulation efficiency was between and %. average particle size of microspheres was between and m. during to in-vitro release studies burst effect was observed with chitosan microspheres. for decreasing the burst effect gluteraldehit, betacyclodextrin and poly ethylene oxide were added to formulations. conclusion: according to our results modified chitosan microspheres are promising vehicles for controlled release somatotropine delivery. cancer immunotherapy using hyperthermia with magnetic nanoparticles and dendritic cells k. tanaka, a. ito, t. kobayashi, t. kawamura, s. shimada, k. matsumoto, t. saida, h. honda department of biotechnology, school of engineering, nagoya university, nagoya, aichi - , japan. e-mail: h d@mbox.nagoyau.ac.jp (k. tanaka) our hyperthermia system utilizes magnetic nanoparticle covered with lipid layer including cationic lipid (magnetite cationic liposomes, mcls) as a heating mediator and necrotic cell death is induced by means of locally generating heat. in this process, heat shock proteins (hsps) are strongly induced and released. dendritic cells (dcs) are potent antigen-presenting cells (apcs) that play a pivotal role in regulating immune responses in cancer, which is in carrying antigens to apcs and in the maturation of dcs by acting as a danger signal. in the present study, we investigated the therapeutic effects of dc therapy combined with mcl-induced hyperthermia on b melanoma. in an in vitro study, when immature dcs were pulsed with b cells heated at • c for min, mhc class i/ii, costimulatory molecules cd /cd , and chemokine receptor ccr in the dcs were up-regulated, thus resulting in dc maturation. c bl/ mice bearing a b melanoma nodule were subjected to combination therapy using hyperthermia and dc immunotherapy. mice were divided into four groups: group i (control), group ii (hyperthermia), group iii (dc therapy), group iv (hyperthermia + dc therapy). complete regression of tumors was observed in % of mice in group iv, while no tumor regression was seen among mice in the other groups. increased ctl and nk cell activity was observed on in vitro cytotoxicity assay using splenocytes in the cured mice treated with combination therapy, and the cured mice rejected a second challenge of b melanoma cells. this study has important implications for the application of mcl-induced hyperthermia plus dc therapy in patients with advanced malignancies as a novel cancer therapy. fermentation of a marine bacterium for the production of cytotoxic compounds vicky webb , els maas , eiichi akaho , hiroto kambara , debbie hulston , anna kilimnik : marine biotechnology, national institute for water and atmospheric research ltd, kilbirnie, wellington new zealand; faculty of pharmaceutical sciences, kobe gakuin university, kobe - , japan marine bacteria are a potential source of novel compounds for the pharmaceutical industry. new zealand marine bacteria were isolated from a variety of sources and initially bacterial supernatants were screened using a cell based mtt cytotoxic assay. two bacteria were chosen for further fermentations in different media to optimise cytotoxic compound production. the media used were selected for their diverse ingredients. they were either carbon rich, nitrogen rich, starch rich or a basic seawater medium. the fermentations were extracted using ethyl acetate and methanol prior to assaying. the results showed that cytotoxic compound production was enhanced ten fold in the starch rich medium compared to the other media. the levels of cytoxicity appeared to be depended on the cell line used, with the epithelial lung carcinoma cell line a being more sensitive to the cytotoxic compounds than the human fibroblast cell line mrc- . isolation and identification of marine bacteria from deep-sea sediments els maas , cara brosnahan , vicky webb , helen neil , phil sutton : marine biotechnology, national institute for water and atmospheric research ltd., kilbirnie, wellington new zealand; oceanography, national institute for water and atmospheric research ltd., kilbirnie, wellington, new zealand polystyrene micro plate activated by utilizing a common co- gamma source with a crotonic acid. the well surface have been studied in term of optical quality, protein (h-igg) binding capacity and stability. a significantly enhanced total capacity and strength of binding to grafted surfaces was demonstrated as compared to passive adsorption of the proteins to untreated surfaces.the majority routine laboratory tests for the measurement of rheumatoid factor (rf) are semi quantitative and in order to achieve accurate, sensitive and specific rf assay, we are developed enzyme linked immuno sorbent assay (elisa) for human igm-rf kit. stable and reproducible binding of antigen (human-igg) to well of micro titer plate is a prerequisite for rf-elisa kit. the principle of the assay was that the antibodies in serum of patients with rheumatoid arthritis (ra), commonly rheumatoid factor (rf) are directed to the fc part of human igg. in most cases rf belong to the igm class, the well of micro titer plate are coated with antigen (h-igm), and antibodies (h-igg) binding to immobilized antigen is detected by adding enzyme conjugate (anti human-igm-hrp-enzyme) to the wells, substrate was used for color reaction.the performance characteristics of the assay was, the coefficient of variation (c.v) of intra and inter assay was . % and . % respectively, the linearity is ranging from to %, and the recovery for three different sera is ranging from to %. the data presented in this paper indicated that the activation of polystyrene micro titer plate by the gamma rays could be use for preparation of igm-rf kit and may be others immunoassay techniques. overcoming the nuclease barrier to gene expression during trafficking of plasmid dna vectors a.r. azzoni , a. tavares , g.a. monteiro , d.m.f. prazeres : centro de engenharia biológica e química (cebq), instituto superior técnico, - lisboa, portugal; instituto gulbenkian de ciência, rua da quinta grande , - oeiras, portugal. e-mail: azzoni@ist.utl.pt (a.r. azzoni) inefficient nuclear delivery of plasmid dna (pdna) vectors is thought to be a bottleneck to gene transfer in gene therapy and dna vaccination utilizing non-viral delivery systems. one of the main barriers found by pdna vectors during trafficking to the nucleus is degradation by a population of endo/exo-nucleases. this barrier may be partially circumvented by shielding the pdna from the nucleaserich cell environment with adjuvants or by using nuclease inhibitors. a different approach that has been studied at the cebq is the generation of pdna vectors that are more resistant to nuclease action a priori. in this work, the nuclease barriers to gene expression are being studied aiming at the generation of pdna with improved resistance to nucleases and thus higher transfection efficiency. by engineering the plasmid labile sequences, new plasmid vectors with an improved resistance to physical-chemical and biological degradation are being generated. this was indicated by an extended half-life of the supercoiled isoforms during storage and when the plasmids were exposed to nucleases present at eukaryotic cell lysates and mice plasma. the intracellular trafficking of the new plasmid vectors through the cytosol of mammalian cells was then assessed by fluorescence in situ hybridisation (fish) and the expression of the reporter protein (egfp) was detected by fluorescence microscopy. identification and evaluation of antibacterial phytochemicals of fishbone fern (nephrolepis cordifolia) rikhia chakraborty , , promod kumar verma : department of cancer biology, lerner research institute, euclid avenue cleveland, oh , usa, department of biotechnology, guru nanak dev university, amritsar, punjab , india. e-mail: riar @rediffmail.com (r. chakraborty) in this study, different aqueous and organic extracts from the fern, nephrolepis cordifolia, were used for screening tests for antibacterial effects. protein and lipid extracts were first tested for antibacterial activity. subsequently, crude extracts of leaves, roots, and stems were prepared in methanol, ethanol, chloroform, hexane, petroleum ether, diethyl ether, and water using optimized standard protocols. each fraction was tested for anti-microbial effect through agar well-diffusion assay, and paper disc method. the antibacterial spectrum against which the fern is active was thus determined. dosagedetermination for optimum activity was also determined for each of the extracts. bacillus and staphylococcus were used as the indicator test-organisms. the results were very encouraging; being effective even at the th day, thus showing that the antibacterial properties were not due to any changes in external factors and physiological effects. ethanol, methanol and chloroform extracts from the subaerial portions had strong anti-microbial properties. agar-well diffusion assay and paper-disc diffusion assay done for different solvent fractions were giving comparable results. . mg was adequate for maximum effect against b. circulans, s. aureus, s. epididermis, and streptococcus sp. . mg was adequate as effective dosage for kleb-siella pneumoniae. mg was required for mycobacterium bovis, e. coli. pseudomonas was not showing any susceptibility. given the broad spectrum of activity, especially towards the gram-negative bacteria, nephrolepis cordifolia is definitely a promising plant having pharmacological importance. for a full interpretation of the present results further investigations are necessary to elucidate the different physical and chemical parameters of the active principles and also to determine the mechanism of action. the present work highlights n. cordifolia as a plant having a broad spectrum of antimicrobial activity, a phenomenon very rarely observed in the plant kingdom. bacteria (escherichia, salmonella, proteus, staphylococci and bacillus) were isolated from hospital soil using selective enrichment and growth on selective and differential medium, viz. macconkey's agar, clyed medium and baird parkers medium. confirmation and species identification was carried out by biochemical and serological tests. from these isolates, three different pathogens were used to study multiple antibiotic resistances. e. coli bj showed resistance to ampicillin, streptomycin and cefurixime. s. typhi and s. aureus showed resistance to ampicillin and cefuroxime. assay using octadisc using e. coli and s. typhi showed a broader resistance pattern to antibiotics including amoxicillin, clavulanic acid, cephalexin, chloramphenicol, ciprofloxacin, and cotrimoxazole. the r plasmid profile was studied to understand the mechanism of drug resistance. to combat the problem of drug resistance, a strategy of combined antibiotic response of cultures were studied. such a synergistic combination would possibly have the effect of overcoming multiple antibiotic resistances. for e.g. kanamycin resistance strains were inhibited in presence of kanamycin and cefotaxime. further, the bactericidal activities of antimicrobials in honey and garlic were also tested. the mic of honey was observed to be %, while that of garlic was between . and %. honey and garlic were also found to inhibit the growth of organisms in the presence of antibiotics. kanamycinresistant e. coli was unable to grow in presence of kanamycin and . % garlic. these traditional agents, long used in ayurvedic system of medicine in india, could be further explored for potent antimicrobial properties. hepatitis b virus (hbv) infection is s global health problem. assays for hbv antigens and antibodies are widely available and standardized. extremely sensitive qualitative pcr kits are also available for detection of hbv in serum. hbv pcr kit may be useful in assessment of occult hepatitis b in hbcab positive alone subjects and carriers. a positive pcr results show presence of virus articles in serum without considering serologic results. but there are differences in efficiency of hbv dna amplification kits. in this study we compare two commercially available hbv pcr kits for evaluation viremia of hbsag positive carriers and hbcab alone positive subjects. material and methods: of the randomly selected subjects serologically examined for hbv, and were positive for hbsag and hbcab alone respectively. both later groups were tested for hbv dna by two commercial kits, hbv pcr detection kit (cinnagen, iran) and hbv pcr test (pazhohesh azma, iran). dna extraction kits recommended by each manufacturer were used for hbv dna extraction. amplicons in both kits were a highly overlapped fragment in conserved sequence of viral genome. results: of the hbsag positive carriers, hbv dna was detected in . and . % using kit and kit respectively. only . % were positive in both kits. in hbcab positive subjects (n = ), . % were positive by kit and all samples were negative when tested by kit . there was a significant difference between two kits. sensitivity of kit and kit were . and . %, respectively. overall, kit increased the detection rate of hbv dna by . %. discussion: our study show there is a significant variation between these two commercial kits especially in hbcab positive subjects that the copy of virus is very low. from these results, it can be concluded that the unstandardized kits have not compatible results, and pcr test interpretation should be done with great care. . the antibacterial activity of the extracts was evaluated based on the inhibition zone using plate diffusion method. most of the extracts were active against both gram positive and gram-negative bacteria, but pseudomonas aerugenosa, bacillus subtilis and sarcina marcescens, were more susceptible to almost all the extracts. finally, further research will be done to elucidate the nature of the active compound and investigate for peptides by using protein gel immobilization bioassay. short interfering rna delivery and gene silencing using polymeric nanocarrier systems k.a. howard , x. liu , d. oupicky , f. besenbacher , j. kjems : inano, university of aarhus, denmark, department of pharmaceutical sciences, wayne state university, detroit, usa the effectiveness of a drug is determined by the ability to migrate through the body and reach target sites in therapeutically relevant levels. nanocarriers for delivery of bioactive agents are being developed at inano to maximise drug payload at target sites. the inclusion of "biological triggers" into the nanocarrier design is used for modulation of cellular nucleic acid trafficking and increased target interaction. chitosan and peptide-based polymers were used to formulate nanocarriers in the size range - nm containing small interfering rnas (sirnas) for gene silencing applications. page analysis showed the structural integrity of the sirna was maintained during particle formation. in systems composed of bioresponsive polymers, nanocarrier disassembly and sirna release under cellular conditions were shown, using atomic force microscopy. the time course for sirna uptake into nih cells was visualised using confocal microscopy. in addition, sirna localisation within cells could be modulated by the composition of the polymer used. the ability of the nanocarrier system to mediate gene expression was investigated in a cell line stably expressing enhanced green fluorescent protein (egfp). furthermore, the various delivery systems were tested in a mouse model stably expressing the egfp protein using both nasal and intravenous delivery routes. the use of animals as a source of organs and tissues for xenotransplantation can overcome the growing shortage of human organ donors. however, the presence of xenoreactive antibodies in humans directed against swine gal antigen present on the surface of xenograft donor cells leads to the complement activation and immediate xenograft rejection as a consequence of hyperacute immunological reaction. the graft of genetically modified organ of a swine depleted of enzyme ␣ , -galactosyltransferase that is responsible for gal antigen origin, would be tolerated with simultaneous administration of medicines decreasing other less severe immunological reactions. to prevent hyperacute rejection it is also possible to modify swine genome by human genes controlling enzymatic cascade of complement or modifying the set of donor's cell surface proteins. for this purpose genetic constructs containing inactivated ␣ , -galactosyltransferase gene, human cd , cd and cd genes controlling complement activation and human genes encoding ␣ , -fucosyltransferase and ␣-galactosidase enzymes modifying cell surface proteins were prepared. these genetic constructs were transfected into the pig foetal fibroblast using lipofection method. after selection, molecular and cytogenetic characteristic of cells with transgene integrated into the host genome were performed. introduction: protease a( a-pro) of coxsackievirus b (cvb ) plays major role in viral replication. in case of infection, viral proteins are being synthesized from viral mrna using host biosynthesis machinery. a-pro of virus, after being synthesized, exhibit two critical functions, cleavage of viral proteins and breaking eif g (eukaryotic initiation factor g-formerly called p ) which leads to host cell translational system shot-off. the enzyme plays essential role in viral replication and cellular damage. to understand pathogenicity of infection and also developing potent and selective inhibitors against picornavirus infection, it is necessary to prepare pure apro enzyme. in this study an expression system with efficient and high yields was obtained. methods: cdna of apro was synthesized using in vitro infection of permissive host through reverse transcription process and was cloned in pet b(+) and since a-pro is a toxic product, naturally before induction its expression will act on the host and damage the cells. for this different hosts were checked and finally, blr(de ) plyss which carries an extra-plasmid for lysozyme expression that minimizes unwanted target protein production (leakage) was selected. on the other hand for biological activity assay, polyclonal antibodies against antigenic sites of p was prepared by synthesizing small peptides, corresponding to antigenic site of p coupling to klh and injecting subcutanously to rabbit. then, the enzyme and its substrate (hela cells lysate that contain p ) were incubated together for different times intervals. results: the recombinant product was confirmed by sds polyacrylamide gel electrophoresis and immunoblot analysis. also p cleavage by apro was assessed by sds-page and western blot analysis. cleavage of p by r a-pro was prominent after h. so recombinat a-pro with good activity was prepared. application of bombyx mori nuclear polyhedrosis virus (bmnpv) bacmid system on production of glycoprotein in larvae of silkworm ayano kageshima, tatsuya kato, misun kwon, enoch y. park department of applied biological chemistry, shizuoka university, ohya , suruga-ku, shizuoka - , japan bombyx mori nuclear polyhedrosis virus (bmnpv) bacmid system was applied on production of glycoprotein in larvae of silkworm. the bacmid system of autographa californica nuclear polyhedrosis virus (acnpv) has already been established and widely used. since the acnpv does not have a potential to infect silkworm we developed the first practical bombyx mori nuclear polyhedrosis virus (bmnpv) bacmid system directly applicable for the protein expression of silkworm. by using this system, the green fluorescence protein and glycoprotein, human ␤ , -n-acetylglucosaminyltransferase were successfully expressed in silkworm larvae not only by infection of its recombinant virus but also by direct injection of its bacmid dna. three different kinds of signal sequences were tested for the secretion of glycoprotein into hemolymph of silkworm. signal peptides of prophenoloxidase-activating enzyme and bombyxin: insulin-like brain secretory peptide showed the highest secretion ratio, % of total expressed ␤ , -n-acetylglucosaminyltransferase was secreted into hemolymph of silkworm. using bacmid system mu/ml of ␤ , -n-acetylglucosaminyltransferase was expressed in hemolymph of silkworm, which was -three times higher than that of insect cell. silkworm is one of the most attractive hosts for large-scale productions of eukaryotic proteins as well as recombinant baculoviruses for gene transfer to mammalian cells. this method provides the rapid protein production in silkworm, is free from biohazard, thus will be a powerful tool for the future production factory of recombinant eukaryotic proteins and baculoviruses. we have established an efficient system for foreign gene expression in lily (lilium longiflorum) pollen in a transient mode. pollen was transformed using agrobacterium via vacuum filtration for min. the pollen germinated for h was analyzed to confirm its foreign gene expression in molecular analysis. mouse fed the transgenic pollen culture for weeks showed immune reaction specific for pollen-derived recombinant protein. and the igg level was highly elevated by one time boosting injection afterwards. the lily pollen system may be suggested as a novel type of biofactory for producing edible vaccine with rapidity. anti-apoptosis engineering with the kc gene obtained from silkworm shin sik choi, won jong rhee, tai hyun park school of chemical and biological eng., seoul national university, seoul - , korea the chinese hamster ovary (cho) cell line producing recombinant human erythropoietin (epo) was manipulated to express the kc gene, which was originally obtained from a silkworm. the expression of kc inhibited serum deprivation-induced apoptosis and increased the cell density and epo expression level per unit cell by five-and two-folds, respectively. an increase in these two factors resulted in a -fold increase in the volumetric productivity of epo. compared with the controls, the oligosaccharide structures of the epo synthesized by the cells expressing kc showed greater homogeneity. the terminal sialylation of the glycans of epo were promoted by the expression of kc . the positive effects of kc expression on the cell viability and productivity were attributable to the stable maintenance of the mitochondrial activity. these results demonstrate that the cho cell line genetically engineered with the kc gene has a great potential for use in the production of therapeutic proteins. evaluation of fucoidan-chitosan hydrogels on superficial dermal burn healing in rabbit: an in vivo study a.d. sezer , f. hatipoglu , z. ogurtan , a.l. baş , j. introduction: healing of dermal wounds with macromolecular agents such as natural polymers is one of the research areas of the pharmaceutical biotechnology. fucoidan is a sulphated polysaccharide which is commonly obtained from seaweeds. although a great number of studies on the different pharmacological properties of fucoidan are present, there is very limited information on the fucoidan-based system used in dermal burns. the aim of this study was to prepare chitosan hydrogel containing fucoidan and to investigate this hydrogel formulation for treatment of dermal burns on rabbits. methods: fucoidan-chitosan hydrogels were prepared as follows: the polymers were dissolved in acidic solution and sonicated for removing the air-bubbles then the gels were stored at + • c for in vivo studies. seven adult male new zealand white rabbits (mean weight, . ± . kg) were used for the evaluation of the gels on superficial dermal burns. the back of the rabbits were depilated and sedated. the wounds were made by circular stamp aluminium caps ( . cm ) at • c. four wounds were formed for each rabbit; (a) was treated with fucoidan-chitosan gel, (b) was treated with fucoidan solution, (c) was treated with chitosan gel (without fucoidan) and (d) as a negative control group. biopsy samples were taken at , and st days at the beginning of the study and each wound site was macroscopically observed and evaluated histopathologically. results: oedema was not observed in all groups after days treatment except controls. after days treatment, fibroplasia and scar were observed on wounds treated with fucoidan-chitosan gel and fucoidan solution. the best regenerate dermal papillary formation and the fastest closure of wounds were observed in group a after days treatment. the wound epithel elongation and thickness values were measured; a ( and m), b ( and, m), c ( and, m), d ( and, m) at the end of the study. conclusion: re-epithelization and contraction of the wound area which was treated with fucoidan-chitosan hydrogel were faster than the other groups. the fucoidan-chitosan hydrogel formulations can be suitable for the treatment of dermal burns. aim: to analyze the neutralizing -related activity of antibodies against e region of hcv, specific polyclonal antibody was raised by immunized rabbits with synthetic peptide that had been derived from e region of hcv with the amino acid sequence e antibody [ghrmawdmm). materials and methods: hyper-immune hcv e antibodies were incubated over night at • c with serum samples from patients positive for hcv rna, with different viral load, ranged - million copes/ml, then incubated min to hepg cells. rt-pcr and flow cytometry were used to study the inhibition binding and entry effect of e antibody. direct immunostaining of e antibody conjugated with fitic and flow cytometry analysis showed reduced the mean fluorescence intensity in the samples pre-incubated with e antibody compared with samples without e . of positive serum samples, ( %) samples showed completely inhibition of infectivity as detected by rt-pcr. conclusion: in house produced e antibody, blocks binding and entry of hcv virion infection to target cells suggesting the involvement of this epitope in virus binding and entry. isolation of these antibodies that block virus binding and entry will be useful in providing potential therapeutic reagents and for vaccine development. tumor necrosis factor beta (tnf␤) is a pleiotropic cytokine mediating its activity through the same receptors as the structurally related tnf␣. shortening of the n-terminal part has been reported to enhance its cytotoxic activity, with the explanation that removal of the flexible n-terminus reduces steric interferences in the receptor binding process. for studies of relationship between the n-terminal protein structure and physicochemical properties, three different forms mettnf␤, his tnf␤ and n tnf␤ were expressed in e. coli. high solubility of n tnf␤ was expected, however, both analogs his tnf␤ and n tnf␤ were predominantly obtained in the form of inclusion bodies (ibs). on the other hand, mettnf␤ was equally distributed between the soluble and insoluble fraction. most probably, the composition of the n-terminal part, such as the exposure of hydrophobic residues in the case of n tnf␤, or a mildly hydrophobic stretch of seven histidines appended to the natural hydrophobic n-terminus in the case of his tnf␤, lead to incorrect folding and force aggregate formation. solubilization of ibs was performed under native and denaturing conditions using various concentrations of nls, urea and gndhcl. mettnf␤ ibs were easily dissolved with . % nls, while his tnf␤ and n tnf␤ demanded denaturing conditions. structural differences in the nterminal part of various tnf␤ proteins were also reflected in protein refolding characteristics and chromatographic behavior. dilution, ultrafiltration and dialysis were used for refolding, and imac was chosen as the main chromatographic step. our results suggest that not only the length of the n-terminal part of tnf␤ but also the composition and exposure of certain amino acid residues affect its physicochemical properties. enzymatic modification of sphingomyelin long zhang, lars hellgren, xuebing xu biocentrum-dtu. e-mail: lz@biocentrum.dtu.dk (l. zhang) due to its major role in maintaining the water-retaining properties of the epidermis, ceramide is of great commercial potential in cosmetic and pharmaceuticals such as hair and skin care products. currently, chemical synthesis of ceramide is a costly process, and developments of alternative cost-efficient, high yield production methods are of great interest. in the present study, the potential of producing ceramide through enzymatic hydrolysis of sphingomyelin (sm) have been studied. sm is a ubiquitous membrane-lipid and rich in dairy products or by-products. in present study, we have optimized the production of ceramide from sm using phospholipase c from clostridium perfringens. water and enzyme amount had the biggest influence on sm hydrolysis in the system. botulinum neurotoxins constitute a family of bacterial toxins for botulism syndrome in human. the toxins bind with high affinity to nerve cells where they cause a complete inhibition and release of neurotransmitters and thereby produce flaccid paralysis. in this work we have reported isolation of the binding domain of type e neurotoxin by pcr and expressed in a proper expression vector. the output of this investigation can be used as a tool to study the mechanism of binding of holotoxins and also can be useful to study the antibody production against botulism syndrome. the synaptobrevin (vamp ) a protein which play a key role in the fusion and exocytosis of the vesicle of mammalian nerves terminals this protein is substrate different serotypes of botulinum neurotoxins. light chain of clostridium botulinum type b, d, f and g cleave end of neurons. due to intraction of clostridium botulinum neurotoxin with vamp , this protein can be used as one of the toolsin the detection of poisings cause by this bacteria in the clinical laboratory using the enzyme with proof reading activity the above gene amplified by pcr technique for the production of the recombinant protein, the prokaryotic expression vectors (pet system) was used. a recombinant protein developed on poly acrylamide gel analyzed by western blotting and eliza. most children with down syndrome (ds) are born to younger mothers (< years). recent reports linking down syndrome (ds) to maternal polymorphisms at the methylenetetrahydrofolate reductase (mthfr) gene locus have generated great interest among investigators in the field. in this study, forty mothers with their affected outcomes and control mothers were included. all mothers were subjected to complete medical and nutritional history with special emphasis on folate intake through food or oral supplementation. estimation of blood homocysteine level was done. also we examined the two polymorphisms in genes encoding the folate metabolizing enzyme methylenetetrahydrofolate reductase (mthfr), namely, c > t and a > c. folic acid intake from food and from vitamin supplements was significantly low (below the recommended daily allowance) in the group of mothers with ds children compared to control mothers (p < . ) using student t-test. blood homocysteine was normal in both control and ds mothers. the prevalence of the two polymorphisms, namely, c > t and a > c in mothers of ds children (case mothers) (n = ) was compared with controls (n = ). frequencies of mthfr genotypes (cc, ct, and tt) at position demonstrated no difference between the case and control groups. genotype frequencies of mthfr at position a (aa, ac, and cc) were different among the case and control mothers. we here report the first study on a possible relation between ds with mthfr a > c genotypes in egypt. our results showed that mthfr a > c polymorphism is a remarkable genetic entity among egyptian females with d.s. children. sufficient folate intake and supplementation is an important preventive strategy in overcoming the risk of nondisjunction. homologous recombination (hr) is the mechanism that permits the creation of genetically engineered strains through gene targeting. in order to further develop gene targeting techniques, notably for higher eukaryotes such as filamentous fungi, it is of crucial importance to fully understand the molecular mechanisms behind mitotic hr. in saccharomyces cerevisiae, a dna double strand break (dsb) is an essential intermediate in meiotic hr. however, as hr occurs at a low rate in mitotic cells, it has been difficult to determine the nature of the event(s) that triggers it. rad is a key protein involved in hr and is evolutionarily conserved from yeast to human. to shed light on the molecular events in hr, we have generated a large collection of defined rad mutant strains in the yeast s.cerevisiae. a screen of these mutants led to the identification of strains that fail to repair dna dsbs, yet are proficient for homologous recombination. this result strongly suggests that dsbs may not be the major cause of spontaneous mitotic hr and gives new perspectives in respect to novel potential gene targeting substrates. we have analyzed these separation of function mutants in a variety of new assays to obtain a more detailed understanding of their controversial phenotype. our latest results will be presented. the outcome of interferone plus ribavirine treatment of hepatitis c virus (hcv) genotype is unfortunately poor. development of alternative therapy for this genotype is of a paramount importance. inhibition of hcv gene expression in vitro by the use of antisense phosphorothioate oligodeoxynucleotides (s-odn) against internal ribosomal entry site (ires) elements were associated with favorable results. to assess s-odn activity, previous studies utilized viral subgenomic or full cdna fragments linked to reporter genes and transfected into adhered cells or in a cell free system. in the present study we utilized hepg cells infected with native hcv rna of genotype . the culture system presented herein was shown to support hcv replication on the following bases ( ) consistent detection of both plus and minus rna strands for weeks in cells and in fresh culture supernatent, ( ) ability of supernatent to infect naive hepg cells ( ) consistent expression of core and e envelope proteins in infected cells throughout the week culture. s-odns against aug translation start site (s-odn- , nt - ) of the viral polyprotein precursor and stem loop iiid within the ires region (s-odn , nt - ) were added to infected cells. intracellular viral replication was monitored by nested rt-pcr of plus and minus strands. the results of these experiments demonstrated that intracellular replication of hcv genotype was completely arrested after h in culture using either s-odn molecule (with more efficacy of s-odn than s-odn ) at concentrations as low as m. the inhibitory effect of s-odn appeared to be specific to hcv replication since equal levels of human glyceralehyde -phosphate dehydrogenase (gapdh) gene expression were noted pre and post supplementation of s-odns. in conclusion, the present study provides evidence antisense phosphorothioate oligonucleotides have potent inhibitory effect on genomic replication of hcv genotype , the most common type in egypt. a sensitive and specific pcr-elisa was developed to detect shigella dysentery in food. the assay was based on the incorporation of degoxigenin-labeled dutp and a biotin-labeled primer specific for shiga toxin genes during pcr amplification. the labeled pcr product were bound to streptoavidin-coated wells of a microtiter plat and detected by an elisa. the elisa detecting system was able to increase the sensitivity of the pcr assay by up to -fold, compared with a conventional gel electrophoresis. the detection limit of the pcr-elisa was . - cfu dependent upon shigella dysentery serotypes and genotypes of shigatoxin. the entire procedure took about h. isolation, cloning, expression and purification of snap- as a substrate of botulinum neurotoxin type a and e m.l. mossavi , f. ebrahimi , j. amani * , h. basiry , z. ahmadi : department of biology, faculty of science, imam hussein university, tehran, iran; department of biology, faculty of medicine, bageatallah university, tehran, iran. e-mail: kpjamani@ihu.ac.ir (j. amani) clostridial neurotoxin inhibit neurotransmitter relase by selective and specific intracellular proteolysis of synaptosomal associated protein of kda (snap- ); synaptobrevin/vamp- and syntaxin. snap- is one of the components that form docking complex in synaptic ends. this protein is substrate for botulinum neurotoxins type a and e. each of these toxin serotypes specifically cleave snap- in particular position and there by block docking and synaptic vesicle membrane fusion and finally prevent neurotransmitter exocytosis and transition of neurotic signals. in order to use the protein as a substrate for detection of different type of clostridium neurotoxin in vitro test, the protein was produced by recombinant technique. the dna encoding snap- was isolated from rat brain by pcr using the two primer the amplified fragment colonel into expression vector pet a.the expression protein was purified by affinity chromatography. confirm by different method the his tag fusion protein was digested with entrokinase. the present work deals with the preparation of pure alpha- antitrypsin (aat) protein from healthy subjects, which can be used in preparing its corresponding monospecific antibody in albino rabbits. this antibody was found very useful in the immuno-diagnosis of pulmonary emphysema. this study has been also concerned with the biochemical changes associated with aat deficiency in pulmonary diseases. to fulfill this work, a group of healthy blood donors was selected for separation of pure aat antigen from blood. the pure aat was used for the preparation of anti-aat, the purity and potency of antibody was checked by titration methods. the biochemical changes were studied in thirty subjects clinically divided into three groups including, control, heavy cigarette smokers with pulmonary emphysema, and non-smoking subjects with pulmonary emphysema. the activity of elastase and hydroxyproline (hp) level as a marker of elastin and collagen breakdown were assayed in bronchoalveolar lavage (bal) fluid. the activity of aat and to inhibit proteases as represented by tryptic inhibitory capacity (tic) was evaluated. serum ceruloplasmin, transferrin and iga level as well as thiobarbituric acid reactive substances (tbars) were estimated in all groups. our data revealed that aat and its tic showed very highly significant decreased levels in all patients with emphysema as compared to control, while the elastase activity and hp level in bal fluid were significantly increased in these patients. serum tbars was significantly increased in such patients associated with increasing level of both ceruloplasmin and transferrin. while, serum iga was significantly increased. furthermore, the biochemical changes were markedly changed in smokers with emphysema than non-smoking subjects. in conclusion, the preparation of anti-aat on the local level is very important where less expensive and less time consuming and can be useful in immuno-diagnosis and prognosis of pulmonary diseases. a new method combined with boosting and projective adaptive resonance theory for analysis of gene expression data from cancer patients hiro takahashi, yasuyuki murase, hiroyuki honda department of biotechnology, school of engineering, nagoya university, nagoya - , japan. e-mail: h d@mbox.nagoyau.ac.jp (h. takahashi) an optimal and individualized treatment protocol based on accurate diagnosis is urgently required for the adequate treatment of patients. for this purpose, it is important to develop that a sophisticated algorithm that can manage large amount of data, such as gene expression data from dna microarray, for optimal and individualized diagnosis. in our previous study, we developed the projective adaptive resonance theory (part) as a gene filtering method and boosted fuzzy classifier with sweep operator (bfcs) as a modeling method. in the present study, we applied the combination of part and bfcs (part-bfcs method) to microarray data of brain tumor (central nervous system tumor) obtained from the website. the method enabled the selection of important genes related to the prognosis of the tumor, i.e., sensitivity for combined therapy with surgery, radiotherapy, and chemotherapy mainly with vincristine, cisplatin, and cytoxan. the constructed model showed about % higher accuracy than that of the conventional method. genomic signal analysis of hiv variability based on rt gene p.d. cristea, rodica tuduce d. otelea biomedical engineering center, university "politehnica" of bucharest, romania. e-mail: pcristea@dsp.pub.ro (p.d. cristea) dna sequences genotyped from clade f hiv- isolates from romanian patients in the laboratory of the national institute of infectious diseases "matei bals", bucharest, romania, have been studied. the symbolic sequences have been converted into digital genomic signals by using a complex quadrantal representation of the nucleotides described earlier. the cumulated phase and unwrapped phase of a complex genomic signal reflect the statistical distribution of bases and base-pairs, respectively. independent component analysis of the genomic signals has been used to characterize the variability of the f subtype hiv strains isolated in romania. the sequenced segment is of (about) base pairs, approximately aligning with the standard sequence of hiv- (accession nc in genbank) over the interval - bp. this segment, which is currently used for the standard identification and assessment of hiv- strains, comprises the protease (pr) gene and two thirds of the reverse transcriptase (rt) gene. only results referring to the analysis of the rt gene region are presented here and used for extracting features of virion isolates and for establishing phylogenetic trees of the studied strains. the analyzed rt encoding segment has the length bp and is located in the second interval ( - bp) of the analyzed dna segment, respectively along the - bp region of nc . taking into account the mutations identified in these sequences, the samples were classified in three groups from the point of view of their resistance to current antiretroviral compounds: sensitive, resistant and multiresistant. the paper presents results for the isolates in which mutations leading to multiple drug resistance have been identified. over expression of secb protein in e. coli enhances the periplasmic expression of human growth hormone m. ghafari , , a. zomorodipour : islamic azad university of jahrom, tehran, iran; national institute for genet eng and biotechnol., tehran, iran. email: maryamghafari @yahoo.com (m. ghafari) among several proteins involved in the secretion pathway of proteins in e. coli, secb plays a key-important role in solubilization of preproteins before processing. in order to increase processing of a human growth hormone precursor (pelb::hgh) which appeared to have problem in processing efficiency, as a possible solution a regulated co-expression of a secb gene was considered. in this regard, we designed an arabinose-regulated secb expressing plasmid compatible with an iptg/lactose-regulated pelb::hgh expressing plasmid. for the construction of the secb expressing plasmid the origin of replication and antibiotic resistant gene (amp) of a pbad vector was replaced by a p a-ori and a kanamycin resistant gene, respectively. the expression and processing of pelb::hgh preprotein in the two-plasmid containing bacteria in a secb over-expression state was compared to that of the pelb::hgh expression in normal bacteria. although a decline in total expression level of hgh during the overexpression of secb was observable, probably due to presence of two different expressing plasmids, but both the processing efficiency of pelb::hgh and the transport of mature protein into the periplasmic space was enhanced during prolonged arabinose induction. current diagnostics and therapeutics for cat allergy are based on cat epithelial extracts. natural allergen extracts contain a mixture of allergenic and non-allergenic components that are difficult to standardize. recombinant allergens can improve diagnosis and de-sensitization against single component. major cat allergen is a heterodimer composed of disulfide linked . and . kda polypeptide chains. both chains of feldi protein were obtained in e.coli system. purification of recombinant proteins was performed in denaturing conditions using immobilized metal affinity chromatography specific for proteins with histidine tag. from ml culture approximately mg protein for feldi chain and mg of feldi chain were obtained. after purification histidine tag was removed by hydrolysis with thrombin. immunological activity of feldi against serum of patients allergic to cat was narrowed to subgroup of patients allergic to feldi protein by surface plasmon resonance. immunological activity of each chain and renatured heterodimer was also tested using immunoprecipitation techniques against serum of population group. subdoligranulum variabile -a novel member of the human gut micro flora with a high prevalence kim holmstrøm, trine møller bioneer a/s, hørsholm, dk- , denmark in we isolated and cultured for the first time a bacterium from a human fecal sample representing a hitherto unknown member of the clostridium leptum rrna supra generic cluster. the c. leptum rrna supra generic cluster represents one of the major phylogenetic lineages within the human gut microbiota, and is characterized by having only a small proportion of its members actually identified by cultivation compared to the estimated numbers of bacteria contained in this group from culture-independent gut flora analyses. s. variabile is an obligate anaerobe gram negative bacterium with a characteristic pleiomorphic coccoid-droplet-like cellular morphology. its closest previously cultivated relative based on a s rdna phylogenetic analysis is faecalibacterium prausnitzii, a rod-shaped and therefore easily distinguishable gram negative bacterium present in high numbers in the human fecal micro flora. based on s rdna sequence we designed a s. variabile specific oligonucleotide probe for use in fish analysis to estimate the prevalence of this "new" bacterium in fecal samples collected from healthy human beings. interestingly, we observed a high proportion of s. variabile present in all tested samples, and in some instances we observed a higher prevalence than the more well-known group of bifidobacteria equally estimated by fish analysis. documentation of these results will be presented. several species of sea cucumbers, long an incumbent of traditional medicines were selected as the source of animal based antibiotic compounds. swabs of the inner surface and coelomic fluid (inner fluid) samples from sea cucumber (holothuria atra jaeger) were taken. thirty strains of bacteria were isolated. these strains were grown in different antibiotic production media. nine human pathogenic bacterial species were used as test agents and they are, k. pneumoniae, mrsa, m. luteus, s. thyphimurium, s. epidermitis, s. saprophyticus, b. subtillis, p. aeruginosa and s. pyogenes; only four bacterial strains showed mild antibiotic activity against s. pyogenes and s. thyphimurium. similar testing on two other species, h. scabra and s. variegatus will be carried out. different media, especially antibiotic production enrichment media will also be used. characterization will be done upon obtaining an antibiotic compound, which shows moderate to high activity against at least one of the nine human pathogens used. for plant based medicines, three rhizomes, "cekor," "jerangau" and "bonglai" were analyzed. solvent extraction using ethanol, methanol and acetone was carried out, at a concentration of - mg per ml solvent. the filtrates were used for the antibiotic testing stage. all the three plant species showed moderate antibiotic activity against m. luteus, s. epidermitis, s. pyogenes and s. saprophyticus. interestingly, the antibiotic activity increased when combinations of the herbal extracts were used. cell-based therapy continues to be a promising avenue for the treatment of duchenne muscular dystrophy, an x-linked skeletal muscle-wasting disease. recently, we have demonstrated that freshly isolated myogenic progenitors contained within the adult skeletal muscle side population (sp) can engraft into dystrophic fibers of non-irradiated mdx cv mice after intravenous transplantation. engraftment rates, however, have not been therapeutically significant, achieving at most % of skeletal muscle myofibers expressing protein from donor-derived nuclei. to improve the engraftment of transplanted myogenic progenitors, an intra-arterial delivery method was adapted from a previous procedure. cultured, lentivirus transduced skeletal muscle sp cells were transplanted into the femoral artery of non-injured mdx cv mice. based on the expression of microdystrophin and gfp transgenes in host muscle, sections of the recipient muscles exhibited % to % of skeletal muscle fibers expressing donor-derived transgenes. further, donor muscle sp cells, which did not express any myogenic markers prior to transplant, express the satellite cell transcription factor pax and the musclespecific intermediate filament desmin after extravasation into host muscle. the expression of these muscle-specific markers indicates that progenitors within the side population can differentiate along a myogenic lineage after intra-arterial transplantation and extravasation into host muscle. given that femoral artery catheterization is a common, safe clinical procedure and that the transplantation of cultured adult muscle progenitor cells has proven to be safe in mice, our data may represent a step towards the improvement of cell-based therapies for dmd and other myogenic disorders. metabolic engineering design of an extracellular hgh synthesis system birgül Şentürk , pınar Ç alık , güzide Ç alık , tunçer h.Özdamar : bre lab, department of chemical engng, ankara university, ankara, turkey; iblab, department of chemical engineering, metu, ankara, turkey. e-mail: ozdamar@eng.ankara.edu.tr (t.h.Özdamar) metabolic engineering design of an extracellular human growth hormone (hgh) synthesis system is based on cloning the dna encoding human therapeutic protein together with the signal dna sequence of an extracellular enzyme gene into a host-vector system. in this context, extracellular protease (subc) signal dna sequence, i.e. pre-signal dna sequence, was fused into the frame in front of hgh mature dna sequence, by the use of four primers designed using pcr-based gene splicing by overlap extension method. b. licheniformis chromosomal dna and plasmid carrying hgh cdna were used as templates in pcrs, respectively, for the amplification of the subc signal dna sequence and hgh mature peptide sequence. for the fusion of two target genes, i.e. mature peptide sequence of hgh and, signal dna sequences were amplified separately by pcrs. the primers used at the ends to be joined were designed as complementary to one another by including nucleotides at their ends that are complementary to portion of the other primer. these products were mixed in the next pcr reaction, where one strand from each fragment contains the overlap sequence at end. extension of this overlap by dna polymerase has yielded the recombinant hybrid-gene; and hybrid-gene serve as template for the continuation of reactions for the increase of the concentration in the microreactors. the hybrid gene fragment was first cloned into puc , and then sub-cloned to pmk e.coli-bacillus shuttle plasmid. thus, a new expression vector with high stability and high copy-number was obtained and transferred into host b. subtilis (spo − ). the metabolic flux distributions calculated by the mass balance based stoichiometric model based on the proposed metabolic reaction network for r-b.subtilis were determined by using time profiles of the substrate, dry-cell, hgh, amino acids and organic acids concentrations as the constraints. on the basis of the intracellular bioreaction rates and the interactions with the bioreactor operation parameters, an in-depth insight will be provided for further metabolic engineering design for the extracellular hgh production in r-b.subtilis. medicines based on polypeptides consisting of and more amino acid residues are widely spread in pharmaceutical market at the present time. practically all polypeptide medicines known are prepared by general chemical synthesis that caused high cost of their production. that's why biotechnological way of polypeptide medicines preparation using recombinant gene expression in bacteria seems to be promising. during our work we design hybrid construction allowing solving a problem of specific cleavage of target polypeptide from the hybrid protein using system of protein splicing from new england biolabs. in case of thymosin ␣ production impac system (intein mediated purification with affinity chitin-binding tag) has been used, where the modified sce vma from s. cerevisiae has been applied as intein. in contrast to other systems, impact allows the preparation of target protein without using of serine protease and other factors that may cleave the hybrid protein. in the presence of thiol reagents, such as dithiothreitol, mercaptoethanol, or cystein the hybrid protein can be site-specifically cleaved to give intein, the target protein and small fragment of nextein. using of this system does not allow to obtain the target protein with ser, cys or thr on n-terminal of protein, because in those cases target protein and n-extein ligation product will be formed. ser is n-terminal amino-acid in thymosin ␣ . it was recently found that some metal ions essentially affect the splicing. we tried to use zncl and we have found that, in case of intein-thymosin ␣ , the maximal yield of target polypeptide and the minimal yield of splicing products are observed in the absence of dithithreitol and in the presence of . - mm zinc chloride in buffers on all stages of thymosin ␣ isolation. the structure of recombinant thymosin ␣ of human was confirmed by the determination of n-and c-terminal amino-acid sequences and by maldi tof mass-spectrometry. mature embryos of five t. aestivum and five t. durum cultivars formed embryogenic callus on two different media. embryos were removed from surface sterilised seeds and placed with the scutellum upwards on a solid agar medium containing the inorganic components of murashige skoog and mg/l , -dichlorophenoxyacetic acid ( , -d) or mg/l naphtalenacetic acid (naa). the developed calli and regenerated plants were maintained on , -d or naa free ms medium. wheat plants can be regenerated via two different systems. there were significant differences in percentage of callus induction and regeneration capacity on the different initiation medium. among the t. aestivum cultivars, yakar had the highest regeneration capacity in both induction medium. in t. durum cultivars, kiziltan gave the highest regeneration capacity in ms + , d medium and yilmaz gave the highest regeneration capacity in ms + naa medium. a strong genotypic effect on the culture responses was found for both induction medium. tumor necrosis factor beta (tnf␤) is a pleiotropic cytokine mediating its activity through the same receptors as the structurally related tnf␣. shortening of the n-terminal part has been reported to enhance its cytotoxic activity, with the explanation that removal of the flexible n-terminus reduces steric interferences in the receptor binding process. for studies of relationship between the n-terminal protein structure and physicochemical properties, three different forms mettnf␤, his tnf␤ and n tnf␤ were expressed in e. coli. high solubility of n tnf␤ was expected, however, both analogs his tnf␤ and n tnf␤ were predominantly obtained in the form of inclusion bodies (ibs). on the other hand, mettnf␤ was equally distributed between the soluble and insoluble fraction. most probably, the composition of the n-terminal part, such as the exposure of hydrophobic residues in the case of n tnf␤, or a mildly hydrophobic stretch of seven histidines appended to the natural hydrophobic n-terminus in the case of his tnf␤, lead to incorrect folding and force aggregate formation. solubilization of ibs was performed under native and denaturing conditions using various concentrations of nls, urea and gndhcl. mettnf␤ ibs were easily dissolved with . % nls, while his tnf␤ and n tnf␤ demanded denaturing conditions. structural differences in the nterminal part of various tnf␤ proteins were also reflected in protein refolding characteristics and chromatographic behavior. dilution, ultrafiltration and dialysis were used for refolding, and imac was chosen as the main chromatographic step. our results suggest that not only the length of the n-terminal part of tnf␤ but also the composition and exposure of certain amino acid residues affect its physicochemical properties. high level expression of recombinant growth hormones in e.coli faces common problems such as protein aggregation and inclusion body formation. discussions are raised whether it is more beneficial to obtain soluble protein but to loose expression rates. here we describe an experiment based on the hypothesis that slower expression should result in at least partially soluble recombinant protein. experiments were performed on bovine, chicken and mink growth hormones. expression rate was controlled externally by adjusting cultivation temperature, media, inducer amount, and both induction and cultivation times. another approach to the problem was performed through genetic manipulation. we changed strong t promoter to e. coli promoter consensus sequence thus reducing expression rate. recombinant growth hormone was still found to form aggregates, even when expressed at extremely low levelsseveral ( - ) percent of total intracellular protein. we developed optimization scheme of insoluble protein production and showed that expression rate minimization is not influencing recombinant growth hormone solubility in vivo thus suggesting an idea of sequence specific aggregation. to optimize the recombinant protein production in small-scale shake-flask system and high cell density fermentation, new tools have been developed at our laboratory which helps to get knowledge about the physiological state of the cell culture. these tools include (i) a quantitative monitoring system for cellular mrnas based on a sandwich hybridization technique, and (ii) a wireless online monitoring tool (senbit), applicable for standard sensors such as ph, po and temperature for the continuous data collection from shake flasks. the senbit system is a new tool supplying valuable information for the optimisation of the expression of recombinant genes in shake flasks and allowing conclusions towards the reproducibility of shake flask cultures. the presentation will focus on use of the sensitive sandwich hybridization technology for the quantitative analysis of process relevant marker genes in different kind of microbial cell cultures with a focus on the production of recombinant proteins. samples from shake flask cultures and high cell density fed-batch fermentations of the yeast pichia patoris, have been analysed. additionally the mrna analysis was combined with the application of the senbit wireless system to study the production of a recombinant protein in shake-flask cultures of p. pastoris. aside from p. pastoris, mrna sandwich hybridization also was used to monitor product expression in fed-batch fermentation of e. coli for the production of a protein with two subunits by sequential induction. quantitative rna analysis as a tool for optimization of tetrameric collagen prolyl -hydroxylase production in e. coli a. neubauer , m. bollok , j. myllyharju , p. collagen prolyl -hydroxylase (p h) involved in the biosynthesis of collagens is an ␣ ␤ tetramer. recombinant expression of p h in e. coli was described recently . the construct for cytoplasmic expression contains the genes of both subunits in one plasmid under control of different promoters. the ␣ subunit forms inactive aggregates, when expressed separately. in mammalian cells the ␤ subunit is available in large excess and keeps the ␣ subunit in a soluble active form. to mimic this in the bacterial system, we induced both genes sequentially. after induction of the ␤ subunit with iptg, expression of the ␣ subunit was initiated with anhydrotetracycline. here we use the analysis of the product mrnas with a bead based sandwich hybridisation assay (sha) (rautio et al., ) for optimization of the fermentation procedure. a high p h activity was obtained if a high mrna level of the ␣ subunit could be maintained over a longer time. the obtained results illustrate the importance of the second induction for a high level expression of the p h tetramer. the cells need to be in a "healthy state" with low metabolic load to react efficiently to the second induction. the data illustrate the optimization of a fermentation process by monitoring mrna levels which is of general interest for optimization of products which are difficult to detect. cell-based therapy continues to be a promising avenue for the treatment of duchenne muscular dystrophy, an x-linked skeletal muscle-wasting disease. recently, we have demonstrated that freshly isolated myogenic progenitors contained within the adult skeletal muscle side population (sp) can engraft into dystrophic fibers of non-irradiated mdx cv mice after intravenous transplantation. engraftment rates, however, have not been therapeutically significant, achieving at most % of skeletal muscle myofibers expressing protein from donor-derived nuclei. to improve the engraftment of transplanted myogenic progenitors, an intra-arterial delivery method was adapted from a previous procedure. cultured, lentivirus transduced skeletal muscle sp cells were transplanted into the femoral artery of non-injured mdx cv mice. based on the expression of microdystrophin and gfp transgenes in host muscle, sections of the recipient muscles exhibited - % of skeletal muscle fibers expressing donor-derived transgenes. further, donor muscle sp cells, which did not express any myogenic markers prior to transplant, express the satellite cell transcription factor pax and the muscle-specific intermediate filament desmin after extravasation into host muscle. the expression of these muscle-specific markers indicates that progenitors within the side population can differentiate along a myogenic lineage after intra-arterial transplantation and extravasation into host muscle. given that femoral artery catheterization is a common, safe clinical procedure and that the transplantation of cultured adult muscle progenitor cells has proven to be safe in mice, our data may represent a step towards the improvement of cell-based therapies for dmd and other myogenic disorders. collagen and its derived product gelatin are attractive mammalian proteins to be used as model for the production of complex heterologous proteins in plants. the availability of a recombinant product will provide a safer, more homogeneous product than the current animal-derived material. the aim of the project is to investigate the feasibility of a production system for the accumulation of recombinant collagen for conversion to gelatin using barley. the -end of the cocksfoot mottle virus (cfmv; genus sobemovirus) genomic rna sequence, called cfmv -element, has been shown to enhance recombinant protein synthesis in barley (wo / , mäkinen et al., ) . the -element will be used to study whether accumulation levels of complex mammalian proteins can be further increased, using collagen as a model that will serve as basis for exploring the expression of other complex proteins. this system can be study the production of barley-derived recombinant collagen for conversion to gelatin. classical swine fever virus (csfv) is an animal pestivirus which can be used as a surrogate model to elucidate the role of envelope glycoproteins of closely related human hepatitis c virus (hcv). the necessity to use the surrogate models for hcv is due to the fact that this virus cannot be grown in vitro cultures. csfv genome codes for three major antigenic glycoproteins which are located in the same cluster of genes; they are designated as e and e (e rns ) and e . glycoproteins form heterodimeric and homodimeric complexes on the external part of viral particles. it is generally accepted that envelope glycoproteins play a major role in the initial stages of viral infection both for csfv and hcv. formation of complexes is needed to effectively infect host cells. we have investigated the formation of glycoprotein dimers by immunoperoxidase monolayer assay and by immunoblotting (western blotting). immunoblotting is a very useful technique in these studies because the complexes are formed via cysteine-cysteine disulphide bonds and they are retained during sds-page under non-reducing conditions. by modifying the glycoprotein genes and by arresting n-glycosylation of e and e we have investigated which factors influence the formation of complexes. it has been found that some glycosylation inhibitors which act at the early stages of glycan chain processing influence, not only glycosylation, but also the stability of e protein, effectively inhibiting the formation of glycoprotein complexes and the yield of the virus. these inhibitors are potential agents for arresting the multiplication and spread of csfv, and its relative -human hcv. recombinant proteins have been produced in a variety of heterologous protein expression systems. eukaryotic unicellular algae have distinct advantages, e.g. it can synthesize complex protein that requires post-translational modification. furthermore, microalgae can be grown in confined environment and thus prevents leakage of genes to the environment. our group has developed a platform technology for the production of recombinant proteins in red microalga porphyridium sp. we have constructed algal transformation plasmid vectors containing a camv s promoter and polya signal site. a streptoalloteichus hindustanus bleomycin-resistant gene was used as the selective marker. we have expressed ovalbumin and hepatitis-b surface antigen (hbsag) as model proteins. transformation was carried out by agitating algal cells and vector dna with glass bead. transgenic lines were selected by growing algal cells on agar plate containing g/ml zeocin. positive transgenic lines were selected by screening the colonies by pcr and confirmed by dna sequencing. expression of ovalbumin and hbsag protein was examined by western blot analysis. ovalbumin was found to be expressed inside the algal cells while small hbsag was secreted into the medium due to presence of signal peptide. these findings indicate that red microalgae are capable of producing heterologous proteins. life-material exhibition as ethical interpretation chang shih-lung biotechnology industry study center, taiwan institute of economic research, taipei , taiwan. email: schang@tier.org.tw in this thesis we aim to explore the medical-related life-material exhibitions within ntu hospital-the humanity building, and taipei mackay hospital-the historical showroom as abecedarian clues to understand the burgeoning phenomenon-medical museums in taiwan. following these clues, we treat the whole context of medical museums, which transform values through situational construction, as the background to interpret the ethical implications of group val-ues that are transformed in the medical profession. in this thesis, we see medical museums, as the social prescription transforming medical profession, format the ritual context of situation ethics with the cultural construction of life-material. multi-disciplinary interactions and visiting itinerary can be transferred to the exploring horizon of research approach through description and interpretation. among them, we observe that humanistic elements have become essential equipment (or mat'eriel) of medical profession. though humanistic equipment (or mat'eriel) has its bottleneck in the museum situation, it can also unblock new possibilities for museum exhibitions, ethical practice or life ethics. as part of a wide research program aimed at developing new antitumoral agents, we present herein a series of stereoisomeric derivatives of fused tetrahydrofuranes (fthf) substituted with diverse protecting groups either at the primary or secondary hydroxyl groups. unprotected derivatives were also synthesised to investigate the influence of substituents on the in vitro activity of fthf. data on the synthesis, chemosensitivity and apoptosis induction by this series of fthf will be correlated to substitution pattern, stereochemistry and protecting groups, as an aid to the rational design of novel antitumour drugs. establishment of in vitro test systems for pulmonary edema resorption by peptide drug candidates dominik geiger, aswin mangerich, rudolf lucas, klaus p. schäfer, inge mühldorfer department of biotechnology, altana pharma ag, konstanz, germany; imc university of applied sciences, krems, austria tnf-␣ was found to up-regulate the rate of lung liquid clearance (llc) in several animal models by the activity of its lectin-like tip domain. this activity can be mimicked by a circular peptide designated tip. tip was shown to induce llc and consequently pulmonary edema resorption in different animal models. in order to study the mechanism of action of tip, we established two in vitro test systems for edema resorption with the human lung epithelial cell line calu- : ( ) the ability of calu- cells for spontaneous dome formation within confluent monolayers was utilized for quantitative examination of tip's activity on active transepithelial fluid transport ("dome assay"). ( ) the effects of tip on bioelectrical properties of polarized cell monolayers were studied by using the transepithelial electrical resistance (teer) technology ("teer assay"). dome assay experiments confirmed that dome formation is a sodium dependent process and that tip is able to increase this process. teer assay experiments proofed that tip acts in a polarized and dose dependent manner. in conclusion, there is strong evidence that the dome and the teer assays are suitable systems for in vitro activity testing of tip and other anti-edema peptide drug candidates and are useful for studies on their mechanism of action. increasing safety concerns in gene therapy result in more stringent regulatory requirements. those cover the complete process chain of cell banking, fermentation, and purification. a wide range of applications for pdna requires gram to kilogram amounts for clinical trials and market supply. economic, productive and robust processes are a prerequisite for low cost of goods (cogs). therefore manufacturer of biopharmaceuticals need to address these considerations by developing new production processes meeting the new standards. boehringer ingelheim austria developed a novel pdna production process suitable for large-scale cgmp production. the process is based on e. coli fermentation. the process contains no components from animal origin. the optimized fermentation process yields up to g pdna/l fermentation volume. for isolation of pdna from e. coli alkaline lysis in glass bottles or stirred tanks is commonly used. cell wall structure is destroyed by a combination of alkaline ph and detergents. in our process alkaline lysis is operated in a closed, continuous system directly connected to clarification without using enzymes. we developed a scalable process for pdna purification based on different chromatographic principles. the capture step is carried out by hydrophobic interaction chromatography followed by anion exchange chromatography as intermediate step. final polishing is carried out by conventional size exclusion chromatography in a group separation mode providing also buffer exchange and desalting for the final formulation. the process results in a pdna drug substance of highest quality containing a low level of impurities (genomic dna, rna, proteins endotoxines) suitable for therapeutic applications. depending on the conditions during fermentation and the used host homogeneities of greater than % or even % are possible, while a high over all yield can be achieved (∼ %). during the development monolithic chromatography supports (cim ® ) were compared with conventional resins and evaluated as potential alternatives. the complete process is monitored by a set of analysis covering cell banking to final purification. new sensitive methods based on hpce, hpiex and fluorometric measurement were developed. whereas many natural amino acids are currently produced by very cost efficient biological processes, the manufacture of methionine is still performed by traditional chemical synthesis. this was mainly due to the poor performances of the currently available producing strains that inhibited the development and commercialization of a biological process to l-methionine. metabolic explorer has recently reinvestigated and developed an efficient biological process that employs an engineered microorganism and utilizes a renewable starting material (corn sugar) as its feedstock and converts glucose into l-methionine. we will describe (a) the general scheme for the engineering of the host organisms and (b) the general approach to maximize carbon and reducing equivalent throughput to l-methionine. to highlight this effort, we will present the global approach developed to improve the process combining metabolic flux analysis, traditional protein biochemistry, molecular biology and fermentation optimisation. saccharomyces cerevisiae is an established 'work horse' of the fermentation industry and modern biotechnology has led to a spectacular expansion of the range of products that can be produced by this yeast. however, for the large-scale sustainable production of chemicals, it is equally important that the range of carbohydrate feedstocks be expanded. especially relevant in this respect is the ability to consume the pentose sugars d-xylose and l-arabinose, which make up a substantial part of plant carbohydrates. wild-type s. cerevisiae strains cannot metabolise d-xylose, but are capable of slowly metabolising its keto-isomer, d-xylulose. therefore, efficient conversion of d-xylose into d-xylulose has long been a key issue in yeast metabolic engineering. non-saccharomyces yeasts that is capable of growing on d-xylose use two enzymes, xylose reductase and xylitol dehydrogenase for this purpose. while both enzymes have been successfully expressed in s. cerevisiae, this is not always compatible with efficient product formation. for example, in the case of ethanol production, the different cofactor specificities of these two oxidoreductases cause massive byproduct formation. theoretically, introduction of a xylose isomerase, which catalyses the interconversion of xylose and xylulose, might circumvent these problems. however, it is notoriously difficult to express bacterial and archaeal xylose isomerases in s. cerevisiae and, until recently, activities of heterologous xylose isomerases expressed in s. cerevisiae were vanishingly low, at least under physiological conditions. a breakthrough was reached when, in , a xylose isomerase gene from the anaerobic fungus piromyces was expressed in s. cerevisiae. while this led to high activities of xylose isomerase, these were not enough to enable fast growth or product (ethanol) formation. in this presentation, we will discuss how a combination of metabolic and evolutionary engineering led to fast and efficient xylose utilization by engineered saccharomyces cerevisiae strains under aerobic as well as anaerobic conditions. furthermore, we will illustrate how evolutionary approaches can be applied to facilitate the utilization of mixed substrates. hyaluronic acid (ha) is a natural and linear polymer composed of ␤- , -n-acetyl glucosamine and ␤- , -glucuronic acid repeating disaccharide units with a molecular weight (mw) up to mda. it is a major constituent of the extracellular matrices and the synovial fluid. in the last decades, various fields of application including cosmetics, ophthalmology, rheumatology, tissue engineering and drug delivery have been explored, owing to the many important biological functions of ha and its unique physico-chemical properties. however, for some specific applications, the relatively high mw of ha is a limiting factor and the availability of low mw fractions would be highly desired. for food applications, low mw ha has been shown to penetrate the gastrointestinal barrier, thereby increasing the ha bioavailability. moreover, low mw ha fractions are able to re-establish the ha content in the skin and can thus be used in cosmetics as anti-aging and anti-wrinkle agents. finally, low mw ha has shown to prevent oxygen free radical damage in granu-lation tissue during wound healing. a range of methods has been described for the depolymerization of ha to low mw fractions. these techniques involve heat treatment, ultrasonication, uv/gamma irradiation, chemical and enzymatic degradation. we present results on a degradation process of ha originating from bacillus subtilis fermentation into well-defined low mw fractions. the process developed in lab scale is safe, well-controlled and produce low mw ha fractions with narrow polydispersity. moreover, the process is readily up-scalable. the low mw ha fractions are being evaluated in various cosmetic applications. metabolic pathway manipulation for improving the properties and productivity of microorganisms is becoming an established concept. metabolic engineering can be defined as the directed improvement of product formation or cellular properties through the modification of specific biochemical reactions or introduction of new ones with the use of recombinant dna technology. a detailed analysis of the physiological means of the different pathways is needed to be able to introduce modifications aimed to the production of not only important metabolites, but also to understand the fundamentals of cell biology. aimed to produce single compounds, metabolic engineering necessarily includes the modification of the cellular pathway(s) as well as the redirection of the energy toward the production itself. the existing metabolic engineering applications are the culmination of more than two decades of global experience developing processes for the production of fine chemicals, vitamins, nutraceuticals and animal nutritional aids such as amino acids. based on the relative low complexity, the first biotechnological applications have been developed from microorganisms. our laboratory has been engaged in this field since different years. yeasts like saccharomyces cerevisiae, kluyveromyces lactis and zygosaccharomyces bailii have been developed for the production of fine chemicals like lactic and ascorbic acids from d-glucose. in this contribution, we will present the last data obtained. since years amino acid production is in the focus of industrial microbiology. l-glutamate and l-lysine are produced with corynebacterium glutamicum, while escherichia coli is used for l-threonine production. the worldwide market of threonine drastically increases: in the amount of threonine produced worldwide was , t and raised to , t in and , t in . concerning predictions of experts the demand of threonine will rise with a two-digit rate of economic growth within the next few years. meanwhile the prices declined. these conditions enforce very efficient production processes. beside the strain development and an optimised downstream procedure the fermentation process is an important target for productivity improvement. strain development is dependent on detailed knowledge of the production strains. with innovative methods we are able to get a close look inside the cells under different culture conditions. these methods have been called 'omics' in recent literature. knowledge about genome, transcriptome, proteome, phosphoproteome, metabolome and fluxome leads to new ideas for strain improvements. data generated by these methods must be based on clearly defined culture conditions. therefore, highly parallel fermentations have to be performed to generate biological parallel samples. cutting-edge technical equipment is the basic requirement for experiments like this. other requirements are optimised sampling for different analysis, technical parallels of analytical steps and a detailed statistical analysis of data. these procedures guarantee distinction between real data and data noise. integration of all these data to a holistic model of the cell is the challenge for the future. by combination of a new strain, process and downstream improvements the plant productivity was increased drastically. we ended up in an optimized, fast and high yield process to scope challenges worldwide market comes up with. rieping, m., hermann, t. ( ); fermentation process for the preparation of l-threonine; wo/ . ) are potentially quite useful biocatalysts, as they allow for the regioselective and stereoselective hydroxylation of activated as well as non-activated carbon atoms. in addition, the large number of members of the p superfamily exhibits a wide diversity of specificities from which a useful biocatalyst may be selected. from a technical point of view, however, they have significant drawbacks. thus, they usually cannot be produced in large quantities nor recovered or stored without a severe loss in activity. their catalytic activity is mostly quite low, and their operational stability leaves much to be desired. most p enzymes require a complex protein/phospholipid machinery for activity, and the final electron donor in the reaction cascade, usually nadph, does require extensive recycling to arrive at a commercially satisfactory process. recently, the use of bacterial cytochromes as hydroxylation biocatalysts has received considerable attention. some of them are natural fusion proteins, which contain the heme and the reductase domains on a single polypeptide chain. they are catalytically much more active compared to, e.g. cytochromes occurring in human tissue. we thus have set out to further improve the technical applicability of these enzymes, and have centered our activities around several bacterial cytochromes. it proved very useful to apply rational mutagenesis and directed evolution to these enzymes, leading to a surprising compatibility of mutant enzymes with a wide variety of substrates. mechanisms for the limited stability of the enzymes were explored, leading to hybrid enzymes with enhanced stability, and the cofactor problem was alleviated using auxiliary enzymes or mediator-based technologies. as a result, a bioreactor based on microbial cytochromes was built and operated for several days. baeyer-villiger monooxygenases represent useful biocatalytic tools as they can catalyze reactions, which are difficult to achieve using chemical means. however, so far only a limited number of these monooxygenases were available in recombinant form kamerbeek et al. ( ) . using a recently described protein sequence motif fraaije et al. ( ) and the available genome sequence information, we were able to identify and overexpress a number of novel bacterial bvmos. one of the overexpressed bvmos was found to be relatively stable as it originates from thermobifida fusca, which grows at ∼ • c. the enzyme was shown to be active on a broad range of substrates, preferring aromatic ketones fraaije et al. ( ) . the best substrate discovered so far is phenylacetone, hence its name: phenylacetone monooxygenase. we have solved the crystal structure of phenylacetone monooxygenase, which represents the first structure of a bvmo malito et al. ( ) . the crystal structure provides insight into the complex mechanism of catalysis mediated by fadcontaining bvmos. by site-directed mutagenesis we have probed the role of several active-site residues. a crucial role is played by an arginine residue. as phenylacetone monooxygenase shares significant sequence identity (> %) with all known nadph-dependent bvmos, many of the observed structural features seem to be conserved within this class of atypical monooxygenases. by homology modeling using the phenylacetone monooxygenase structure, catalytic properties of other baeyer-villiger monooxygenases can be explained or predicted. screening for fungal baeyer-villiger monooxygenases l. butinar , j. friedrich , v. alphand : laboratory of biotechnology, national institute of chemistry, ljubljana si- , slovenia; groupe biocatalyse et chimie fine cnrs fre , université de la méditerranée, marseille, france the asymmetric form of the baeyer-villiger (bv) oxidation (transformation of ketones into lactones) is an important challenge for organic chemistry since the obtained lactones are valuable building blocks for synthesis of countless biologically active products. to date, enzymatic or microbial bv oxidations appears as more successful than their chemical counter-parts. (ten brink et al.) whereas most active bv monooxygenases are produced by bacteria (among which the well-studied enzyme of acinetobacter calcoaceticus), only a few fungal strains expressing bvmo were described (alphand et al., carnell and willetts) . in order to increase the number of available biocatalysts which perform such an asymmetric biotransformations, a screening of fungi belonging to major groups of zygo-, ascoand basidiomycetes was conducted using bicycloheptenone as testsubstrate. surprisingly, a large number of the tested fungi were able to transform the substrate into one to four different lactone isomers. the yields, the enantio-and regio-selectivity of the reaction depended on the fungal strain. alphand, v., furstoss, r., . j. mol. catal. b , - . carnell, a., willetts, a., . biotechnol. lett. , - . ten brink, g.j., et al., . pyranose oxidase (p ox) is a periplasmic enzyme that widely occurs in basidiomycetes. it catalyses the c- oxidation of several aldopyranoses to the respective -keto derivatives, transferring electrons to molecular oxygen to yield h o . p ox is of interest for carbohydrate conversions, as its reaction products ( -keto sugars) can be attractive intermediates in the production of food ingredients. we cloned the gene encoding p ox, and subsequently amplified a cdna clone by rt-pcr. the cdna was inserted into a bacterial expression vector and successfully expressed in e. coli. properties of the heterologous protein were compared to those of the native enzyme showing that they are essentially identical. both the native as well as the recombinant enzyme were used in biotransformations of sugars. recently, the d-structure of this tetrameric enzyme was elucidated. based on structural information, several enzyme variants containing point mutations were constructed and further characterised. two of these variants (e k and e r) displayed improvements in stability and certain kinetic properties thus making them attractive for biocatalytic applications. lactones are important compounds for the fragrance and flavour industry. right now the production of lactones is dependant on the import of crude materials from tropical countries. in this project, we want to tackle the manufacture of lactones via a biocatalytic route using p monooxygenases. cytochrome p monooxygenases catalyse the oxyfunctionalization of non-activated c-atoms. cyp a from bacillus megaterium, cyp a and cyp a from bacillus subtilis are soluble fusion proteins comprising p monooxygenase and fad/fmn reductase domains in one polypeptide chain. all three enzymes are highly homologous fatty acid hydroxylases. especially, cyp a also known as p bm- is well characterized and shows high activity compared to other p monooxygenases. the aim of the work is to change selectivity but conserve the high activity that is typical for those enzymes. using methods of structure modelling, rational protein design and directed evolution new mutants of these enzymes with changed regioselectivity are obtained. products of conversion with monooxygenases are intermediates in the production of lactones. the interface of biology and materials science has led to new materials with unique structural and functional properties, and new process technologies with the ability to produce, from "bottoms up", a wide range of biomimetic structures. these materials and their designs have broad application as catalysts, sensors, and devices for use in synthesis, cell and tissue engineering, bioanalysis and screening, and nanoelectronics. we have focused on the generation of sugar-based nanostructures, complete with tailored selectivities and biocatalytic activities at the molecular and nanoscales. these include biocatalytically-generated carbohydrate derivatives that selfassemble with high precision to give novel architectures with functional and responsive properties. izumoring: a strategy for total production of rare sugars ken izumori rare sugar research center, kagawa university, kagawa - , japan. e-mail: izumori@ag.kagawa-u.ac.jp we found a new enzyme, d-tagatose -epimerase (dte), that epimerize all ketohexoses at c- position. this epimerase catalyze not only between d-tagatose and d-sorbose, but also d-fructose = dpsicose, l-sorbose = l-tagatose, and l-psicose = l-fructose. this new enzyme offered us a useful key tool to connect all ketohexoses using hexitols as intermediates. the figure shows that all eight ketohexoses can be connected with dte and polyol dehydrogenases (pdh) in a ring. using this ring, we can easily find the pathway to transfer d-fructose to d-tagatose via d-psicose using dte and pdh. various aldose isomerases transform ketohexoses to the corresponding aldohexoses. so, we can connect all aldohexoses with ketohexoses using the enzyme. finally, all hexoses, ketohexoses, hexitols and aldohexoses are connected using enzyme reactions in a ring structure (not shown). this kind of strategy is effective also on transformation of tetroses and pentoses. now, we can produce all monosaccharides; tetroses, pentoses and hexoses by enzyme reac-tions. the bioproduction strategy of all rare sugars (monosaccharides that are rare in nature) is illustrated using ring form structures named as izumoring. we have already succeeded to produce d-psicose in large scale and are now in the progress of mass production of various rare sugars from natural and cheap sugars using izumoring. bioprocess development for chiral intermediates christian wandrey institute of biotechnology, forschungszentrum jülich gmbh, jülich d- , germany chiral alcohols, diols, amino alcohols and chiral acids (e.g. hydroxy acids and amino acids) play an important role in pharma and agro synthesis. in the past such chiral intermediates were obtained by racemic resolution via chiral reductions using prochiral precursors. here the problem of cofactor regeneration arises. this problem could be solved by enzyme-coupled or substrate-coupled cofactor regeneration using formate or isopropanol as reducing agent. alternatively, whole cell bioreductions were developed where glucose is used as the reducing agent. in recent years "designer microorganisms" were developed in which oxidoreductases (e.g. alcohol dehydrogenases) were over-expressed in escherichia coli. in such cases, cofactor regeneration was achieved intracellularly with isopropanol as the reducing agent or by coexpression of a formate dehydrogenase, so that once again format could be used for reduction. another route to obtain chiral intermediates is a fermentative approach using classical pathways (like the aromatic amino acid pathway). here, the pathway is interrupted after the intracellular production of chorismate. new chiral intermediates can be obtained by over expression of additional genes, which catalyzed the production of chorismate derivatives leading to cyclohexadiene-transdiols and the corresponding amino cyclitols. the last example can be regarded as an example of industrial biotechnology where glucose is used as starting material (white biotechnology). here bioprocess development is carried out in an integrated approach, in which molecular biochemical engi-neering cares for the optimal intracellular metabolic network and the classical biochemical engineering cares for the optimal environment of the cell in a fermenter. examples will be given which reach (in cooperation with industrial partners) up to kilogram scale. biotransformations are usually involved in just one or very few separate reactions in organic syntheses. the development of a cell-free "system of biotransformations" (sbt), in which a set of enzymes acts in a coordinated fashion in a one-pot synthesis, lead to increased catalytic complexity, selectivity and yield, as well as facilitated operation at reduced costs. the example chosen to prove the usefulness of the sbt-approach is the production of dihydroxyacetone phosphate (dhap). dhap, a c -compound from glycolysis, is an important precursor for asymmetric c-c-bond formation. so far, the production of dhap is difficult and expensive. for the construction of the dhap-producing sbt, e. coli's glycolysis is isolated from the metabolism to an as large as possible extent by the construction of a multi-ko-mutant. a culture is grown in an appropriate medium, homogenized in the production buffer, and used as the catalytic system. high production yields can be achieved since the production pathway is almost completely isolated from the metabolic network. the employed dhap-producing sbt provides not only a path from glucose to the product, but also an integrated atp-regeneration and nad-recycling system. in first experiments with a tpi-ko-mutant, a dhap-production yield on glucose of % could be achieved, without optimizing the system. the system remained active for more than h. up to now, atp cannot be applied in catalytic concentrations, but has to be present in equimolar amounts to glucose. the production yield could be increased by % through the addition of phosphate ions as substrate to the reaction, enabling the system to utilize atp more efficiently. these experiments indicate that the sbt-approach is viable and a large potential remains to improve the dhap-producing sbt. for some years novozymes have manufactured a pectate lyase for scouring of textile as an ecological alternative to the traditional harsh chemical treatment, and recently, we at novozymes discovered additional applications for pectate lyases. however, to be commercially attractive more robust pectate lyses had to be developed. in this paper, we will demonstrate how we for two different pectate lyases have improved their stability significantly. as the two enzymes are quite similar in sequence and structure, it was a new discovery for us to find that different concepts of protein engineering had to be used in our attempt to stabilize each individual pectate lyase. the stability of one enzyme was improved by substitutions in the internal of the structure whereas the stability of the other pectate lyase was primarily improved by changing surface residues. starting with knowledge from structural analysis, we have applied rational based protein engineering resulting in few selected variants. also random based protein engineering combined with screening of hundreds of thousands variants was used. in conclusion: the project team showed that by synergistic use of the two approaches, we were able to move faster towards a solution and eventually we succeeded finding new stabilized pectate lyase variants, applicable for new business areas. the importance energy independence as a national goal equals or exceeds that of the moon landing in . the development of a new industry to produce fuel ethanol from woody biomass would increase national security, improve employment and the environment, and provide substantial relief from the debt of imported petroleum. costs associated with the rapid development of this new industry (∼$ . billion per year) could be paid by re-assigning cent per gallon from existing federal gasoline taxes, a small price to pay for future energy independence. the corn-to-ethanol industry continues to make a remarkable contribution to our liquid fuel needs through expansion. today, one row of every six rows of corn is converted into fuel ethanol in the u.s. however, this expansion will be limited to - % of total automotive fuel by the economics of corn costs and production. corn can do more! corn stover is the single most abundant agricultural residue in the us and can be used as a feedstock to produce - gallons of ethanol per dry ton. further expansion with other biomass feedstocks such as agricultural and municipal residues (lignocellulose, woody biomass) could produce over billion gallons of fuel ethanol annually according at a recent joint report by the usda and doe (april, ) . current technology has been demonstrated at pilot scale for the production of fermentable sugars from hemicellulose by dilute acid hydrolysis and for the hydrolysis cellulose using fungal cellulose enzymes. biocatalysts such as recombinant escherichia coli have been developed and demonstrated for the efficient conversion of all sugar constituents of biomass to ethanol. a national goal for the full-scale deployment of current technology to produce biomass-based fuel ethanol will allow the us to reduce imported petroleum by %. together with increased efficiencies of hybrid vehicles, energy independence could be achieved within - years. similar gains could be realized by many nations around the world to provide new manufacturing and employment, redistributing wealth and ensuring a cleaner, healthier environment. bioethanol production using thermophilic bacteria marie just mikkelsen, birgitte k. ahring emab, biocentrum-dtu, lyngby, denmark the industry of bioethanol production is facing the challenge of redirecting the process from fermentation of relatively easily convertible but expensive starchy materials, to complex but inexpensive lignocellulosic biomass. on lignocellulosic hydrolysates, gram-positive thermophilic bacteria have unique advantages over the conventional ethanol production strains. the primary advantages are their natural broad substrate specificities, and in some strains, a high tolerance to lignocellulosic hydrolysates. moreover, ethanol fermentation at high temperatures also has the advantages of high productivities and substrate conversions, low risk of contamination and facilitated product recovery. some thermophilic bacteria naturally produce primarily ethanol from most sugar monomers present in lignocellulosics, but modifications are still necessary to increase ethanol yields. the release of useable sugars from lignocellulose biomass for industrial fuel-ethanol fermentation is often facilitated by a weak acid hydrolysis step. as a consequence, inhibitors such as furfural and -hydroxymethylfurfural (hmf) are formed as degradation products of xylose and glucose, respectively. moreover, the fermentative end-product of ethanol is also inhibitory. these, and other inhibitors present an environment, which elicits the expression of stress-related genes in saccharomyces cerevisiae. recently, s. cerevisiae genes have been identified as important in furfural stress tolerance. when furfural is present, yeast with these genes disrupted grows poorly compared to wild-type yeast. a sub-class of these genes suggests that yeast grown under furfural-induced stress may rely upon similar pathways as cells grown under various other stresses, including oxidative, heat, and sorbate. to investigate this link further, we analyzed stress-induced phenotypes such as ros activity, dna damage, and membrane damage in wild-type and mutant yeast exposed to furfural or hmf stress. moreover, we investigated whether overexpression of this sub-class of genes would provide protection from furfural-induced stress and oxidative damage. micro-organism to be used in fermentation of lignocellulose hydrolyzates should preferably have three characters: (a) high ethanol tolerance, (b) resistance to inhibitors found in the hydrolyzate, and (c) a broad substrate utilization range, since the hydrolyzate contains several sugars. in addition to the possibility of controlling the level of potential inhibitors, fed-batch fermentations also permit the parallel uptake of several different monomeric sugars. two strains of saccharomyces cerevisiae, cbs (a commonly used laboratory strain) and tmb (a strain isolated from a spent sulfite liquor fermentation plant), were characterized in batch and fed-batch fermentation of a dilute-acid hydrolyzate from spruce. the strains had different abilities to ferment spruce hydrolyzate. the study suggests that the furan reduction capacity of a yeast strain is a key factor for its performance in fermentation of lignocellulosic hydrolyzate. polyketides constitute a structurally highly diverse group of natural products that possess broad ranges of pharmacological properties and represent a major source for novel cancer therapeutics. however, these compounds may be sub-optimal in regard of activity, selectivity, availability and unwanted side effects. in addition, the sustainable production of these valuable metabolites can be a challenge. studying the molecular basis of the biosynthetic pathways may set the basis for improving the production and for rationally engineering derivatives with altered bioactivity profiles, e.g. through targeted knockouts, mutasynthesis ziehl et al. ( ) , and swapping of pathway genes. our results in elucidating and manipulating the biosynthesis of selected antitumoral polyketide metabolites from bacteria (aureothin, chartreusin) and fungi (cytochalasines, rhizoxin) are presented. analyses at the genetic and biochemical levels provided new insights into several unusual biosynthetic features, e.g. non-linear polyketide assembly for the nitroaryl-substituted polyketide aureothin he and hertweck ( , ) , an oxidative rearrangement cascade in the chartreusin pathway xu et al. ( ) , and a fungal iterative pks-nrps hybrid synthase schuemann and hertweck ( ) involved in cytochalasin biosynthesis. the most surprising result was obtained from elaborating the biogenesis of the antimitotic agent rhizoxin from rhizopus sp., which allowed for a significant improvement in large-scale production partida-martinez and hertweck ( ). he, j., hertweck, c., chem. biol. , , - chem. bio. chem. , , glycopeptides such as vancomycin and teicoplanin are the drugs of last resort for the treatment of severe infections caused by antibiotic resistant gram-positive bacteria. glycopeptides inhibit the peptidoglycan biosynthesis by binding as dimers to the d-ala-d-ala termini of the cell wall precursors. amycolatopsis balhimycina synthesizes the vancomycin-type glycopeptide balhimycin, whose structure and biological properties greatly resemble vancomycin and which only differs by its glycosylation pattern. using a "reverse genetics" approach we have identified the -kb gene cluster encoding the biosynthesis of balhimycin. by a combination of genetics, biochemistry and analytical organic chemistry, we were able to elucidate the biosynthetic pathway and to assign functions to almost all genes of the cluster. the biosynthesis starts with the pathway-specific provision of the non-proteinogenic amino acids ␤-hydroxytyrosine (␤-ht), hydroxyphenylglycine (hpg) and dihydroxyphenylglycine (dpg) which form together with (n-methyl)-leucine and asparagine the heptapeptide backbone of balhimycin. dpg is synthesized via a polyketide synthase mechanism (pksiii) similar to that known from plant chalcon/stilben synthases (pfeifer et al., ) . for the ␤-ht synthesis three genes are essential which form an operon (puk et al., ) : bpsd, an nrps binds a tyrosine molecule, which is then hydroxylated by the p monooxygenase oxyd. the perhydrolase bhp is required for the release of ␤-ht. subsequently bhaa, a nadh/fad-dependent halogenase catalyzes the chlorination of ␤-ht to form chloro-␤-hydroxytyrosine (puk et al., ) , which is needed to stabilize the dimerization. the amino acids are linked by non-ribosomal peptide synthetases (recktenwald et al., ) , and the aromatic side chains are interconnected by p monooxygenases; a series of reactions which lead to the first antibiotically active intermediate. inactivation of the oxygenase genes revealed the order of the cyclization steps (bischoff et al., ) : the oxygenases act in a stepwise fashion in the sequence oxyb, oxya and oxyc. the resulting cross-linked heptapeptide is then finally modified by methylation and glycosylation. the biosynthesis is regulated by the strr-type regulator bbr, which was shown to bind in front of different operons of the balhimycin gene cluster. this ensures the coordinated expression of the biosynthetic genes. the non-producing mutants, defective in the supply of the non-proteinogenic amino acids, were used as recipients in cloning experiments as well as in approaches of precursor-directed biosynthesis by feeding chemically synthesized alternative precursors. thus, novel balhimycin derivatives were generated (weist et al., (weist et al., , . bischoff et al., . angew. chem. int. ed. , - . pfeifer et al., . j. biol. chem. , - . puk et al., . j. bacteriol. , - . puk et al., . chem. biol. , - . recktenwald et al., . microbiology , - . weist et al., . angew. chem. int. ed. , - . weist et al., spectroscopy guided discovery of novel bioactive microbial natural products thomas ostenfeld larsen, michael edberg hansen cmb biocentrum-dtu, technical university of denmark, lyngby, denmark the task of finding novel bioactive natural products is usually bioassay driven. often a certain type of compound (e.g. polyketide, alkaloid) turns out to be active in an assay. when having generated a promising hit in a bioassay the normal procedure in the drug discovery process usually is to produce a large number of structurally analogous compounds either by traditional chemical synthesis or by combinatorial chemistry in order to study structure activity relation-ships and to find even more active lead compounds. alternatively to chemical synthesis of analogues nature can be explored for structurally similar compounds by uv-spectroscopy guided screening. this work will present a new method for the systematic and automated computer assisted search of full uv spectra in large number of datafiles for both dereplication of known and discovery of new natural products based on the use of the new mathematical algorithm x-hitting. exploring the substrate spectrum of the antibiotic producing bacteria saccharopolyspora erythraea p. krabben, p. oliveira, f. baganz, j. ward department of biochemical engineering, university college london, london wc e je, uk knowledge of substrate utilisation capabilities play an important role in the development of genome scale metabolic models (borodina et al., ) and refinement of first generation annotations. furthermore, knowledge of the product formation during catabolism of different substrates provides valuable information about the distribution of metabolic fluxes and thereby forms a basis for rational strain improvement. we present here, data on the substrate utilisation capabilities and the corresponding product formation of s. erythraea. this analysis will help in improving the production of erythromycin and provide clues to the activation of the cryptic secondary metabolic pathways present in the s. erythraea genome. reference borodina, i., et al., ( ) . genome-scale analysis of streptomyces coelicolor a ( ) modeling of growth and product formation on complex media containing multiple substitutable substrates is a challenge. complex media offers the organism multiple choices of carbon and nitrogen substrates including free amino acids, peptides, soluble and insoluble proteins in addition to the defined sources such as glucose and ammonium sulfate. we present a structured model that accounts for growth and product formation kinetics of rifamycin b fermentation in a multi-substrate complex medium. the model considers the organism to be an optimal strategist with a mechanism to regulate the uptake of the substrate combinations. further, we assume that the uptake of a substrate depends on the level of a key enzyme, which may be inducible. the model also considers control parameters as fraction of flux through a given metabolic branch. the control parameters are obtained using a simple multi-variable constrained optimization. the model parameters were rigorously estimated via a specifically designed experimental plan. the model correctly predicts the experimentally observed growth and product formation kinetics and the regulated simultaneous uptake of the substitutable substrates under different fermentation conditions. the model and the model parameters provide useful insights into the growth and product formation strategy of this industrially important process. this presentation will describe the experimental results, the model development and the relevant model parameters for a. mediterranei s . the recent surge in oil price and the increasing concern on our environment have generated much interest in the production of chemicals from renewable resources. succinic acid, also called as amber acid, is a four-carbon dicarboxylic acid, which can be used as a precursor of numerous products including biodegradable polymers, green solvents, pharmaceuticals, and bulk and fine chemicals. a new capnophilic bacterium named mannheimia succiniciproducens mbel e was isolated from the rumen of korean cow. this bacterium can produce large amounts of succinic acid along with some other metabolites such as lactic, formic and acetic acids. we have completely sequenced the genome of m. succiniciproducens and charaterized its genome content in the context of metabolic pathways. we then constructed the genome scale in silico metabolic network for metabolic flux analyses, and carried out metabolic flux analysis under varying environmental conditions. based on the in silico analyses results, we selected several target genes to be manipulated for enhanced succinic acid production. detailed results of metabolic engineering based on genome-scale information will be reported. we have been developing tools for inverse metabolic engineering in order to identify gene targets that improve the phenotype of industrial strains and cells for medical applications. to this end, we create genomic fragment libraries from a source organism and use it to transform the host organism. cells are properly selected in environments that favor the phenotype of interest and genes enriched in these cells are sequenced and used in follow up transformations of cells with specific genetic backgrounds. this overall strategy is complemented with additional tools for modulating gene over-expression, gene deletion, and high throughput clone isolation. we will demonstrate applications of this strategy to the identification of gene targets for improved xylose assimilation in recombinant saccharomyces cerevisiae and improved lycopene production in escherichia coli. based on assumed reaction network structures, nadph availability has been proposed to be a key constraint in ␤-lactam production by penicillium chrysogenum. in this study, nadph metabolism was investigated in glucose-limited chemostat cultures of an industrial p. chrysogenum strain. enzyme assays confirmed the nadpspecificity of the dehydrogenases of the pentose-phosphate pathway and the presence of nadp-dependent isocitrate dehydrogenase. pyruvate decarboxylase/ nadp-linked acetaldehyde dehydrogenase and nadp-linked glyceraldehyde- -phosphate dehydrogenase were not detected. although the nadph requirement of penicillin-gproducing chemostat cultures was calculated to be . - . -fold higher than that of non-producing cultures, activities of the major nadph-providing enzymes were the same. isolated mitochondria showed high rates of antimycin a-sensitive respiration of nadph, thus indicating the presence of a mitochondrial nadph dehydrogenase that oxidizes cytosolic nadph. the presence of this enzyme in p. chrysogenum has important implications for stoichiometric modelling of central carbon metabolism and ␤-lactam production and may provide an interesting target for metabolic engineering. bacteria and mammalian cells have been traditionally used as hosts for commercial recombinant protein production. however, in recent years, the insect cell-baculovirus system has emerged as a potentially attractive recombinant protein expression vehicle. although flow cytometry has been used widely for analysis of mammalian and microbial cells, there is very little information on applications of this powerful technique in insect cell culture. here we compared cell ratiometric counts and viability (propidium iodide and calcein am) of sf- cell cultures using flow cytometry to those determined by more traditional methods using a haemocytometer and the trypan-blue exclusion dye. flow cytometry has also been used to monitor various parameters during cultures of sf infected with the recombinant autographa californica nuclear polyhedrosis virus (acnpv) containing the inserted nucleic acid sequence amfp coding for am-cyan coral protein, which emits natural green fluorescence. complete elucidation of the genetic control of a metabolic flux requires the availability of fine-grained expression levels of the gene(s) of interest. we developed a collection of promoters of varying strength for tuning gene expression in the yeast s. cerevisiae. engineered promoters were obtained through random mutagenesis of the constitutive tef promoter. eleven mutated promoters were selected by fluorescence-activated cell sorting (facs) spanning gradually increasing activities between about and % compared to the native tef promoter. data were also confirmed at the level of mrna via rt-pcr. by introducing selectable markers in front of the different tef promoter mutations, we provided plasmid collections, which can be directly used to amplify promoter replacement cassettes for genomic integration of the fine-grained promoter collection in front of any yeast gene. l-arabinose, widely distributed in plant kingdom, is a component of plant cell wall. l-arabinose does not abundantly exist in free state in plants, but usually in corn hull, sugar beet pulp, gum arabic, mesquite gum, as the polysaccharide such as arabinoxylan and arabinogalactan. to produce arabinose from agricultural wastes, we screened arabinogalactan degradable strain from compost. thereafter, putative arabinase gene from this strain was cloned (b ts - ). as a result of spectrometric assay using -nitrophenyl ␣l arabinofuranoside, recombinant showed - -fold higher activity than wild type e. coli strain. after enzymatic reaction with corn fiber, b ts - produced . g/l of l-arabinose, which was detected on hplc. however, the enzyme activity was very low. so, we are transferring the gene into expression vector system. further characterization study and enzyme engineering to enhance the activity toward corn fiber will be presented in poster. there are only a limited number of hypersaline areas all over the world, which include several locations in turkey such as van lake located in eastern region of turkey. isolation and identification of halophilic and hyperhalophilic microorganisms from such locations is essential for the determination of biodiversity in turkey. high-level production of extremozymes from these microorganisms has also many economical advantages due to their stability at extreme reaction conditions. proteolytic enzymes are the most important group of enzymes produced commercially. of these, proteases produced by alkalophilic microorganisms are investigated not only in scientific areas such as protein chemistry and protein engineering but also find wide application in food, pharmaceutical, leather and detergent industries. in this study, microorganisms isolated from van lake were screened for the presence of extracellular alkaline protease activity. the optimum screening temperature and ph were determined as • c and ph . , respectively. one of the isolates that could grow at - % salinity reached highest levels of extracellular alkaline protease activity. this best producer, which was identified as the halotolerant bacillus pumilus, was found to produce alkaline protease both in the presence and absence of nacl. to improve enzyme production yields, culture conditions such as medium composition, growth ph and temperature were optimized. the effect of different carbon sources, organic and inorganic nitrogen sources on the production of alkaline protease was studied. whereas a mixture of inorganic and organic nitrogen sources induced high protease production, use of only an organic nitrogen source supported poor enzyme production. halotolerant bacillus pumilus produced maximum alkaline protease activity when maltose, yeast extract and sodium nitrate were used as carbon source, organic and inorganic nitrogen sources, respectively. this project was supported by tubitak through project tbag - t . in the market of biochemical products a very important role is played by heterologous proteins production, and despite recent advances in mammalian cells exploitation, yeasts can still present advantages as host systems. among them, the spoilage yeasts belonging to the zygosaccharomyces genus have become, due to some peculiar properties, significantly attractive. in particular, z. bailii is characterized by acid resistance, osmotolerance to high sugar and ethanol concentration combined with high biomass yield. despite still little is known about its genetics and cellular biology, our group is working on its development and exploitation for recombinant productions with an integrated approach coupling physiological study with the creation of molecular tools for heterologous proteins production. we previously described and did a patent application regarding the first techniques necessary to transform this yeast and to express and secrete different proteins derived from different sources. here we present and discuss the last advances in optimization of heterol-ogous protein expression in particular, on one side we present a reproducible strategy for target gene deletion, leading to the first z. bailii auxotrophic mutant, and on the other we show the improvement of gene dosage and plasmid stability by building a set of multicopy expression vectors based on the sequences of the z. bailii -like endogenous plasmid psb and an integrative plasmid. all the known ␥-butyrolactone autoregulator receptors are highly conserved in the dna binding motif present in their n-terminal portions and have been proposed to play roles as transcriptional regulators in antibiotic production and/or morphological differentiation. previously, kim et al. reported that the cloned scar in streptomyces clavuligerus has several characteristics of the autoregulator receptors in the genus streptomyces. in this study, to clarify the in vivo function of scar, a scar-disrupted strain was constructed by means of homologous recombination after introducing a scar-disruption construct via transconjugation from e. coli. no difference in morphology was found between the wild-type strain and the scar disruptant. however, the scar disruptant showed a . -fold higher production of clavulanic acid than the wild-type strain. the phenotype was restored to the original wild-type phenotype by complementation with intact scar. therefore, the autoregulator receptor, scar, acts as a negative regulator of biosynthesis of clavulanic acid but plays no role in cytodifferentiation of s. clavuligerus. lactate dehydrogenase catalyses the production of lactate from pyruvate. it is the first target for many researches on lactic acid producer microorganisms like rhizopus oryzae. in the present study based on the known sequences of r. oryzae ldha and ldhb genes skory ( ), they were cloned and expressed in a citric acid producer fungus aspergillus niger. the aspergillus niger strains expressing ldha or ldhb gene resulted in increased production of lactate in aspergillus niger. among transformants tested ldha and ldhb expressing strains were found to have higher lactate dehydrogenase activity compared to wild type in the conditions tested. the highest specific activity obtained with ldha transformants was only . times of the wild type while this was times for one of the transformants expressing ldhb. in addition to increased lactate production citric acid production was also increased. however, gluconic acid production ceased in the ldha or ldhb expressing a. niger strains. the production of lactic acid in a. niger transformants and lactate dehydrogenase a and lactate dehydrogenase b enzymes are being investigated in the chosen strains. selection of n source suitable for production of rhodococcus sp. biomass for the purposes of microbial transformation of ␣h-epoxypregnanolone ( ␣h) and -epoxy-pregnenolone ( ) into their ␣-hydroxy-derivatives was carried out. three dehydrated and three non-dehydrated n sources were tested. the transformation reaction was carried out in phosphate buffered medium containing g l − of the steroid substrate and . g l − cells. the steroids were determined by hplc. the transformation resulted in formation of three derivatives appearing in the reaction medium in the sequence: - -keto-; ␣-hydroxy-and ␣, ␤-hydroxy-epoxy-pregnenolone. a strong influence of the n source on the hydroxylating activity of the biomass was observed. triptose (difco) gave a cell depot actively hydroxylating ␣h without any significant accumulation of the - -keto-derivative. the most effective accumulation of hydroxylated derivatives of was observed with biomass grown on freshly prepared meat extract while the commercial products triptose (difco), meat extract (difco) and lactalbumin (flika) gave valuable information about the dynamics of the transformation process. biobleaching of kraft cellulose pulp by poliporus versicolor aysun ergene , nazif kolankaya : kırıkkale university, faculty of science and literature, department of biology, yahsihan, kırıkkale, turkey; hacettepe university, faculty of science, department of biology, beytepe, ankara, turkey the suitability of culture supernatant from poliporus versicolor for use in the biobleaching of kraft cellulose pulp was investigated. p. versicolor was found to grow on mycological broth ( % soytone, % d-glucose and . % cellulose pulp). maximal extracellular ligninase production was detected after day ( nkat). the optimum biobleaching conditions are • c and ph: . , with days. in this condition p. versicolor decreased the kapa number from . to . and increased brigthness from to . in day treatment. xylanase production, purification and characterization from a soil isolate, bacillus m- ayşegül ersayin , aytaç kocabaş , b. zümrüt ogel , ufuk bakir : biotechnology department, middle east technical university, ankara, turkey; food engineering department, middle east technical university, ankara, turkey; chemical engineering department, middle east technical university, ankara, turkey, . e-mail: ubakir@metu.edu.tr (u. bakir) xylan is a major component of the plant cell wall, representing up to % of the dry weight. xylan molecule is a complex polymer consisting of a ␤-d- , -linked xylanopyranoside backbone substituted with acetyl, arabinosyl and glucuronosyl side chains. hydrolysis of the xylan backbone is mainly catalysed by endo-␤- , -xylanases (ec . . . ). many bacterial and fungal species are able to utilize xylan as a carbon source. interest in the enzymology of xylan hyrdolysis has increased because use of xylanases in bioconversion of agricultural wastes to valuable products like single cell protein, xylo-oligosaccharides and fuel, in bio-bleaching processes, food and animal feed industries. in this study, xylanolytic nature of a soil isolate bacillus spp., bacillus m- , has been shown. bacillus m- produced multiple xylanases when grown on a liquid medium containing agricultural wastes as the sole carbon source. various agricultural wastes including corn-cobs and cotton waste, with and without pretreatments were used to maximize enzyme production. the major xylanase having molecular weight of kda upon sds-page and a pi of . upon ief was partially purified by liquid chromatographic techniques -fold with % recovery, including gel filtration, ion exchange and hydrophobic interaction chromatography. enzymes are important constituents in the laundry detergents due to their contribution to shortening washing times, reduction of energy and water consumption by lowering washing temperatures, provision of environmentally friendlier wash-water effluents and fabric care. however, they can loose a significant part of their activity in the chemically-hostile detergent matrix over a time period of several weeks. therefore, improving the storage stability of enzyme granulates is the main challenge in the development of a new product. the complexity of the detergent matrix implies the presence of a complicated mechanism involved in the inactivation of the enzymes. a combination of factors, such as oxidation by h o released by the bleaching agents, humidity, high temperature, autolysis of enzymes, high local ph in a granule, oxygen, and other detergent components, plays a role in the activity loss. an experimental investigation on the inactivation of the solid-state enzyme during storage has been initiated. the release rate of h o from the bleaching agent, sodium percarbonate, was determined using a simple and accurate method for measuring the gas phase h o concentration. the deactivation kinetics of pure enzyme was determined as a function of gas phase h o concentration and humidity. the preliminary results indicate that humidity plays a significant role in the inactivation mechanism of the detergent enzyme due to a possible increase in the mobility of the enzyme molecule and the surface area exposed to destructive agents. the effect of main granulate ingredients on the stability of the enzyme was investigated and the extent of the protection of each component was estimated. the study is important for the revealing of the phenomena occurring in the detergent matrix during storage. understanding the inactivation mechanism provides a valuable tool for the development of more effective protective coatings and stabilizers. the use of biosurfactants in cosmetic industry has attracted great attention of biotechnological researchers because they consist of two inexpensive, renewable and easily accessible starting agricultural materials: sugar and oil/fat. carbohydrate based products are non-toxic, biocompatible and biodegradable. in addition, the enzymatic processes present many advantages in comparison with the chemical methods, which employ high temperatures in the presense of alkalin catalysts, high-energy consuption and low selectivity of products. sugar esters present vast application, such as for antibiotics, biomaterials, surfactants, cosmetics and so on. we investigated the synthesis of sugar vinyl esters, using protease-catalysed transesterefication method applying protease from bacillus subtilis. sucrose . m and vinyl ester . m has been mixed in dimethylformamide at rpm of agitation. at first, we have studied the effects of protease from bacillus subtilis concentrations ( , , and mg/ml) as catalyst. afterwards, the influence of the temperature ( and • c). after that, the influence of the molar ratios ( : ; : ; : m/m) between vinyl laurate (ch (ch ) cooch ch ) and sucrose. subsequently, we investigated the effects of water amount, using , , and % of water in dmf. the conversion ratios of sucroseto-sucrose esters were determined decreasing sucrose measurement with hplc. the results showed that the best conditions to produce high activity on the enzymatic reaction was by using mg/ml of protease from bacillus subtilis at • c, molar ratio of : (vinyl laurate:sucrose) and adding % of water in dmf. finally, we succeeded in the characterization of vinyl sugar ester, which was produced after hours of reaction by c nmr. the results confirmed the c substituted sugar mono-ester ( -o-vinyl lauroyl sucrose). effects of oxygen transfer on ␣-amylase production by b. amyloliquefaciens nurhan güngör, güzide Ç alık bre lab, department of chemical engng, ankara university, ankara, turkey ␣-amylase (e.c. . . . ) a commercially important enzyme used in food, textile, detergent, brewing, paper, and animal feed industries; hydrolyses ␣- , glucosydic bonds in amylose, amylopectin, and related polysaccharides. optimum medium composition and influence of bioreactor operation parameters on ␣-amylase production with high yield and selectivity were determined together with the metabolic flux distributions using b. amyloliquefaciens (nrrl b- ), which is found to be a good producer of the enzyme. systematic investigation of oxygen transfer in relation with the metabolic fluxes for ␣-amylase is not available in the literature. shake-flask experiments were conducted in . dm air-filtered bioreactors in orbital shakers with agitation and heating controls (b. braun, certomat-bs ). laboratory-scale bioreactors were composed of agitation, heating, foam, dissolved-oxygen and ph-controlled . and . dm systems (b. braun, biostat q; and chemap). after separation of the cells with a sorval rc s ultracentrifuge, ␣-amylase activity was measured by the dns method bernfeld ( ) . amino acid concentrations were determined with a hplc (waters), pro-tein and organic acid concentrations were measured with a hpce (waters, quanta e) Ç alik et al. ( ) . oxygen transfer characteristics in the bioreactor systems were calculated using the dynamic method rainer ( ) . in the mass flux balance-based analyses, a pseudo-steady state approximation for the intracellular metabolites and the accumulation rates of the extracellular metabolites measured throughout the fermentations in considerations of the biochemical feature of the system were used to acquire the flux distributions. in laboratory scale, the effects of different c sources, i.e. glucose, fructose, maltose, lactose and soluble starch; n sources, i.e. (nh ) hpo , (nh ) so , and nh cl and/or their concentrations; and the operation parameters, ph and temperature on cell growth, substrate utilization, ␣-amylase and by-product concentrations, and ␣-amylase activity were investigated. in pilot scale, the fermentation and oxygen transfer characteristics of the bioreaction system together with the metabolic fluxes were determined. bernfeld, p., . methods in enzymol. : - . Ç alık, p., Ç alık, g.,Özdamar, t.h., . enzyme microb. technol. , - . rainer, b.w., . geldanamycin is a benzoquinone ansamycin produced as a secondary metabolite by the actinomycete, streptomyces hygroscopicus var. geldanus, in submerged culture. it is a broad-spectrum antibiotic and exhibits an anti-tumour activity through its interaction with the heat shock protein family of chaperone proteins. the optimal recovery of geldanamycin from fermentation broths is the focus of the presented work. the application of adsorbent resins was assessed and the viability of developing a solid phase extraction process for geldanamycin was determined. it was found that recovery of geldanamycin from fermentation broth was possible using adsorbent resins and the use of resins facilitated the recovery of a product stream of high purity. the composition of the fermentation broth had an impact on the performance of the resins and it was found that assessing performance on the basis of experimentally derived data was more apt than studying the kinetics of adsorption alone. adsorptive processes are, by their nature, difficult to optimise and this was found to be the case when optimising the recovery of geldanamycin from partially clarified fermentation broth. considerable effort was required to optimise geldanamycin adsorption, via examining the effect of environmental conditions and process system configuration, and geldanamycin desorption, via examining the effect of environmental conditions and investigating selective elution patterns. it is well known that halophilic eubacteria synthesize compatible solutes in order to face the high ionic strength environment in which they proliferate. these biomolecules are gaining more and more importance as biotechnological tools in a wide array of applications, and the recently developed novel bioprocesses enabled large-scale production of these compounds and therefore commercial distribution. however, there is still interest in the optimization of the production process of sole hydroxyectoine that was demonstrated to have a superior stabilization capacity. in this research project, we optimized growth conditions of marinococcus m to obtain high yield of hydroxyectoine. their production proved faster in the batch experiments at a higher oxygen supply, even if the stationary phase was comparable in all cases. the mf experiments showed a final biomass which was -fold that obtained in the corresponding batch process. in addition the monitoring of compatible solutes production showed that in the last h experiment hydroxyectoine accounted for - % of the total content, accumulating up to - % of the cell dry weight. studies for improving downstream process for ectoine and hydroxyectoine recovery showed that short permeabilization cycles in water are effective in a temperature range between • c and • c using a ratio : /biomass:water. moreover, we evaluated the ability of ectoine to stabilize lactic acid bacteria during freeze-drying and to protect human cells from heat stress. in particular, the compatible solutes were added to the medium of confluent keratinocytes before subjecting the cells to heat stress, or lps insult. rt-pcr and western blot analysis demonstrated the hsp b' gene over-expression in heat stressed human keratinocytes treated with ectoine. finally, we demonstrated that even at low concentration ( mm) these compatible solutes are able to diminish cell death in lactic acid bacteria due to lyophilization procedure. among all existing alternative energy sources, biomass-derived bioethanol is especially advantageous since it is clean, sustainable and potentially inexpensive. the actual production of bioethanol is divided into a pre-treatment step, an enzymatic hydrolysis step and a fermentation step. while fermentation has been practiced by humans for centuries, our knowledge of enzymatic hydrolysis is still limited. nevertheless, it is well accepted that hydrolysis is a synergism among three classes of enzymes, ␤-glucosidase, endoglucanase and cellobiohydrolase. furthermore, a complete and efficient hydrolysis is only achieved when the enzymes are in the correct proportion. the common enzyme proportions have so far been based on the natural enzyme abundance as produced by the microorganism or mainly been determined by a trial-and-error approach. in this study, however, we used metabolic control analysis (mca) as a modelling tool to gain fundamental knowledge about enzymatic hydrolysis and to design an optimal enzyme mixture. using gepasi, a free software, the degree of control of each reaction step or each enzyme towards the overall hydrolysis can be calculated. our hypothesis is that the amount of each enzyme used for hydrolysis should be proportional to the degree of control of the enzyme. with mca, a significant amount of time, labour and reagents can be saved on developing hydrolysis enzyme mixture. furthermore, this study should demonstrate the usefulness of mca on understanding enzyme-catalyzed reactions outside the cell. process optimization for fed-batch fermentation of bacillus thuringiensis subsp. israelensis arindam chaudhury, gopinathan c department of biotechnology, university of calicut, calicut, kerala india. e-mail: g achaudhury@umassd.edu (a. chaudhury) bacillus thuringiensis (bt) is a desirable biopesticide because of its low cost and lack of toxicity. the use of bt in developing countries is limited due to process complications and economic non-feasibility of the fermentation process. in the present study, we have shown how regional production, using inexpensive alternatives for carbon and protein sources, can effectively reduce the cost of mass production of bt. while using alternative media supplements, the biomass production, nor the larvicidal activity was hampered. in addition, the positive effects of sparged aeration and the indispensable role of yeast extract were also proved. this work provides the first experimental proof of delineating the sporulation process and delta-endotoxin production. the role of various buffering agents and additives in increasing biomass production and early sporulation were also investigated. for the production of coenzyme q (coq ), an electron carrier in the respiration chain with antioxidant activity. with decrease of dissolved oxygen level from to %, the intracellular coq content increased about -fold, yielding mg per g-dry cell weight at % dissolved oxygen level. azide significantly increased the intracellular coq content, with the highest value of . mg per g dry cell weight in the presence of . mm of sodium azide. however, dnp (up to m) and h o (up to m) did not affect the intracellular coq content, indicating proton gradient release and oxidative stress do not affect the synthesis of coq . these results show that restricted electron flux by limited oxygen supply and the addition of azide increases the intracellular coq content. fourier transform infrared spectroscopy (ft-ir), combined with in situ heat sterilizable attenuated total reflection (atr) probes, constitutes a promising and versatile technique for on-line monitoring of bioprocesses. the ft-ir enables rapid determinations of the medium composition without the requirement of sample withdrawal and preparation. in this work the concentration levels of the substrates glycerol and methanol were monitored on-line in pichia pastoris cultivation. partial least squares (pls) models were used for obtaining the concentration readings. the glycerol concentration measurement proved to be very reliable and reproducible in the glycerol batch phase. however, the on-line information regarding the glycerol concentration was not utilized for any process control purposes. on the other hand, the availability of on-line information about the methanol concentration proved to be crucial for the successful implementation of the cultivations. the temperature strategy in the methanol fed-batch phase utilized temperatures as low as • c. in order to keep the metabolic activity at a reasonable level the culture was therefore pushed towards the maximal substrate consumption rate, rather than being a conventional substrate limited fed-batch. as a consequence methanol accumulation occurred on occasions. without on-line information about the concentration this accumulation, if sustained, would have resulted in a poisoning of the culture, either from methanol itself, or perhaps more importantly from formaldehyde. therefore, it can be concluded that the ft-ir/atr instrument was very useful in this application. jørgensen department of chemical engineering, denmark technical university, building , dk- lyngby, denamrk. e-mail: fpd@kt.dtu.dk (f.p. davidescu) modeling biochemical reaction network in microorganisms still represents a challenge due to the very large number of enzyme catalyzed biochemical reactions, to the very complex system and to the many feed-forward and feedback regulation mechanisms. the presented approach to model such a system is based on the stochastic grey-box modeling framework proposed by kristensen et al. ( ) . this methodology consists of parameters estimation based on a prediction error method followed by different statistical tests for parameter significance and for model (in-) validity. the methodology furthermore allows estimation of unknown functional relations, e.g. kinetic rates. a set of experimental data zangirolami ( ) obtained during continuous cultures of a high enzyme producing aspergillus oryzae strain. the oxygen concentration was decreased stepwise and the substrate concentration was modified from one experiment to other. a model proposed by agger et al. ( ) is investigated on these data. the primary interest is to develop a physiologically feasible model, also at the low oxygen concentrations often found in industrial practice. microbially produced secondary metabolites such as antibiotics have tremendous economic importance. streptomyces spp. have long been identified as sources of antibiotics and chemotherapeutic compounds, synthesising over bioactive compounds. geldanamycin is a novel chemotherapeutic agent produced by streptomyces hygroscopicus var. geldanus in submerged fermentation. initial studies have focused on optimisation of media design through understanding and controlling metabolic routes of biosynthesis within the cell. geldanamycin is a by-product of the shikimate or aromatic amino acid biosynthesis pathway. stimulation of this pathway and concomitant production of geldanamycin is achievable through amino acid control. increasing concentrations of primary carbon source greatly influence biomass generation and product formation, as does the inclusion of cations such as magnesium and calcium to the fermentation media. optimisation of production media through balancing minerals, nitrogen, and carbon sources has significantly improved antibiotic yields in shake flask cultures and the development process will be extended into pilot scale through the use of bioreactors. microbiology and biotechnology research group, school of life sciences, napier university, edinburgh, eh dt, scotland. email: m.el-mansi@napier.ac.uk (m. el-mansi) synopsis: during growth of corynebacterium glutamicum on glucose or other glycolytic intermediates, pep carboxylase fulfils an anaplerotic function as it replenishes intermediary metabolism with biosynthetic precursors that are essential for growth and glutamate production. under these conditions, pep carboxylase plays a central role and this in turn is characterised by a high flux control coefficient thus rendering this enzyme an ideal target for metabolic interventions. further analysis in silico revealed that any increases in the concentration of the enzyme was accompanied by increases in flux through the enzyme itself as well as glutamate formation, presumably as a consequence of sustaining a high intracellular level of ␣-ketoglutarate; the immediate precursor for glutamate biosynthesis. a combined approach to enhance periplasmic expression of human growth hormone in escherichia coli, using a modified signal peptide from alpha amylase gene of bacillus licheniformis s.k. falsafi , , a. zomorodipour : islamic azad university of jahrom, iran; department of mol genet. national inst for genet eng & biotechnol., tehran, iran. e-mail: soheil falsafi@yahoo.com (s.k. falsafi) the alpha amylase gene signal peptide, originated from a strain of bacillus licheniformis, was shown to be able to transport its native protein, when expressed in e. coli the competence of the fusion protein being processed and translocated through the inner membrane is highly dependent on the amino acid sequences in the signal peptide. therefore, in order to increase the expression efficiency of bla signal peptide, we reconstructed the bla signal peptide coding fragment with the following modifications. two rare codons of arg (cgg) and arg (cga) and codons for leu (tta) and pro (cct), in the signal peptide were substituted with their corresponding e. coli major codons. two other changes, including phe (ttc) → leu (ctg) and ala (gcg) → met (atg), were also introduced to increase the processing efficiency. the hgh-expressing plasmid equipped with the modified bla (blaf ) was subjected for further expression analysis in a t -based expression system. the results obtained from the protein patterns of the induced bacteria indicates in high expression level of hgh preprotein (hgh::blaf ) followed by efficient transfer of the mature hgh to e. coli periplasm. (ip ) has been demonstrated to have a wide range of health benefits such as prevention and therapy of various cancers, amelioration of heart disease, and prevention of renal stone formation as well as complications from diabetes. on the hand, lower phosphorylated forms of inositol, especially inositol trisphosphate (ip ) and inositol tetrakisphosphate (ip ) are important signal transduction molecules within the cells both in plants and the animal kingdom. it has been hypothesized that at least the anticancer function of ip is mediated via these lower inositol phosphates. the diversity and practical unavailability of the individual myo-inositol phosphates preclude their investigation. phytases, which catalyze the sequential hydrolysis of phytate, render production of defined myoinositol phosphates in pure form and sufficient quantities. different phytases may result in different positional isomers of myo-inositol phosphates and therefore different biochemical properties. phytases differing in ph optima, substrate specificity, and specificity of hydrolysis have been identified in plants and microorganisms. in this paper the dephosphorylation pathway of the novel phyfauia was compared to other bacterial phytate degrading enzymes. preliminary results have shown that phyfauia converted ip into ip (myoinositol , , , , pentakisphosphates) and another isomer, which is yet to be elucidated. characterization of the novel ␤-peptidyl aminopeptidase (bapa) from sphingomonas sp. - w that cleaves synthetic ␤peptides birgit geueke, hans-peter e. kohler environmental microbiology, eawag, duebendorf, switzerland. e-mail: birgit.geueke@eawag.ch (b. geueke) non-natural peptides, which are capable of evoking a specific biological response, are currently receiving much attention. oligomers of ␤-amino acids (␤-peptides) are representing a group of pharmaceutically interesting peptides because of their very high stability towards enzymatic degradation and their ability to mimic the structure of naturally occurring biologically active peptides. the pharmaceutical potential on the one hand and the high stability on the other hand aroused interest for studies on the environmental fate and the degradation behaviour of this class of compounds. a novel bacterial strain (sphingomonas sp. - w ) that was capable of degrading short ␤-peptides was isolated from an enrichment culture. the ␤peptide degrading enzyme was purified and its gene sequence was determined (bapa). the gene encodes a ␤-peptidyl aminopeptidase (bapa) of amino acids that is synthesized as preprotein with a signal sequence of amino acids. it belongs to the n-terminal nucleophile (ntn) hydrolase superfamily and is the first peptidase that is capable of cleaving amide bonds in ␤-peptides composed of synthetic ␤-amino acids. the biochemical properties of recombinant bapa were investigated regarding its substrate specificity and possible application in the synthesis of ␤-peptides. to produce efficient strains of agaricus bitorquis (quel.) saccardo, which are resistant to high temperatures p. guler, a. ergene, s. tan kirikkale university, faculty of science and literature, department of biology, yahsihan-kirikkale in this study, the culture mushroom agaricus bitorquis (quel.) sacc. the growth of the mycelium and the fructifications under high temperature is examined. the spores taken from the mushrooms that were collected from nature were grouped as a, b, c, d, e. the spores were inoculated into malt extract agar and incubated at • c and primer mycelium was produced. the mycelium discus taken from primer mycelium in mm diameter were inoculated into the center of malt extract agar and incubated at , , , and • c separately. during the incubation period the growth of the mycelium were measured. during the growing period the radial growth speed of the mycelium were taken as criteria. the best mycelium growth for all groups was seen at • c. at • c the e group mycelium and at • c other group's mycelium did not grow. these temperatures were determined as thermal lethal point for the groups. from all the mycelium produced from all temperatures spawn was prepared and with the results taken from these, spawn calendar is prepared. in this research, the spawn was inoculated to compost with mixing system and separately put in culture rooms, temperatures as and • c. at this level the culture mushroom production techniques were used. the harvested mushrooms were inspected morphologically. at this morphological inspections the cap width, cap tissue thickness, stalk thickness and stalk long ness was taken as criteria. in the study the best growth was seen at d group mushrooms and this group mushrooms tyrosinase's activities were measured and graphics were made. introduction: viral contamination of biological products; cause many problems in viral diagnostic laboratories, blood transfusion organizations, and biological producers. bovine viral diarrhea virus (bvdv), from the pestivirus genus, is the most common viral contamination in (fetal) bovine serums (fbs). also, bvdv used as a module, for study hepatitis c virus inactivation due to its similarity in structure and genome. pulsed uv lights (puvls) have this potential to inactivate known and unknown or reemerging viruses as well as prions. two puvl with the wavelengths of and nm, were produced by q-switched nd + :yag laser in its third and forth harmonic, respectively. the energy of each pulse for nm was . mj/cm and for nm was . mj/cm . bvdv were produced and titrated in mdbk cell line. mdbk and fbs were already checked for non-cytopathic or cytopathic pestiviruses, using related ag-elisa kit. bvdv suspended in solution with the dilution of : before exposure. the quartz tube with the minimum uv-absorption in compare with air, used as a container for exposed solutions. calculation of the virus titer, . tcid /ml, was done based on the reed and muench method. bvdv suspended in pbs was exposed into the . - j/cm of puvls with the wavelength of nm and also, was exposed into the . - . j/cm of puvls with the wavelength of nm. furthermore, bvdv suspended in fbs was exposed into the , , and j/cm of puvls with the wavelength of nm and also, was exposed into the . , . , . , and . j/cm of puvls with the wavelength of nm. results: the minimum dose for inactivation of bvdv suspended in pbs with the and nm wavelengths of puvls, were and . j/cm , respectively. also, the minimum dose for inactivation of bvdv suspended in fbs with the and nm wavelengths of puvls, were and j/cm , respectively. to evaluate the fbs quality to support cell culture, treated fbs with the dose of . j/cm of nm puvls was used to grow vero cell line in successive passages. the viability of cells in two study groups was identical. the statistical evaluation of two treated groups showed no significant difference, in passages. conclusion: because inexpensive equipment can be used to produce puvls capable of handling different volumes of biologics with operational ease, this viral inactivation technique is cost effective for relevant industries. the procedure has the potential to be combined synergically with other inactivation method. puvls offer a new, nonadditive and chemically safe alternative for the treatment of fbs to inactivate adventitious viruses and to preserve the biological activity necessary for the propagation of cell culture. characterization and gene cloning of the g-resorcylic acid decarboxylase for application to selective production of g-resorcylic acid y. iwasaki , y. ishii , k. kino , k. kirimura : dept. appl. chem., sch. sci. eng., waseda univ., tokyo, japan; bme, asmew, waseda univ., tokyo, japan. e-mail: iwasaki@moegi.waseda.jp (y. iwasaki) for selective production of ␥-resorcylic acid (␥-ra, , -dihydroxy-benzoic acid) from resorcinol (re, , dihydroxybenzene) under mild conditions, we screened various microorganisms and found the reversible ␥-ra decarboxylase (rdc) as a novel enzyme applicable to carboxylation of re to form ␥-ra, in a bacterial strain rhizobium radiobacter wu- ) . rdc catalyzed the decarboxylation of ␥-ra, and regio-selective carboxylation of re to form ␥-ra, without formation of ␣-ra and ␤-ra. the molecular weight of rdc was estimated to be kda by gel-filtration, and that of the subunit was determined to be kda by sds-page, suggesting that rdc is a homotetrameric structure. the gene encoding rdc was sequenced, and a site-directed mutagenesis study revealed that the two histidine residues at positions of and in rdc are essential for the catalytic activity of rdc. through the reactions using e. coli cells highly expressing rdc, . mm ␥-ra was produced from mm re at • c for h, with a yield of %. ishii, y. et al., . biochem. biophys. res. commun., , - . laccase biosynthesis in stirred fermenters teresa jamroz, stanislaw ledakowicz, barbara sencio department of bioprocess engineering, technical university, lodz pl - , poland industrial applicability of enzymes is closely related to development of efficient methods of their production. currently, significant interest in lignolytic enzymes, including laccase, has been observed. laccase is an enzyme applied in various industrial branches and environmental processes. broad laccase applicability induces researchers to develop urgently efficient methods for its commercial production. laccase (ec. . . . . p-diphenol oxidase) is produced by cap mushrooms from the class basidomycetes. this is the so-called white rot fungi which in natural conditions appears on both living and dead wood. as shown in the practice of biotechnological processes, high-efficient strains have low resistance to destructive factors in bioreactors. hence, to preserve a proper morphology and physiological state of an organism, strictly determined culture conditions must be obeyed. this is very important in the case of basidomycetes for which submerged culture in the liquid phase is not a natural habitat. results of studies on laccase production from cerrena unicolor family are discussed. cultivation of active biomass was carried out in stirred tank and rotating disc bioreactors of different volume (b. braun of working volume dm ; fas- of working volume . dm ). experiments in both fermenters were made at impeller revolutions and min − , on a modified lindberg substrate. significant differences in the rate and yield of laccase production were reported. an almost three times higher values of laccase activity were obtained in the b. braun fermenter, at rotational speed min − . to retain suspended cells in bioreactor a filtration process can be used. the biomass is concentrated by withdrawing cell-free culture broth. if the desired product is dissolved in the broth (extracellular production), the procedure enables the continuous harvest in the cellfree permeate. an application test of filtration system for suspended biomass of aspergillus niger in submerged single stage continuous culture was presented in this report. the system is easy to construct and there is a possibility of its sterile exchange during cultivation. the culture medium contained the following substances (g/dm ): white sugar, . ; nh no , . ; mgso · h o, . ; kh po , . ; feso · h o, . . fermentations were carried out in the lab bioreactor biomer . the bioreactor was a standard cstr (continuous stirred tank bioreactor) with working volume of dm . high citric acid concentration in culture medium (p = . g/dm ), high yield of citric acid (y p/s = . %) and high efficiency coefficient (k ef = . ) were observed in single stage continuous culture with biomass retention. oxygen transfer regulates benzaldehyde lyase production in e. coli pınar Ç alık, iblab, department of chemical engineering, metu, ankara, turkey. e-mail: pcalik@metu.edu.tr the effects of oxygen transfer rate on benzaldehyde lyase (bal) production by puc ::bal carrying recombinant escherichia coli on a defined medium with . kg/m glucose were investigated in order to fine-tune the bioreactor performance, in v = dm batch bioreactors at five different conditions with the parameters at, i.e. q o /v r = . vvm and n = , , , min − and; q o /v r = . vvm and n = min − . the concentrations of the product and by-products amino acids and organic acids were determined in addition to bal activities. medium oxygen transfer rate conditions and uncontrolled ph operation at ph o = . are optimum for maximum bal activity, i.e. u/cm at h, and productivity and selectivity. on the bases of the data, response of the intracellular bioreaction network of r-e. coli to oxygen transfer conditions were analysed using a mass-flux balance based stoichiometric model that contains metabolites and reaction fluxes. the results reveal that metabolic reactions are intimately coupled with the oxygen transfer conditions. oxygen transfer rate showed diverse effects on the product formation by influencing metabolic pathways and changing metabolic fluxes. metabolic flux analysis was helpful to describe the interactions between the cell and the bioreactor by predicting the changes in the fluxes and the rate controlling step(s) in the metabolic pathways. therefore, knowing the distribution of the metabolic fluxes during the growth, and bal and by-product formations provide new information for understanding physiological characteristics of the r-e. coli, and reveals important features of the regulation of the bioprocess and opens new avenues to successful application of metabolic engineering. saprophytic mycobacterium strains belong to the best known microorganisms which have been applied to the pharmaceutical industry for the production of steroid drugs. the mycobacterial cell wall is the permeation barrier to chemical compounds, including lipophiles. using isoniazid (inh), the inhibitor of the mycolic acids biosynthesis, we were able to demonstrate increased ad production and susceptibility to antimicrobial agents. the process of sterol transformation and products accumulation was monitored using gas chromatography. isoniazid was shown to intensify ␤-sitosterol side-chain degradation by mycobacterium sp., and accumulation of -androstene- , -dione (ad) and , -androstadien- , -dione (add), which are the starting materials in the biotechnology of medically important steroids. to confirm these results, the sensitivity of the bacteria to antimycobacterial drugs was performed. the minimum inhibitory concentration mic of rifampicin and erythromycin decreased markedly in the presence of inh. this work was supported by grant nr p c of the committee for scientific research. for the purpose of high-throughput screening and to reduce experiments with animals in pharma biotechnology biosensor systems gain importance. the principle of a biosensor is the combination of cultured cells and a sensorchip device, which allows the monitoring of cellular activity. in contrast to traditional analytics with a biosensor you can measure on-line cellular activity change caused by an effector as well as the restored activity after privation of the effector (re-native activity). cmos technology can be used for the realisation of various biological sensorchips such as adhesion sensorchips, metabolical sensorchips and electrophysiological sensorchips. the standard cmos technology allows a high reproducibility of the chips, the integration of electronic components on the chip, which reduces the amount of external devices and the combination of different sensors on one chip. in cooperation with the semiconductor company micronas and the biotech company bionas we have realised different types of cmos sensorchips to measure adhesion of a cellular monolayer with interdigitated electrodes (ides), metabolical activity via acidification with ion-sensitive field effect transistors (isfet) and sponteneous neuronal network activity with passive palladium electrodes. microbial agents have been applied to the different stages of pulp and paper processing. the work presented describes a study on the effect of applying ligninolytic enzymes, such as a laccase plus mediator system, on a variety of different types of pine and eucalyptus pulps and subsequently subjecting these to different ageing processes. industrial pulps were obtained from different portuguese pulp and paper companies. the pulps used were ( ) unbleached pine pulp from portucel tejo; ( ) unbleached eucalyptus pulp from portucel setúbal; ( ) bleached eucalyptus pulp from portucel setúbal; and ( ) pulp made from recycled paper from renova s.a. several types of handsheets were produced with different grammage namely, and g/m . the prepared handsheets were subject to an aging sequence in three different chambers: ultraviolet radiation (wavelength of nm), temperature ( • c) and moisture ( %); and thick saline fog at a concentration of % and temperature of • c. in order to evaluate the effect of moisture cycles and temperature, two aging sequences were used for each type of handsheet. in the first, the moisture varied ( , and %), while the temperature was held constant ( • c); in the second the temperature varied ( , and • c) and the moisture was held constant ( %). following the aging phase, the handsheets were subject to several chemical (viscosity and index kappa) and physico-mechanical (colour, tensile breaking strength, stretch and the bursting strength) tests in order to characterize the effect of the aging conditions. results will be presented describing the effect of application of the laccase-mediator system on the optical and mechanical properties of the prepared handsheets. fundação para a ciência e a tecnologia, project pocti/ agr/ / . aspergillus niger is a filamentous fungi widely used in industry. its growth as freely dispersed hyphae leads to an increase in the medium viscosity and to problem of mass transfer, especially oxygen transfer. oxygen acts both as final electron acceptor in the mitochondrial chain and as nutrient for the biosynthesis of unsaturated fatty acids and sterols. thereby, a lack of oxygen affects the nadh/nad ratio, the atp production, the growth and has a strong influence on the physiology of the microrganism. in the present study, the metabolic changes of a. niger in response to a lack of oxygen was investigated using oxygen limited chemostats combined with nitrogen pulse. under these conditions, the main consequence of a sudden decrease of oxygen availability is an increase in the mannitol production. this work showed that the mannitol biosynthesis, involving the enzyme mannitol- -p dehydrogenase, helps the reoxidation of nadh when the final electron transport acceptor, oxygen, is limiting. investigation of the lipase activity of the bacteria isolated from olive mill wastewater sevgi ertugrul , nur koçberber , gönül dönmez , serpil takaç department of biology faculty of science ankara university beşevler ankara turkey; department of chemical engineering faculty of engineering ankara university tandogan, ankara, turkey the bacteria that could grow on media containing olive mill wastewater (omw) were isolated and their lipase production capacities were investigated. the strain possesing the highest lipase activity among strains grown on tributryin agar medium was identified as bacillus sp. the effect of ph on the lipase activity of the strain was investigated in tributryin medium and ph was found to be the optimal. the liquid medium composition was improved by adding different carbon sources and fatty acids into tributryin medium -omitted tributryin -to increase the enzyme activity. the cultivations were performed at • c and ph . lipase activity of the bacillus sp. was measured spectrophotometrically through the hydrolysis of p-nitrophenol palmitate. among the media containing different compositions of tricapryn, trimyristin, tributyrin, triacetin, tween , glycerol-trioleate, glycerol-trioctanoate, glycerol-tridodecanoate, omw, glucose, and whey; the medium consisted of % whey + % glycerol-trioleate was found to give the highest lipase activity. cultivation of bacillus sp. in the optimum medium at ph = and • c for h was resulted in the extracellular and intracelluar lipase activities of and u/ml, respectively. this study was supported by ankara university biotechnology institute (project no: - and - ) . under different abiotic stresses, cell growth and metabolic activity are highly influenced in all types of living organisms medium osmolality is usually one of those factors affecting different types of biological systems in different ways. however, even in the same organ of higher eukaryotes the degree of osmoregulation mechanism is highly variable in different types of cells. therefore, studying the effect of osmotic stress on mammalian cell is very important subject for particular cell line. the effect of hyperosmotic pressure on the kinetics of cell growth of and metabolic activity of mesenchymal stem cells (mscs) and two industrially important cell lines, hybridoma cells and human embryonic kidney cell (hek) were investigated in batch cultures at different osmotic pressures in the range from to mosm kg − . in case of mscs cells, the maximal specific growth rate [] of . [h − ] associated with the highest specific glucose consumption rate [-q gluc ] of . × − [mol cells − h − ] was obtained in medium of mosm kg − . in case of hybridoma cells, osmotic pressure showed not only influence on the kinetics of cell growth and metabolism but also on the monoclonal antibody production. the maximal mab production was obtained in case of cells cultivated under osmotic pressure of mosm kg − . further increase in osmotic pressure resulted in significant reduction in growth rate as well as mab production. on the other hand, hek cells were more sensitive to osmotic pressure in industrially used serum free medium and the addition of serum decreased the inhibitory effect of high osmotic pressure on the cells. gustavo g. fonseca , , andreas k. gombert , elmar heinzle , christoph wittmann : biochemical engineering, saarland university, saarbrücken, germany; chemical engineering, são paulo university, brazil kluyveromyces marxianus cbs is a potentially interesting yeast strain characterized by a high capacity of conversion of substrate into biomass. however, this yeast has been only marginally studied so far. therefore, we performed a metabolic characterization in batch and chemostat cultures at dilution rates of . , . and . h − . the specific rate of o consumption (qo ) increased with dilution rate from . to . mmol (g dw) − h − . the respiratory coefficient remained almost stable around . for all metabolic states investigated. even at the dilution rate of . h − , which is close to the maximum growth rate of the strain of . h − , no significant overflow metabolism was observed. the concentration of extracellular metabolites increased with the dilution rate, but remained below % of the carbon consumed as glucose. all carbon balances closed near % underlining the consistency of the data. in contrast to s. cerevisiae the respiratory capacity of k. marxianus cbs is not strongly influenced by the dilution rate in aerobic chemostat or batch cultures, indicating its high potential for biomass-directed applications. a thermostable l-arabinose isomerase for enzymatic production of d-tagatose o. hansen, f. jørgensen, p. stougaard department of enzyme technology, bioneer a/s, kogle allé , dk- hørsholm, denmark. e-mail: och@bioneer.dk (o. hansen) d-tagatose, an isomer of d-fructose, is a low-calorie bulk sweetener with a sweetness equivalent to sucrose. d-tagatose has obtained gras approval for use as a food ingredient, and is currently produced by chemical isomerization of d-galactose, which may readily be obtained by hydrolysis of lactose. structurally, d-galactose is closely related to l-arabinose, and it has previously been shown that some variants of l-arabinose isomerase (araa) may catalyze the conversion of d-galactose to d-tagatose, in addition to the metabolic conversion of l-arabinose to l-ribulose. we have screened a number of bacterial araa enzymes for their ability to catalyze the isomerization of d-galactose to d-tagatose. the best enzyme was found in the thermophilic bacterium thermoanaerobacter mathranii (dsm ). the araa gene of t. mathranii was cloned, sequenced and expressed in e. coli. amino acid sequence comparisons of the t. mathranii sequence and other known araa sequences showed a relatively low sequence identity of about %, indicating a distant phylogenetic relationship to the other members of the l-arabinose isomerase group. the t. mathranii enzyme was thermostable with optimal activity at • c and it required manganese ions. unlike other araa variants, the t. mathranii enzyme showed k m values in the same order of magnitude for l-arabinose and d-galactose, suggesting that this enzyme is a versatile isomerase capable of isomerizing structurally related aldoses. the enzyme was immobilized by chemical cross-linking of a crude e. coli cell homogenate, and the immobilized enzyme efficiently converted d-galactose into d-tagatose. currently, we are developing an enzymatic method for industrial production of d-tagatose using the immobilized enzyme. the agricultural production can be negatively affected by different pest insects (pi) and the use of chemical insecticides (chi) has been the traditional method for controlling pi during decades. nevertheless, there are various ecological implications due to the extensive application of chi. a viable alternative for the use of chi in certain agro-systems, is the use of entomopathogenic nematodes (epn) of the genera steinernema and heterorhabditis that are natural pathogens for different pi; besides, the presence of a symbiotic bacterium is necessary for an effective entomopathogenic activity can take place. the nematode/bacterium complex does not represent a risk for the environment. different authors propose that the best alternative for the massive production of epn is the submerged culture within bioreactors; nevertheless, more research is required to have really robust processes. particularly, information regarding the actual hydrodynamics during epn production and its relation with the epn productivities are scarce, among other aspects. the present study deals with the hydrodynamic characterisation during the production of the epn steinernema carpocapsae and its symbiont bacterium xenorhabdus nematophilus, in submerged monoxenic culture in an internal-loop air-lift bioreactor (v l = . l) using two culture media: one of them containing whey and the other one, agave juice, aguamiel, (agave spp.). process viscosity of the culture broth was determined along the time, exhibiting a maximum value of mpa s. moreover, it was determined that the hydrodynamic conditions were always located within the laminar region (re < ). at the experimental conditions tested, it can be inferred that the epn productivity are more sensitive to changes in the culture medium composition than on the prevailing hydrodynamic conditions during the fermentations. bacillus thuringiensis is a gram-positive bacterium used as a biological pest control agent. moreover, it is able to produce, several biologically active molecules such as bacteriocins and hydrolytic enzymes among which chitinases that play double roles, fungicide and improving the insecticidal effect of b. thuringiensis deltaendotoxins. a newly isolated b. thuringiensis subsp. kurstaki strain bupm , was shown to produce a novel bacteriocin named bacthuricin f . the highest bacteriocin activity was found in the growth medium and evidenced in the late exponential growth phase. upon purification of bacthuricin f , the specific activity was increased -fold. this bacteriocin was heat-stable up to • c and resisted up to ph . . its molecular mass, determined by mass spectrometry was . da. direct n-terminal sequencing of bacthuricin f revealed the following sequence: dwtxwsxl. the latter was unique in the databases. bacthuricin f was active against bacillus species while it had little or no effect on gram-negative bacteria. the bacteriocin produced by the b. thuringiensis strain bupm respond to both criteria of thermostability and stability to low phs. thus, it could be used as a source of bacteriocin active against related species of bacillus harmful for agricultural products and as food preservative. the other example of antimicrobial compound produced by b. thuringiensis is a chitinase. we describe the selection of b. thuringiensis high chitinase-producing strain bupm , and the characterization and the heterologous expression of a novel chitinase encoding gene. the cloning and sequencing of the corresponding gene named chi showed an open reading frame of bp, encoding a amino acid residue protein. both nucleotide and amino acid sequences similarity analyses revealed that the chi is a new chitinase gene, presenting several differences from the published chi genes of b. thuringiensis. the identification of chitin hydrolysis products resulting from the activity, exhibited by chi through heterologous expression in e. coli revealed that this enzyme is a chitobiosidase. the addition of the sequence of chi to the few sequenced b. thuringiensis chi genes might contribute to a better investigation of the chitinase "structure-function" relation. cloning and characterization of s-adenosyl-l-methionine synthetase from pichia ciferrii dscc - kwon-hye ko , gee-sun yoon , gi-sub choi , joo-won suh , yeon-woo ryu s-adenosyl-l-methionine(sam) has an important role for dna methylation and cell signaling. sam was synthesized from methionine and atp by sam synthetase and play an pivotal function in the primary and secondary metabolism of cells. recent studies have revealed in the effect of sam in case of morphological differentiation in both eukaryotes and prokaryotes. the p. ciferrii produces large quantities of sphingoid base. tetraacetylphytosphingosine(taps), which is a precursor of sphingolipid, could be used for the production of pharmaceuticals and cosmetics. we isolated sam gene from p. ciferrii and cloned it into expression vector for e. coli and p. pastoris, respectively. an . kb sam-s gene fragment was isolated by low-strigency pcr using degenerated primer. by the analysed primary sequence deduced from dna sequence, this gene included conserved domains similar with other well-known sam synthetase. first of all, sam synthetase gene cloned pgem-t vector and subcloned into histidine tagging system to purify the expressed protein using metal chelating resin. typical characteristic analysis of this enzyme is underway. metabolic networks offer a large variety of different synthesis pathways starting from cheap substrates and leading to interesting high-value compounds, i.e. metabolites. in case an interesting pathway can be disconnected from the remaining metabolic network, the perforated cell or the crude extract could be used for a one-pot multi-step synthesis of the desired compound. pathway isolation, achieved by deletion of genes encoding gene products enabling side reactions, interferes with the viability of the organism, which is a requisite for the production of the system of biotransformation (sbt). in this work, a rational systems biology-derived approach is presented for the design of a sbt. it is illustrated for a sbt allowing for production of dihydroxyacetone phosphate. the design procedure comprises three steps: (i) a production pathway is identified in the metabolic network of e. coli. the e. coli pathway is complemented by two additional enzymes in order to obtain a fully energy and redox balanced production pathway. (ii) an optimal combination of gene knockouts is designed and a suitable growth medium composition is identified, both by a model-based approach: flux balance analysis of a genome-scale metabolic network is used to predict enzyme expression within the wild-type organism on different media, while a mixed-integer optimisation is employed to identify viable mutants as this approach strongly depends on the quality of the fba prediction, available regulatory information on the usage of metabolic pathways and thermodynamic constraints were taken into account. (iii) thermodynamic analysis of the obtained, partially branched sbt reaction cascade revealed the extent of loss in yield by the remaining side reactions. in summary, this systems biology-driven approach potentially enables the substitution of a elaborative multi-step synthesis process by a one-pot enzyme reaction cascade. institute of industrial biotechnology, inha university, incheon - , korea. e-mail: leecg@inha.ac.kr (c.-g. lee) algal biotechnology is drawing increasing interest due to its potential as a source of valuable pharmaceuticals, pigments, carbohydrates, and other fine chemicals. currently, its application is being extended to the areas of wastewater treatment and agriculture. however, lack of suitable photobioreactors (pbrs) makes the cost of algally-derived compounds higher than those derived by chemical synthesis and thus has prevented widespread use of algal cultures. the culture of algae prior to the late s was apparently restricted to laboratory scale operations. experiments on outdoor algal mass production began in the late s with nearly concurrent development of experimental culture facilities in germany and the united states. for the next two decades, outdoor mass culture of algae was undoubtedly the hottest topic in the algal biotechnology area. recent developments of high-density pbrs enable the production of valuable biologically active compounds by algal mass cultures. however, light is almost always the limiting factor in high-density photobioreactors. key factors for successful photobioreactors will be discussed and various photobioreactors will be analyzed and compared for their advantages and disadvantages. the new techniques, such as pigment redcution and application of flashing light and lumostatic operation, will be discussed for possible solutions to overcome the light limitation in high-density microalgal cultures. a quick and reliable method for screening fungal transformants for specific genetic modifications is essential for many molecular applications, for example when one tries to develop a transformation system for a new fungal host. southern analysis is laborious and time consuming. several colony hybridisation methods have been developed for the analysis of a large number of transformants. unfortunately these methods suffer from different disadvantages such as non-specific binding, a limited usability to screen for specific integration and the fact that these procedures always take a few days (van zeijl et al., ) . recently, methods for pcr-based analysis of fungal transformants have been described. most of these methods require high quality dna. many methods for dna extraction from fungi have been described in the past few years. these methods often are tedious, time consuming, costly or limited to a small number of samples each time. most of the available protocols include the growth of mycelium in a liquid culture, followed by freeze-drying or maceration in liquid nitrogen and grinding of the frozen material to break the cell walls (cassago et al., ) . lately a few methods have been described to isolate dna from fungi suitable exclusively for pcr and appropriate for the simultaneous treatment of a large number of samples. some methods also describe the direct use of mycelium (cooke et al., ) in the pcr-reaction mixture. we compared the applicability of a few rapid dna extraction methods for myrothecium gramineum and tested the resulting dna samples on there suitability for pcr-applications. myrothecium gramineum is a filamentous ascomycete used in ongoing research as a new cloning and expression host. five methods were tested. in four of these methods dna was extracted from mycelium (goodwin et al., and aljanabi et al., ) or spores (ferreira et al., and xu et al., ) prior to pcr. a fifth assay used mycelium straight in the pcr-reaction mixture. only this last method seemed useful for myrothecium to isolate dna suitable for pcr. fragments up to bp were amplified. cheese whey is a liquid effluent from cheese-making processes. there is an increased interest in the economic utilization of whey produced by the dairy industries, because the whey is a pollutant, due mainly to its lactose content. the goal of this work was to find the most suitable values of some fermentation parameters for lactic acid production from whey by a lactic acid bacterium, lactobacillus helveticus (atcc ). the effects of lactose content, temperature, ph and the supplementation with yeast extract were investigated using surface response methodology. a composite central design was used with three center points, making a total of operational conditions. the region of maximum production is outlined by the following intervals: temperature around • c; lactose concentration between and g/l; concentration of yeast extract between and g/l; ph between and . . fermentation studies on a continuous fermentative process coupled to a vacuum flash evaporator were carried out in lab scale equipment. the phases of this work consisted in an assembly and instrumentation of the prototype and elaboration of a supervisory system coded in labview . , which allows the data acquisition and control through personal computers. the experiments in continuous fermentation used saccharomyces cerevisiae and sugar cane molasses as substrate. the analytical follow up was done through analysis of total reducing sugars, ethanol, glycerol, dry mass and viable cells. the system worked for months uninterruptedly, producing an alcoholic solution at the condenser with • gl. the fermentation operated with concentrations of ethanol at • gl, which is a weakly inhibitory value for the yeast of the process, even when fed with concentrated cane molasses, containing up to g/l of sugar. the result meets the initial goal, which was to operate the system with low level of ethanol and to guarantee high productivity, even in high concentrations of sugar in the feeding. the results showed that system productivity was superior to that of the conventional continuous process. lactic acid (la) is a versatile chemical, used as an acidulant, flavor and preservative in the food, pharmaceutical, leather and textile industries, and for production of biodegradable poly lactic acid (pla). l(+)lactic acid is the only optical isomer for use in pharmaceutical and food industries because human body is only adapted to assimilate this form. in this research, lactic acid production was improved on l fermentor. in our experience, among six strains of lactobacillus were examined for the production of l(+) lactic acid, lactobacillus casei ssp. casei atcc was selected as a highest l(+) lactic acid producer. optimized medium used for lactic acid production contained (per l) g glucose, g whey powder and g corn steep powder. for a homofermentative process, ph . was found to be optimal. in order to avoid product inhibition, the produced lactic acid was neutralized using calcium hydroxide. maximum production and productivity of lactic acid in batch system, were g and . g/lh, but in fed batch system, after feeds of glucose, production and productivity increased up to g and g/lh. saleh a. mohamed molecular biology dept., national research centre, cairo, egypt an extracellular polygalacturonase (pgii) from trichoderma harzianum was purified to homogeneity by two chromatography steps using deae-sepharose and sephacryl s- . the molecular weight of t. harzianum pgii was , da by gel filtration and sds-page. pgii had isoelectric point of . and optimum ph of . . pgii was very stable at the ph . . the extent of hydrolysis of different pectins by enzyme was decreased with increasing of degree of esterification (de). pgii had very low activity toward nonpectic polysaccharides. the apparent km value and kcat value for hydrolyzing polygalacturonic acid (pga) were . mg/ml and s − , respectively. pgii was found to have temperature optimum at • c and was approximately stable up to • c for min of incubation. all the examined metal cations showed inhibitory effects on the enzyme activity. , phenanthroline, tween , tween , triton x- and sds had no effect on the enzyme activity. the rate of enzyme catalyzed reduction of viscosity of solutions of pga or pectin was higher three times than the rate of release of reducing sugars indicating that the enzyme had an endo-action. the storage stability of the enzyme in liquid and powder forms was studied, where the activity of the powder form was stable up to one year. these properties of t. harzianum pgii with appreciable activity would be potentially novel source of enzyme for food processing. tarek m. mohamed, biochemistry division, chemistry department, tanta university, tanta, egypt preparation of peroxidase from horseradish, which could be used for commercial applications such as diagnostic kits, was occurred through a simple reproducible method consisting of extraction, ammonium sulphate precipitation, filtration through non-binding protein filter and lyophilization. the purification method was developed allow the preparation of mg of enzyme from kg of horseradish roots. the one mg of enzyme contains units of peroxidase. this value is similar to that produced by sigma ( - unit mg − powder). the final preparation is salt free reddish brown powder with free ammonia content less than . g − units. the rz value (a /a ) of the enzyme, which is a good criterion of purity and heme content, was . . the lyophilized enzyme was stable at − • c for at least one year. the liquid form of the enzyme in presence of . % sodium azide was stable up to days at • c, while it lost most of activity at room temperature in the same period. the properties of horseradish peroxidase including km, optimal ph and temperature, activation energy, thermal stability and effect of different compounds were studied. the applicability of this enzyme in determination of serum glucose was performed. the analysis of glucose in human sera gave results using the kit containing the prepared peroxidase similar to those obtained with a commercial glucose kit. lactobionic acid production using lactose oxidase: from laboratory to l scale mikkel nordkvist , per munk nielsen , peter budtz , john villadsen : center for microbial biotechnology, technical university of denmark, dk- lyngby, denmark; novozymes a/s, dk- bagsvaerd, denmark; chr. hansen a/s, dk- hørsholm, denmark. e-mail: mnq@biocentrum.dtu.dk (m. nordkvist) currently, lactobionic acid is mainly a high-price specialty product used, e.g. in solutions for organ stabilization. however, lactobionic acid can also be used as a biodegradable cobuilder in detergents, and it has several applications in food technology. with lower production costs it has the potential to become a bulk chemical. the kinetics for the oxidation of lactose to lactobionic acid by a new carbohydrate oxidase was studied in a l bio-reactor with control of ph, temperature, and dissolved oxygen. the byproduct hydrogen peroxide has a negative influence on the lactose oxidase enzyme, and hence additional experiments were made with addition of catalase to remove hydrogen peroxide, thereby also providing extra oxygen. on the basis of the experiments in l scale, experiments were performed in a l reactor equipped with a new system for dispersion of air to supply the necessary oxygen for the oxidation. the aeration system in the large scale reactor was able to supply oxygen sufficiently fast to give the same production rate, at low values of the air flow rate and the energy input, as was obtained in the high-performance laboratory reactor. the non-characterized gene previously proposed as d-tagatose -epimerase from agrobacterium tumefaciens was cloned and expressed in escherichia coli. the expressed enzyme was purified by affinity chromatography on histrap hp, desalting chromatography on hiprep / , and gel filtration chromatography on sephacryl s- hr with a final specific activity of . u/mg. using maldi-tof-ms, the native protein was estimated to have a molecular mass of , da and a monomeric structure. the purified enzyme exhibited maximal activity at • c and ph . without the addition of metal ions and at • c and ph . with . mm mn + . among various metal ions, mn + was the most effective divalent cation for d-fructose epimerization activity. the addition of mn + significantly increased the thermal stability and the epimerization activity with other ketoses such as d-psicose, d-tagatose, d-ribulose, d-sorbose, and d-xylulose. the activity, substrate affinity, maximum velocity, and catalytic efficiency (k cat /k m ) of the enzyme for d-psicose were higher than those for d-tagatose, which suggests that the enzyme is not d-tagatose -epimerase but d-psicose -epimerase. the equilibrium ratio between d-psicose and d-fructose was : at • c with . mm mn + . when the enzyme was used at u/ml, dpsicose was produced at g/l from g/l d-fructose containing mm mn + after min, corresponding to a conversion yield of . %. the role of ammonium ions in glucosamine formation during the citric acid fermentation process by aspergillus niger m. papagianni , f. wayman , m. mattey : department of hygiene and technology of food of animal origin, school of veterinary medicine, university of thessaloniki, thessaloniki , greece; department of bioscience, university of strathclyde, glasgow, g xw, uk. e-mail: mp @vet.auth.gr (m. papagianni) stoichiometric modeling of the citric acid fermentation process by aspergillus niger, in -l stirred tank reactor, indicates that nh + ions combine with a c-containing metabolite inside the cell to form a nitrogen compound which is then excreted by the mycelium. the close correlation between calculated and experimental profiles indicates that this metabolic process is rapid and takes place before the c-structure of the glucose has been greatly altered by glycolysis or the pentose phosphate pathway. hplc analysis identified glucosamine as the product of this relationship. a clear effect of medium concentration of nh + on glucosamine formation was observed when fermentations carried out with optimal and sub-optimal ammonium concentrations. (nh ) so addition when medium nitrogen was depleted, enhanced the formation of new cells from the tips of fragmented hyphae and led to glucosamine accumulation in amounts depending to pulse concentration. the fungus reacts in excess ammonium by converting it to glucosamine, to be utilized later when a regeneration process takes place with fragmentation of vacuolated hyphae and subsequent regrowth, depending always on the culturing conditions. however, depending on carbon and ammonium concentration in medium, glucosamine can be secreted in concentrations as high as g/l. about microorganisms originated from traditional korean food origin were screened for efficient palatinose production. an isolate designated fmb was exceptionally efficient in sucrose-palatinose conversion activity. conversion of sucrose into palatinose by fmb was much faster than a reference strain of erwinia rhapontici. fmb is a gram negative, facultatively anaerobic, motile, noncapsulate, and straight rod-shaped bacterium producing acid from glucose. based on api and s rdna analyses, fmb was determined to be enterobacter sp. the maximum conversion of % sucrose to palatinose and trehalulose by enterobacter sp. fmb was achieved within h. the preliminary dna sequencing result of the gene corresponding to sucrose isomerase of enterobacter sp. fmb revealed that it showed % similarity to that of klebsiella sp. (??). within the scope of an r&d project developed in collaboration with leather tanning portuguese industrial partners a screening of new proteases to be used in the industrial process was performed. a bacillus subtilis strain isolated from alkaline spent purge liquor was shown to be a promising protease producer. microorganism growth was studied for optimisation of temperature, agitation, ph and medium composition either for biomass or proteases production. optimal growth temperature is different for maximum biomass growth ( • c) and optimal proteolytic activity ( • c) yielding biomass specific growth rate of . and . h − , respectively. the achieved proteolytic activities were . and . u/ml of protease, respectively. optimised medium composition ( g/l beef extract, g/l yeast extract, g/l peptone and . g/l cacl ) yielded a specific growth rate of . h − and . ku/l of protease, in shake flask experiments. bioreactor experiments (from to l) with the selected medium were performed at • c in order to test aeration rate ( and vvm), stirring ( - rpm) and ph (uncontrolled, controlled at and ). best protease activity was u/ml in l bioreactor without ph control at rpm and vvm. the proteolytic extract was characterized and compared to commercial bates. results indicate that these proteases can be employed in the purge phase of the leather tanning process in industry. the gram positive bacterium bacillus megaterium is known for its capacity to produce exoenzymes including amylases, proteinases, and penicillin amidase at industrial scale. here, we describe the development of various vectors for the production and export of recombinant heterologous proteins employing b. megaterium signal peptides. the target gene can be cloned directly adjacent to the signal peptide coding sequence (bart et al., ) . this arrangement allows for a correct n-terminal sequence of the mature protein after processing by the signal peptidase sipm. using this newly developed protein production and export system lactobacillus reuteri levansucrase (van hijum et al., ) was secreted in significant amounts (∼ mg/l) into the growth medium. fusion of the recombinant levansucrase to affinity tags allowed one-step purification of the recombinant protein from the growth medium. however, fused peptide tags resulted in a decreased secretion of the fusion protein. . mg his -tagged levan-sucrase were purified per litre of culture. the system was further enhanced via coexpression of a gene for the signal peptidase sipm (malten et al., a) and deletion of the gene for the extracellular protease nprm. developed new tools allow for various strategies of integrated high level production, export and purification of heterologous proteins in b. megaterium. methods for high-throughput screening of secreted enzymes are under development. the determined sequence of the b. megaterium genome, studies using high-cell density cultivations (malten et al., b) and proteome data from batch fermentations implicate new targets for directed genetic optimization of b. megaterium production and secretion strains. novel strong and inducible promoters are currently under investigation. toru matsui , naoya shinzato , hisashi saeki , hitoshi matsuda : center of molecular biosciences, university of the ryukyus, okinawa - , japan; japan energy co., japan. e-mail: tmatsui@comb.u-ryukyu.ac.jp (t. matsui) optically active epoxides are considered as the potential intermediate for chiral drugs synthesis. although s-styrene oxide (so) have been extensively investigated using styrene monooxygenase from pseudomonas sp., microbial production of r-so with high enantiomeric excess was hardly examined. in this study, r-so producing bacteria from styrene was screened using various alkene assimilating bacteria. r-so with the highest ee (ca. %ee) was obtained using ethene utilizing bacteria, identified as mycobacteirum sp., while produced relatively lower at around %ee when using propene utilizing bacteria. the alkene monooxygenase gene homologue sequence amplified from the genomic dna revealed a significant similarity to that of etnabc. these bacteria also showed stereoselective degradation of racemic so, suggesting that the produced so might be further stereoselectively degraded to increase the ee. the ethene utilizing bacteira produced not only r-so but also s-epichrolhydrin at high ee.when using arylchloride as the substrate. this research was supported by nagase science and technology foundation. the secretion efficiency of the escherichia coli sec pathway is dependent on the growth phase but not on protein size f.j.m. mergulhão, g.a. monteiro centro de engenharia biológica e química, instituto superior técnico, av. rovisco pais, - lisbon, portugal. e-mail: filipem@alfa.ist.utl.pt (f. mergulhão) the secretion efficiency of the escherichia coli sec pathway was evaluated through the expression of green fluorescent protein and human proinsulin fusion proteins. translocation to the periplasm is dependent on the growth phase of the bacterial culture and the highest secretion efficiency is attained in mid-exponential phase. secretion performance is independent of protein size ( - kda) and even when the amino acid composition of the secreted proteins is very similar, the amino acid distribution within the protein can affect translocation. in silico prediction analysis suggests that proteins that are prone to form ␣-helix structures are more efficiently translocated. culture medium composition plays an important role on secretion performance with the highest secretion results being obtained in minimal medium. streptokinase is a common fibrinolytic drug. that is used in thrombolytic therapy for long time. to compare with another thermbolytic drugs like tpa, it has lot of advantages. in this present research dna was extracted from s. equisimilis h a for the first time in iran. streptokinase gene was amplified by using two forward primers and one reverse primer. a common restriction enzyme, bamh-i, was used for cloning. both ends of the pcr products (full length: bp and mature section: bp) and the restriction site on mcs of pqe- vector were digested. in this study, pqe- expression vector was used with high level expression ability for production of recombinant fusion streptokinase with simplifying the purification by employing affinity-metal chromatography method. in addition, the cloning results were controlled by double digestion and sequencing. takesono oxidation of short-chain iso-alkanes was studied with propanegrown resting mycelia of scedsporium sp. a- . isobutane was oxidized to tert-butanol, but not to isobutanol. isobutanol was used for growth, but both isobutene and tert-butanol were not used for growth. isopentane was oxidized to -methyl- -butanol, -methyl- -butanol, and -methyl- -butanol but not to -methyl- -butanol. -methylpentane was oxidized to -methyl- -pentanol, -methyl- pentanol, and -methyl- -pentanol but not to -methyl- -pentanol or -methyl- -pentanol. -methylpentane was not oxidized. oxidation of branched alcohols was also studied. application of nadph-dependent . -diketo-gluconic acid reductase for production of l-ascorbic acid claudia pacher , : division of food biotechnology, department of food sciences and technology, boku, university of natural resources and applied life sciences, muthgasse , a- vienna, austria; research centre applied biocatalysis, petersgasse , a- graz, austria. e-mail: claudia.pacher@boku.ac.at ascorbic acid is an organic acid with various applications in the food and pharmaceutical industries. at present, the majority of commercially manufactured vitamin c is synthesized via the reichstein process, which is highly energy-consuming, involves considerable quantities of organic solvents and gives an overall yield of about %. therefore, during the past decades a lot of research was done to develop biotechnological alternatives for the synthesis of reichstein intermediates by enzymatic or fermentative means, which show some advantages regarding costs and environmental friendliness. one of the fermentation routes runs via . -diketo-d-gluconic acid ( . -kdg), produced by pectobacter (erwinia) cypripedii. this compound has been reduced by a nadph-dependent . -diketogluconic acid reductase (dkr) from corynebacterium glutamicum to the key intermediate -keto-l-gulonic acid ( -klg) before chemical rearrangement leads to the final product. for economical reasons we wanted to express dkr heterologously. based on our long term experience with coenzyme regeneration we wanted also to perform the reaction in homogeneous solution. the spent coenzyme of nadph-dependent dkr has been regenerated by a second isolated nadp-dependent enzyme like glucose dehydrogenase. we describe here the recombinant production, purification and characterization of dkr and the results of enzymatic -klg formation by using the recombinant enzyme in a homogeneous system with conjugated coenzyme regeneration. grp residing in endoplasmic reticulum (er) functions as a molecular chaperon by associating transiently with incipient proteins as they traverse the er and aiding in their folding and transport. furthermore, the protein can also be induced under various stress condition such as glucose starvation, inhibition of protein glycosylation by tunicamycin, blockage of vesicular trafficking by brefeldin a and er-calcium-atpase pump inhibition by thapsigargin. thus, substances that directly down and up-regulate grp transcription are expected to be useful for treatment of cancer and alzheimer's disease, respectively. in the course of our screening program to obtain substances, which regulate grp expression, we first constructed an assay system monitored by the expression of a reporter gene. hela cells, which are transformed with luciferase gene under the control of grp promoter designated as hela c cells, respond sensitively to luciferase grp induction by er stress such as treatment of tunicamycin. by using this screening system, we isolated pyrisulfoxin as an up-regulator of grp , and valinomycin, citreoviridin and alternariol as down-regulators. detailed studies on other biological activities were now under way. pdh is an enzyme that was described only several years ago in a number of ecologically related litter-decomposing fungi (agaricales, gasteromycetales). it catalyzes the c- and/or c- oxidation of several aldopyranoses to the respective keto sugar derivates. pdh shows a very broad substrate range, oxidizing almost all major sugar components of wood polysaccharides, and is implicated to play a role in lignocellulose degradation. agaricus bisporus, the white button mushroom, is an economically significant agricultural crop. the cultivation, which is done by solid-substrate fermentation on straw-and hay-based composted substrate, is sometimes seen as one of only few economically feasible methods for bioconversion of lignocellulosic agricultural waste material. deeper insight in the physiological role of pdh may provide help for mushroom growers to increase yield, improve quality or make new sources of raw materials utilizable. we amplified a fragment of the pdh gene with degenerated primers derived from internal peptide sequences. the screening of a genomic library led to the isolation of the pdh gene. subsequently we amplified a cdna clone by rt-pcr and investigated the transcriptional regulation by different carbon sources on a defined minimal medium. optimization of monoclonal antibody production processes with simulation and scheduling tools demetri petrides, charles siletti intelligen inc., scotch plains, nj , usa. e-mail: dpetrides@intelligen.com (d. petrides) this presentation will review the state of the art in batch process simulation and scheduling tools and their applications in the design and debottlenecking of integrated biopharmaceutical processes. a systematic methodology will be presented for identifying and eliminating size, time, and throughput bottlenecks that limit production in single and multi-product facilities. the methodology will be illustrated with an industrial case study dealing with the optimization of a multi-product facility that produces therapeutic monoclonal antibodies (mabs). mab processes are characterized by a long bio-reaction time (e.g., . - weeks for fed-batch operation and - months for perfusion operation). the cycle time for processing a lot in the recovery and purification train typically takes - days. consequently, one way of increasing throughput is by installing extra bioreactors that operate in staggered mode and utilize the same recovery train. the result is that multiple batches may be at different stages of completion at any given time. since cleaning equipment (e.g., cip skids) and buffer preparation and holding tanks are shared by multiple steps across many batches, this type of operation leads to time/scheduling bottlenecks that constrain the cycle time and the throughput of a process. the problem becomes more challenging in the context of multi-product facilities and when constraints imposed by the limited availability of resources are considered. our methodology and its computer implementation will illustrate how to systematically identify and eliminate such bottlenecks. the industrial case study will provide a real world example of the methodology. application of two stage continuous cultures of aspergillus niger for citric acid biosynthesis jerzy j. pietkiewicz, malgorzata janczar, wladyslaw lesniak food biotechnology department, university of economics, wroclaw - , poland. e-mail: jerzy.pietkiewicz@ae.wroc.pl (j.j. pietkiewicz) the aim of the work was application test of submerged two stage single stream continuous cultures of aspergillus niger for citric acid production from sucrose. studies were carried out in lab fermenters with working volume of dm . the bioreactors were standard cstrs. in two stage continuous cultures (tscc) high mycelium growth and high citric acid production were observed in the first bioreactor. there was almost four times lower growth of biomass rate and about three times lower citric acid production rate in the second bioreactor. studies on influence of dilution rate in race from . to . dm /(dm h) on course and efficiency of tscc showed, that the highest citric acid yield (y p/s = . %), high volumetric rate of its production (r pc = . g/(dm h)) and the highest biosynthesis efficiency coefficient (k ef = . ) were obtained with dilution rate d = . dm /(dm h). there was also high citric acid concentration (p = . g/dm ) and low residual sugar concentration (s k = . g/dm ) in the medium flowing out the second bioreactor in those cultures. the beta-galactosidases in commercial use are of different origins and yeast and fungal lactases present the greatest interest. the yeast lactases present neutral optima ph and are suitable for the hydrolysis of lactose in milk. in this work, the aim was to study the influence of aeration in the production of beta-galactosidase in batch fermentations with kluyveromyces marxianus atcc . the medium composition for culture was as follows (in g/l): lactose pa , yeast extract , (nh ) so and kh po . the fermentations was carried out at • c, ph . , at rpm starting with an initial cellular concentration of × cels/ml, with different aeration rates. the cells were disruped with chloroform % (v/v) as solvent. the enzymatic activity was determined as initial rate of lactose hydrolysis at defined conditions. the studies have revealed the importance of aeration on kluyveromyces marxianus in the growth and beta-galactosidase synthesis. the enzymatic activity of fermented medium with . vvm was % higher than one without aeration. furthermore, the cellular growth was faster in the aerobic fermentation than in the anaerobic one. the aeration has taken an important place in the enzymatic synthesis and in the cellular growth, however the results have shown that the aeration rate increase of . - . vvm has not implied a increase in the cellular growth neither in the enzymatic reached activity. the lactose presents in the milk has a solubility of only % at • c, and a high percentage of the world population presents intolerance to this sugar, due to the low or absence of the activity of the lactase enzyme in the organism. to minimize such problems, the most viable alternative for nourishing dairy products is the enzymatic hydrolysis of milk, although it is an expensive process due to the high cost of the beta-galactosidase enzyme. an alternative that has been greatly studied is the immobilization of this enzyme, originated from many different sources. there are several immobilization procedures for this enzyme, however, a procedure considered ideal was not obtained yet. the objective of this work was to study the immobilization process of beta-galactosidase from aspegillus oryzae in sodium alginate with commercial gelatin. in the immobilization process was studied the glutaraldehyde influence for , and %, in the presence of commercial gelatin at the concentration of % at the immobilization medium. the activities of the immobilized enzymes were obtained in a stirred micro-reactor, at the temperature • c, ph . with a gl − lactose solution in acetate buffer. the experimental results showed that the immobilized biocatalyst that presented the larger initial activity was the one obtained at the immobiliza-tion medium that contained % of glutaraldehyde. after daily determinations of enzymatic activities, a fall of , and % was verified in the enzymatic activities for the immobilized biocatalysts using glutaraldehyde at , and %, respectively, however, in all cases, the enzymatic activity reached the half of their initial activity after determinations. hydrolysis of sucrose by immobilized beta-fructofuranosidase in silica eloízio júlio ribeiro, ubirajara coutinho filho faculdade de engenharia química, universidade federal de uberlândia, uberlândia - , brazil. e-mail: ejribeiro@ufu.br (e.j. invertase, known as beta-fructofuranosidase (ec . . . ), plays a catalytic role in the conversion of sucrose into glucose and fructose. it is largely used in the food industry to prevent the crystallization of sucrose in sugar mixtures and can be used in enzyme reactors for hydrolysis of sucrose. the objective of this work was to study the kinetic of sucrose hydrolysis by immobilized betafructofuranosidase in a continuous recirculation reactor, evaluated the enzyme stability and determine the effective half-life of immobilized enzyme. invertase was covalently immobilized on sillanized controlled pore silica. nonlinear fitting were used to determine the kinetic parameters for substrate and product inhibition observed in the enzymatic hydrolysis of sucrose. the kinetics studies of immobilized invertase were carried out in a continuous recirculating reactor. the half-time of enzyme inactivation (t / ) was calculated from the initial rates of the remaining enzyme activity. the model of inhibition by substrate and product adequately represented the enzymatic hydrolysis. the fructose effect was competitive inhibition (k f = . . − mol/ml) and the glucose effect was noncompetitive inhibition (k g = . . − mol/ml). the effective diffusivity of sucrose into the support was shown to be the same as for sucrose in dilute solution ( . × − cm /s at • c). the half-time of enzyme inactivation (t / ) was h. controlled pore silica showed to be an excellent immobilizing support. the immobilized invertase was very stable at temperatures lower than • c. the intrinsic parameters (k i , k f , k g and v m ) were shown to be similar to the apparent values. the low permeability of mycobacterial cell wall envelopes is a result of the unique composition and organization of the cell wall lipids. the permeability of mycobacterial cell wall can be changed by means of partial disintegration of its compounds. the aim of present work was to characterize the changes in the cell wall skeleton (cws) and non covalently bound free lipids under the influence of isoniazid, the inhibitor of mycolic acids biosynthesis. fatty acid (fames) and mycolic acid methyl esters (mames) obtained from all tested preparations were analyzed by gc/ms analysis. the analysis of free lipids and cws revealed distinct changes in the composition of the frac-tions obtained from the cells exposed to action of the isoniazid. the changes in the quantity of fatty acids in the inh-treated cells indicates that inh interferes with the synthesis of lipidic compounds of the mycobacterial cell wall also. the decreased amount of covalently bound mycolic acids in the cws is responsible for the enhanced penetration of hydrophobic compounds through the cell wall. this work was supported by grant nr p c of the committee for scientific research. barbara sencio, teresa jamroz, stanislaw ledakowicz department of bioprocess engineering, technical university, lodz, poland the enzyme laccase (ec. . . . . p-diphenol oxidase) is a subject of research in many centres dealing with improvement of bioprocesses with the use of different white rot fungi species. most strains that produce this enzyme in vitro require inductors initiating its biosynthesis. when cerrena unicolor was applied, it was found that the strain produced laccase very efficiently without additional toxic compounds. to specify optimum conditions for laccase production in a submerged culture, research was undertaken to obtain the most efficient inoculum c. unicolor. the goal of this research was to determine the effect of form and incubation time of inoculum on enzymatic activity of the laccase producing strain. the experimental inoculum was the mycelium prepared on a solid and liquid substrate. basing on results obtained, it was found that the laccase yield was the highest in the cultures where the mycelium was grown on a solid substrate. maximum activity of the c. unicolor strain was achieved on the th day of culture, and the amount of laccase produced was higher by, ca. % as compared to the mycelium obtained from the liquid substrate. results of these experiments were used to continue studies on the impact of inoculum age. experiments were carried out using an inoculum incubated for - weeks at the temperature • c in a certomat bs shaker at rpm. the best results in the c. unicolor strain culture were achieved using a -day-old inoculum. effect of alcohol treatment on hydrolytic activity of candida rugosa lipase serpil takaç, a. ezgiÜnlü department of chemical engineering, institute of biotechnology, ankara university, tandogan, ankara, turkey candida rugosa lipase (crl) was treated with , , and % concentrations of methanol (m), ethanol (e), -propanol ( p) and -butanol ( b) to investigate the changes in its hydrolytic activity toward p-nitrophenylacetate. the treatment included the following steps at + • c: (i) stirring crl with phosphate buffer for h; (ii) treating the solutions with alcohols; (iii) stirring treated-crl for h; (iv) centrifugation at , rpm for min; (iv) dialysis the supernatant against bidistilled water for h. the activity of crls was followed for h at • c in the presence and absence of isooctane. the enzyme activity was measured spectrophotometrically and the protein concentration was measured by lowry's method. it was found that the recovered protein did not change considerably with the type of alcohol; however, decreased with alcohol concentration. in the presence of isooctane, specific activities of the untreated and treated-crls increased compared with those obtained in the absence of isooctane. b-crls and e-crls showed higher activities than m-crls and p-crls whereas untreated-crl exhibited higher activity than m-crls, e-crls and p-crls. the highest and the lowest activities were obtained with % b-crl and % p-crl, respectively. the changes occur in the structure of crl after treatments were investigated by electrophoretic analysis. this study was supported by ankara university biotechnology institute (project no: ) . different genera, species and strains of microorganisms were found to posses different cryoresistance. optimal ways for cryopreservation of microorganisms-producers of antibiotics, microorganisms, used in food industry, agriculture and veterinary have been developed. it was demonstrated, that non-lethal damages could occur in cryopreserved microorganisms after their returning to physiological culture conditions, which were manifested in streptomyces' hypha fragmentation, that of cyanobacteria's, streptococci's chains as a result there was an increase in a number of colony-forming units, a reversible inhibition of microorganisms' proliferative activity in bacillus thuringiensis and lactic streptococci, stimulation of the enzyme processes and antibiotic production. non-lethal damages are repaired during microorganism culturing in the first passage. the cause of non-lethal damages is a reversible inhibition of biosyntheses of protein and nucleic acids respiratory activity. the repairing of non-lethal damages is accompanied by the production of stressproteins, different from heat shock proteins. effect of ph in the -propanol treatment of candida rugosa lipase on its enantioselectivity in the hydrolysis of racemic naproxen methyl ester serpil takaç, a. ezgiÜnlü department of chemical engineering, ankara university, tandogan, ankara, turkey candida rugosa lipase (crl) was treated with -propanol ( p) at the ph values of . , , , . , and to investigate the changes in its enantioselectivity in the hydrolysis of racemic naproxen methyl ester. the treatment included the following steps at + • c: (i) stirring crl with different buffer solutions to maintain the desired ph values for h; (ii) treating the solutions with % p; (iii) stirring treated-crls for h; (iv) centrifugation at , rpm for min; (iv) dialysis the supernatant against bidistilled water for h. hydrolyses of racemic naproxen methyl ester to form s-naproxen were performed in shaking flasks at rpm and • c for h in isooctane-phosphate buffer solution biphasic system using treated-crls with the activity of . u. the concentrations of the enantiomers of naproxen methyl ester and naproxen were determined with hplc. it was found that the treatment ph has an important role on the enantioselectivity and conversion. the highest enantiomeric excess for the substrate, for the product, enantiomeric ratio, and con-version were obtained with crl treated at ph . as , , and %, respectively. these values were followed with p treated crl at ph as , , and %. however, lower enantiomeric excesses, conversions and enantiomeric ratios were obtained at the treatment ph values between . and . the effects of fatty acids, nitrogen (as nh no ), phosphorus (as kh po ), ph value, manganese (mn + ), iron (fe + ) and methanol concentration on growth and production of oxalic acid from post refining fatty acids by a mutant of aspergillus niger xp in submerged fermentation experiments was studied. of the a. niger strains screened, a. niger xp was identified as the best oxalate producer on lipids. the influence of the ph on oxalic acid formation shows that the maximum production rate and higher concentration of product are observed at the ph ranging from to . with a medium containing g fatty acids/l, the production reached a maximum of g oxalic acid/l after days. the addition of . % (w/v) methanol to seed culture increased the product yield and concentration of oxalic acid but decreased the amount of an undesired by-product (citric acid). under this condition, the maximum oxalate productivity ( - g/l days) was maintained for - days of fermentation. other results of the experiments show that supplementation of the production medium with manganese and iron enhances oxalate production. fatty acids proved to be a very good substrate for oxalic acid production by a. niger xp giving excellent yields and productivity at low ph. the results are very promising as they may lead to cheap alternative processes for oxalic acid production from renewable lipid resources. department of food engineering, middle east technical university, ankara , turkey. e-mail: banuy@metu.edu.tr (b. yalcindag) laccase (e.c. . . . , p-benzenediol:oxygen oxidoreductase), which is an enzyme belonging to the multi-copper oxidase family, catalyzes the oxidation of a broad variety of polyphenols with a preference for p-isomers, which are converted to p-quinones. fungi generally contain several laccases which have been found to be involved in delignification, melanin synthesis and pathogenesis. laccase has also important potential application areas especially in food and chemical industries. after aspergillus fumigatus genome data were released, research on functional analysis of laccases has been initiated in our laboratory. laccase genes of aspergillus nidulans, ya and tila, and laccase and multi-copper oxidase genes of aspergillus fumigatus, abr and abr , were used to analyze a. fumigatus genome for laccases. this sequence analysis resulted in probable laccase genes, one of which was the previously cloned abr gene. in this study, one of these genes (aflac ) was further characterized. after sequence alignment and characterization studies, aflac was predicted to have bp having six introns, which makes the protein amino acid long, and the predicted protein sequence showed % homology with the dihydrogeodin oxidase of aspergillus terreus and % homology to the laccase of botryotinia fuckeliana. aflac gene is found within an uncharacterized gene cluster containing genes with homology to glutathione-s-transferase, polyketide synthase, o-methyl transferase, and others. the information obtained from sequence analysis was employed in designing pcr-primers to amplify the aflac gene, followed by cloning onto pan - and pan - vectors for heterelogous expression in aspergillus sojae. in addition, by the use of rt-pcr, aflac cdna will be cloned and expressed in escherichia coli. furthermore, gene silencing studies will be performed to enlighten the function of aflac and associated gene cluster. stability of growth rate of photosynthetic cells is an important factor in designing of effective photobioreactors especially in long term operations. in our experiments, in order to keep operational parameters almost constant, a semi-continuous culture method was developed. in this method, a part of culture broth containing grown cells was repeatedly replaced by fresh medium at a predetermined time interval. the replacement of broth with fresh media could keep the cell concentration, volume of broth and distribution of light intensity constant at initial values throughout the cultivation. it was shown that in one side illumination with a halogen lamp, if the ratio of the light intensity at the front side of a flat plate photobioreactor to that at the rear side was kept lower than , the growth rates was sustained in constant levels. however, at higher ratios the growth was followed by rapid decrease after - h. supplemental illumination with a fluorescent lamp from the rear side of the flat plate photobioreactor could sustain almost stable growth rate. beside of the illumination conditions, increased ferrous ion concentrations in medium could keep the stability of growth rate even in unstable illumination conditions, while consumed ferrous ion was slight. glutathione (gsh) plays a pivotal role in protecting cells from by-products generated by oxidative metabolism. these characteristics make this active tripeptide an important drug for the treatment of liver diseases and is of interest in the food additive industry, therapeutics and sport nutrition. in the first part of the research, a screening was carried out among yeast strains, to find out those able to accumulate higher gsh intracellular levels. two saccharomyces cerevisiae strains proved to be the best gsh producers ( . %dw), in every samples the presence of s-adenosyl-methionine in traces ( . % dw) was also evidenced. s-adenosyl-methionine (sam) plays a role in the immune system, maintains cell membranes, participates in detoxification reactions and in the manufacture of brain chemicals and cartilage. the second part of the research was aimed at increasing, in a post fermentative procedure, gsh levels present inside the cells at the end of the growth phase. moreover, time course of sam intracellular levels, to be related with accumulated gsh, was also monitored. cells were then suspended in an appropriate solution containing mineral salts, glucose and the aminoacids, precursors of the two studied molecules. according to this procedure, gsh intracellular levels reached . % dw after h incubation. moreover, gsh levels can be related to sam production (up to . % dw). the presence, in several samples, of intermediate metabolites, such as cystathionine and omocysteine, proved the establishement of an intracellular equilibrium between gsh and sam; this behaviour represents a promising starting point for the set-up of a microbial process for the simultaneous production of the two studied molecules. three acetate mutants of y. lipolytica yeasts, which varied in colony morphology (rough and smooth), were employed for continuous citric acid production from glucose and fructose syrup in a membrane reactor with cell recycle. the strains were compared for their product yields, specific acid production rates and ratios of citric acid to isocitric acid. experiments shoved that glucose syrup was a better substrate for citric acid production by y. lipolytica. citric acid concentration in the effluent ranged from to g/l, depending on the yeast strain used. all y. lipolytica strains produced very low amounts of isocitric acid. its concentration did not exceed g/l. based on the results of these experiments, smooth strain awg- was found to be the most suitable for citrate production both from glucose and fructose syrup during long time continuous processes ( h). in the steady state, the highest citrate productivity ( . g/lh) was obtained with this strain, when the feed medium contained g/l of glucose and dilution rate (d) was d = . /h. supplementation of the feed medium with bacto-pepton improved the productivity, citric acid yield and stability of the continuous process in the cell recycle fermentation system. for textile dyeing with natural dyes, indigo has an almost unique position as the most blue natural dye. due to the importance of indigo, considerable research has been conducted to replace the chemical synthesis of the dye by an application of biotechnological methods. therefore, we investigated several characteristics of natural indigo derived from polygonum tinctorium and its dyeing properties using silk fabrics, such as washing, perspiration, and light fastnesses. this work was financially supported by program for cultivating graduate students in regional strategic industry from korea industrial technology foundation. glycine oxidase is the product of the yjbr gene of bacillus subtilis that was predicted by sequence homology to be a flavoprotein similar to sarcosine oxidase. glycine oxidase catalyzes the oxidative deamination of various primary and secondary amino acids (e.g. sarcosine, n-ethylglycine, and glycine) and d-amino acids to form the corresponding ␣-keto acids and hydrogen peroxide. previous investigations reported on the cloning and production of the glycine oxidase gene in escherichia coli was up to u/g cell. the present works has improved the expression of the recombinant his-tagged glycine oxidase by -fold by using pet a and rosetta cells under the optimal iptg, temperature and time of induction. the protein obtained represented % of total soluble proteins in crude extract. the enzyme was purified to near homogeneity using imac with a % recovery and with and specific activity of . u/mg protein. the enzyme was active towards glycine, sarcosine and different d-amino acids, having in general, a basic ph optimum. the kinetic parameters were also studied, showing a km range from . to mm. the enzyme was immobilized, and used to obtain pyruvic acid (␣-keto acid) from d-alanine with a good yield. the enzymatic synthesis of lipophilic derivatives of various natural antioxidants including flavonoid glycosides, as well as derivatives of cinnamic acid, was performed using various immobilized lipases in ionic liquids such as -butyl- -methylimidazolium tetrafluoroborate (bmim-bf ) and -butyl- -methylimidazolium hexafluorophosphate (bmim-pf ). the influence of various reaction parameters on the catalytic behavior and the selectivity of lipases was pointed out. a response surface methodology was applied for the optimization of the yield and the productivity of the biocatalytic process. the antioxidant activity of the biocatalytically prepared lipophilic derivatives of natural antioxidants, as expressed on cu + -induced oxidation of low-density lipoprotein (ldl) and total serum, was investigated. process strategy for reduction of proteolysis in pichia pastoris fermentations jan weegar , john dahlbacka , noora sirén , niklas von weymarn , kaj fagervik : faculty of chemical engineering,Åbo akademi university, finland; laboratory of bioprocess engineering, helsinki university of technology, finland. e-mail: jan.weegar@abo.fi (j. weegar) the yeast pichia pastoris is a popular host organism for production of recombinant proteins. it is, however, common that the products are degraded by proteases towards the end of the fermentation, resulting in productivity and purity decreases. proteolysis of recombinant proteins in p. pastoris fermentations is affected by the temperature and ph of the growth medium. in this study, it was shown that decreasing the temperature from to • c during the induction phase effectively prohibited proteolysis. on the other hand, the temperature decrease resulted in a reduced maximal methanol consumption rate, which subsequently resulted in a culture highly sensitive to residual methanol. the temperature was slowly decreased according to a predetermined trajectory. as the temperature reached values below • c, the methanol concentration had to be closely monitored and the substrate feed rate adjusted in order to prohibit methanol poisoning as well as to maintain the culture as a substrate limited fed-batch. measurement of protease concentrations revealed that proteases were present at • c, but at this temperature the proteolysis rate was evidently effectively reduced. the recombinant protein produced could almost totally be recovered (i.e. high purity) with this process strategy compared to a constant high temperature culture ( • c) where severe breakdown of the product was observed. with the growing of an ecological conscience in the public opinion, more and more industrial processes are analyzed for a possible ecologically beneficial alternative. for the production of ascorbic acid, which is nowadays done by the reichstein process, a lot of research was done to develop biotechnological alternatives for the synthesis of reichstein intermediates by enzymatic means, which show some advantages regarding costs and environmentalfriendliness. besides of two-stage fermentation, our approach is to design a tailor-made organism that produces -keto-l-gulonic acid, which is the direct precursor of ascorbic acid from glucose or gluconic acid and which can easily be converted to the final product by conventional methods. erwinia (pectobacter) cypripedii, which is a natural producer of , -diketo-d-gluconic acid, was selected as a suitable host for a , -diketo-d-gluconate reductase from corynebacterium glutamicum. to increase the yield of the desired compound we investigate two -keto-reductases in the host organism that diminish the yield of -keto-l-gulonate by reducing the compound to l-idonic acid, or by metabolisation of intermediates. these two enzymes were investigated with vari-ous biochemical and molecular biological methods, which will be presented. overproduction of bioinsecticides by heat and salt stress and control of dissolved oxygen in cheap media of bacillus thuringiensis nabil zouari, dhouha ghribi, samir jaoua laboratoire des biopesticides, centre of biotechnology of sfax, tunisia, bp:k, sfax, tunisia bioinsecticides based on preparations of spores and insecticidal crystal-proteins (icps) produced by the bacterium bacillus thuringiensis (bt) proved to be a high tool for fighting some agricultural pests and vectors of diseases. however, the use of bt preparations as commercial insecticides would be prohibitively expensive because it is not easy to reach cheap overproduction of icps during large-scale fermentation. here, we report possibilities to improve delta-endotoxins production as a consequence of responses of bt strains to low levels of heat and salt stress. each stressor results differently in the improvement of delta-endotoxins production, but both were shown to be most efficient at the beginning or the midexponential phase of the cultures which become resistant at the stationary or the sporulation steps. heat stress caused increase of % of synthesis yields of the sporulating cells, in contrast, salt caused increase of % of spores counts, corresponding to % toxins production improvement. combined effects of both stressors lead to toxins production improvement of %, yield improvement of %. we focused on the overcome of carbon repression catabolite, closely related to oxidative metabolism, by an adequate control of dissolved oxygen in the cheap media we formulated for bt insecticides production. we showed that an equilibrium between the high density of vegetative cells and their ability to synthesize toxins during their sporulation was necessary to take into account. % increase of icps production was reached into l fermenter combination of mutagenesis, heat and salt stress and oxidative metabolism control allowed more than % improvement of delta-endotoxins. these results are of great importance in practical point of view, since high bioinsecticides concentrations could be produced without decrease of the yields of their production. mechanism and function of the intramembrane-cleaving protease rhomboid marius lemberg , javier menendez , christopher koth , matthew freeman : mrc laboratory of molecular biology, cambridge, uk; ontario center for structural proteomics, toronto, canada rhomboids are a family of intramembrane serine proteases that are widely conserved throughout evolution. among diverse functions discovered so far, rhomboids participate in intercellular signalling, parasite invasion, membrane dynamics and bacterial quorum sensing, making them potentially valuable therapeutic targets. the identification of physiological substrates and of selective inhibitors will be key towards their evaluation as drug targets. we have developed an in vitro cleavage assay to monitor rhomboid activity in the detergent solubilised state, enabling the first isolation of a highly pure rhomboid with catalytic activity. analysis of purified mutant proteins suggests that rhomboids use a serine protease catalytic dyad instead of the previously proposed triad, and gives insights into subsidiary functions like ligand binding and water supply. oligosaccharides and -keto-glycosides. availability of the enzyme is, however, hampered by the very slow growth and low production rates of the fungus. cloning of the encoding gene and production of the protein by heterologous expression are therefore a prerequisite not only for any biotechnological application, but further scientific investigations as well. on the basis of peptide sequences degenerated primers were designed, and the resulting pcr fragment was used as a probe to isolate the gene from a genomic library. two very similar genes encoding previously uncharacterized proteins were also found, and flanking regions were amplified using rage-pcr. furthermore cdna clones of all genes were isolated by rt-pcr. for textile dyeing with natural dyes, indigo has an almost unique position as the most blue natural dye. due to the importance of indigo, considerable research has been conducted to replace the chemical synthesis of the dye by an application of biotechnological methods. therefore, we investigated several characteristics of natural indigo derived from polygonum tinctorium and its dyeing properties using silk fabrics, such as washing, perspiration, and light fastnesses. this work was financially supported by program for cultivating graduate students in regional strategic industry from korea industrial technology foundation. the behavior of phytate degrading enzymes isolated from malaysian zea mays root in rice bran media anis shobirin meor hussin , abd-elaziem farouk , hamzah mohd salleh , ralf greiner : biomolecular engineering research group, department of biotechnology engineering, kulliyyah of engineering, international islamic university malaysia, jalan gombak, kuala lumpur, malaysia; centre for molecular biology federal research centre for nutrition and foods, haid-und-neu-straße , d- karlsruhe, germany phytate degrading enzymes catalyze the step-wise release of phosphate from phytate, the principle storage form of phosphorus in plant seeds and pollen. they are widespread in nature, occurring in plants and microorganisms, as well as in some animal tissues. phytate-degrading enzymes have been studied intensively in recent years because of the great interest in such enzymes for phytate degradation and their application for animal feed and human health. from over isolate screened isolates of phytate degrading enzymes, three isolates from the root malaysian maize plantation have shown phytate degrading activity. the production of phytate degrading enzyme was studied using different concentrations [%, w/v] of rice bran media during different stages of cultivation. the dephosphorylation of phosphate from rice bran phytate has shown regulatory effect on the secretion of bacterial phytases. in this conference, we will present data for the characterization of the enzymes. in this paper we present the properties of a phytase purified from a bacterium isolated from malaysian wastewater, which might find application as an animal feed supplement. the phytase described herein is a periplasmic enzyme. the phytase was purified about fold to apparent homogeneity using ion-exchange chromatography and gel-filtration with a recovery of % referred to the phytatedegrading activity in the crude extract. the enzyme exhibits an activity of about u mg − . gel filtration of the native enzyme on a calibrated sephacryl s- column gave a molecular mass of the phytase of , ± da with elution position being measured by determination of enzyme activity. lower molecular mass species or higher molecular mass aggregates were not observed. the phytase appeared homogeneous by polyacrylamide gel electrophoresis under non-denaturing conditions at ph . and . and gave a single protein band upon sds gel electrophoresis after coomassie staining of the gels. these results indicate that the phytase could be regarded as homogeneous. the estimated molecular mass after sds-page indicated that the phytase having a molecular mass of , ± da. consequently, this enzyme is a monomeric protein. the purified enzyme had a single ph optimum at ph . and was virtually inactive above ph . . at ph . , % and at ph . , % of the activity at optimal ph was observed. the effect on enzyme stability was studied in the ph range . - . at • c. within days the phytase did not lose any activity in the ph range from . to . , but at ph values below . a rapid decline in activity was observed. at ph . , % and at ph . , % of the initial activity was lost during h. in the range of temperatures studied, - • c, the optimum temperature for the enzyme was found to be • c. the apparent activation energy was estimated at ph . from the slope of log v max versus /t. the data showed excellent linearity from to • c. the arrhenius activation energy for the hydrolysis of phytate was calculated to be . kj/mol. in order to check thermal stability, the purified enzyme was incubated at different temperatures, cooled to • c and assayed using the standard phytase assay. the enzyme lost no activity in min at temperatures up to • c. when exposed for min at • c, it retained % and at • c % of the initial activity. in order to determine the substrate selectivity of the purified phytase, several phosphorylated compounds in addition to phytate, were used for k m and v max estimation by detecting the release of the phosphate ion during hydrolysis using formation of a soluble phospho-molybdate complex in an acidic water-acetone mixture. only phytate was identified as a substrate. the kinetic parameters for the hydrolysis of phytate were determined to be k m = . mmol l − and k cat = s − at ph . and • c. like other bacterial phytatedegrading enzymes, the purified enzyme showed substrate inhibition. the activity of the purified enzyme was inhibited at substrate concentrations > mm. the study of the effect of metal ions on enzyme activity showed that none of them had an activating effect when used at a concentration between − and − m. mg + , ca + , mn + , co + , ag + , hg + , and cu + had little or no effect on enzyme activity, while zn + , fe + , and fe + showed strong inhibitory effects. the reduced phytate-degrading activity in the presence of fe + and fe + is attributed to a lower phytate concentration in the enzyme assay because of the appearance of a fe-phytate precipitate. when compounds which tend to chelate metal ions, such as o-phenanthroline, edta, oxalate, citrate or tartrate, were tested for their effect on enzyme activity, it was noted that none of them was inhibitory at a concentration from − to − m. fluoride, a known inhibitor of different phytate-degrading enzymes from bacteria and the hydrolysis product phosphate as well as its structural analogs molybdate, wolframate and vanadate were found to be strong inhibitors of the purified enzyme. flouride inhibited the hydrolysis of phytate with a k i value of mol l − . several fusion strategies have been developed for the expression and purification of small antimicrobial peptides (amps) in recombinant bacterial expression systems. in the present work, we investigated the use of the baculoviral polyhedrin (polh) protein as a novel fusion partner for production of a model amp (halocidin subunit; hal ) in escherichia coli. the recombinant hal amp could then be hydroxylamine cleaved from the fusion protein and easily recovered by simple dialysis and centrifugation. this was facilitated by the fact that polh was soluble in the alkaline cleavage reaction but became insoluble during dialysis at a neutral ph. importantly, recombinant and synthetic hal peptides showed nearly identical antimicrobial activities against e. coli and staphylococcus aureus, which were used as representative gram-negative and -positive bacteria, respectively. these results demonstrated that baculoviral polh can provide an efficient and facile platform for production or functional study of target amps. extensive industrial and food additive applications of succinate have attracted much effort towards finding an environment-friendly alternative to the petrochemical production processes. it is very attractive to engineer s. cerevisiae for succinate production because of its generally regarded as safe (gras) status, ease of genetic manipulation and fermentation. we approached this metabolic engineering problem with a two-step methodology combining modern computational as well as molecular biology tools. in the first step we identified potential metabolic engineering targets leading to high succinate yield and productivity, with the aid of genome scale metabolic model and a bi-level optimization framework using flux balance analysis and quadratic programming. in the next step, various deletion mutants are being constructed and characterized for physiology and succinate production. results from these experiments then will be used to improve the predictions in computational models. so far, we have constructed a saccharomyces cerevisiae mutant deleted in sdh , which encodes a major subunit of sdh-complex converting succinate to fumarate in mitochondria. the physiology of sdh mutant has been characterized in aerobic and anaerobic batch cultivations and in glucose limited chemostat at dilution rate as low as . h − . in aerobic batch fermentations, the mutant showed reduced maximum specific growth rate as compared to the wild-type, and it was incapable of growing on ethanol as sole carbon source, as predicted from the model. interestingly, the mutant showed much higher specific growth rate in anaerobic conditions, close to the wildtype strain. moreover, the chemostat cultivations indicate that the critical dilution rate of the mutant is below . h − . this opens further opportunities to investigate interesting behavior of the mutant and the underlying regulatory processes to improve our understanding of yeast mitochondrial metabolism. department of chemical engineering, yıldız technical university, davutpaşa campus, esenler/istanbul, turkey. e-mail: dkilic@yildiz.edu.tr (d.k. apar) lactose is the dominant carbohydrate in milks which are, in turn, the only significant natural sources of lactose. a large number of people do not digest lactose properly due to a lack or inactivity of the intestinal beta-galactosidase and they suffer from intestinal dysfunctions -gas, abdominal pain and diarrhea -if their diet contains lactose. moreover, lactose is a sugar with a high bod, low sweetness, low solubility, when compared to the products of its hydrolysis (glucose and galactose) and being a hygroscopic sugar has a strong tendency to adsorb flavours and odours. the hydrolysis of this sugar is very attractive towards the improvement of processes for the production of ice cream and other refrigerated dairy products and it could be very interesting for the development of additives for animal and human alimentation. the enzymatic hydrolysis of lactose is carried out by beta-galactosidases, enzymes that are widely distributed in nature, appearing in micro-organisms, plants and animal tissues. the present investigation describes the effects of the sonication process parameters on enzymatic hydrolysis of milk lactose and enzyme stability. bandelin sonopuls sonicator was used for the lactose hydrolysis experiments. ␤-galactosidase enzyme used is produced from kluyveromyces marxianus. the reactions were carried out in ml of milk. the process variables for the sonicator are duty cycle, acoustic power and enzyme concentration. the amount of residual lactose concentration (g/l) and residual enzyme activity (%) against time were investigated versus process variables. beside of this; the mathematical models depending on the operating conditions were also derived by using the experimental data of lactose concentration and enzyme activity. kinÖzbek department of chemical engineering, yıldız technical university, davutpaşa campus, esenler/istanbul, turkey. e-mail: dkilic@yildiz.edu.tr (d.k. apar) over the last decade, the use of plant protein hydrolysates alternative to animal protein hydrolysates in human nutrition has broadly expanded. protein hydrolysates are often used in different nutritional formulations, such as supplementation of drinks to enhance their nutritional and functional properties, or special medical diets. there are many processes which employ protein hydrolysis and hydrolytic products. among these processes, the use of enzymes allows selective hydrolysis of protein and produces a potentially safer and more defined material. in the present study, the effect of the temperature, ph and viscosity on the hydrolysis of corn gluten was investigated using a stirred batch reactor system. the corn gluten was hydrolysed by using neutrase enzyme, a bacterial protease produced by a selected strain of bacillus amyloliquefacien. the reactions were carried out in . l of aqueous solutions containing % (w/v) corn gluten and . % (v/v) enzyme. the degree of hydrolysis (%) and soluble protein concentration depending on the time were investigated at the temperatures , , , , and • c; and at the ph values . , , . , and . to investigate the effect of viscosity, the various amounts of glycerol was added to the reaction solutions to produce viscosities in the range of . - . cp. the degree of hydrolysis (dh) was computed by using ph-stat method. for the soluble protein determination in the hydrolysates samples, the folin-lowry method ( ) was used. polyhydroxyalkanoates (pha), one of the most promising bioplastics for the partial replacement of synthetic polymers like polypropylene, are polyesters produced by bacteria as intracellular storage reserves of carbon and energy. the industrial production of pha is achieved by pure cultures in its natural state or using genetically engineered organisms. the main obstacle to the replacement of synthetic plastics by biopolymers is their great cost difference. research on the field of biopolymers synthesis using mixed cultures and waste organic carbon sources as substrates prove to decrease substantially the production costs of pha. the optimization of pha production under aerobic feeding conditions (adf) was achieved recently in our group, obtaining the highest value of pha content stored by mixed cultures ( . % of cell dry weight). in this work only a homopolymer of polyhydroxybutyrate (phb) was obtained and since it is a highly crystalline and brittle material its application field is limited. the mechanical and thermal properties of pha can be varied to a great extend by adjusting the monomer composition. the incorporation of different monomeric units, other than hb, in the polymer chain, originates copolymers with improved mechanical properties. optimization of pha production from propionate by a mixed culture was studied varying the carbon and ammonia concentrations. propionate only, acetate alone or a mixture of acetate and propionate were tested. copolymers of hydroxybutyrate and hydroxyvalerate, p(hb/hv), with different compositions were obtained. consequently polymer properties could be manipulated by feeding the selected volatile fatty acid composition. the pharmaceutically important plant species of glycyrrhiza sp. (called licorice) is an important commercial product used as a natural sweetener, anti-inflammatory, anti-cancer and anti-diabetic agents. agrobacterium rhizogenes transformation system was used for the hairy root cultures of licorice g. uralensis. after inoculation of aseptic stem segments the ability of hairy root formation was scored for a period of weeks. mean transformation frequency ranged from % (for up to % (for ). some transformed genotypes showed significant differences in roots weight, flavonoids and glycyrrhizin (gl) production. the cotransformation rate of licorice intact explants cultivars with lba tl-dna and the s gus gene showed an average of more than %. these obtained root cultures were additionly elicited with extracts of biotic elicitor acremonium sp. (endomycorhizal fungus), and were used as an in vitro system to metabolites production. the transformed and elicited hairy roots of g.uralensis were obtained by infection of a. rhizogenes have produced gl at an yield of . % dry weight on the period of culture as a days. according to tentative analyses the hairy roots cultures of glycyrrhiza species produced flavonoids (liquiritigenin and liquiritigen). more high levels ( . g/l) of the total flavonoids production have been identificated on the strains which transformed by lba . this study involved any difference among elicitor treatments and incubation periods for the optimal meabolites production. clearly, the selection of an effective agrobacterium strain for the production of transformed root cultures is highly dependent on the plant species, and must be determined empirically. production, purification and characterization of scytalidium thermophilum phenol oxidases didem sutay , ufuk bakir , zumrut b. ogel : chemical engineering department, middle east technical university, inonu bulvari, ankara, turkey; food engineering department, middle east technical university, inonu bulvari, ankara, turkey. e-mail: ubakir@metu.edu.tr (u. bakir) phenol oxidases (pos) are a group of enzymes which are responsible for oxidation of various phenolic compounds in the presence of molecular oxygen. there are different types of pos present in nature and three major groups of these enzymes are laccases (e.c. . . . , p-benzenediol: oxygen oxidoreductase), catechol oxidases (e.c. . . . , o-diphenol oxidoreductase) and tyrosinases (e.c. . . . , monophenol monooxygenase). another group of enzymes, peroxidases (e.c. . . . ), can also be considered as a member of po family. pos have very wide substrate range and final oxidation products of these substrates are quinones, which are highly reactive molecules and polymerize into brown, red or black waterinsoluble compounds. pos are very common in nature, they can be found in almost all plants, animals and microorganisms. pigmentation and protection from the environment are main functions of these enzymes. pos have different applications in food, pharmaceutical, textile industries and waste-water treatment systems. the objective of this study was po production by the thermophilic fungus, scytalidium thermophilum, purification and characterization of the enzyme. for this purpose, enzyme production was performed either in a shaker-incubator or a temperature, ph and dissolved oxygen controlled l bioreactor (probiotem) to optimize enzyme production medium composition and bioreactor parameters. as the carbon and nitrogen sources, % glucose and . % yeast extract were determined as the optimal concentrations, respectively. copper, gallic acid and tannic acid were determined to increase enzyme production. purification was performed by using membrane and chromatographic techniques. hydrophobic, ion exchange and gel filtration columns were used by using a fplc system;Äkta prime (amersham biosciences). especially the phenyl sepharose tm high performance column appeared to be very efficient for po purification from scytalidium thermophilum. purified po have been characterized by electrophoretic techniques and kinetic studies. isolation of lipolytic microorganisms from subtropical soils. cloning, purification and characterization of a novel esterase from strain pseudomonas sp. cr- núria prim, cristian ruiz, cristina bofill, f.i. javier pastor, pilar diaz department microbiology, university of barcelona. av. diagonal , microorganisms or their enzymes are used in a wide range of biotechnological activities such as polymer hydrolysis, synthesis of added-value compounds, sample decontamination, etc. thus, there is an increasing interest for isolating new enzymes and new enzymeproducing organisms for their use in industrial conversions (cherry and fidantsef, ) . among these enzymes, lipases, esterases, cellulases, xylanases and pectinases play an important role in many biotechnological processes like those related to pulp and paper processing. three samples of subtropical forest soil from puerto iguazú (argentina) were used for the isolation of autoctonous microorganisms growing in an organic matter-rich environment. a total of pure cultures of bacteria and fungi were obtained and their hydrolytic activities on polysaccharide and lipidic substrates were assayed using olive oil, tributyrin, cholesterol esters, xylan, cellulose and pectin as substrates. among the isolates analysed, were active on one or more of the substrates evaluated, and of them degraded all substrates. nearly half of the strains displayed lipolytic activity, whereas the number of strains active on xylan, cellulose, pectin and cholesterol esters, was much lower. the strains bearing the highest hydrolytic activities were selected and stored for further characterization (ruiz et al., ) . among them, strain cr- , one of the most active isolates on tributyrin, was selected for identification and characterization of its lipolytic system. lipolytic strain cr- was identified by morphological, physiological and phylogenetic tests, as a pseudomonas sp., closely related to p. fluorescens. sds-page and zymogram analysis (diaz et al., ) of cell extracts and supernatants from the strain revealed a complex lipolytic system consisting of at least two lipolytic enzymes. sequence alignment and clustering of previously described pseudomonas lipases and esterases allowed the design of different sets of primers for the isolation of the lipase/esterase coding genes. a gene coding for an esterase with homology to family vi bacterial lipases (arpigny and jaeger, ) was isolated and cloned in escherichia coli. the cloned enzyme was further purified and characterized, showing preference for short fatty acid esters and displaying a typical michaelis-menten kinetics, with no interfacial activation. the substrate profile, together with the kinetic behaviour and sequence similarity of the cloned enzyme to family vi bacterial esterases, allowed to identify this enzyme as an esterase and was named esta . maximum activity was achieved on muf-butyrate at • c and ph . , suggesting that it could be of interest for biotechnological purposes. microbial xylitol production from agricultural wastes has recently attracted much attention from industries because it has potentials to realize the cheaper production of xylitol with low environmental impact (tada et al., ) . in order to realize the effective xylitol production by a xylose utilizing yeast, the oxygen supply is a key for maximizing xylitol yield over consumed xylose (y xl ) because the intracellular xylitol metabolism is strongly influenced by the amount of available oxygen. in the present work, we tried to apply a metabolic reaction model in order to determine the optimal oxygen transfer rate (otr) in a fermentor for maximizing xylitol yield. corn cob hydrolysates containing g-xylose/l was employed as medium for xylitol production by computer-controlled batch cultures using candida magnoliae (ferm p- , aist). a metabolic reaction model considering main xylitol metabolisms including glycolysis, pentose-phosphate pathway, tca cycle and cell synthesis was developed. the model allows to estimate various intracellular metabolic flux distributions including a xylitol production rate. the oxygen uptake rate to maximize the ratio of xylitol production rate over xylose consumption rate corresponds to the otr condition to maximize a xylitol yield over xylose consumed. based on the metabolic reaction model, the otr was optimized by a linear programming, the optimal otr and the maximum xylitol yield were estimated as . mmol o /l h and . g-xylitol/g-xylose, respectively. the experimental verification using the optimal otr demonstrated that the xylitol yield was greatly improved to . g-xylitol/g-xylose from . g-xylitol/g-xylose in our previous study. expression of a bacterial sugar phosphate transporter in s. cerevisiae to release l-glycerol -phosphate accumulated by metabolic engineering almut popp, huyen thi thanh nguyen, ulf stahl, elke nevoigt department of microbiology and genetics, university of technology, berlin, germany. e-mail: a.popp@lb.tu-berlin. de (a. popp) in contrast to glycerol, its phosphorylated precursor l-glycerol- phosphate (l-g p) is retained by the plasma membrane. therefore, engineered yeast strains accumulate l-g p in the cytosol resulting in low overall yield of the desired product and laborious downstream processing. a suitable sugar phosphate transporter in the yeast plasma membrane would overcome these limitations. the glycerol- -phosphate transporter (glpt) of e.coli is an antiporter and naturally mediates the uptake of l-g p in exchange with inorganic phosphate. we assume that this transporter would also mediate the excretion of accumulated l-g p into a phosphate-rich medium if it was present in the plasma membrane of engineered yeast. despite many inconsistencies in codon usage, we were able to express the bacterial glpt gene in yeast. expression was monitored by a c-myc tag added to the c-or n-terminal hydrophilic tail, respectively. the quantity of the construct with n-terminal tag clearly exceeds the quantity of the construct with c-terminal tag. both gene products are located in the endoplasmic reticulum, as shown by immunofluorescence microscopy. obviously, yeast's transmembrane protein sorting machinery does not recognise it as a substrate for the secretory pathway. metabolic flux analysis of c-and p-limited shikimic acid producing e. coli gaspard lequeux , louise johansson , jo maertens , peter vanrolleghem , gunnar lidén : biomath, ghent university, coupure links , gent, belgium; department of chemical engineering, lund university, p.o. box , lund, sweden. e-mail: gaspard.lequeux@biomath.ugent.be (g. lequeux) metabolic flux analysis (mfa) was applied to decipher why plimited e. coli fermentations are more optimal for shikimic acid production in comparison with glucose-limited fermentations. as mfa allows obtaining insight in the intracellular flux distribution over different pathways by only measuring net production and consumption rates of metabolites, under the condition that parallell pathways are removed. to this end a detailed metabolic network model was created and checked for consistency, dead-ends, and parallell pathways. several fermentations were performed at different dilution rates (ranging from . to . h − ) and different limitations (phosphate and glucose). the e. coli strain used was w with genetic modifications in the aromatic amino acid pathway to enhance shikimic acid production. for each dilution rate, a metabolic model was solved. this way, the evolution of each flux can be followed with respect to the dilution rate. the p-limited cultures showed a better yield which can be explained by the diminished excretion of dehydro-shikimic acid (as is known from literature) and a reduction of the hydrolysis of atp. takasumi hattori, kuniki kino, kohtaro kirimura department of applied chemistry, school of science and engineering, waseda university, tokyo, japan. e-mail: takasumi@suou.waseda.jp (t. hattori) alternative oxidase is a terminal oxidase in respiration chain, which is a branched chain of cytochrome pathway, and inhibited by salicylhydroxamic acid (sham), but not by cyanide. the citric acid-producing fungus aspergillus niger wu- l has a cyanide-insensitive and sham-sensitive respiration catalyzed by the alternative oxidase (kirimure et al., ) and did not produce citric acid when cultivated with sham (kirimure et al., ) . in this study, the transcript levels of alternative oxidase gene (aox ) (kirimure et al., ) and activities of alternative oxidase under the conditions of citric acid production were examined during days-cultivation. the amount of aox mrna was determined by northern blot analysis, and the specific activity of alternative oxidase as that of duroquinol oxidase. the transcript level and the activity were highest at days at log-phase, decreased during and days, and thereafter maintained at low levels. however, the transcript and alternative oxidase activity was constitutively detected during whole the cultivation periods under the conditions of citric acid production. the sequence analysis of aox chromosomal dna revealed the presence of potential binding site of cyclic amp responsive element (cre), stress responsive element (stre) and heat shock factor (hsf) in its upstream region. these results indicated that the expression of alternative oxidase was regulated in the transcription level and alternative oxidase contributes as the main respiration chain during citric acid production. kirimura, k., et al., . curr. genet. , - . kirimura, k., et al., . biosci. biotechnol. biochem. , - . trichoderma strains are considered to be among the most useful fungi in industrial enzyme production, agriculture and bioremediation. metabolic versatility displayed by these fungi makes it a very amenable source of new gene products. functional analysis of candidate genes goes by two complementary ways: gene overexpression and loss-of-fuction mutants generation. a few expression systems are available mainly to direct constitutive gene expression in catabolite repression conditions. following a genomic approach, we have recently cloned some gene promoters with high expression in glucose in trichoderma harzianum cect . a main goal in a gene functional analysis is the generation of knock-out mutants. up to date, there is no reference about successful gene disruptions in t. harzianum mainly due to a very low homolog recombination frequency. rna-mediated gene silencing has been shown as an efficient tool to diminish or totally abolish specific gene expression. especially those strategies based on the use of hairpin constructs allow rapid and easy generation of strains with reduced levels of mrna from genes of interest. we have used the t. harzianum cect tss promoter to direct the expression of a hairpin dna construct that induced the appearance of small-interfering rnas (sirnas) and the silencing of the uida reporter gene in a previous uida overexpressing strain. reduced levels of mrna and gus activity correlated with the presence of sirnas. this is the first report on rna-mediated gene silencing in trichoderma and constitutes a useful and promising tool for functional genomic studies in fungal systems. analysis of the metabolic response of escherichia coli to quantitative modulations of the glucose- -phosphate dehydrogenase based on c-labelling experiments cécile nicolas, fabien létisse, stéphane massou, philippe soucaille, jean-charles portais. e-mail: fabien.letisse@insa-toulouse.fr (f. létisse) microorganisms have an efficient capacity for adapting their metabolism in response to genetic or environmental changes, and understanding metabolic robustness has become an emergent issue. part of the robustness originates from the network organization of metabolic systems, where the interplay between all available biochemical reactions provides alternative mechanisms for compensating the perturbations. recently, c-metabolic flux analysis ( c-mfa) has been applied to escherichia coli knock-out mutants lacking key enzymes to determine the phenotypic effects of structural changes in the metabolic network, providing further evidences for compensatory phenomena. the aim of our on-going work is to understand how the central metabolism in e. coli responds to quantitative alterations at a specific key-point of the metabolic network. the glucose- -phosphate dehydrogenase (g pdh), a key enzyme in the central metabolism for which the effects of deleting the gene (zwf) has been already described (zhao et al., ) , was chosen as the target. to this aim we have generated a set of expression mutants, i.e. mutants having each a fixed level of expression of the zwf gene. four different levels of expression, leading respectively to g pdh activity ; . ; . and times higher than in the wt strain, have been obtained. for each mutant, transcriptomics analysis will be carried out and compared to both the zwf-and wt strains to detect changes in the network structure, and the distribution of fluxes will be measured using c-mfa. the flux maps obtained for the various strains will be compared to evaluate the quantitative response of the central metabolic network to imposed and increased g pdh activity. metabolic control analysis will be applied to provide insights onto the sensitivity of the measurable metabolic fluxes to g pdh activity. combination of transcriptomics and fluxomics approaches will provide information on the nature and extent of the compensatory mechanisms. because the activity of a single enzyme is tuned at different levels in knock-out and expression mutants, this investigation provides a situation that mimics gene-level regulation of metabolism. , j., et al., , metab. eng., , . with the depletion of the world's petroleum supply, there has been an increasing worldwide interest in ethanol as an alternative, nonpetroleum source of energy. this fact caused increased interest in the new ethanol technology fermentation process research. as reported before, bacteria zymomonas mobilis possesses more advantages than saccharomyces cerevisiae, microorganism used for ethanol production in industrial scale. for that reason, we have focused on the fermentation studies of this facultative bacterium in free and immobilized form. the immobilization of the cells into polyvinylalcohol (pva) hydrogel lens-shaped capsules lentikats, improved the batch fermentation process efficiency nine times. starch, the substrate considered as one of the best of renewable energy source is considered as fuel ethanol feedstock. due to z. mobilis disability of maltose, maltotriose and dextrin utilization, the starch has to be converted into glucose monomers. this pre-fermentation step can overcharged whole ethanol production process. this ineffective part of the process was resolved with immobilization of glucoamylase into lentikats. the system with immobilized enzyme and cell was stabile in continuous mode for days without any significant change in the system efficiency. the combination of cell and enzyme immobilization can significantly improve the efficiency and the cost of ethanol production in industrial scale. fermentation of an inhibitory dilute acid-hydrolysate from spruce using a fed-batch procedure combined with cell-reuse andreas rudolf, gunnar lidén department of chemical engineering, box , lund university, se- lund, sweden. e-mail: andreas.rudolf@chemeng.lth.se (a. rudolf) a well-controlled addition of hydrolysate to the fermentation has proved very efficient in reducing yeast inhibition due to compounds formed during lignocellulose hydrolysis. furthermore, using high cell mass concentrations has been another way of avoiding the negative impact of the inhibitory compounds. if possible, the yeast should therefore be re-used in the process. in the present work a dilute-acid hydrolysate from spruce was fermented using a fed-batch procedure with reutilization of yeast. the fermentation procedure worked satisfactorily, with more than % of fermentable sugars consumed in each of the four consecutive fed-batch fermentations performed. the ethanol yields on fermentable sugars reached . g/g. there was continued cell growth in the repeated fed-batch experiments, with an average cell yield on fermentable sugars of . g/g. in contrast, only about % of the fermentable sugars were consumed within h, when the fermentation of the hydrolysate was run in a batch process. the work shows the potential to re-use the yeast in a suitably designed process. metabolic engineering is defined by bailey in his seminal paper as "the improvement of cellular activities by manipulation of enzymatic, transport, and regulatory functions of the cell with the use of recombinant dna technology". the manipulation of these functions ultimately results in the manipulation of metabolism, which is the purpose of many biotechnological processes. metabolic engineering has sought its methods and tools in mathematics and the physical sciences, and later in information technology (it) leading to the proliferation of bioinformatics. in this work we propose a novel approach to metabolic engineering that regards it as a business process reengineering (bpr) endeavour. hammer and champy in their celebrated book define bpr as "the fundamental rethinking and radical redesign of business processes to achieve dramatic improvements in critical contemporary measures of performance, such as cost, quality, service and speed". our thesis is that metabolic engineering with its goal of reengineering the metabolism of the microorganism, is equivalent to business process reengineering (bpr) in business and management. indeed this is essentially what metabolic engineering does to the cell through the use of recombinant dna technology, which can be viewed as a radical redesign of the metabolic process. after all, it causes changes that cannot be attained otherwise, and whose purpose is to achieve dramatic improvements in cellular activities such as the increase in production of some metabolites by orders of magnitude. the cost incurred by the process, which is metabolism in this case, can be, for example, energy requirements or change to a cheaper substrate. in this study we elucidate this parallelism between the two with emphasis on modelling of metabolism and how the concepts of business process modelling can be applied to it. the purpose is not to produce a model, but rather to introduce the modelling methodology and demonstrate its utilisation and benefits and outline its limitations and challenges. we believe that the novelty of our work lies in applying a new paradigm in approaching metabolic engineering that has not been considered previously. thermophilic ethanol production from wheat straw hydrolysate in continuous culture tania i. georgieva, birgitte k. ahring biocentrum-dtu, technical university of denmark, building , dk- lyngby, denmark. e-mail: tig@biocentrum.dtu.dk (t. georgieva) ethanol production from lignocellulosic biomass has attracted widespread attention as an unlimited low cost renewable source of energy to transportation fuels due to increasing petroleum use and air pollution towards greenhouse gases. wheat straw available as agricultural residue has been considered as a potential lignocellulosic substrate for industrial bioethanol production. a major technical obstacle to commercialize bioethanol production form lignocellulose (such as wheat straw) is associated with a lack of microorganism able to rapidly and efficiently ferment both hexose and pentose sugars into ethanol and to tolerate the inhibitors present in undetoxified hydrolysates. currently used industrial mesophilic microorganisms (saccharomyces cerevisiae and zymomonas mobilis) are excellent ethanol producer from glucose, however, they are not able to ferment other sugars such as xylose, which is the second most abundant sugar in lignocellulose. thermophilic anaerobic bacteria have been considered for ethanol production from lignocellulosic biomass as an alternative to mesophilic ethanol producing strains, predominantly because of their abilities naturally to ferment the whole diversity of sugars found in lignocellulosic biomass. an increase attention to thermophilies for ethanol production have also arise from broad spectrum of advantages regarding industrial scale ethanol fermentation such as high growth and metabolic rates, low oxygen solubility, reduced risk of reactor contamination, and cost savings via mixing, cooling and facilitated product recovery. in addition, simultaneous co-fermentation of glucose and xylose in a single operation unit could substantially reduced the ethanol production cost. research has been attempted to study the potential of using a thermophilic anaerobic bacterium for continuous ethanol fermentation of lignocellulosic biomass, with particular emphasis on effectiveness of our strain to ferment undetoxified wet oxidized wheat straw hydrolysate with respect to sugar (glucose and xylose) conversion and ethanol yield. the experiment was carried out in a lab-scale reactor operated at • c with wheat straw hydrolysate as a substrate in concentrations from to wt.% equivalent to total sugar mixture of - g/l. wheat straw hydrolysate (woh) [ g/l wheat straw, . % dry matter (dm)] was prepared using wet oxidation pretreatment process followed by enzymatic saccharification with commercial enzymes mixture of celluclast . , and novozym (novozymes, denmark). both xylose and glucose sugars were simultaneously converted to ethanol. the sugar utilization was higher than %, and high ethanol yields were achieved. reactor shows good long-term performance ( days) in terms of operation stability and reactor contamination. maltotriose is the second most abundant fermentable sugar in wort and due to incomplete fermentation, residual maltotriose in beer may cause problems in the brewing industry. to study genes that might improve utilization of maltotriose we used a library with dna from brewer's strains and a laboratory strain and identified a new transporter encoded by mtt . mtt gave lager strain a the ability to grow on yp/ % maltotriose in the presence of mg/l of the respiratory inhibitor antimycin a. this transporter gene shares % similarity with mph and mph , % similarity with agt and % similarity with mal and mal . moreover, mtt shares even higher similarity ( %) with the s. pastorianus mty gene (m. salema-oom, unpublished, ncbi accession number aj ). purified radiolabeled maltotriose and radiolabeled maltose were used to study sugar uptake of lager strains a and ws / , and of a containing mtt or mtt alt, a more efficient, altered version of this gene lacking the basepairs from the end and containing base-pairs of vector sequences. these transport studies show that mtt and, especially, mtt alt encode maltose transporters with relatively high activity towards maltotriose compared to maltose. this study is part of a multi-disciplinary project, funded by the european union (contract no. qlk -ct- - ) focusing on the development of high-gravity resistant brewer's yeast strains. metabolic engineering of l-phenylalanine pathway in bacillus subtilis yasemin demirci , pınar Ç alık , güzide Ç alık , tunçer h.Özdamar : bre laboratory, department of chemical engineering, ankara university, ankara, turkey; iblab, department of chemical engineering, metu, ankara, turkey. e-mail: ozdamar@eng.ankara.edu.tr (t.h.Özdamar) metabolic engineering design of a recombinant l-phenylalanine (phe) production system is based on coordinated overexpression of the flux-controlling genes in the aromatic-amino acid pathway. based on the insights gained by the work carried out in our laboratories (Özçelik et al., ) , aroh for the reaction r at the branch-point chorismate that connects the preceding reactions of the aromatic group amino acid pathway to the proceeding reactions towards phe, was predicted as the first-, and aroa for dahp synthase (r ) predicted as second-metabolic engineering sites. aroa gene was cloned next to aroh, by the use of four primers designed using pcr-based gene splicing by overlap extension method. the genes were amplified separately by pcrs. the primers used at the ends to be joined were designed as complementary to one another by including nucleotides at their ends that are complementary to the portion of the other primer. these products were mixed in the next pcr reaction, where one strand from each fragment contains the overlap sequence at end. extension of this overlap by dna polymerase has yielded the recombinant two-gene product; and the two-gene product serve as template for the continuation of reactions for the increase of the concentration in the micro-reactors. the two-gene fragment was first cloned into puc , and then sub-cloned to pmk e. coli -bacillus shuttle plasmid. the new expression vector with high stability and high copy-number was obtained and transferred into host b. subtilis. the metabolic flux distributions calculated by the mass balance based stoichiometric model based on the metabolic reaction network for r-b.subtilis were determined by using time profiles of the substrate, dry-cell, phe and other amino acids, and organic acids concentrations as the constraints. on the bases of calculated intracellular fluxes of recombinant b. subtilis carrying pmk ::aroa::aroh, an in-depth analyses of the metabolic engineering design will be presented. ozçelik,İ., Ç alık, p., Ç alık, g.,Özdamar,t.h., . metabolic engineering of aromatic group amino acid pathway in bacillus subtilis for l-phenylalanine production. chem. eng. sci. ( - ), - . the % respiratory-deficient nuclear petite amylolytic saccharomyces cerevisiae npb-g strain capable of excreting a hybrid protein possessing both ␣-amylase and glucoamylase enzyme activities was generated and its employment for direct fermentation of starch into ethanol was investigated under both shake flask and controlled bioreactor cultivation conditions. when compared with a standard host strain, higher ethanol concentrations and yields were achieved with the nuclear petite strain under both cultivation conditions. further improvement in ethanol production was achieved by the use of an initial glucose supplement. comparison of the ethanol fermentation performances of the respiratory-deficient npb-g and the parental respiratory-sufficient wtpb-g strain showed an increase of, ca. % in both ethanol production yields and ethanol productivities with the respiratory-deficient strain. response surface methodology (rsm) was used as a statistical tool to optimize the initial yeast extract and starch contents of the medium, which resulted in a substantial increase in the stability of the expression plasmid in both strains with concomitant improvement in their amylolytic potentials. high ethanol yields on substrate values of the bioreactor cultures, that were very close to the theoretical yield, indicated that the amylolytic respiratory-deficient strain developed in this study was very effective in the direct fermentation of starch into ethanol. establishing a biotechnology educational framework to support a knowledge-based economy in puerto rico rosa buxeda, lorenzo saliceti-piazza industrial biotechnology program, university of puerto rico, mayagüez campus, mayaguez , puerto rico. e-mail: rbuxeda@uprm.edu (r. buxeda) industrial biotechnology has been identified as a major thrust area of economic development within the past five years for the island of puerto rico. the portfolio of biotechnology manufacturing investments in the island has passed the two billion dollar mark. world known companies like amgen, abbott and eli-lilly lead these investments, which have catalyzed a strong technology transfer to the island. a strong collaboration between industry and academia was needed to provide a well-trained workforce for these company startups. as a result, the university of puerto rico, mayagüez campus (upr-m) developed four initiatives which are part of the educational pipeline in biotechnology. these are: (i) an industrial biotechnology program, a -year bs degree which contains a novel curriculum with courses from science and engineering with undergraduate research and industrial internships as part of the degree requirements; (ii) an industrial biotechnology learning center, which provides customized biotechnology and bioprocessing training modules, including lectures and hands-on experiences to train and develop the workforce needed in the biotechnology manufacturing plants; (iii) a biotechnology summer camp, which addresses the high school student population and its main purpose is to educate and advise on the different career paths that can be followed in the field of biotechnology; and (iv) a biotechnology center for research and training in bioprocessing, that will address the development of corporatesponsored research projects to strengthen links between industry and academia in order to build up a knowledge-based economy. our paper will describe each initiative in detail as well as its outcomes and impact on puerto rico's knowledge-based economy goals. isolation of acid phosphatase from sweet potato and immobilization using different adsorbent d. omay, y. güvenilir, n. deveci istanbul technical university, department of chemical engineering, maslak , istanbul, phosphatase enzymes occur in a wide range of plant and animal tissues. they catalyze the hydrolysis of phosphate bonds in organic phosphates, between the phosphate group and rest of the molecule. immobilization of enzymes and biological compounds is currently gaining importance due to its wide variety of applications in the food and pharmaceutical industries and also its biomedical applications. it was reported that enzymes can be activated by complexation with polysaccharides such as chitin or chitosan. the aim of this experimental study was determined as partial purification and isolation of acid phosphatase enzyme and its immobilization. the purification was realized by applying centrifugation, ammonium sulfate precipitation and dialysis respectively. the specific activity of the supernatant was . u/mg and after % saturation this value increased . u/mg. furthermore, acid phosphatase was investigated using different adsorbent (chitin, chitosan, synthetic zeolite and raw zeolite) and evaluated the storage stability and re-usability of the immobilized acid phosphatase. it was estimated that, acid phosphatase activity was shielded the ratio of , , , and % by using raw zeolite, synthetic zeolite, chitin and chitosan respectively under h operation condition. Øyvind m. jakobsen , , michael c. flickinger , svein valla , trond e. ellingsen , trygve brautaset : department of biotechnology, norwegian university of science and technology, norway; sintef applied chemistry, sintef, norway; biotechnol. institute, department of biochemistry, molecular biology and biophysics, university of minnesota, usa aerobic methylotrophs can utilize one-carbon (c ) compounds such as methane and methanol as a sole c-source for growth and energy. the majority of research on these organisms has focused on their biochemical novelity and commercial viability. for the industrial production of bulk products such as the amino acids lysine and glutamate raw material costs and abundance are important, and c sources are thus attractive compared to sugars. bacillus methanolicus can secrete up to g/l of glutamate upon methanol growth at • c (thermotolerant and methylotroph) and mutants producing - g/l of l-lysine have been selected. we study the genetics for conversion of methanol into biosynthesis of glutamate and lysine, and in the present report we unravel the regulation of genes impor-tant for the consumption and tolerance level for c compounds by b. methanolicus. b. methanolicus has a methanol dehydrogenase gene (mdh) for oxidation of methanol into formaldehyde and a ribulose monophosphate (rump) pathway for assimilation of formaldehyde. we recently discovered that mdh and five rump genes are carried by natural plasmid pbm in this bacterium and this represented the first documentation of plasmid-dependent methylotrophy in any microorganism. we here use real-time pcr to analyse the regulation of plasmid-and chromosomally located rump genes, in cells upon methylotrophic and non-methylotrophic growth. high methanol concentrations in the growth medium is cell toxic and the mechanisms for this sensitivity of b. methanolicus is poorly understood. our results indicate that plasmid pbm plays a fundamental role for this trait as well and the impact of our results on the biotechnological applications of this bacterium is discusssed. department, national research centre, dokki, cairo, egypt adding a proteolytic enzyme extraction from jack fruit (artocarpus integrifolis) in combination of fermentation process in low fat yogurts manufacture was tried to improve yogurt flavour and rheological properties. experimental yogurts milk contained control, . (t ), . (t ) and . (t ) units/ml milk from crude extracts of plant proteinase. the ph of the product treated with crude proteinase was lower than the control. however, the rate of acidity development during storage slightly increased with increasing the addition of crude proteinase level and progress of storage period of yogurt. the proteolytic activity of all yogurts gradually increased until the end of storage period ( days). yogurts made from milk treated with crude proteinase preparations were less firm compared with control at all storage periods, where t showed more less firm after days of storage being . g/ g. generally, increasing units of plant proteinase preparations decreased the firmness. on the other hand, yogurt made from milk pretreated with plant proteinase had higher syneresis, and apparent viscosity than the untreated product. the greatest viscosity was found in t and t of and mpa s respectively, compared with control of mpa s at days storage. the results indicated that there is an inverse relationship between the amount of units of crude proteinase preparations and susceptibility of yogurt to syneresis. the t gained the highest scores ( points) followed by the control ( . points) after days of storage, while yogurt of t showed a low scoring being . from the foregoing results, it is recommend to use jack fruit (artocarpus integrifolis) as a source of plant proteinases and utilize it to develop a high quality yogurt at a level of . units of plant proteinases/ml milk. the penicillium chrysogenum oat gene encoding a class iii omega-aminotransferase has been cloned and characterized. this enzyme that converts lysine into -aminoadipic semialdehyde is important in providing -aminoadipic acid, a precursor of penicillin and other ␤-lactam antibiotics. the enzyme is related to ornithine- -aminotransferases and to lysine- -aminotransferases encoded by the lat gene located in the bacterial cephamycin gene clusters. expression of oat is induced by lysine, ornithine and arginine and repressed by ammonium ions. area-binding consensus sequences and an -bp direct repeat associated with arginine induction in emericella (aspergillus nidulans) have been found in the oat promoter region. deletion of the oat gene resulted in the loss of omegaaminotransferase activity. the deletion mutants were unable to grow on ornithine or arginine as sole nitrogen sources and showed a reduced growth on lysine. complementation of the deleted mutant with the oat gene restored growth on ornithine, arginine and lysine to the levels of the parental strain and omega-aminotransferase activity. the strong expression of oat gene after induction by the basic amino acids may provide additional -aminoadipic acid for the formation of the -aminoadipyl-cysteinyl-valine tripeptide for ␤-lactam biosynthesis. morphological characterisation of two high producing strains of penicillium chrysogenum carrying a disruption in the nadph-dependent glutamate dehydrogenase k. rueksomtawin, j. thykaer, h. noorman, j. nielsen center for microbial biotechnology, biocentrum-dtu, technical university of denmark, dk- lyngby, denmark. e-mail: kr@biocentrum.dtu.dk (k. rueksomtawin) metabolic engineering has proven to be useful in optimisation of ␤-lactam production, e.g. constructing superior strains with multiple copies of the ␤-lactam gene cluster. it is however, of equal importance to gain insight into other aspects of the metabolism to establish a general overview in order to apply metabolic engineering for further improvement of the production strains. in that context, the redox metabolism is essential, as it functions as a tightly controlled connection between the different parts of the metabolism. in order to investigate this role of the redox metabolism in more detail, the gdha-gene, encoding the nadph-dependent glutamate dehydrogenase, was disrupted in two industrial strains of penicillium chrysogenum. during physiological characterisation of the two strains it became apparent that considerable changes in the morphology had occurred due to the genetic alteration. since the morphology is an important parameter in process optimisation, an examination of the morphology of the two strains was undertaken. in this work, the morphological differences between the gdha-disrupted strains and the reference strains were comprehensively investigated both during growth on solid media and submerged growth in a flow-through growth chamber. with the advance development of computerized image analysis techniques, the key morphological properties of the individual hyphal elements were quantified. in comparison to the reference strains, the disruption of the gdha gene resulted in a morphological change from short hyperbranched hyphal elements to long elongated hyphal elements with less branches. in the parallel studies with aspergillus nidulans and its corresponding gdha-deleted strain, no difference in the morphology was observed. polyketides (pk) represent one of the largest groups of natural products and are found in fungi, bacteria and plants. since many useful polyketides either originate from sources that are difficult or even impossible to cultivate or are produced in inadequate amounts, we are interested in expressing polyketide synthases (pkss) in heterologous hosts. saccharomyces cerevisiae, aspergillus niger and aspergillus nidulans were chosen as initial hosts, because the techniques necessary to cultivate and manipulate these strains genetically are well established. -methylsalicylic acid synthase ( -msas) was chosen as a model pks. replicative plasmids carrying the genes encoding -msas from penicillium patulum and phosphopantetheinyl transferase (pptase), respectively, were transformed into s. cerevisiae. in addition, an integrative vector was designed and the gene encoding -msas was integrated in the yeast chromosome. batch cultivations on galactose minimal media were performed. the results are presented and in particular the effect of expression mode and type of pptase (bacterial versus fungal) is discussed. furthermore, the progress of the work on expressing -msas in a. niger and a. nidulans using an integrative vector system is presented and discussed. the valorisation of functionalized chemicals from biomass resources compared to the conventional fossil production route ben brehmer, wageningen ur agrotechnology & food sciences, workgroup: valorisation of plant production chains, p.o. box , aa wageningen, the netherlands. e-mail: ben.brehmer@wur.nl at some undisclosed point in the foreseeable future, cheap fossil fuels resources will become depleted and the industry will be forced to pursue more difficult reserves with increasingly high extraction costs. most alternatives available and under research do not consider price as the main motivation for replacement, but focus solely on sustainability and environmental benefits. sustainability is an interesting word as there are enough fossil resources scattered around the world to be sustainable in quantity but not sustainable in price. seeing that fossil fuels are derived from prehistoric biomass it is not at all presumptuous to assume that every application and product can be replaced by the biomass of today. in fact, many highly specialised pharmaceutical chemicals already have a biomass origin. yet, not all of the uses of fossil fuels need to be replaced by a comparable carbon based source, such as biomass. energy and transportation in particular do not necessary need to rely on carbon cleavage, whereas practically all of the petrochemicals contain a carbon backbone. the main stipulation in substituting fossil-based chemicals with bio-based chemicals is availability and cost. it is proposed that already today, by utilising existing, recently developed and developing technology, it is economically advantageous for many chemicals to derive from biomass, in particular the functionalized chemicals. the only way to validate this conjecture is to develop a complete comparative life cycle analysis. as opposed to a traditional lca, the "multicriterion" developed here will revolve around energy flows and process efficiency in terms of exergy. the aim is to assess the optimum route with the best production options along the whole production chain while determining any possible limiting factors. using this tool, a systematic production matrix relating several logical source crops and a few key chemicals of varying derivative levels can be created and compared to the conventional fossil routes. combined with economic considerations and some unambiguous environmental factors, the investigation will provide all the information relevant to the industry. the goal is to create an objective and reliable simulation system ratifying the economic and environmental feasibility of exploiting biobased chemicals today and indicate the steps necessary for further improvement. biosynthesis of multi-enzymatic preparation from aspergillus niger ibt- useful in textile fabric treatment rita pyc , jadwiga sojka-ledakowicz , tadeusz antczak , joanna lichawska : institute of technical biochemistry, the technical university of lodz, lodz - , poland; textile research institute, lodz - , poland among many methods of producing enzymatic preparations, i.e. by liquid surface fermentation -lsf, submerged fermentation -smf or solid state fermentation -ssf, this last is most advantageous. cultivation in solid state means fermentation on a matrix formed by industrial and agricultural wastes. most often filamentous fungi -due to the lack of available water in the foundation -are the efficient, competitive microorganisms applied in solid state bio-conversion. the aim of research works carried out at the institute of technical biochemistry of the technical university of lodz and at textile research institute, lodz was defining optimal conditions for biosynthesis and testing the possibility of applying multi-enzymatic preparation from aspergillus niger ibt- in the treatment of woven fabrics made of natural cellulose fibres. as the result of biosynthesis optimization process, malt sprouts, wheat barn and beet pulp were selected as the best media to obtain enzymes of high pectinolytic activity maintaining at the same time high activity of cellulolytic enzymes and xylanase. the highest obtained activities reached: pm for total pectinolytic activity, j/ml for endoglucanase and j/ml for endoxylanase and they were . - . times higher than those achieved before optimizing process. performed research works demonstrated that the optimum activity of applied enzymatic system is obtained in the range of ph . - . . woven fabrics made of flax and cotton fibre blends, subjected to bio-pre-treatment, were evaluated with reference to their sorption properties. comparative evaluation of liquid sorption by woven fabrics allowed to notice efficient enhancement of fibres' sorption capabilities after pre-treatment using enzymes system from aspergillus niger ibt- . this offers the possibility of substitution of alkali scouring of linen-cotton woven fabrics before their bleaching by bio-treatment. the phylum actinobacter includes many bacteria of industrial importance both for accumulation of primary and secondary metabolites. both primary and secondary metabolites are dependent on precursors and cofactors that are provided by the central carbon metabolism of microorganisms. there are alternative pathways for catabolism of carbon either via the embden meyerhof parnas (emp) and pentose phosphate (pp) pathway or through the entner doudoroff (ed) pathway. the emp pathway is energetically more favorable and has therefore been presumed to be the dominating route for carbon metabolism in bacteria producing secondary metabolites. however, primary metabolism is poorly studied for most actinobacter species as focus traditionally has been on secondary metabolism. with the aim to gain more knowledge about the diversity of central carbon metabolism within the phylum actinobacter, different strains were collected from various sources and strain collections. the strains were grown in minimal medium with supplement of standard vitamin solution and [ - c] glucose as carbon source. the c-labeling patterns of proteinogenic amino acids were determined by gc-ms analysis. through this method, the fluxes in the central carbon metabolism during balanced growth were estimated and pathways for carbon metabolism were determined. in particular the labeling patterns of alanine and valine were of interest as they are derived from pyruvate and therefore can be used to distinguish between whether glucose is metabolized through the ed pathway or the emp pathway. nobacter, amycolatopsis balhimycina produces the glycopeptide balhimycin. the balhimycin aglycone is identical to the aglycon of vancomycin, which is a commercial glycopeptide. as a. balhimycina appears to be accessible to genetic modifications and the biosynthetic cluster responsible for balhimycin production is published, this genetically well-characterised academic strain can serve as a platform strain for production of vancomycin analogues derived through combinatorial biosynthesis. the understanding of the physiology of this microorganism is essential for the efficient accumulation of potentially commercial secondary metabolites. the bacterium is capable of growth in a fully defined minimal medium, with the production of balhimycin. the strain was grown at either nitrogen or phosphate limitation and balhimycin accumulation was followed at these conditions. flux analysis of balhimycin production by amycolatopsis balhimycina based on c labelling experiments was performed. the zygomycetes blakeslea trispora and phycomyces blakesleeanus accumulate beta-carotene, ubiquinone (coenzyme q), various different sterols, and other terpenoids, all of them produced via the mevalonate pathway. these fungi are used or could be used for the industrial production of these terpenoids, edible oil, chitosan, and various organic acids. by measuring the specific radioactivity of terpenoids made from radioactive mevalonate, leucine or acetate in the presence of excess glucose in wild types and mutant strains we have concluded that these fungi have separate subcellular compartments for the production of carotene, sterols and triacylglycerols. the terpenoid moiety of ubiquinone is synthesized in the same compartment as ergosterol. these compartments contain separate pools of all their common metabolites, beginning from acetyl-coa. mevalonate carbon atoms do not find their way back to general metabolism, i.e., these fungi lack the "shunt" pathway. the compartments are regulated independently. the very large variations in carotene content caused by many environmental and genetic changes are not accompanied by variations in the ubiquinone content. the ubiquinone content increases when the cultures grow on leucine or acetate as carbon sources and is not affected by illumination. phycomyces, but not blakeslea, increases the production of ubiquinone in presence of oligomycin. lincomycin, produced by streptomyces lincolnensis, is important, clinically used antibiotic. its gene cluster consists of putative open reading frames with biosynthetic or regulatory functions (lmb genes) and three resistance genes (lmra, lmrb, lmrc). the organization of transcription units was determined. the analysis of the lincomycin biosynthetic gene transcripts in various cultivation stages revealed the genes with putative regulatory functions which are transcribed in early stages of cultivation. previous analysis of biosynthetic pathways of lincomycin and functionally different anthramycin antibiotics (anthramycin, sibiromycin, tomaymycin, mazetharmycin and porothramycin) indicates that the genetic information on the lincomycin and anthramycin biosynthesis should share common elements (genes), both biosynthetic and regulatory. hybridization experiments demonstrated presence of several analogues of lmb genes involved in the biosynthesis of anthramycin produced by streptomyces refuineus and porothramycin produced by streptomyces albus. effect of various calcium salts on the erythromycin production by saccharopolyspora erythraea m. rostamza , a. noohi , j. hamedi * : department of biology, faculty of science, science and research branch, islamic azad university, tehran, iran; microbial biotechnology lab., department of biology, faculty of science, university of tehran, tehran, iran. e-mail: jhamedi@khayam.ut.ac.ir (j. hamedi) calcium carbonate has the positive effect on the erythromycin, however, because of its low water solubility, caused to clogging the spargers of fermenters and fouling the microfilters. in this research, various soluble calcium salts was added to the fermentation medium and their effect the growth of saccharopolyspora erythraea and erythromycin production was studied in the complex medium consisted soy bean meal, dextrin and starch as major ingredients. the fermentation conditions were rpm, days at • c. the results obtained showed that there is no significant difference between erythromycin concentrations in the medium containing calcium lactate and calcium carbonate. however, erythromycin concentrations in the other calcium salts containing media were less than to calcium carbonate containing medium. optimum concentration of calcium lactate for erythromycin production was g/l. lincosamides and its derivatives are clinically important antibiotics. comparison of gene cluster coding for lincomycin biosynthesis and newly identified cluster of genes for celesticetin biosynthesis revealed new information on functions of several genes common for both biosynthetic pathways. the celesticetin gene cluster was identified by screening the cosmid library of streptomyces caelestis with heterologous probes based on lincomycin biosynthetic genes involved in a part of biosynthesis shared by both antibiotics. sequence analysis of the cluster revealed putative orfs, out of which are lincomycin biosynthetic genes analogues, four are specific for celesticetin biosynthesis and one codes for resistance. the gene cluster is bounded with transposase genes on both sides. in order to clarify function of three putative regulatory genes of lincomycin biosynthesis, the insertional inactivation with the pcr targeting system in streptomyces lincolnensis was done and resulted in differently reduced production of lincomycin. larsen cmb biocentrum-dtu, technical university of denmark, kgs. lyngby, denmark we present a new algorithm called "x-hitting" for automatic identification of novel bioactive compounds based on full spectroscopic characters of highly complex mixtures of natural product. one of the most dramatic advances in recent drug discovery has been the increase in screening capacity throughput and data handling. therefore, analysis has become the bottleneck in the drug discovery process. the algorithm presented here is investigated and demonstrated on identifying potentially new bioactive compounds. in addition method is shown to have a high performance for automatic identification of known structures. these tasks are referred to as new-hitting and cross-hitting, respectively. finally, the receiver operating characteristics (roc) is introduced to the research field as an important tool for evaluating the performance of the "compound predictor". through examples it is shown, that known cross-hits are identified with high proficiency, and that the new-hitting works on finding new targets represented by analogues and structurally new compounds. a gene encoding a ␥-butyrolactone autoregulator receptor that have a common activity as dna-binding transcriptional repressors, controlling secondary metabolism and/or morphological differentiation in streptomyces was cloned from a natamycin producer, streptomyces natalensis, and its function was evaluated by in vivo. pcr using the primers designed from two highly conserved regions of streptomyces autoregulator receptors gave a -bp band, the sequence of which revealed high similarity to the expected region of a receptor gene. by genomic southern hybridization with -bp insert as a probe, a -bp intact receptor gene (sngr) was obtained from s. natalensis. in vivo to clarify the function of sngr, a sngrdisrupted strain was constructed, and a phenotype was compared with the wild-type strain. the sngr disruptant started natamycin production h earlier and showed a . -fold-higher production of natamycin than the wild-type strain. in addition, sporulation was earlier and -fold abundance. the phenotype indicates that the autoregulator receptor protein of s. natalensis acts as a primary negative regulator of the biosynthesis of natamycin and is related to regulation of sporulation. exploring the biocatalytic potential of the novel thermostable baeyer-villiger monooxygenase: phenylacetone monooxygenase daniel e. torres pazmiño , gonzalo de gonzalo , gianluca ottolina , giacomo carrea , dick b. janssen , marco w. fraaije , biochemical laboratory, groningen biomolecular sciences and biotechnology institute, university of groningen, nijen- baeyer-villiger monooxygenases (bvmos) represent useful biocatalytic tools as they can catalyze reactions which are difficult to achieve using chemical means. however, only a limited number of these atypical monooxygenases are available in recombinant form. using a recently described protein sequence motif, a putative bvmo was identified in the genome of the thermophilic actinomycete thermobifida fusca. the nadph-dependent and fad-containing monooxygenase is active with a wide range of aromatic and aliphatic ketones and sulfides. genetic and kinetic data suggest that phenylacetone is the physiological substrate of the enzyme. previously, it was reported that this bvmo exhibits only a moderate enantioselectivity with (r,s)-␣-methylphenylacetone. this poster we will show an overview of the biocatalytic potential of the enzyme as explored so far. interestingly the enzyme has been found to perform highly enantioselective oxidations with a range of ketones and sulfides. this again indicates that this novel thermostable oxidative biocatalyst can be a useful tool for the synthesis of chiral building blocks. the enzyme s-adenosylhomocysteine hydrolase (adohcyase) catalyzes hydrolysis of s-adenosylhomocysteine (adohcy), an inhibitor of transmethylation reactions, into adenosine (ado) and homocysteine (hcy). the catalysed reaction is reversible, and the equilibrium is strongly displaced in direction of the synthesis of adohcy when reaction occurs in vitro. nevertheless, its biotechnological application resides in the synthesis of antivirals. for it, we selected between different producing microorganisms of the enzyme, the gram positive bacterium corynebacterium glutamicum. after designing the specific oligonucleotides, the gene was expressed in escherichia coli using the expression system pet a+ with iptg. the electrophoretic analysis under denaturing conditions, shows a clear induction and over-expression of a protein with a mw of kda. on the other hand, the immobilization of this recombinant enzyme in a solid support allows to use it as a catalyst for the synthesis of adohcy. the enzyme was purified by imac thanks to the presence of n-terminal × his tag end, and immobilized in eupergit c for the optimization of the production of adohcy, a product of high value. sequencing, cloning, expression, purification and characterization of a novel cytochrome p monooxygenase from rhodococcus rubber luo liu, rolf d. schmid, vlada b. urlacher institute of technical biochemistry, stuttgart university, allmandring , stuttgart, germany. e-mail: itbvur@itb.unistuttgart.de (l. liu) the cytochrome p monooxygenases are heme-containing proteins, which catalyze a wide range of oxidative reactions (werck-reichhart and feyereisen, ) . a monooxygenation activity was observed for the strain rhodococcus ruber dsm . a p -like gene fragment was amplified by pcr using degenerated primers. for identification of regions that flank this p -like dna fragment, the method "directional genome walking using pcr" was applied (mishra et al., ) . the full size gene encoding a cytochrome p enzyme was amplified by pcr from genomic dna and cloned into the vector pet a(+) for heterologous expression in escherichia coli bl (de ) cells. the enzyme was purified using metal affinity chromatography. the primary protein structure suggests, that this enzyme is a natural self-sufficient fusion protein consisting of a ferredoxin, a reductase and a p monooxygenase. the reductase activity was determined using an exogenous electron acceptor cytochrome c. the reductase domain of this p monooxygenase showed a strong preference for nadph over nadh. the substrate spetrum was investigated. in the presence of nadph the p enzyme shows hydroxylation activity towards -ethoxycoumarin, naphthalene, indene, acenaphthene, toluene and fluorene. mishra, r.n., singla-pareek, s.l., nair, s., sopory, s.k., reddy, m.k., . directional genome walking using pcr. biotechniques , - . werck-reichhart, d., feyereisen, r., . cytochromes p : a success story. genome biol. ( ), . - . . selective production of monoglyceride consisted of conjugated linoleic acid by penicillium lipase yomi watanabe , , yoshie yamauchi-sato , toshihiro nagao , satoshi negishi , tadamasa terai , takashi kobayashi , rolf d. schmid , yuji shimada : osaka municipal technical research institute, osaka, japan; stuttgart university, stuttgart, germany; the nisshin oillio group, ltd, yokosuka, japan; osaka institute of technology, osaka, japan conjugated linoleic acid (cla) is a group of c fatty acid (fa) containing two conjugated double bonds. it is expected to prevent cancer, adipositas, atherosclerosis etc, and is commertially available in the primary form of free fa, containing almost equal amounts of cis, trans-and trans, cis-cla. it is therefore desired to be converted to a palatable form. for this purpose, we have previously proposed two enzymatic ways to convert cla to monoglyceride, an emulsifier, with penicillium camembertii lipase; ( ) sequencial esterification-glycerolysis, ( ) esterification at low tem-perature. these methods, however, are time-and energy-consuming. esterification of cla with glycerol under ambient pressure by the lipase produces equal amounts of mono-and diglycerides. in contrast, it was newly found that the reaction under reduced pressure supressed the formation of diglycerides and achieved to produce % monoglyceride at % esterification. improving the thermal stability of cellobiohydrolases i (cel a) from t. reesei by site directed evolution frits goedegebuur , lydia dankmeyer , peter gualfetti , brad kelemen , edmundo larenas , paulien neefe , pauline teunissen , colin mitchinson : genencor international bv, archimedesweg , cn leiden, the netherlands; genencor international inc., page mill road, palo alto, ca , usa genencor international has been working to produce improved enzyme products for economic conversion of ligno-cellulosic biomass to fermentable sugars. most of this work was performed under a subcontract with the u.s. department of energy for cellulose cost reduction for biomass conversion. cellulolytic biomass conversion is performed in nature by a complex mixture of enzymes. cellobiohydrolases play a key role and all effective cellulase mixtures contain a large excess of cellobiohydrolases over endoglucanases, suggesting that it is the exoglucanase activity that is limiting. the fundamental dependence of reaction rate on temperature predicts that large increases in performance, and decreased enzyme cost, would be achieved if the enzymatic conversion could be operated at elevated temperatures. industrial strains of trichoderma reesei produce cellulases at very high levels and low cost. however, t. reesei cbh (hypocrea jecorina cel a) does not have sufficient stability to survive and perform at high temperatures. this poster shows the thermal stability improvement in t. reesei cbh by site directed evolution. sites with increased thermal stability properties were combined and evolved in high temperature stable cbhi variants. the evaluation of lipases as biocatalysts for organic chemistry can be carried out, at laboratory scale, by using soluble enzymes for biotransformations in aqueous media. however, the industrial exploitation of such an enormous potential should require a suitable protocol for immobilization of lipases. the binding of lipases on suitable pre-existing supports should greatly improve the performance of industrial reactors allowing us a continuous use or re-use of such interesting biocatalysts. in addition, lipases, like most enzymes, are not perfect chemical catalysts. lipases may be unstable and they may not have the optimal activity nor the optimal enantio or regioselectivities. in this way, immobilization of lipases, together with its relevance for the performance of each different industrial reactor, could be also used as a tool to improve and optimize some of these parameters. that is, immobilization of lipases, far from an already solved problem, constitutes an exciting field of research in the promising area of industrial bio-organic chemistry. in this work we would like to present useful immobilization methods of several lipases. the lipase immobilised were used for enzymatic hydrolysis of peptidomimetics of structure a. type of immobilzation used can changed the enantioselectivity of biocatalyst prepared. the absolute configuration of products b and c obtained in enzymatic reactions were assigned by chemical correlation. two-component flavin-dependent monooxygenases form an interesting class of flavoenzymes. they consist of two separate proteins; a monooxygenase component, which catalyses an oxygenation reaction in the presence of reduced flavin, and a flavin reducing component, which reduces flavin (fad or fmn) using nad(p)h as an electron donor. a well-known example of this class of monooxygenases is styrene monooxygenase (otto et al., ) . due to the ability to form enantiopure epoxides, which are relevant building blocks for the pharmaceutical industry, styrene monooxygenases form a valuable class of enzymes for biocatalysis. while screening a metagenomic library for oxidative enzymes, an indigo-producing clone was found. sequencing the particular clone revealed an inserted fragment of environmental dna encoding a two-component monooxygenase ( many investigations over the recent years have been directed to the production of natural aroma compounds. through biotransformation and bioconversion, aroma compounds considered as "natural" can be produced starting from monoterpenes, generating high value products as rose oxide. rose oxide is found in small amounts in some essential oils such as bulgarian rose oil and geranium oil. (−)-rose oxide is an impacting flavor compound and has a small threshold: . ppb. application of agro-industrial residues in bioprocess on one hand provides alternative substrates, and on the other hand helps solving pollution problems, that might be caused by the disposal of this residue in nature. the liquid cassava waste, is originated by the pressing of cassava roots. it is considered as a "harmful" pollutant waste due to its high organic charge and presence of cyanide. on the other hand, can be considered rich in nutrients that can be used in other applications. it was found that sporulated surface cultures of penicillium sp. were able to convert citronellol into cis-and trans-rose oxides. other bioproducts were , -dimethyl- -octen- , diol and , -dimethyl- , -epoxy- -octanol. no chemical oxidation or auto-oxidation products were detected in liquid control broths. the experiments were conducted at • c and rpm. when the medium was cassava, the production of rose oxide, , -dimethyl- octen- , -diol and , -dimethyl- , -epoxy- -octanol were insignificant reaching trace amounts. but when the mycelium developed in cassava medium and than transferred to mineral medium (citronellol as c-source) the concentrations of rose oxide increased dramatically, reaching mg/l for the cis-isomer and mg/l for trans-isomer. a mechanistic mathematical model of enzymatic degradation of avicel and phosphoric acid swollen cellulose (pasc) has been proposed. the model is based on the degree of polymerization (dp) of starting substrate, and follows its decline with time, to the final end product -glucose. three enzyme classes, namely, endoglucanase (eg), cellobiohydrolase (cbh) and ␤-glucosidase (bg) are all individually incorporated in the model. the model is, additionally, taking into account cooperative action of the involved enzymes, as well as effects of enzyme inhibition by end-products, cellobiose and glucose. to be able to describe the complex process of enzymatic hydrolysis with a set of differential equations certain assumptions needed to be introduced. those assumptions represent the simplification of an up-to-date knowledge of both substrate and enzyme structure, but also enzyme mode of action. for example, one of the often asked questions is: "what is happening with shorter cellooligosacharides (dp and up) laying on the surface of cellulose chain? are they being adsorbed to the core cellulose chain or partly solubilized to a hydrolysis broth?" to give answers to these questions and confirm mathematical modeling real enzyme hydrolysis data are needed. in this work, four well characterized, highly purified mono-component enzymes from humicola insolens (two eg and two cbh) and one bg from aspergillus niger were used to hydrolyze avicel and pasc. by careful choice of catalyst, some enzyme specific characteristics like presence or absence of cellulose binding domain will also be incorporated into the model. hydrolysis experiments were initially performed by distinct mono-component enzymes, to confirm the basic characteristics of each of the enzyme classes. soluble hydrolysis products (dp - ) were analyzed by hplc and detection of non-soluble, higher-dp polysaccharides was performed by technique of polysaccharide analysis using carbohydrate gel electrophoresis. optimisation of halogenase enzyme activity k. muffler , m. retzlaff , k.-h. van pée , r. ulber : technische universität kaiserslautern, germany; technische universität münchen, germany; technische universität dresden, germany. e-mail: muffler@rhrk.uni-kl.de (k. muffler) halogenases provide the opportunity of a regioselective and stereospecific halogenation of organic subtrates in contrast to the class of haloperoxidases. these enzymes allow a gentle synthesis of halogenated organic molecules and are capable to halogenate in specific positions (hammer et al., ; keller et al., ) , whereas traditional organic synthesis often failures or mainly leads to byproducts (hasegawa et al., ) , e.g. halogenation of tryptophan in other positions than position . our current research is focussed on tryptophan- -halogenases, because the -br/cl-tryptophan could be applied as a pharmacologically attractive precursor of serotonin. in our work we describe the optimization of an enzyme assay respectively the enzyme activity. for this purpose we use a genetic algorithm. the responsible gene of the fadh dependent enzyme was cloned from streptomyces sp. origin into a pseudomonas fluorescens strain. however, the optimization procedure was done with the purified his-tagged tryptophan- -halogenase, which was easily obtained from the crude extract of the lysed cells by application of immobilized metal affinity chromatography. the application of algorithms allows an optimization of the multidimensional search problem leading to a global optimum in the search space in contrast to the traditional used one-factor-at-a-time method. latter often failures, because this method does not reflect possible influences the parameters can have on each other. effect of organic solvent type on the enantioselectivity of candida rugosa lipase in the hydrolysis of racemic naproxen methyl ester in biphasic reaction system serpil takaç, deniz mutlu department of chemical engineering, institute of biotechnology, ankara university, tandogan, ankara, turkey hydrolysis of racemic naproxen methyl ester to produce s-naproxen was carried out in the biphasic system using isooctane, cyclohexane, hexane and toluene with candida rugosa lipase after stirring in the phosphate buffer (ph . , . m) for h at + • c and centrifuging. the hydrolyses were carried out in shaking flasks for h at rpm and • c with the initial substrate concentration of . m. the concentrations of the enantiomers of racemic naproxen methyl ester in organic solvents and those of naproxen in buffer solution were determined with hplc. it was found that the enantiomeric excess for substrate (ee s ), enantiomeric ratio (e) and conversion (x) decreased in the following order: isooctane > cyclohexane > hexane > toluene. the enantiomeric excess for product (ee p ) was found to be the same for isooctane, cyclohexane and hexane where the lowest ee p was obtained in toluene. the highest ee s , ee p , e and x values achieved in isooctane at the residence time of h were , , , and %, respectively. this study was supported by ankara university biotechnology institute (project no: ). fusarium fujikuroi (gibberella fujikuroi mating group c) produces multiple secondary metabolites such as gibberellins and bikaverin. gibberellins are terpenoid hormones that induce growth and regulate various stages of development in plants. they have numerous applications in agriculture industry. bikaverins are polyketides that have toxicity against different organisms because they inhibit respiration. regulation of polyketide and gibberellin synthesis by nitrogen has been intensively studied in fusarium but little is known about their regulation by carbon source. our main interest is to understand the regulation of biosynthesis of these compounds in f. fujikuroi imi . to investigate this regulation the organism was grown in high nitrogen medium under submerged conditions and then transferred to nitrogen-free media having various concentrations of different carbon sources. the gibberellin production was not affected significantly. on the other hand bikaverin was synthesized enormously when sucrose was used as the only carbon source. high production in sucrose required a minimal amount of the sugar, but did not change appreciably above this threshold along a wide range of concentration. the bikaverin synthesis was repressed when glucose coexisted with sucrose in the medium. the effect of the c source on the expression of key genes, cps/ks (copalyl diphosphate synthase/kaurene synthase) for gibberellin and pks (polyketide synthase) for bikaverin biosynthesis is currently under investigation. ment of dormancy and loss of viability in seeds with the passage of time, it lacks any systematic propagation from seeds and is typically propagated through rhizomes. this restricts large scale cultivation of this plant. in vitro propagation of plants is an effective means for rapid multiplication of species, in which conventional methods have limitations. in the pesent study we have analysed the role of various growth promoters and the effects of dark and light incubation on germination of n. alba seeds. the results indicate that in vitro propagation of n. alba from seeds can be applied as an efficient method of multiplication. the study was funded by the state planning commisson (dpt) turkey and the university of ankara vide projects no. . this research was performed for developing of biological treatment process of odor gas such as mek, h s, and toluene, which is generated from the food waste recycling process. to establish the operational conditions of odor gas removal by small-scale biofiltration equipment, it was continuously operated by using toluene as a treating odor object. when the odor treating microorganisms were adhered to fibril form biofilter, high removal efficiency over % was obtained by biofilm formation. at ppm of inlet odor gas concentration and s of retention time, the removal efficiency was and % in first stage reactor and second stage reactor, respectively. however, the removal efficiency remained over % at the operational conditions above s of retention time. post soviet countries are going through the transition stage and are extremely sensitive to new technology, economics or social changes and globalization processes. that is why decision-making and system of regulation of the use of gmo's are very sensitive to number of factors. three levels of factors, the most influential to decision-making: global, regional and local; are identified at the research paper. global level depends on external policy of leaders of gmo regulations. usa and eu have the biggest influence on transition countries, though their positions completely differ. as usa was leader in inventing gmos, it is lobbing newly created biotechnological industry. in eu and other european countries lobbing of biotechnological industry was not as strong as in usa. thus, their national law is stricter. world trade organization and international agreements are also part of global level. regional level. geographical position of country is also very important because every regulation system depends a lot on regulation that is implemented in neighboring countries. countries do not exist in vacuum; they are linked territorially, politically, economically and socially with neighboring states. regulation systems of the transition countries in the eastern europe can be divided into three types: those who have no system of regulation of gmo (belarus, romania, hungary and ukraine); who approved some variety that are treated as safe to the market (poland, moldavia and georgia) and who approved all of the gmos (bulgaria, croatia and russia [before ]). but even if a country declares not to use gmo it is rather difficult to control import of such products, because of the lack of the testing laboratories, corruption of the state employees, agreements on intellectual property, and institutional country problems. in spite of global and regional tendency of gmos related regulation the most important part is the local level, namely the national regulation system. depending on national level we are choosing priorities at higher levels. as an example of post soviet countries ukraine is taken, as ukraine is one of the largest countries and one of the biggest exporters of agricultural products in europe. research includes the analysis of attitude to gm product, their potential risks and benefits of three categories that influence decision-making the most: farmers, gm experts and non-governmental organizations. recombinant microorganism development for extracellular benzaldehyde lyase production hande kaya , pınar Ç alık , tunçer h. ozdamar : iblab, department of chemical engineering, metu, ankara, turkey; bre laboratory, department of chemical engng, ankara university, ankara, turkey. e-mail: e @metu.edu.tr (h. kaya) in this study, the extracellular production of the benzaldehyde lyase (bal, ec . . . ) that catalyses the synthesis the enantiopure -hydroxy ketones for drug syntheses, by bacillus sp., was aimed. for this purpose, the signal dna sequence of an extracellular bacillus enzyme, i.e., serine alkaline protease, was fused in front of the bal gene (accession number ax ) from pseudomonas fluorescens biovar i, using pcr-based gene splicing by overlap extension (soe) method. b. licheniformis (dsm ) chromosomal dna was used as sap gene (accession number x ) template for the synthesis of sap signal sequence. bal gene was amplified by using the plasmid carrying bal gene, puc ::bal. thereafter, the signal peptide of sap with its own promoter was fused in front of the bal gene by soe method. the hybrid gene first cloned into puc plasmid, thereafter sub-cloned into pbr , pmk and phv shuttle vectors. the escherichia coli-bacillus plasmids carrying the hybrid gene pre(subc)-bal was transferred into bacillus subtilis npr− apr−, and bacillus licheniformis. the influence of the host bacillus species on bal production on a defined medium with glucose was investigated in bioreactor systems. for each of the recombinant (r-) bacillus species, effects of initial glucose concentration on cell growth and bal production were investigated; and, physiological differences and similarities between the wild-type and r-bacillus species are discussed. thereafter, the benzaldehyde lyase production capacities of recombinant e. coli and b. subtilis are compared in terms of cell concentration and bal volumetric and specific activities. for the comparison bal gene was cloned into prseta vector which is under the control of strong t promoter and expressed in e. coli bl (de ) plyss strain. the variations in by-product distributions with each recombinant organism and yields are also discussed. phosphoketolases (ec . . . ) are thiamine diphosphate (thdp)-dependent enzymes, that play a crucial role in the pentose phosphate pathway (ppp) of heterofermentative and facultative homofermentative lactic acid bacteria, and of the d-fructose phosphate shunt of bifidobacteria. reports affirm that cellulomonas flavigena can use the ppp when is cultured under anaerobic conditions. a genomic library of c. flavigena constructed in -zap express vector was screened. four positive clones were isolated, in vivo excised and the resulting pbk-cmv phagemids, each containing a . kb insert, were characterized by restriction analysis and dna sequencing. the open reading frame (orf) of the dxylulose -phosphate/d-fructose -phosphate phosphoketolase gene, xpkl, was located from nucleotide to . the xpkl orf encoded a amino acids-residue polypeptide (xpkl) with a calculated molecular mass of , da, a value coincident with that estimated by comparative sds-page (about , da). a putative ribosome binding site (gggagc) is present - nucleotide upstream of the translational start of the xpkl polypeptide. the c. flavigena xpkl polypeptide sequence was % identical to dxylulose -phosphate/d-fructose -phosphate phosphoketolase from bifidobacterium sp., bifidobacterium gallinarum, gloeobacter violaceus and bifidobacterium adolescentis. this analysis also revealed highly conserved regions. lactococcus lactis is a main diacetyl-producing bacteria by citrate metabolism in dairy products. the transport of citrate in these bacteria is dependent on citrate permease that is encoded by citp gene. previous studies of the citqrp operon in escherichia coli mutants showed that citp message is considerably stabilized in rnase iii mutant. so, in the context of the citrate metabolism research, the characterization of the lactococcal rnase iii enzyme is very important for the dairy industry. rnase iii is an endoribonuclease which has an important role in rrna processing and control of gene expression. with the aim of studying lactococcal rnase iii we have cloned the rnc gene from l. lactis ssp lactis il in the broad host range pls rgfp vector. this plasmid includes the gfp gene, encoding the green fluorescent protein (gfp), cloned under the control of the pm promoter, that is inducible by maltose. maltose induction of the lactococcal rnc expression showed a -fold increase of rnc transcription from this plasmid. activity assays for lactococcal rnase iii were standardized using crude extracts and a substrate specific for b. subtillis rnase iii. the results showed that this substrate was specifically cleaved by lactococcal rnase iii and its activity induced by maltose. lac-rnc was cloned in pet vector and the corresponding six-histidine-tagged rnase iii protein was overproduced in e. coli bl (de ) strain by iptg induction. the protein was purified by affinity chromatography using hplc system and was shown to be active by in vitro activity assays using the lac-rnase iii specific substrate mentioned above. we have also cloned lactococcal rnc gene and studied its expression in an e. coli rnc deletion mutant ( rnc). complementation assays performed in e. coli demonstrate that the lactococcal rnase iii (lac-rnase iii) is able to process rrnas and to regulate the levels of polynucleotide phosphorylase (pnpase). these results demonstrate that the lactococcal enzyme is able to substitute the ec-rnase iii not only in the rrna processing, but also in the processing of mrnas. the amount of lactococcal rnc transcript in an e. coli rnc strain was . -fold higher than in the wild type strain, suggesting that the e. coli rnase iii triggers the degradation of the heterologous rnc mrnas. the results obtained have shown that lac-rnase iii is an interesting enzyme for biotechnological purposes. objectives: the pharmaceutical and food industry has an increasing demand for selectively glycolized active agents. in our application isomaltose can be synthesized by immobilized dextransucrase, which transfers a glycosyl residue from sucrose (substrate) to glucose (acceptor). as the reaction proceeds, isomaltose can act as an acceptor and is converted into undesired follow-up products called isomalto-oligosaccharides, imos. we investigate on two approaches to avoid imo formation, selective adsorption of isomaltose (ergezinger et al., ) and the co-entrapment of dextranase adsorbate, which breaks imos down to isomaltose. results and conclusions: the first part of our research concerns the adsorption of dextranase on bentonite, which complies with langmuir model. at complete saturation ( . g g − ) our immobilisate exhibits an activity of , u g − . a kinetic analysis does not reveal significant differences between the adsorbed and free form of enzyme (k m,bentonit : . ± . m versus k m,free : . ± . m). thus, bentonite displays a high binding capacity paired with favorable kinetic properties. beyond that we investigate the activity of dextransucrase in co-immobilisates, which is reduced during coimmobilization due to interactions with the adsorbate. among various co-immobilisates, the one containing dextranase bound to preblotted bentonite imparts the highest activity ( % as compared to control: immob. dextransucrase). the molar yield coefficient of coimmobilisates y isomaltose/sucrose surpasses coefficient of control by %. further on we will characterize mass transfer of dextranase substrate into alginate matrix as well as bentonite-dextransucrase interactions. and kefir. a second approach is to use yeast as a production organism to produce natural folates for fortification. here we investigate and discuss the folate content in skq n, a diploid strain of saccharomyces cerevisiae, when cultured in different media and at different stages of growth. the aim is to gain a basal knowledge of the folate production profile, forms of folate produced and degree of leakage to the surrounding medium, in relation to the culturing medium and physiological state of the cells. danisco innovation, danisco a/s, langebrogade , po box , dk copenhagen k, denmark we at danisco a/s (copenhagen, denmark) have revealed a new starch degrading pathway by the discovering several new enzymes and metabolites in fungi and algae. these new enzymes include glucan lyases, dehydratases and tautomerases, which proved to be useful in biocatalysis. these new metabolites proved to be useful as both antioxidants and antimicrobials for food and non-food applications. this pathway is named as anhydrofructose pathway of starch and glycogen degradation. this technology is referred to as the anhydrofructose technology. diet is evolving from nourishing populations via providing essential nutrients to improving health of individuals through nutrition. modern nutritional research focuses on health promotion and disease prevention, on protection against toxicity and stress, and on performance improvement. as a consequence of these ambitious objectives, the disciplines "nutrigenetics" and "nutrigenomics" have evolved. nutrigenetics asks the question how individual genetic disposition, manifesting as single-nucleotide polymorphisms (snps), copy-number polymorphisms (cnps) and epigenetic phenomena, affects susceptibility to diet. nutrigenomics addresses the inverse relationship, i.e. how diet influences gene transcription, protein expression and metabolism. the mid-term objective of nutrigenomics is integrating genomics (gene analysis), transcriptomics (gene expression analysis), proteomics (global protein analysis) and metabolomics (metabolite profiling) to define a "healthy" phenotype. the long-term deliverable of nutrigenomics is personalised nutrition for maintenance of individual health and prevention of disease. the major challenges for -omics in nutrition and health still lie ahead of us, some of which apply to -omic disciplines in general while others are specific for -omic discovery in the food context: (i) the integration of gene-and protein expression profiles with metabolic fingerprints is still in its infancy as we need to understand how to (a) select relevant sub-sets of information to be merged, and (b) resolve the issue of the different time-scales, at which transcripts, proteins and metabolites appear and act; (ii) the definition of health and comfort is less of a clear-cut case than the one of disease; (iii) -omics in nutrition must be particularly sensitive: it has to reveal rather many subtle than a few abundant signals to detect early deviations from normality; (iv) in the food context, health cannot be uncoupled from pleasure, that is, food preference and nutritional status are interconnected. transcriptomics serves to put proteomic and metabolomic markers into a larger biological perspective and is suitable for a first "round of discovery" in regulatory networks. metabolomics, the comprehensive analysis of metabolites, is an excellent diagnostic tool for consumer classification. the great asset of this platform is the quantitative, non-invasive analysis of easily accessible human body fluids like urine, blood and saliva. this feature also holds true to some extent for proteomics, with the constraint that proteomics is more complex in terms of absolute number, chemical properties and dynamic range of compounds present. proteomics in the context of nutrition and health has the potential to (a) deliver biomarkers for health and comfort, (b) reveal early indicators for disease disposition, (c) assist in differentiating dietary responders from non-responders, and, last but not least, (d) discover bioactive, beneficial food components. independent of the context of application, proteomics represents the only platform that delivers not only markers for disposition or condition but also targets of intervention: the only way to intervene in a biological condition and to modulate its outcome is interfering with the proteins involved. it is evident that not only comprehensive analyses with one discovery platform (lateral integration of information) are required but also vertical integration between different -omic levels are indispensable for a deeper understanding of disposition, health, environment and diet (desiere, ) . a major "vertical integration issue", to date unresolved, is given by different timescales of transcript production, protein expression and metabolite generation (nicholson et al., ) . the transcript machinery usually responds fast to an external stimulus (seconds to minutes), the proteins may be expressed within minutes to hours (and have a halflife from minutes to even months) and metabolites vary significantly during the day and depend on latest dietary input. this means that data, which seem to correlate qualitatively (e.g. reflecting the same pathway), may not necessarily be related time-wise. rather, they may represent different responses at different time points and, possibly, to different stimuli. comprehensive -omic analyses is an essential building block of "systems biology", which can be defined as follows (clish et al., ) : systems biology is the comprehensive analysis of the dynamic functioning of a biological system (cell, organ, organism or even ecosystem) at gene, protein and metabolite (or higher organizational) level, achieved by comparison of two defined biological states of this system, typically before and after perturbation. while a comprehensive list of components (genes, proteins, metabolites) of a given biological system is a pre-requisite for this kind of research, the main reasoning for the "system view" is that only information on the interactions between the components gives clues to function of the entire network. a systems biology approach has recently demonstrated the power of proteomics to dissect immunity and inflammation. toll-like receptor recognition and signalling was elucidated and showed, how bacterial "barcodes" are read and interpreted in order to trigger an adapted immune response (aderem and smith, ) . in order to address some of the challenging objectives of -omics-driven nutritional research, we have addressed (a) the effect of early antibiotic administration on the maturation of intestinal tissues, (b) protein discovery in human milk, (c) the effects of polyunsaturated fatty acids on gene expression and lipid profile in the liver, and (d) biomarkers for intestinal stress. (a) antibiotics and gut maturation: the effects of early administration of antibiotics on intestinal maturation were assessed at the gene expression level in a rat model. (b) human milk: rapid enrichment and iterative, consolidated identification of immunologically relevant milk proteins was achieved through the employment of restricted-access media and a tailored proteomic strategy (labéta et al., ; lebouder et al., ; panchaud et al., ) . (c) fatty acids and liver transcriptome/lipidome: epidemiological studies have correlated higher intakes of poly-unsaturated fatty acids (pufas) with lower incidence of chronic metabolic disease. the molecular mechanisms regulated by pufa consumption were examined assaying the liver transcriptome and lipid metabolome of mice fed a control and a pufa-enriched diet (mutch et al., ) . (d) gut stress markers: as a first step, we catalogued protein expression along the jejunum, ileum and colon of the rat intestine and found gut segment-specific proteins (marvin-guy et al., ) . the innovative combination of a neonatal separation model with proteomic analysis allowed us to study, whether early life psychological stress may impact the adult gut neuromuscular protein expression and the approach revealed specific protein biomarkers. omics for engineering lactic acid bacteria willem m. de vos wageningen center for food sciences, wageningen university, the netherlands. e-mail: willem.devos@wur.nl. url: http://www.wcfs.nl/ lactic acid bacteria (lab) are high at-rich gram-positive bacteria that have a well-established record in industrial food fermentations where they contribute to conservation, flavour and texture. in addition, several lab are used as food-grade hosts for the production of enzymes, peptides or metabolites. finally, lab are exploited in functional foods that contribute to the health and well-being of the consumer. a variety of metabolic engineering approaches have allowed for the improvement of many attributes of lab. these approaches have been facilitated by the possibility of uncoupling of growth and metabolite production in lab, the wealth of genetic tools that allow modulation of gene expression in a dynamic range, and the determination of several complete lab genomes (de vos et al., ) . we have developed lactobacillus plantarum as a paradigm for lab engineering by experimental and modelling approaches, the application of functional and comparative genomics, and the implementation of other post-genomics avenues (kleerebezem et al., ; smid et al., ; de vos et al., ) . examples of optimizing the production of vitamins and other cofactors, the impact of these engineering approaches on the global transcription and metabolite profiles, and determining the l. plantarum activity in the human host will be discussed. solanum tuberosum (potato) is the fourth major crop worldwide and used for food, feed and biotechnological applications. to fully realize the biosynthetic potential for production of starch, protein and metabolites, we conducted an extensive quantitative profiling of the expressed genes of mature potato tuber. a total of , sage (serial analysis of gene expression) tags of nt representing , different tags were analyzed. the tags seen or more times were assigned a tentative function by comparison to homologous genes. contrary to the transcript profile of rice seedlings (gibbings et al., ) the storage organ of potato is not dominated by transcripts encoding storage proteins. transcripts for four types of protease inhibitors, a metallothionein and a lipoxygenase were more prominent than patatin isoforms. the lactic acid bacterium lactococcus lactis is used extensively in the production of fermented milk products. during cheese production the bacterium experiences many changes in its immediate environment, as a result of its own reactions. the most severe change is the accumulation of lactic acid, which changes the ph of the medium until growth is totally inhibited. we have focused upon a survey of these dairy related stress responses, as a means of constructing more robust strains. when l. lactis starter cultures are produced in rich media, they will experience an initial period with purine limitation after being added to the milk substrate, a stress condition that in several studies have been found to induce cross resistance towards a number of other stresses. we have analyzed both general purine nucleotide (atp and gtp) and specific gtp limitation in chemi-cally defined medium, using both proteomics and transcriptomics. the differential expression analyses were performed with a custom designed dna microarray of pcr amplified probes. the two stress conditions resulted in very different stress responses, both at the transcriptomic and proteomics level. from a new study on the temporal expression pattern of l. lactis during growth in milk, we present preliminary data showing differential expression of genes and proteins of the purine stress stimulon as well as other stress stimulons. cell physiology of the yeast saccharomyces cerevisiae glucose repression mutants ∆snf , ∆snf and ∆snf ∆snf was studied in batch and glucose limited chemostat cultivations. detailed physiological studies were performed on cells grown in batch using glucose, galactose, or glucose-galactose mixture as a carbon source. during growth on glucose-galactose mixtures it was shown that after glucose was consumed, galactose consumption remained repressed for about h in ∆snf or ∆snf mutants, and for more then h in ∆snf ∆snf mutant, whereas it only lasted h in wild-type cells. the global transcriptional response in the glucose repression mutants was studied using chemostat cultures. s. cerevisiae wild type and the mutants were grown in glucose limited aerobic chemostats at a dilution rate of . h − . biological triplicates were performed for each strain. to identify transcriptional responses of the glucose repression mutants, statistical tests, clustering method and a model-driven analysis method were used. the global transcription data analysis experiments showed that genes involved in hexose transport, carbohydrates metabolism, respiration, and signal transduction were differently expressed in ∆snf and ∆snf mutants comparing to wild type cells. combination of gene expression data and gene-scale metabolic model indicated changes in the metabolic sub-networks among studied glucose repression mutants. genomics technologies have recently been introduced into food and nutrition science for identifying targets of molecular actions of nutrients as well as non-nutrient components of foods. changes in the transcriptome, proteome and metabolome have been determined for assessing the molecular actions of zinc as an essential micronutrient and of flavonoids in processes such as colon carcinogenesis and atherosclerosis. zinc is essential for the structural and functional integrity of cells and plays a pivotal role in the control of gene expression. zinc deficiency effects in human cells and an animal model (rats) were analyzed by microarrays and showed that a low intracellular zinc concentration caused major alterations in the steady-state mrna levels of several hundred target genes-dependent on the tissues studied including liver, brain, muscle, intestine and kidney. proteome analysis from the same samples by d-page followed by peptide mass fingerprinting via maldi-tof-ms identified similarly a large set of proteins with altered expression level but allowed a common theme of action to be identified. although pleiotropic in first view, the obtained pattern of zinc-affected genes/proteins may represent a reference for defining the zinc regulon in mammalian cells. flavonoids occurring in large number in plant species are considered protective agents in a variety of processes including inflammation and cancer development. we have studied the effects of around selected flavonoids in a screening program and identified for compounds such as flavon, genistein or quercetin by genomics technologies their putative mode of action in colon cancer models and endothelial cells. as part of a collaborative effort employing human endothelial cells and blood mononuclear cells from a human intervention trial with soy isoflavones (genistein/daidzein) the effects of the flavonoids on the stress-response to oxidized ldl and homocystein was analyzed. a set of markers of anti-inflammatory and anti-apoptotic activity was identified for genistein and daidzein and cell biological studies confirmed that both compounds prevented programmed cells death in stressed endothelial cells. in the food processing industry, unwanted presence of extremely heat-resistant bacterial endospores creates major problems due to their capability to survive classical thermal treatments and their ability to subsequently germinate and form actively growing vegetative cells. screening of spoilage isolates using genomic typing techniques to visualise putative genome-based biomarkers allowed us to classify strains according to the degree of thermal resistance of their spores. in addition, we showed that sporulation in the presence of ingredients rich in calcium ions promotes thermal resistance of developing spores and correlates with the expression of specific (marker) genes (see oomes and brul, ) . finally, the molecular program that forms the basis of spore germination has been assessed using genome-wide expression analysis. noticeably genes involved in dna-repair were transiently expressed in germinating wild-type spores. also, surprisingly, it was found that spores contain significant levels of ribosomal and messenger rnas. degradation of these rna molecules upon spore thermal injury was found to be characteristic for their thermal resistance and predictive for their subsequent outgrowth behaviour. this finding is currently being patented. the information on spore presence, predictions of their thermal resistance and process survival chances, is used to structure a process management system to facilitate optimal food quality assurance and allow for real time analysis in case of the need for quality control. the information on spore presence, predictions of their thermal resistance and process survival chances is now being integrated. this is used to formulate the conditions for a process management system with state of the art food production quality assurance, which allows for real time analysis in case of the need for quality control. the interplay of the pectinase spectrum of aspergillus niger as revealed by dna microarray studies elena martens, jac benen, johan van den berg, peter schaap fungal genomics group, laboratory of microbiology, wageningen university, dreijenlaan , ha wageningen, the netherlands. e-mail: elena.martens@wur.nl (e. martens) the saprobic fungus aspergillus niger is an efficient producer of extracellular enzymes many of which show carbohydrate modifying activities. these enzymes have gras status and therefore are widely used in the food and feed industry. after determination of the genomic sequence of a. niger by dsm, it was estimated that only a fraction of the potential of secreted enzymes is currently characterised. database mining using the proprietary genome sequence has resulted in the identification of more then genes encoding enzymes involved in the depolymerisation of the back bone and the site chains of the complex polysaccharide pectin. additional enzymatic activities required to remove methyl and acetyl esters, present in pectin were also observed. by using dna microarrays we have sought to gain insight into the complex regulation of all the genes involved in pectin degradation. a. niger was cultivated on sugar beet pectin and on the monomeric constituents of pectin, viz. galacturonic acid, rhamnose and xylose. subsequently the corresponding transcriptomes were analysed. we will report on our findings concerning the regulation of the expression of the genes involved in the degradation of pectin and its main constituent-galacturonic acid and the consequences for the interplay of the encoded (novel) enzymes. since reactive oxygen species (ros) are formed in all living organisms a wide range of antioxidative enzyme systems are present to keep the system in balance. when an animal is slaughtered the cellular anti-oxidative capability is reduced, resulting in an accumulation of ros followed by an increased oxidation of dna, lipids and proteins. generally lipid oxidation is a well-known problem, causing increased rancidity during prolonged storage, of especially fatty fish. the implications of protein oxidation are, however, more unclear also in respect to quality decay of fish. protein oxidation causes a wide variety of amino acids modifications, where of the most studied is carbonylation of proline, argenine, lysine or threonine. these carbonyl groups can be labelled with , -dinitro-phenylhydrazine. combining two-dimensional gel electrophoresis with immunoblotting enables the detection of carbonyl groups for each single protein. the results presented here, reveal that both during frozen storage and tainting of rainbow trout protein oxidation/carbonylation increases, furthermore there is an increase in oxidation/carbonylation for distinct proteins. anne-marie neeteson european forum of farm animal breeders, benedendorpsweg , wl oosterbeek, the netherlands society is concerned about food, animal welfare, food safety, new technologies, scientists and industry. these elements are all present in genomics for farm animals. therefore, it is important to build awareness in scientists and industry, start a dialogue with stakeholders and society, and to be transparent in a pro-active way. this paper will address the issues at stake for scientists and industry, when it comes to genomics and animal health. it will combine the results of imperical, ethical and sociological efforts in three eu funded projects. (c) the proper use of genomics in relation to infectious diseases in production animals, and the role of the scientist in the development in new technologies in this field, are being addressed in european animal disease genomics network of excellence (ead-gene, http://www.eadgene.org/). some observations are that: ( ) genomics does not concern changing the gene. however, acceptability of any discovery dealing with living beings and edible products, is not obvious just like that! animals have a symbolic and emotional load. ( ) genes are still related to eugenics, in the mind of people. genes as such cause reluctance, but it is seen as positive if the use of medication can be reduced, and if animals will be better resistant to disease. ( ) consumers are in favour of consumer education, compulsory labelling and the imposition of minimum standards. the inclination to pay more for foods produced according to desired standards relates closely to income level. ( ) animal welfare is the major issue citizens mention as a concern. the focus of breeding organisations on productivity should be counterbalanced by serious attention to the animal's needs in order to avoid unnecessary negative impact on the welfare of the animals. ( ) when technical specialists and lay people communicate, they tend to use different languages: they use the same words, but with rather different interpretations. so transparency of breeding practices and clear definitions of terminology will be essential for effective communication among all stakeholders. ( ) food safety and human health are the major concern for most people, when it comes to making a choice. during the latest decades research within the field of animal genomics has in general been following the same strategies as those used within the field of human genomics, although with much less resources. the porcine genome has been characterized intensively through the development of linkage maps, comparative maps and physical maps. until a few years ago it had not been anticipated that it would be possible to embark on whole genome sequencing of animals genomes. however, because of technological developments and much lower costs for sequencing, several animal genomes have now been assembled/are on the way to being assembled. the initial step towards sequencing the porcine genome was taken by the sino-danish pig genome project. the efforts within this project have now generated approximately . million genomic shotgun sequences and . expressed sequence tags (ests). the shotgun sequences have been included in a three-species alignment to make an initial evolutionary analysis. the results show that pig is much closer to human than mouse is. the ests represent -end sequences from a total of non-normalized cdna libraries. based on assembly and annotation of the ests the structure of the porcine transcriptome has been analysed. the relevance of assembling the porcine genomic sequence is justified both from the perspective of sustainable animal breeding and from the fact that the porcine model is an important research platform because of the anatomical, physiological, biochemical and metabolically similarities with man. examples of functional genomic studies both aimed at sustainable animal breeding and aimed at exploiting the pig as a model for medical studies will be discussed. genomics refers to global, systematic and high throughput approaches that allow collecting large amount of data and thus offer new possibilities for analysis and understanding biological processes. we will present some new knowledge related to reproduction in farm animals resulting from three different strategies. ( ) functional analysis of gene and protein expression: the transcriptome and the proteome analysis allowed to identify new genes and proteins whose expression is associated with processes of ovarian follicular growth and atresia as well as oocyte maturation in bovine and porcine species, maturation of spermatozoa in the different compartments of epididymis. farm animals produce food as cost effectively as possible, however this may have negative side effects for their health and welfare. trade off processes between production on one hand and reproduction and health on the other hand play a crucial role. the principles of selective breeding for the best of naturally occurring variation has proven to be able to balance an increased level of production and quality of life for the animal. every year, the economic value of the genetic gain achieved by the breeders and carried over to the producers is . % of the economic value of eu farm animal production. consequently, a conservative estimate of the gain from animal breeding is, every year d . billion in europe. recent developments, such as the sequencing of the genomes of the human, chicken and cow, together with high throughput laboratory techniques, means that there are new opportunities to enhance quality of life. the goal of this paper is to give an overview of the options offered by genomics for enhanced quality of life with focus on identifying relevant gene variants and technologies for large scale tracking and tracing. selective breeding for the best of naturally occurring variation remains the same as in traditional systems, but by pinpointing the relevant gene variants along genomics it is possible to identify directly the animals best selected for high production without comprising health and welfare. the combination of full genome sequences, software tools, study of functional physiological processes cost-effective high-throughput snp genotyping and comparative mapping have the (proven) potential to identify relevant gene variants, e.g. pork color, boar taint, general disease resistance. functional mutations have direct option of application in breeding programs. unfortunately this is not the case for genetic markers due to cost of genotyping and inconsistent phenotypic effects. new technologies for snp genotyping are cost effective and enable large scale genotyping ( . of animals/day). a selection of the best technology and strategic use of these opportunities enable tracking and tracing. the application of this technology offers new opportunities for quality of life, both for animal and humans. background: studies have shown that prebiotic and probiotic consumption alters the gastrointestinal flora, modulates the immune system, inhibits genotoxicity and has a protective effect on colon carcinogenesis. however, the effect of synbiotic consumption on these parameters in subjects at risk of colon cancer has not until now been investigated. aim: to determine if a synbiotic (prebiotic and probiotics together) modulates cancer risk biomarkers in human subjects at risk of colon cancer. methods: a -week randomised, double blind, placebo controlled, ethically approved trial of a food supplement containing lactobacillus gg, bifidobacterium bb- and raftilose synergy (prebiotic) was performed in colon cancer subjects who had undergone 'curative resection'. faecal and blood samples were obtained before (t , week ) midway through (t , weeks) and following intervention (t , weeks). rectal biopsies were obtained at t and t . the effect of synbiotic consumption on the faecal flora was assessed using standard plate count techniques. genotoxic damage was measured in single cells derived from biopsies using the comet assay. fw was prepared by diluting faeces : in dmem, ultracentrifugation and sterile filtration. the genotoxic (comet assay) and cytotoxic potential (almar blue assay) of fw was determined. peripheral blood mononuclear cells were isolated from blood and cytokine production in vitro assayed by elisa. natural killer cell cytotoxic activity and the phagocytic and respiratory burst activity of monocytes and granulocytes in whole blood were determined by flow cytometry. results: in the synbiotic group faecal numbers of bifidobacteria significantly increased (p < . ) and lactobacilli increased although not significantly (p = . ) while coliforms decreased (p < . ). enterococci, clostridium perfringens and bacteroides were unaffected. in the placebo group bifidobacteria decreased (p < . ), the other bacterial groups were unaffected. in biopsies genotoxic damage was increased in the placebo group at t versus t (p = . ) but was unchanged in the synbiotic group. the genotoxic and cytotoxic potential of fw was unaltered. synbiotic consumption significantly increased (p < . ), ifn-␥ production by pbmcs but il- , il- , il- , and tnf-␣ production was unaffected. natural killer cell, phagocytic and respiratory burst activities were unaltered. conclusion: synbiotic consumption did not have a strong immunomodulatory effect on the systemic immune system in this study, nor did it influence the genotoxic and cytotoxic potential of fw. however, synbiotic consumption altered the composition of the gut flora to a more beneficial composition as well as protecting against genotoxic damage in vivo, suggesting a protective effect of synbiotics against colon carcinogenesis. emmaÅrsköld, malin svensson, halfdan grage, peter rådström, ed w.j. van niel applied microbiology, lund institute of technology, lund university, p.o. box , sweden lactobacillus reuteri is used today in a variety of dairy products as a probiotic bacterium. several lactic acid bacteria have the ability to produce different kinds of exopolysaccharides (eps), which have the potential to be used as an alternative biothickener. however, the yield of eps is too low to be profitable in the food industry. to optimise the environmental conditions for eps formation a l. reuteri strain was chosen for a factorial design study. the factors used in this experiment were temperature ( - • c), ph ( . - . ) and sucrose concentration ( - g/l); for each factor three different values were chosen. the strain was grown in batch mode using a semi-defined medium at constant ph and sucrose as the carbon source. the results obtained with fermentations revealed that the highest eps formation was found at • c, ph . and g/l of sucrose. sucrose did not further affect the eps formation above a concentration of g/l. temperature and ph were significant for the eps formation, but only temperature was significant for growth. a central composite design study was chosen for further optimization of the ph and sucrose concentration for maximum eps formation. also the gene expression of the sucrase enzyme responsible for eps formation was investigated using qrt-pcr. the data were used to develop a model for growth and eps formation. dendritic cells (dc) play a pivotal immune regulatory role in the th , th and treg cell balance. dc are present in the gut mucosa and may thus be target for modulation by gut microbes. here, we screened a large panel of human gut-derived lactobacillus and bifidobacterium spp. for dc polarizing capacity: bone marrow-derived murine dc were exposed to lethally irradiated bacteria and cytokine and dc surface markers were analyzed. substantial differences were found among strains in their capacity to induce proinflammatory cytokines, while the differences for anti-inflammatory cytokines were less pronounced. bifidobacteria were weak il- , il- and tnf-␣inducers, while both strong and weak cytokine-inducers were found among the strains of lactobacillus. remarkably, strains weak in il- induction inhibited il- , il- and tnf-␣ production induced by otherwise strong cytokine-inducing strains, while il- production remained unaffected. those lactobacilli with greatest capacity to induce il- were also most effective in up-regulating surface markers. surface marker up-regulation was however reduced in the presence of weak il- -inducing strains. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th /th /treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. cell surface-associated glycolytic enzymes from lactobacillus plantarum v mediate adhesion to human epithelial cells and extracellular matrix proteins s.m. madsen, j. glenting, a. vrang, p. ravn, h.k. riemann, h. israelsen, m.r. nørrelykke, a.m. hansen, m. antonsson, s. ahrné, h.c. beck bioneer a/s, hørsholm dk- , denmark among the main selection criteria of lactic acid bacteria for probiotic use, the ability to adhere to intestinal epithelial cells, mucus, or extracellular matrix proteins is considered important. using a proteom based approach we identified a group of novel surface proteins that are non-covalently bound to the cell wall of the probi-otic bacterium lactobacillus plantarum v. surface proteins were extracted and analysed by gel electrophoreses followed by mass spectrometry analysis. the surface proteins included glycolytic enzymes like, e.g glyceraldehyde -phosphate dehydrogenase, which usually is a typical intracellular enzyme. a collection of lactobacillus species was screened and the phenomenon of surface-associated glycolytic enzymes was found in many of the analyzed species. this is to our knowledge the first example of surface-associated glycolytic enzymes in probiotic bacteria. however, in pathogenic bacteria these enzymes are well known and their surface localization is involved in adhesion to human epithelial cells and invasion. we suspect that the presence of these enzymes on the surface of probiotic bacteria could prevent adhesion of pathogenic bacteria and possibly also involve other probiotic activities such as immune modulation. binding studies showed that the surface-associated glycolytic enzymes of lb. plantarum v were able to bind to caco- intestinal cells and extracellular matrix proteins like fibronectin. scientific and industrial interest on exopolysaccharides (eps) synthesized by microorganisms and on their chemico-physical properties has been quickly growing in the last years. many strains of lactobacilli produce eps allowing them to adhere to human mucosae and therefore to have probiotic effects such as the stimulation of immune response and even antitumoral activity of these molecules has been claimed. futhermore prebiotic actions of eps beneficially affect the human host health improving the properties of indigenous microflora. in this research we have studied two novel interesting lactobacilli strains: lactobacillus plantarum dsmz and a particular human isolated strain of lactobacillus crispatus that have probiotic potentialities and are good eps and l(+)-lactic acid producers. the aim of this work has been to characterize these strains and their metabolites (eps, organic acid and bacteriocins), to state them as probiotics and to study their adhering ability on human cells. we have studied the physiology of l. plantarum and l. crispatus in shaking flasks as well as their optimal fermentation conditions to obtain high cell density cultures suitable for use as starters in the food industry and eventually for probiotic preparations. fermentation experiments have been performed in a bioreactor equipped with microfiltration (mf) modules using a semidefined medium and various carbohydrates in different culture conditions (aeration, temperature). the kinetic of eps production has been followed and according to results both strains have shown a growth-related production ranging from to mg/l. in vitro studies concerning the ability of these strains to adhere to human mucosae are in progress as well as the structural characterization of the exopolisaccharides. previous studies have shown that compression coating improves the storage stability of freeze-dried lactobacillus acidophilus, although this stability is related to the degree of cell injury, which in turn is related to the compression pressure used. compression coating has also been found to improve the survival of freeze dried l. acidophilus during exposure to simulated gastric fluid (sgf). the aim of the present work is to create a compression coated l. acidophilus formulation, with targeted release at the terminal ileum and beginning of the colon in the human gastrointestinal tract. dissolution studies were performed using a phosphate buffer with a ph of and . , to simulate gastric fluid and intestinal fluid (sif), respectively. cell viability was monitored using multi-parameter flow cytometry (mpfc), together with traditional cfu/ml counts. mpfc was used to identify live, dead and stressed cell populations, using the fluorescent stains propidium iodide (pi), , -dihexylocarbocyanine iodide (dioc ( )) and to-pro- . results show that an enteric coating material, eudragit l - , is both suitable for compression coating, and enhancing the survival of cells when exposed to sif. pectin usp has also been shown to promote targeted release of the cells. the opium poppy papaver somniferum contains more than tetrahydrobenzylisoquinoline-derived alkaloids. it is the source of the narcotic analgesics codeine and morphine, which accumulate in specialized internal secretory cells called laticifers. in the aerial parts of the plant, the laticifer cells are anastomosed, forming an articulated network. laticifers are found associated with the vascular bundle in all plant parts. the morphinan alkaloids morphine, codeine and thebaine are found both in roots and in aerial plant parts and specifically accumulate in vesicles within laticifers. the benzo[c]phenanthridine alkaloids sanguinarine and -hydroxysanguinarine are found in root tissue. the syntheses of sanguinarine and of the tetrahydrobenzylisoquinoline latex alkaloid laudanine are completely understood at the enzyme level. nearly all enzymes of morphine biosynthesis have also been described. in more recent years, cdnas encoding enzymes of alkaloid biosynthesis in p. somniferum have been isolated and characterized. the cell-specific localization of several of the enzymes of morphine, sanguinarine and laudanine biosynthesis has also been described. with knowledge of many of the gene of alkaloid formation and their sites of expression, the metabolic engineering of p. somniferum for tailored alkaloid profiles is now being undertaken. an agrobacterium tumefaciens-based transformation and a regeneration protocol have recently been developed specifically for narcotic tasmanian cultivars. the various cdnas encoding genes of alkaloid biosynthesis in p. somniferum are being systematically reintroduced into the plant to achieve engineered plants with altered alkaloid profiles. the first results have now been obtained with sense and antisense genes stably expressed in a regenerated tasmanian cultivar. the ultimate goal of exploiting the genes of alkaloid biosynthesis is to produce transgenic medicinal plants of specific alkaloid content that would facilitate commercial production and improve our understanding of the factors that regulate biosynthesis as well as provide experimental systems with which to investigate the ecological role of alkaloids in planta. integrated transcript and metabolite profiling for gene discovery in plant natural product pathways richard a. dixon, lahoucine achnine, bettina deavours, mohammed farag, marina naoumkina, lloyd w. sumner plant biology division, samuel roberts noble foundation, ardmore, ok , usa the rich diversity of chemical structures found in the plant kingdom arises in large part from a limited number of basic chemical scaffolds (e.g. terpene, polyketide) that are modified by a limited number of chemical substitution types (hydroxylation, glycosylation, acylation, prenylation, o-methylation, etc.) . much of the diversity is brought about by the substrate-and/or regio-specificities of the substitution enzymes. in contrast to the large collections of gene sequence and transcript level data available on-line, little detailed information exists on the plant (secondary) metabolome. promiscuity of substrate specificity in vitro may complicate attempts to assign functions to genes of secondary metabolism accessible to researchers through various cdna library collections. using the isoflavonoid and triterpene pathways in medicago species as examples, we describe how integrated metabolite and transcript profiling approaches can aid functional genomics, help explain metabolic regulation, and provide tools for assessing the impacts of genetic modifications in plant secondary metabolism. focused and non-targeted approaches were used to assess the impact associated with introduction of new high flux pathways in arabidopsis thaliana by genetic engineering. transgenic a. thaliana plants expressing the entire biosynthetic pathway for the tyrosine derived cyanogenic glucoside dhurrin as accomplished by insertion of cyp a , cyp e , and ugt b from sorghum bicolor accumulated % dry-weight dhurrin with marginal inadvertent effects on plant morphology, free amino acid pools, transcriptome and metabolome. in a similar manner, plants expressing only cyp a accumulated % dry-weight of the novel tyrosine derived glucosinolate, p-hydroxybenzylglucosinolate with no morphological pleitropic effects. in contrast, insertion of cyp a plus cyp e resulted in stunted plants, transcriptome alterations, accumulation of numerous new glucosides derived from detoxification of intermediates in the dhurrin pathway, and in loss of the brassicaceae specific uv protec-tants sinapoyl glucose and sinapoyl malate as well as kaempferol glucosides. the accumulation of new glucosides in the plants expressing cyp a and cyp e , was not accompanied by induction of glycosyltransferases, demonstrating that plants are constantly prepared to detoxify novel xenobiotics. the pleiotrophic effects observed in plants expressing sorghum cyp a and cyp e were complemented by retransformation with s. bicolor ugt b . accordingly, insertion of high flux pathways directing synthesis and intracellular storage of high amounts of natural products is achievable in transgenic plants with marginal inadvertent effects. arabidopsis thaliana-distinct function in gene transcription dynamics? jeppe madura larsen, brian stougaard vad, søren mølgaard, kell andersen, mads n. davidsen, klaus d. grasser department of life sciences, aalborg university, aalborg, denmark in recent years it has been shown that introns to some extent can regulate expression in the eukaryotic cell. insertion of introns in the utr in arabidopsis genes has shown to increase gene expression at both rna and protein level. in order to investigate if utr introns have distinct characteristics, we analyse these in the well annotated arabidopsis thaliana genome published by the arabidopsis genome initiative. , loci annotated with full-length cdnas were analysed and loci ( . %) containing utr introns were isolated. we studied if the genes containing these introns showed different patterns in alternative splicing and gene function (gene ontology classification) compared to the remaining genes not containing this intron type. of the isolated loci ( . %) contained only one utr intron and these were used for further analysis, where it was investigated if the utr introns had a characteristic size distribution. genes containing transcripts with utr introns were more subjected to alternative splicing ( . % versus . %) and had a tendency to be more involved in cell regulatory functions compared to genes without this intron type. it was also found, that utr introns was characteristically size-distributed. we identified thee predominant sizes of approximate , and bp compared to only one for orf introns. this suggests widespread multiple splicing events in utr introns. the results presented here suggest that utr introns have distinct characteristics and function in gene transcription dynamics. cytochrome p monooxygenases appear to be involved in the biosynthetic pathways of a large variety of primary and secondary metabolites in microbial, animal and plant cells. in particular, cytochrome p scc catalyzes the conversion of cholesterol into pregnenolone-the precursor of all steroid hormones in mammalian steroidogenic tissues. cytochromes p are also involved in the biosynthesis of different plant steroid derivates that play important role in regulation of plant growth and development. therein, investigation of possible influence of cytochrome p scc expression on plant regulatory system is of a great interest. this report devoted to the investigation of transgenic tobacco plants, which have been generated by the transformation with recombinant plasmid pgbp f constitutively expressing cyp a cdna of the bovine cytochrome p scc . the transgenic state of the plants was confirmed by southern blot analysis. transgenic plants are phenotypically different from the control ones. in particular, they obtained a substantially higher growth rate and are larger than wild type plants. we have demonstrated that incubation of fragments of the transgenic plants leaves in [ c]-labeled cholesterol containing medium results in formation of the radioactively labeled product with chromatographic mobility corresponding to pregnenolone. the presence of this metabolite in the steroid fraction of lipid extracts obtained from the transgenic plants leaves was confirmed by gas chromatography mass spectrometry (gc-ms) method. the data obtained indicate that cytochrome p scc synthesized in transgenic plants displays its specific catalytic activity. biotechnological production of glucose isomerase enzyme with streptomyces olivochromogenes for production of fructose syrup from hydrol m. hashemiravan, a. sadat barikani department of food science and technology, azad university (pishva, varamin unit), institute of food science and agriculture, tehran, iran the use of glucose isomerase for isomerization and production of fructose syrup was performed by selected industrial strain of streptomyces olivochromogenes ptcc . growth of microorganism and production of enzyme in different culture media was studied, and effects of different parameters such as phosphate and aeration was evaluated. the growth of microorganism at • c, caused a production of high amount of enzyme. the production of enzyme was considered in two culture media (a and b). medium (a) was selected for the higher production amount of enzyme. the highest amount of enzyme production was seen in medium a, which was . giu/ml, after h. the use of baffles in culture flasks, increased the amount of enzyme production, four times more. the production of enzyme was increased, . times more, in phosphate deficient medium. the cells containing enzyme (intra cellular glucose isomerase) was separated by centrifuge, and extraction and release of enzyme was performed by ultra sonication, that is a physical-mechanical method. this method released about . % of total intracellular enzyme. the best length of time for sonication was found to be min. experiments showed that optimum ph and temperature of the enzyme were . - and • c, respectively. the highest activity of the enzyme was observed at ph . for up min. at the time of isomerization reaction the existence of magnesium ions showed to be necessary and omission of this ion cause a decrease of enzyme activity in isomerization process, but this effect was not necessary for the enzyme activity results showed that treatment of glucose syrup at temperatures of , and • c, by the enzyme, caused %, % and % of isomerization, respectively. efficiency increase of high acetic acid production with the use of acetobactereace iranian native strains mutation m. hashemiravan , a. alirezasadat barikani : department of food science and technology, azad university (pishva, varamin unit) institute of food science and agriculture, iran; young iranian researchrer's club, tehran, iran the first step in the present research is the isolation of acetobactereace native strains from fruits (such as grape or apple) and fresh vinegar. this separation has been done with the use of effective isolation methods and optimized mediums. ten strains were isolated effectively, the bio chemicals test were performed, each of them were detected, classified and nominated with a special code. two methods have been used for mutation: (a) mutation with the ultraviolet radiation; (b) mutation with nitrous acid. in the first method the microbial cells were treated with us radiation for different periods of , , and s, and the effect of the uv mutation was assessed. as a result, the period of s was determined as the optimum mutation periods. in the second method, the microbial cells were first washed with the acetate buffer . m with the ph of . , then ml nitrous acid . m was added and was mixed for - min. finally the sampling was done in the periods of , , , and min and was transferred to a plate containing the medium of ethanol-phenol red-agar. the two methods have been compared with each other. each method has its own advantages and disadvantages. the mutation pathway in method (b) is more stable and conducted, while mutation with the uv radiation method, change the position of thiamine-cytosine bases absolutely randomly. finally, the best mutant site was inoculumed with the medium containing alcohol %, acetozyme gz . g/l, acetozyme d g/l, acetic acid . % and l double distilled water. the acetic acid was produced in the rate of g/ ml. also the mutant strains were detected with scanning electron microscope (sem) and interesting photographs were taken from mutant cell. the performed experiments were planned with the use of taguchi statistical method (qualitek ). this research has been performed in the irost as the thesis of ph.d. in food biotechnology industry. isotachophoresis has almost exclusively been applied for contracting and stacking samples ions before zone electrophoretic separation of proteins. this study attempts to apply microfluidic isotachophoresis (itp) as a high resolution analytical method for proteins. beta-lactoglobulin and other milk proteins with slightly different pi were labelled with fluorescent red and analysed by the micralyne tk system using microfluidic glass chips, either with simple cross (sc) or double cross (tt) injection or designed d-itp-cze chips with double tt injection and sc for transfer to the second dimension cze channel, efficiently non-covalently coated with . % (w/v) epoxy-polydimethylacrylamide to lower electroendoosmosis. capillary zone electrophoresis (cze) in borate or phosphate buffer was reproducibly perform for more than consecutive runs using upchurch tm reservoirs glued to the wells to enable larger buffer volumes and greater run-to-run stability. finally, isotachophoretic anionic separation of the proteins were done using phosphate (ph . ) or chloride (ph . ) as leading ion and -amino-caproic acid (ph . ) as terminating ion. the effect of narrow cut ampholytes as spacers needs further investigations. the perspective aim is to combine the migrating itp separated zones with second dimension capillary zone electrophoresis as a new microfluidic proteomic danalysis. genotypical differences affecting the response of pisum sativum to differing boron/iron applications e.e. hakki, u. zeynep, m. hamurcu, a. tamkoc, m.b. babaoglu, s. gezgin department of field crops, faculty of agriculture, selcuk university, kampus, konya , turkey. e-mail: eehakki@selcuk.edu.tr (e.e. hakki) boron and iron are among the microelements required for the proper development of the vegetative and generative tissues of plants. though iron is present in high amounts in almost all soil types, its bioavailability to crops is extremely reduced, hence most of the plants face an iron defficiency problem and while on one side crop productions effected, on the other hand the nutrition problems come up to human through contagious nutritional chains. boron is also among the most problematic micronutrients of the major crop plantation areas of turkey. both defficiency and toxicity problems exist in a total of about % of the central anatolian soil where pea is among the legumes cultivated. application of varying levels of boron and iron combinations in greenhouse and the analysis of plant aquisition via icp-aes as well as determination of the effects of the element combinations both in morphological and molecular levels are the aim of our studies. the genetic bases of the response differences of plant genotypes to b and/or fe, were investigated through the applications of molecular marker techniques. considerable growth rate and stem size differences were detected within the parents (wild-type versus cultivar) and the f plants. the presence of efficient genotypes to high micronutrient levels are expected to help us increase the cultivation of the crop in problematic areas as well as in exploring the molecular bases of the microelement uptake mechanisms. increase in sulfite production by accelerating sulfate uptake in brewing yeast t. fujimura, y. kodama, y. nakao, n. nakamura, w. miki institute for advanced technology, suntory ltd., mishima-gun, osaka - , japan sulfite plays a role as an antioxidant, which stabilizes beer flavor. therefore, it is important to control the sulfite concentration during fermentation. sulfite is produced as an intermediate in the sulfate assimilation pathway in yeast. we have already reported that over-expression of a lager yeast-specific ssu gene, encoding a sulfite efflux pump, leads to increase of sulfite production (fujimura et al., ) . in the present work, we have clarified that there are two types of sul genes (scsul and non-scsul ) each encoding a high affinity sulfate permease in lager brewing yeast. eighty percent and % identity are found by comparing the dna sequences and the deduced amino acid sequences, respectively. a comparative functional analysis of the two genes has been performed aimed at achieving further increases in sulfite production by accelerating sulfate uptake. over-expression of scsul and non-scsul has been achieved by transformation of lager brewing yeast, saccharomyces pastorianus. experiments have been done with and without expression of non-scssu . the resultant transformants have been evaluated by fermentation tests in wort. over-expression of either scsul or non-scsul failed to show significant effect on sulfite formation. a combination of over-expression of non-scsul and non-scssu resulted in two-fold higher sulfite production compared with overexpression of only non-scssu and four-fold higher compared with the parental strain. these results suggest that the non-sc-gene types significantly contribute to sulfite production in lager brewing yeast. , t., et al., . functional phytases (myo-inositol hexakisphosphate phosphohydrolase) catalyse the release of phosphate from phytate (myo-inositol hexakisphosphate), the predominant form of phosphorus in cereal grains, oilseeds and legumes. possible applications of phytases have been suggested in animal nutrition to increase mineral bioavaliability and to decrease phosphate pollution in area of intensive life stock management and in human health. zea mays is one of the cereals that contain high amount of phytate as the major phosphate storage compound. over bacteria were isolated and screened for phytases from the halosphere, rhizosphere and endophyte of malaysian maize plantation. the phytase activity of the isolates was screened by a modification of the ammonium molybdate method. the highest extracellular phytase activity was detected from bacteria that isolated from the endophyte of the maize root. in this paper, results for isolates chosen for media, temperature and ph optimization will be presented. production of plant proteinase from jack fruit seeds (artocarpus integrifolis) and its influence on rheological and sensory characteristics of low fat yogurt el-sayed el-tanboly dairy sciences department, national research centre, dokki, cairo, egypt adding a proteolytic enzyme extraction from jack fruit (artocarpus integrifolis) in combination of fermentation process in low fat yogurts manufacture was tried to improve yogurt flavour and rheological properties. experimental yogurts milk contained control, . (t ), . (t ) and . (t ) units/ml milk from crude extracts of plant proteinase. the ph of the product treated with crude proteinase was lower than the control. however, the rate of acidity development during storage slightly increased with increasing the addition of crude proteinase level and progress of storage period of yogurt. the proteolytic activity of all yogurts gradually increased until the end of storage period ( days). yogurts made from milk treated with crude proteinase preparations were less firm compared with control at all storage periods, where t showed more less firm after days of storage being . g/ g. generally, increasing units of plant proteinase preparations decreased the firmness. on the other hand, yogurt made from milk pretreated with plant proteinase had higher syneresis, and apparent viscosity than the untreated product. the greatest viscosity was found in t and t of and mpa s, respectively, compared with control of mpa s at days storage. the results indicated that there is an inverse relationship between the amount of units of crude proteinase preparations and susceptibility of yogurt to syneresis. the t gained the highest scores ( points) followed by the control ( . points) after days of storage, while yogurt of t showed a low scoring being . from the foregoing results, it is recommend to use jack fruit (artocarpus integrifolis) as a source of plant proteinases and utilize it to develop a high quality yogurt at a level of . units of plant proteinases/ml milk. an effective process for the chemical-biotechnological utilization of distilled white lees was studied. a first treatment with hydrochloric acid allowed the solubilisation of tartaric acid. the influence of temperature, amount of hcl and reaction time were considered through an experimental design. under the optima conditions g/l from white distilled lees and . g/l from red distilled lees were recovered. the tartaric acid was precipitated as calcium tartrate so that it can be isolated from the rest of the raw material compounds. the solid residue was used as an economic nutrient for lactic acid production by lactobacillus pentosus using trimming wastes as substrate. the lactic acid concentrations and volumetric productivities achieved were similar to those obtained using distilled lees without tartaric acid recovery as nutrient. toasted wine was traditionally produced in galicia, northwest of spain. nowadays this technique is being recovered. grapes after harvesting are air dried in order to concentrate sugars, acids and flavor compounds. raisings are pressed to obtain a must with high sugars concentrations. two different grape wines were prepared concentrating the sugars up to and brix, respectively. in order to get a better knowledge of the problems involved, synthetic media simulating the grape musts were prepared. theses musts were used to optimize the initial sugar concentration, the amount of nutrients required, the optimum temperature to carry out the fermentation and the influence of the type and amount of yeast. under the best conditions some fermentations with grape must were carried out to produce wines with intense aroma and flavor notes and high residual sugar concentrations. in this studies, bacillus sp. e strain was isolated from koreanstyle fermented soybean paste and it was producing the biological response modifier (brm). the brm activated the b cell selectively. it was identified the bacillus licheniformis e . the brm was purified by ion-exchange chromatography and gel filtration. chemical properties of brm: molecular weight of brm was estimated to be about , , da. sugar content of brm was . % (w/w) and glucosamine ( . mol%) was the high level. protein content of brm was . % (w/w) and serine ( . mol%) was the high level. infra-red absorption spectrum was showed the characterization of glycoprotein. biological properties of brm: the brm which isolated from fermented soybean paste was similar to that of bacillus licheniformis e by immuno-fluorescence assay. we confirmed that the brm was capsular substance of b. licheniformis e . potato nitrogen concentrate (pnc) is a highly viscous liquid with high complex nitrogen content produced from the protein-fraction in potato starch extraction. the concentrated extract is rich in minerals and ␣-amino nitrogen. although ␣-amylase nowadays is mainly produced exploiting bacillus production systems there is still considerable demand for fungal ␣-amylase from aspergillus oryzae origin. the aim of the experiments to be reported here was to investigate, if pnc can replace commonly used complex nitrogen sources in the production of fungal ␣-amylase. the following data have been measured in pnc pretreated by diluting to / and clarifying by centrifugation. total-n: . g n/l; ␣-amino-n: . % (w/v) (as glycine); soluble protein (bradford): . mg/l (as bsa); total carbohydrates: . g/l; reducing sugars: . g/l; dry weight: . % (w/w). in the following experiments nitrogen sources were replaced on the basis of their ␣-amino nitrogen content. the carbon source for all experiments was maize starch. the formation of ␣-amylase by a. oryzae atcc in shake flasks -using pnc (centrifuged or not), yeast extract, malt extract, casein hydrolysate or meat extract -was compared to "standard" cultivation with corn steep liquor. the experiments showed only small differences in ␣-amylase titers using complex nitrogen sources. no remarkable differences were observed in the resulting biomass. in general no differences in enzyme productivity and biomass formation could be seen after h of incubation. especially the bench top bioreactor experiments indicated an optimal fermentation time of about h. cultivations of a. oryzae atcc were carried out in bench top bioreactors. comparing cultivations in a medium with pnc as the sole complex nitrogen source to one containing csl as such no significant differences both in the formation and amount of ␣-amylase and the fungal growth were observed. thus pnc might be able to replace complex nitrogen sources such as csl or even the more expensive yeast extract and casein hydrolysate in fungal amylase production systems. . ) is involved in the metabolism of inositol and catalyzes the conversion of d-glucuronic acid to l-gulonic acid with nadph as a cosubstrate posterior to the oxidation of inositol to glucuronic acid by the enzyme inositol oxygenase. although the yeast sporobolomyces oryzicola (nakase and suzuki, ) is not able to grow on inositol as the sole carbon source, intracellular glucuronate reductase can be found in cells grown in a medium containing d-glucuronic acid. the enzyme could be a useful tool in the design of a specific quantitative assay for glucuronic acid, e.g. in so called energy drinks. the organism was grown in media containing either glucose and glucuronic acid or only glucuronic acid and difco yeast nitrogen base. whereas growth on both media was similar in shake flask culture, hardly any growth in either medium was observed in bench top bioreactors. the influence of dissolved oxygen tension was investigated and the relevant data will be shown. the formation of intracellular glucuronate reductase activity by sp. oryzicola is inducible by media containing glucuronic acid. no activity is found in cells grown in a medium containing only glucose as the carbon source. besides the activity against d-glucuronic acid, activities against -ketogluconate and -at very low levelsagainst galacturonic acid and the lactone of glucuronic acid were detected. the enzyme activity is stable up to • c. the ph has relatively low influence on the activity against glucuronate, whereas the reduction rate of -ketogluconic acid is optimal at ph . - . with significantly lower values at ph . and . , respectively. data on the kinetics of the conversion of both glucuronate and -ketogluconate will be shown. nakase, t., suzuki, m., . j. gen. appl. microbiol. , - . the multiple nutritional and functional impacts of food fermentation on human health have been widely accepted (reddy and pierson, ; hugenholtz et al., ) . however, the related role of the involved microorganisms to the nutritional effect from the fermented food is still not well defined and the mechanisms involved are still largely unknown. the present study was to investigate iron bioavailability in carrot juice fermented by two selected lab strains, l. pentosus fsc and ln. mesenteroides fsc . after digestion by gi enzymes, the juice was supplied to fully differentiated caco- cells to study iron uptake and transepithelial transport by caco- cells from the digested juice. our data revealed strain specified changes in iron bioavailability in carrot juice fermented by these two strains. after in vitro digestion with pepsin and pancreatic-bile enzymes, the best yield of soluble iron was from ln. mesenteroides fsc fermented juice. surprisingly, the l. pentosus fsc fermented juice yielded about five times higher uptake iron as compared to fresh juice, while ln. mesenteroides fsc fermented juice was not significantly different from the fresh juice. interestingly, the transepithelial transferred iron across the cell line was however better from ln. mesenteroides fsc fermented juice than from l. pentosus fsc fermented juice. to summarise, our study showed that level of soluble iron after in vitro digestion does not necessary indicate iron absorption, especially in the case of lab fermented food. data on improved iron uptake from l. pentosus fsc fermented juice indicated exiting of promoter(s) for iron absorption in such juice that is not related to the production of organic acids and lowering ph effect. peng zhang, herve vanderschuren, martin stupak, wilhelm gruissem institute of plant sciences, zurich, the tropical root crop cassava (manihot esculenta crantz) is a major source of food for approximately billion people worldwide. in sub-saharan africa, more than million people rely on cassava as their major source of dietary energy. in many parts of africa and latin america, cassava leaves are a vegetable source for daily uptake. cassava is grown mostly by poor farmers under marginal environmental conditions and in areas where few other crops can sustain competitive yields. the crop is therefore fundamental for subsistence farming and food security, but it is also very susceptible to stresses common in the areas and conditions where it grows. in many parts of africa, reliable cassava production is strongly impacted by infections with the african cassava mosaic geminiviruses (cmgs), a rapidly spreading disease that causes large yield losses. in the coastal areas of east africa, cassava production now is threatened by another devastating disease, cassava brown streak disease (cbsd). cassava plants are also frequently attacked by many pests, such as cassava hornworm and stemborers. several reports also indicate that greater leaf longevity, especially under drought conditions, could be important for increasing yields and/or the stability of production in cassava, as well as improve the access to an important nutrient source. conventional breeding efforts have attempted to address the constraint to cassava production, but with limited success. the new tools of biotechnology can change this situation by offering new approaches to the challenges of cassava. these new technologies have the potential to make cassava much more productive, a better source of nutrients, and profitable to grow, hence, greatly contributing on the sustainable development of tropical agriculture. recently we have developed biotech cassava with value-add traits, including resistance to cassava mosaic virus, prolonged leaf life and insect resistance. new strategies are also explored to increase protein content of cassava storage roots. we are currently undertaking pilot studies with two teams of leading scientists and experts for projects to test acmv-resistant transgenic cassava lines in africa and lines with extended leaf retention at ciat, colombia under field conditions. this development of substantially equivalent improved transgenic cassava lines is part of a larger study to analyze the need, effectiveness and biosafety of biotech cassava for agricultural production. the goal of the pilot studies will be the development and coordination of a broader project that produces important and novel scientific results, valuable information on the need and impact of biotechnology at the subsistence farming level, and a sound scientific basis for the development of guidelines for biosafety assessments and release of transgenic organisms into the environment and agricultural production in africa and latin american countries. this study was conducted to reveal the effects of different pretreatments on obtaining haploid plants by using the anther culture in pepper capsicum annum l. cultivars demre sivrisi and sirena. buds were collected at uninucleate microspore stage. anthers collected from buds were cultured in ms medium containing different hormones and hormone concentrations. experiment results revealed that when sirena anthers were pre-treated cold at + • c for h and kept in darkness at • c for a period of week gave good results. in the case of demre sivrisi anthers were pre-treated cold at + • c for h and kept in darkness at • c for a period of week gave good results. on the other hand no cold pretreatment to anthers resulted with low embryo formation. similar results were also observed on the anthers kept at • c for week as callus was produced in some petri dishes but no regeneration was observed. as a conclusion, since no cold pretreatment to anthers resulted with low embryo formation it is possible to say that cold pretreatment should be applied to anthers in pepper another culture studies. the yeast d. hansenii ufv- was tested in this work in batch experiments in synthetic media at constant initial substrate concentration ( g l − ) under variable oxygenation conditions. to get additional information on its fermentative metabolism, a stoichiometric network was proposed on the basis of the general knowledge available in the literature on xylose metabolism in pentose-fermenting yeasts and the specificities of xr and xdh activities in d. hansenii and checked through a bioenergetic study performed using the experimental data of product and substrate concentrations. it can be stressed that under strongly oxygen-limited conditions xylitol production was negligible, whereas under semi-aerobic conditions maximum xylitol production (p max = . g l − ) and yield (y p/s = . g g − ) were obtained. a progressive decrease in these parameters was observed under fully aerobic conditions, suggesting that xylitol-producing yeasts require limited oxygen conditions, which is species-dependent. the proposed model, which utilizes the experimental specific rates of substrate consumption and product formations, allows estimating the main bioenergetic parameters. besides, it proved to be an effective tool to investigate different metabolic situations and showed how they can influence the flux distribution of the carbon source and the bioenergetics of this biosystem. the effect of disinfectants on fungi anne svendsen, pernille skouboe bioneer a/s, hørsholm dk- , denmark prevention of mould spoilage of foods can only be carried out successfully, if the species, which are actually spoiling the food product, are known. a very limited number of fungal species has been associated with the spoilage of each food category. proper disinfection of production facilities is very important to avoid mould spoilage. resistance of moulds to disinfectant treatments are known and different species have shown different response to the same disinfectant. to obtain proper disinfection it is important to know the resistance of the spoilage fungi against different disinfectants. in this study the effect of disinfectants on the spoilage fungi of cheese, rye bread, liver paté and fruit juice was investigated. in collaboration with five food companies the dominating species responsible for spoilage of each food product were isolated and used for testing. commercial disinfectants and disinfectants "under development" were tested. tests were performed in suspension and on surfaces, the methods used were modified after en and en . considerable variability in fungicidal effect among the species was observed. some disinfectants were ineffective at low temperature. some disinfectants showed different effect in suspension and on surfaces, resulting in an effective kill in suspension and almost no effect on surface. the identification of effective disinfectants in the food industry includes: ( ) testing against the specific spoiling species of the food product, ( ) testing on surface, not only in suspension, ( ) test parameters adapted to the food manufacturing plant. intensified research efforts in recent years confirm the major importance of the microbial flora in the gastro-intestinal tract for human health. ingestion of prebiotic oligosaccharides increases the number of the desirable bacteria like bifidobacteria and lactobacilli in the colon. we are looking at beta-galactosidases from lacto-bacillus spp. for the production of galacto-oligosaccharides (gos) because we speculate that the enzymes of probiotics will form gos with high prebiotic potential. in this present study, purified betagalactosidases of selected lactobacillus strains were used for the production of gos from lactose. different enzyme reactor set-ups, both discontinuous and continuous, were tested and compared. temperatures up to • c and ph values between and . were required for satisfactory enzyme stability during the process. enzyme source, substrate concentration and the level of substrate conversion were found to be critical process parameters for gos yields and composition. yields of up to % (w/w) of total sugars were achieved when the initial lactose concentration was g/l. capillary electrophoresis (ce) and hplc with pulsed amperometric detection were the analytical tools for investigating the influence of reactor type, enzyme source and conversion level on gos composition. the prebiotics market is increasing rapidly and is expected to more than double until to about million d world-wide. therefore, the development of enzymatic processes on an industrial scale is a high priority goal of our research. starter addition does not always succeed in improving standardisation and quality of the complex sensory properties of traditional fermented foods. in many cases the added strains do not grow as well as the environmental strains present in the production plant. here, a method of geometric simplification (by dichotomy) of a complex ecosystem found on a raw milk livarot ( strains) was tested on cheese curd. by a limited number of cultures, successively, out of , out of and out of strains were selected on the basis of two criteria (i) respect of the taxonomic proportion, (ii) generation by the daughter ecosystems of an odour close to the one of the mother ecosystem. finally a sub-ecosystem of strains gave an odour similar to the one of the more complex mixture. the use of molecular methods (pcr-sscp) permitted to follow the main species growing. mother and daughter ecosystems were characterized by sensory analysis and gc-ms. probably because of an important redundancy of the strain functions, the method was very efficient. this method may permit to improve a lot the set up of mixture of strains and species used in fermented food industry. effect of the dilution rate on the exopolysaccharide production by bifidobacterium longum atcc c. shene, m. rubilar, s. bravo universidad de la frontera, chemical engineering, av. francisco salazar , casilla -d temuco, chile exopolysaccharides (eps) producing lactic acid bacteria are used in dairy industry (cheese and yogurt) due to the rheological properties that these compounds confer to the products. preliminary results also suggest the use of eps as health-promoting (anti-tumor and immunostimulatory actions) ingredients. bifidobacteria are grampositive bacteria natural inhabitants of the gut of warm-blooded animals and man. a number of investigations have shown that bifidobacteria promote host health mainly because of the reduction proliferation of some pathogenic bacteria through acid synthesis. in this work results obtained in the experiments carried out to test the capability of b. longum atcc to synthesize eps are presented. continuous culture fermentations were carried out at dilution rates between . and . h − . composition of the culture media was that of the mrs broth. biomass concentration presents higher values ( . - . g l − ) at dilution rates between . and . h − . biomass growing at these rates is difficult to pellet and adheres to the fermentor walls behavior that was not observed at other growth conditions. eps from cultures grown at these rates were preparated and fractionated. authors wish to thank the chilean conicyt for the economical assistance given through the project fondecyt . high pressure-low temperature (hplt) inactivation processes were performed on bacillus subtilis vegetative cells at various conditions. at atmospheric pressure, lowering the temperature to as low as − • c was found to have minor anti-microbial effects. upon application of high pressure various phase transitions occurred in the microbial suspensions under study. after pressure treatment at - mpa, cells were plated under optimal conditions to assess cell viability. treatments at - mpa and − • c were the most effective in inactivation. in these cases, ice i-iii solid-solid phase transition was observed. in addition, we hypothesised that intracellular thawing (solid-liquid phase transition) had already occurred while the extracellular surrounding was undergoing solid-solid phase transition. this double effect is suggested to be key in mediating the observed large drop in viability. we speculate that more cells survived after treatment at − • c compared to the same treatment at − • c because both the extra-and intracellular surrounding remained fully frozen. at − • c a solid-solid phase transition was observed when pressure was higher than mpa. a metastable state of ice i was observed at mpa treatment. results from the current study will be presented (see also shen et al., ifset in press) . the data call for a mechanistic evaluation of the effects of hplt as an anti-microbial treatment. such data are currently being gathered and will be used in defining optimal hplt process conditions for the food industry. the influence of saccharomyces cerevisiae, kloeckera apiculata and candida pulcherrima mixed cultures on the selected alcohols formation during model fermentation pawel satora, tadeusz tuszynski department of fermentation technology and technical microbiology, food technology faculty, agricultural university, cracow, poland. e-mail: psatora@ar.krakow.pl (p. satora) for the study five yeast species were chosen, isolated from successive stages of plum fruits spontaneous fermentation: from the beginning (candida pulcherrima, kloeckera apiculata, saccharomyces cerevisiae w ), middle (s. cerevisiae w ) and final fermentation (s. cerevisiae k ). to characterize the potential influence of yeast mixed cultures on the selected alcohols formation, wick-erham synthetic medium ( % glucose) was fermented by mixed cultures of two and three yeast species. after distillation, ethanol, propanol, isobutanol, isoamyl alcohols, hexanol and -phenylethanol were determined using gas chromatography. findings were compared with the results obtained after monoculture fermentations. the use of mixed cultures resulted in increasing of glucose utilization rate, ethanol and fusel alcohols formation (except propanol) and decreasing of methanol synthesis. the samples fermented using two yeast species characterized higher (about %) amount of volatile compounds in relation to monocultures. it takes note of especially high level of ethanol (av. . g/dm ), methanol ( . mg/dm ) and isoamyl alcohols ( . mg/dm ). the positive feature of triple cultures using was limitation of methanol and fusel alcohols synthesis that was accompanied by relatively high ethyl alcohol production (av. . g/dm ). the consumption of sugar syrup becomes increasingly significant in industrial processes due to economic advantages and the easy of use. the production of sucrose syrup using enzymatic hydrolysis represents the safest alternative, once the reaction does not produce any toxic or undesirable substance. this work consists on the production of sugar syrup by immobilized inulinase from kluyveromyces marxianus, with two alternatives process: (a) syrup enriched with fructooligosaccharides or solely with glucose and fructose. the process is comprised by the following stages: production and purification of the enzyme in optimized conditions, immobilization of the enzyme in solid support and the conversion of sucrose in a fixed bed bioreactor with the immobilized enzyme. the final composition of the product can be a mixture of glucose, fructose, sucrose and fructooligosaccharides or a mixture of fructose and glucose, according to the operational conditions. the bioreactor can be operated continually for approximately months with the same biocatalyst. the product from this process is ideal for applications in the food products such as sweet, candies, chocolates, yogurts, etc. besides, the prebiotics properties of the fructooligosaccharides, is a beneficial stimulant of the intestinal flora, which gives to the product a functional property. studies on plant microbial interactions using azotobacter sp. as bio-inoculants towards soil fertility baljeet singh saharan faculty of biotechnology, jcdm college of engineering, sirsa , india. e-mail: baljeet.saharan@gmx.de, baljeet br@yahoo.co.uk (b.s. saharan) high nitrogen fixing, phytohormone producing isolates of azotobacter, azospirillum, acetobacter and pseudomonas were used as inoculants on wheat and cotton with varying doses of nitrogen under field conditions. bio-inoculants were selected on the basis of yield, dry weight and survival rate of bacteria under field conditions. seeds of wheat variety wh were treated with different biofertilizers using nitrogen level of , and kg ha − and one level of p, i.e. kg ha − in field along with control. under field conditions, maximum yield was obtained with azotobacter chroococcum e at ( ± . kg ha − ) as well as kg ha − ( ± . kg ha − ) followed by a. chroococcum ht ( ± . kg ha − ) and avk ( ± . kg ha − ). whereas, with kg ha − highest yield was observed with mac ( ± . kg ha − ) followed by e ( ± . kg ha − ) and avk ( ± . kg ha − ). maximum height at kg ha − was observed with mac inoculation ( . ± . cm) followed by avk ( . ± . cm) and ht ( . ± . cm). various chosen strains were tested with desi (hd ) and american cotton (h ) under similar pot and field conditions as for wheat in the following season. plant height and yield were determined at the time of harvesting whereas survival rate was monitored at various intervals of time. survival rate of inoculated bacteria was determined after , , and days. highest survival rate was observed in mac (( . ± . ) × ), which decreased after and days, respectively. ( . ± . ) × , ( . ± . ) × with mac and ( . ± . ) × and ( . ± . ) × with ht , respectively. maximum boll weight was with avk ( . ± . g boll wt. plant − ) followed by pseudomonas ( . ± . g), ac ( . ± . g) and ala ( . ± . g) boll no. plant − was maximum with ala and avk ( ± . plant − ) followed by pseudomonas ( ± . plant − ). maximum height and dry matter was obtained with pseudomonas ( . ± . cm) and avk ( . ± . cm) with variety hd under field conditions. net saving of % nitrogen was observed using a. chroococcum (e and avk ) bioinoculants for wheat and cotton, respectively. to characterize the antioxidative properties of tempeh-fermented food prepared from vicia faba (l. kontu) with the use of rhizopus oligosporus, the sulfhydryl groups content and surface aromatic hydrophobicity of albumins were investigated. the results obtained for tempeh albumins were compared with raw vicia faba and bovine serum albumin (bsa). these results indicate that tempeh fermentation increased antioxidative activity of albumins. the measurements of antioxidative activity were carried out with the use of , -diphenyl- -picrylhydrazyl (dpph) and , -azinobis-( ethylbenzothiazoline- -sulfonic acid (abts). the albumins of faba bean-tempeh have possessed much higher activity for scavenging free radicals as measured with the dpph and abts ( . % and . %) than raw seeds ( . % and . %) and bsa ( . % and . %), respectively. it has been also found that tempeh fermentation process increased . times sulfhydryl groups content ( . m/mg of albumins) as compared to raw seeds ( . m/mg of albumins). the tempeh albumins have possessed lower surface aromatic hydrophobicity than raw seeds ( . fi and . fi, respectively). orange peel characterization and generation of fermentable sugars solutions for the biotechnological production of food additives b. rivas , j.m. domínguez , p. torre , j.c. parajó : department of chemical engineering, vigo university (campus of ourense), polytechnic building, as lagoas, ourense, spain; department of chemical and process engineering, genoa university, via opera pia , genoa, italy. e-mail: brivas@uvigo.es (b. rivas) the citrus processing industry generates in mediterranean area around millions tonnes of orange peel as byproduct from the extraction of citrus juices in industrial plants. in order to avoid ecological problems and provide an extra profit, this residue was studied in order to generate a suitable substrate for the fermentation process oriented to the production of food additives. orange peels were characterized and the data collected allowed quantifying a % of this waste. soluble sugars ( . %), cellulose ( . %) and pectin ( . %) were identified as more important fractions. this material was submitted to two hydrolysis techniques, prehydrolysis (with diluted sulfuric acid) and autohydrolysis (with water) under different experimental conditions. autohydrolysis was selected as the most appropriate technique for the production of suitable fermentation media. finally, the liquors obtained at • c and liquid:solid ratio of g/g, containing . g/l of sugars, without additional nutrients, were employed to citric acid production by aspergilus niger cect (atcc , nrrl ) . the influence of the addition of calcium carbonate and methanol were studied. under the best conditions an effective conversion of sugars into citric acid was attained, showing the viability of the production of fermentable solutions from this industrial waste. today there is an increasing interest in using high gravity fermentation in brewing. high-gravity fermentation involves production of beer wort of up to • p or even higher and results in beer that has more consistent product quality. the main aim of this study is an increased understanding of how brewer's yeast respond to the various stress factors imposed during high gravity beer fermentation and the consequences these stress factors have on the gene regulation and its consequences on the metabolite levels (both intra-and extracellular). higher attenuation of the wort will be achieved by two different techniques: by the addition of highly fermentable adjuncts such as sucrose or glucose syrups and by mashing with addition of microbial enzymes such as pullulanases and glucoamylases. in the first part of the study model fermentation conditions are established, where the sugar uptake and product formation can be studied in details. characterization of the carbohydrate profile is analyzed by hplc. as flavour changes may occur at higher gravities, it is important to study changes in formation of secondary metabolites, especially esters. transcriptome and metabolome analysis will be used to establish how the stressful conditions prevailing under high gravity fermentations may influence the secondary metabolism in saccharomyces cerevisiae. furthermore, analytical aroma characterization of final beer will be studied by spme and gc-ms. detailed analysis of the effect of different stress factors on the cellular response using dna arrays and metabolite profiling will be carried out. dna arrays will be employed to evaluate if specific metabolic pathways are up-regulated or down-regulated as a consequences of the stress factors. naringin, a bitter compound that occurs in citrus fruit juices, may be converted to a nonbitter form by enzyme hydrolysis. the enzymatic complex naringinase was produced in aspergillus niger cect cultures with naringin as inducer (pérez-mateos et al., ) . crude extracts from a. niger and purified naringinase from penicillium decumbens were immobilized into a polymeric matrix of polyvinyl alcohol (pva) hydrogel cryostructured in liquid nitrogen. the operating stability of the pva-naringinase beads was tested using synthetic citric juice (gray and olson, ) . immobilized enzymes reduced % the naringin content at • c and ph . . furthermore, immobilized preparations from aspergillus and penicillium could be re-used through six cycles ( h) remaining % and % catalytic efficiency, respectively. financial support from "ministerio de ciencia y tecnología" and feder (no. agl - /ali). gray and olson, . j agric. food chem. , - . pérez-mateos, et al., ( ), . otimizing the fermentation broth for tanase production by a new isolated strain paecilomyces variotii vania battestin, gláucia pastore, gabriela macedo department of food science, unicamp, p.o. box , campinas, cep - são paulo, brazil tannase is an inducible enzyme that catalyses the breakdown of ester linkages in hydrolysable tannins, resulting in gallic acid and glucose. the fermentation broth can use by-products as wheat bran, rice or oats, adding tannic acid. the use of by products or residues rich in carbon source for fermentation purposes an alternative to solve pollution problems that can be caused by an incorrect environmental disposal. in the present study we have optimized the production of an extracellular tannase by a new isolated paecilomyces variotii using response surface methodology. the first step was to identify the variables having a significant effect on enzyme production. the variables evaluated were temperature, residues ratio (coffe: wheat bran), concentration of tannic acid, salt solution during , and days of fermentation time. results showed that temperature, residues ratio (coffe: wheat bran) and tannic acid had significant effects on tannase production. commercial wheat bran (cwb) and coffe rusk residues (cr) were used as solid substrate. for fermentation the medium was composed by, cwb:cr were mixed with distilled water and transferred into ml capacity erlenmeyers flasks and auto-claved at • c for min. the medium was then inoculated with spores ( . × ) and the flaks were incubated at • c. tannase was assayed according to the methodology of mondal et al. ( ) . according to the statist analyses, the optimum conditions to produce tannase was the range of temperature ( - • c); tannic acid ( . - %); residues percent (coffe: wheat bran) ( : ) and days fermentation time. the enzyme production increased . times more enzyme production than that was obtained before this optimization. yeast and lactic acid bacteria are two major microbial groups of the most fermented products. a large variety of fermented foods and beverage are made by the activities of both yeast and lactic acid bacteria, simultaneously or successively. during the spontaneous mixed fermentation of lactic acid bacteria and yeast population, it is extremely difficult to control microbial species due to the complexity of the microorganism involved. therefore, we have compared the antimicrobial activity of chitosan against two lactic strains, lactobacillus plantarum and lb. brevis, and yeast strains, saccharomyces cerevisiae to investigate the possible use of non toxic biopolymer chitosan for selective control in mixed culture. the lactobacilli were more sensitive to the inhibitory activity of chitosan than s. cerevisiae. the results suggest the possible use of low molecularweight-chitosan for the control of food fermentation in which both groups of organisms frequently occur together. the effect of vegetable oils on astaxanthin production of phaffia rhodozyma and xanthophyllomyces dendrorhous csaba vágvölgyi, gyöngyi lukács, miklós takó, Árpád csernetics, tamás papp department of microbiology, faculty of sciences, university of szeged, p.o. box , astaxanthin ( , -dihydroxy-␤,␤-carotene- , -dione) is one of the most important carotenoid product. it is used primarily as food colorant and animal feed additive. their effective antioxidant properties linked to a preventive action on various types of cancer and an enhancement of the immune response could lead to expanded commercial applications. among the natural microbial source available, the closely related red pigmented yeasts phaffia rhodozyma and xanthophyllomyces dendrorhous are of great biotechnological interest. these yeasts have desirable properties as biological sources of pigment, including rapid metabolism and producing high cell densities in fermentor, but the commercial production of astaxanthin is limited by the relatively low content in wild-type strains. the purpose of this study was to determine whether the different vegetable oils had an effect on the carotenoid production in p. rhodozyma. effects of media supplemented with corn germ oil, wheat germ oil, sesame-seed oil, palm oil, pumpkin-seed oil, coconut grease, olive oil (extra virgin), olive oil (sanza), sunflower-seed oil and cottonseed oil were tested. studies were performed on both a phaffia and a xanthophyllomyces strain. yeast was grown in yeast-pepton-glucose liquid medium complemented with the appropriate vegetable oil in different concentrations ( . - , v/v, %) . after four days the total carotenoid production was determined spectrophotometrically, and it was referred to dry cell mass. palm oil increased significantly the carotenoid production of the phaffia strain, while a similar effect on the xanthophyllomyces strain could be observed with coconut grease. composition of carotenoid compounds in the strains was determined by thin layer chromatography. lutein is considered a nutraceutic compound that has developed an increasing interest since it is one of the two carotenoids that are located in the macula of the human eye. its consumption is associated with the prevention of age related macular disease (amd). industrially, lutein may be produced using a saponification step of a mixture of lutein diesters that are previously extracted with hexane from natural sources. our proposal is to improve the process by catalyzing the same reaction using microbial lipases during the extraction step with hexane. additionally, the use of supercritical fluids represents an extension of enzymology in non-conventional media with process and environmental advantages. this work was developed using extracts from marigold flower (tagetes erecta) in hexane and supercritical carbon dioxide (sc-co ) where the lutein esters were hydrolyzed by two commercial lipases: lipase b from candida antarctica (novozym ) and lipase from mucor miehei (lipozyme rm m). in particular, we focused our interest in the role of water in the system. interestingly our results show an inverse dependence of the initial reaction rate with respect to the initial water activity (awi) for both lipases, a phenomena that seems to be related to the partition of substrates and products in the solid support)and the hexane phase as a function of water. when sc-co was used as solvent an increase in the consumption rate of lutein diesters occurred, reaching conversions of % in h. for hexane, the same conversion was reached after h. this result suggests a significant effect of the media on the reaction that can be related to shifts in the partition of compounds that bring the substrates in closer contact with the enzyme. this work also demonstrates that lutein hydrolysis seems to be another potential application of commercial immobilized lipases in the food/nutraceutical market. the enzyme of interest in this work is ␤-galactosidase from lactobacillus sp. (ec . . . ). ␤-galactosidases catalyze the hydrolysis and transgalactosylation of ␤-d-galactopyranosides (such as lactose). an attractive biocatalytic application is found in the transgalactosylaction potential of these enzymes which is based on the catalytic mechanism of ␤-galactosidases. the products of transgalactosylation, galacto-oligosaccharides, are non-digestible carbohydrates which meet the criteria of 'prebiotics' and therefore have attracted increasing attention. to produce these 'prebiotic' galactooligosaccharides, an inexpensive and efficient process is desired. immobilization of the enzyme ␤-galactosidase on an insoluble support is an attractive tool to make the process of lactose conversion more economical because the enzyme can be recovered and reused during continuous operation. in this present study, we aimed at immobilizing ␤-galactosidase from lactobacillus sp. by covalent linkages on two solid supports which are commonly used for protein immobilization: chitosan and eupergit c. the protein-binding capacity, the immobilization yield, ph and temperature dependency of activity and stability, and the kinetic parameters of immobilized enzymes were studied. higher activity retention of the immobilized enzymes over a broader ph range and at higher temperatures compared to those of the free enzyme was observed. the immobilized enzymes were evaluated in terms of transgalactosylation activity and stability for a to introduce of foreign genes for the important crop plants such as rice, we need a reproducible efficient procedure for regeneration of the calli through somatic embryogenesis. for this intention, we established the best callus induction medium for tarom mahalli and deilamani cultivars and created the method that the regeneration frequency was reached to %. calli were induced from scutellar tissues of mature seeds on ms medium supplemented with three level of , -d ( , . and mg l − ) and n medium supplemented with five level of , -d ( . , , . , and . mg l − ). for deilamani cultivar the best medium was n with . mg l − , -d and for tarom mahalli the same medium with mg l − were the best. in subculture media, sucrose was used instead of maltose. for regeneration analysis of plantlets, we used two-factorial experiment in base of crd; one factor was regeneration media with six levels (ms medium supplemented with five amount of kinetin and: naa (mg l − ) [( : ), ( : ), ( : . ), ( : ), ( : ), respectively] and . mg l − , -d and mg l − bap). other factor was dehydration process with three levels (without dehydration, dehydration with two layers of filter paper for min [prior to transfer to the regeneration medium], and third factor was factor of with substitution of sucrose with maltose [after weeks; sucrose: maltose]). we conclude that maltose due to changing in osmolarity proceeding can elevate the regeneration frequency to %. therefore, type of carbon source is critical in callus induction and regeneration. márová ivana, hrdličková jana, kubešová jitka, kočí radka, vidláková tereza faculty of chemistry, brno university of technology, purkyňova , brno, carotenoids are the most widespread natural pigments with important biological activities and applications mainly in food and feed industry. at present many ways including genetic engineering are developed to reach higher production of naturally formed carotenoids using microbial producers. in this work cloning and expression of crt gene cluster from pectobacterium carotovorum in recipient bacterial strain e. coli dh ␣ as well as in yeast strain s. cerevisiae was tested. plasmid vector phsg with inserted crt genes was used for transformation of chemically competent e. coli dh ␣ cells, while in s. cerevisiae shuttle vector paur was used. transformants were selected based on resistance to antibiotics, formation of orange-coloured transformant colonies, analysis of recombinant plasmid size and lc/ms analysis of carotenoids produced by recombinant cells. the yield of individual carotenoids (lutein, beta-carotene, lycopene) obtained from various bacterial transformants was several fold higher than in natural producer (lutein: . - . g/g of d.w., beta-carotene: . - . mg/g of d.w.). the highest yield obtained in transformed strain was . g/g of lutein and . g/g of beta-carotene. the yield of biomass and carotenoids in. transgenic s. cerevisiae was comparable to some industrial red yeast strains ( . mg of total carotenoids + mg ergosterol/l; g/l of biomass). so, transgenic yeasts could be suitable for large scale production of carotenoids and/or enriched biomass, while transgenic bacterial producers are perspective above al for high production of rare carotenoids as lutein or lycopene using transformation by specific genes of crt gene cluster. this work was supported by the project msm of the czech ministry of education, youth and sports. two forms of grape seeds, whole and powdered forms, were heated at four different temperatures- , , and • c. after heating, grape seeds were extracted with % ethanol ( . g grape seed/ ml of % ethanol), and total phenol contents (tpc), radical scavenging activity (rsa) and reducing power of the extracts were determined. thermal treatment of grape seed increased the antioxidant activity of extracts. the maximum tpc and rsa of whole grape seed extract (wgse) were achieved when the seeds were heat-treated at • c for min, while that of powdered grape seed extract (pgse) were at • c for min, and were greater than that of the non-treated control. according to the gc-ms analysis, several low-molecular-weight phenolic compounds were newly formed in the wgse heated at • c for min. these results indicated that antioxidant activity of gse was affected by heating conditions (temperature and time) and physical conditions of grape seeds at the time of heat treatments. analysis of the unexpected phenotypic consequences associated with plant transformation jonathan latham, allison wilson, ricarda steinbrecher econexus, , canon frome court, ledbury hr td, uk. e-mail: jrlatham@gn.apc.org (j. latham) transgenic plants often exhibit unexpected phenotypes. such phenotypes could arise from pleiotropic effects associated with the transgene, or they could arise from other sources. a recent econexus report underlined the potential for the process of plant transformation to result in genetic damage to the transformed plant (genome scrambling-myth or reality? transformation-induced mutations in transgenic crop plants: http://www.econexus.info/). the report showed that mutations arising at the site of transgene insertion are often substantial, frequently resulting in loss or rearrangement of chromosomal dna and insertion of multiple superfluous dna fragments. unintended mutations were also documented at other locations in the plant genome. such transformation-induced mutations could provide an explanation for unexpected phenotypes in transgenic plants. we decided to survey regulatory documents and the scientific literature for instances of unexpected phenotypic consequences arising in transgenic plants. this poster documents the preliminary results of our survey. it is intended to assess the range and frequency of unexpected consequences and to examine whether there is sufficient data available to determine their origin. it is our belief that investigating the origin of these unexpected phenotypes should be a principal aim of biosafety research. biotechnological production of metabolites such as carotenoids could be of high interest because of their antioxidative and antimutagenic activities in human body. production of these metabolites by microbial cells is dependent on cultivation conditions. so, presence of exogenous stress in cultivation environment could stimulate biosynthetic pathways of desired metabolites. two non-conventional yeast strains, rhodotorula glutinis and sporidiobolus salmonicolor, were chosen for study of carotenoid production useful in feed industry. hydrogen peroxide, sodium chloride and/or their combinations were used as exogenous stimulators of carotenoid pathway. presence of exogenous stress led to important overproduction of pigments as well as of supplementary studied substances (ergosterol, glycerol). higher adaptability of yeast cells was observed not only in cultivations with one type of stress. combination of stress factors in cultivation media induced significant increase of pigment formation. moreover, under controlled conditions in laboratory fermentor s. salmonicolor produced about eight-fold amount of ␤-carotene ( g/g) in medium with % nacl and mm h o than in control sample. similar result was observed in r. glutinis cultivated in presence of % nacl in inoculation medium only ( g/g of ␤-carotene). the use of stressed biomass of red yeasts in feed industry could have positive effect not only in animal and fish feeds because of high content of physiologically active substances, but it could influence nutritional value and organoleptic properties of final products for human nutrition. this work was supported by the project msm of czech ministry of education. the culture ph significantly affects mycelial growth and morphology, exopolysaccharide (eps) formation, and their molecular properties during submerged cultures of a medicinal mushroom ganoderma lucidum. when the culture ph shifts from to , mycelial growth ( . g/l) and eps production ( . g/l) were favorable compared with other ph-control strategies. the mycelial morphology was also significantly varied upon culture ph: a feather-like pellets were found when the ph was controlled shifting from to at day , which was regarded as undesirable morphological form for eps production. compositional analyses revealed that the ratios and chemical compositions of the eps formed in bottom or top fractions of ethanolic precipitates were significantly different upon culture ph. the molecular characteristics of the eps were further investigated using a size exclusion chromatography/multi-angle laser light scattering (sec/malls) system. plant ␤-n-acetyl-hexosaminidase (hex) (ec . . . ) is reported to have diverse physiological roles like fruit ripening, degradation of reserved glycoproteins in germinating seed and chitin-elicited lignification. in this paper we report the purification and characterization of hex from korean ginseng roots. after extraction with citrate-phosphate buffer, hex was purified to homogeneity using ion exchanger chromatography, hydrophobic interaction chromatography and gel filtration. its molecular weight was determined using gel filtration and mass spectrometer. enzymatic parameters were studied with -methyl-umbelliferyl-n-acetylglucosaminide as substrate. the effect of heat stress and weak organic acids on escherichia coli and a comparison of its recovery by the plate count method and flow cytometry monica s. talsania due to the importance of microbiology for human health, methods have been developed to enumerate viable bacteria. dilution plating is seen to be the 'gold standard' for proof of a cells viability. however, the success of this method relies on post sampling growth, which is limited by our ability to grow cells in the laboratory. additionally, stressed or sub-lethally damaged cells, remain undetected. single cell measurements can provide rapid detailed physiological information, and the assessment of population heterogeneity. this work compared the recovery of stressed e. coli as measured by the number of cfu/ml and by multi-parameter flow cytometric analysis. weak organic acids and high temperature-short time processing (htst) were used to stress the cells both methods commonly used during food preservation. it was shown here that flow cytometry is a powerful tool for the enumeration and detailed analysis of any non-culturable microbial population, which is important because cytotoxic compounds and heat stresses used in food preservation often have a growth inhibiting effect but not necessarily a lethal one. paul g. kovalenko molecular biology & genetics nasu, zabolotnogo str. , kyiv, ukraine the pharmaceutically important plant species of glycyrrhiza sp. (called licorice) is an important commercial product used as a natural sweetener, anti-inflammatory, anti-cancer and anti-diabetic agents. agrobacterium rhizogenes transformation system was used for the hairy root cultures of licorice g. uralensis. after inoculation of aseptic stem segments the ability of hairy root formation was scored for a period of weeks. mean transformation frequency ranged from % (for up to % (for , ). some transformed genotypes showed significant differences in roots weight, flavonoids and glycyrrhizin (gl) production. the cotransformation rate of licorice intact explants cultivars with lba tl-dna and the s gus gene showed an average of more than %. these obtained root cultures were additionally elicited with extracts of biotic elicitor acremonium sp. (endomycorhizal fungus), and were used as an in vitro system to metabolites production. the transformed and elicited hairy roots of g. uralensis were obtained by infection of a. rhizogenes have produced gl at an yield of . % dry weight on the period of culture as a days. according to tentative analyses the hairy roots cultures of glycyrrhiza species produced flavonoids (liquiritigenin and liquiritigen). more high levels ( . g/l) of the total flavonoids production have been identificated on the strains which transformed by lba . this study involved any difference among elicitor treatments and incubation periods for the optimal meabolites production. clearly, the selection of an effective agrobacterium strain for the production of transformed root cultures is highly dependent on the plant species, and must be determined empirically. ayse gul nasircillar akdeniz university, biology, akdeniz univ. faculty of art-science, biological department, antalya, turkey mature embryos of five t. aestivum and five t. durum cultivars formed embryogenic callus on two different media. embryos were removed from surface sterilised seeds and placed with the scutellum upwards on a solid agar medium containing the inorganic components of murashige skoog and mg/l , -dichlorophenoxyacetic acid ( , -d) or mg/l naphthalenacetic acid (naa). the developed calli and regenerated plants were maintained on , -d or naa free ms medium. wheat plants can be regenerated via two different systems. there were significant differences in percentage of callus induction and regeneration capacity on the different initiation medium. among the t. aestivum cultivars, yakar had the highest regeneration capacity in both induction medium. in t. durum cultivars, kiziltan gave the highest regeneration capacity in ms + , -d medium and yilmaz gave the highest regeneration capacity in ms + naa medium. a strong genotypic effect on the culture responses was found for both induction medium. the glycolytic enzyme triosephosphate isomerase (tpi), which catalyses the interconversion of the triosephosphates dihydroxyacetone phosphate (dhap) and glyceraldehyde- -phosphate (gap), was studied for its control on glycolysis and mixed acid production in lactococcus lactis il . we constructed a number of l. lactis strains in which the tpi activity was modulated from % to % of the wild-type level. the enzyme was found to be present in high excess with % tpi activity supporting % of the wildtype glycolytic flux, and with % of the wildtype tpi activity the glycolytic flux was essentially unchanged. measurements of the upstream metabolites glucose- -phosphate (g p), fructose- , bisphosphate (fbp) and dhap were essentially unchanged for tpi activities from % to %, and only in the strain with % tpi activity we observed a significant increase in the intracellular dhap concentration. homolactic product formation was preserved throughout the interval of tpi activity studied, though a small increase in the amount of acetate and formate production was observed in the strain expressing tpi at the lowest level ( % tpi activity). the finding of an increased mixed acid pattern under intracellular conditions with a high dhap concentration is in contrast to earlier data from literature, which indicated that the triosephosphates play an important role in regulation of pyruvate metabolism in l. lactis with a negative effect on the mixed acid flux. we have recently shown that alcohols induce the adhesion of l. monocytogenes at low temperatures, presumably accompanied by enhanced exopolysaccharide (eps) production. however, little is known about the mechanisms involved in the formation of biofilm and eps by l. monocytogenes. in the present project, we show that deletion of selected regulatory and up-regulated genes did not abolish attachment, though the degree of alcohol-induction in some cases was affected. we are applying bioinformatics to search for homologues in l. monocytogenes of known eps genes from various gram positive bacteria. this has revealed candidate genes involved in the synthesis of eps, such as genes encoding glycosyltransferases. moreover, we are at present performing dna microarray analysis for the egde strain grown at • c in the presence of . % isopropanol. this data should, combined with the bioinformatic results, give us a good indication of the genes involved in alcohol-induced surface attachment. repetitive-pcr (rep-pcr) was applied in research on non-starter lactic acid bacteria (nslab) in cheese. we first showed that strains previously differentiated by pulsed field gel electrophoresis (pfge) also could be differentiated by rep-pcr. this was partially due to slight changes in the pcr conditions that allowed reproducible amplification of - kb bands. more than bands were obtained for most strains. a clear differentiation was also obtained between lactobacillus paracasei, lactobacillus plantarum, lactobacillus curvatus and lactobacillus danicus (a new species found in danish and estonian cheeses and traditional starter cultures). we found that this technique is highly reproducible, e.g. identical profiles in three different pcr-machines, two different dna isolation procedures, and different trained personnel. we applied the developed rep-pcr technique to confirm that survivors after heat treatment, were the actual strains introduced and not due to post-pasteurization contamination. we also showed that when we added a cocktail combination of five strains as protecting cultures to cheese, two to three members of this cocktail was dominating the cheese nslab microflora. in control cheeses without the cocktail in most cases other strains dominated, but in a few cases we were able to show cross-contamination between cheese vats. these data indicate that the rep-pcr will be useful to follow development of adjunct cultures as well as provide a reproducible subspecies (e.g. strain) differentiation. rep-pcr is a much quicker and less labour requiring procedure than pfge, and is apparently a much more reproducible technique than what has been seen for rapd. dynamic modeling of lactococcus lactis metabolism and its dynamic behavior for lactate secretion and regulatory characteristics jinwon lee, ui sub jung, hye won lee department of chemical and biomolecular engineering, sogang university, seoul, south korea, - . e-mail: jinwonlee@sogang.ac.kr (j. lee) dynamic metabolic model for lactococcus lactis has been developed in order to analyze a time-dependent behavior of lactate secretion mechanism and probe its regulatory roles. the model was used to compare and analyze the lactate metabolism through in silico simulation and in vitro experimental measurements most of all pyruvate branch point seems to play a major role in producing lactate, and the results of metabolic control coefficient analysis recommend to increase lactate dehydrogenase activity and to decrease nadh oxidase activity. for obtaining more realistic data, we have added some measured flux data including some intermediate metabolites. by combining the simulation results and experimental measurements, we could establish more reliable and robust systematic lactate secretion model. in addition, an efficient parameter estimation method was used to test the exactness of the reported kinetic parameters. what to choose -the fast or the detailed -strategy to get informative profiles of secondary metabolite produced by fungi in culture. chemo-diversity and lead discovery calls for high throughput techniques, but do we need columns will direct infusion esi-ms (dims) do the job. the latter may give matrix effects and lacks resolution resulting in loss of information, while lc-ms analysis takes time and challenge the data processing. results from nano-esi dims and lc-ms analyses of the same extracts important penicillium species are compared. these results illustrate advantages and problems using these techniques for rapid profiling of fungal secondary metabolites, reviling that matrix effects in dims do not seriously hampers detection of important metabolites while the specificity and certainty, for e.g. de-replication is much higher in lc-ms. phenotypic classification of fungi is essential in food biotechnology ulf thrane center for microbial biotechnology, biocentrum-dtu, søltofts plads , technical university of denmark, dk- kgs. lyngby, denmark. e-mail: ut@biocentrum.dtu.dk fungi are of great importance in food and food production. the intended use of fungi as cell factories for production of food ingredients is an upcoming issue in food biotechnology; however, this brings up a possible contamination with mycotoxins as a major issue. a reliable identification of the producer strains is crucial as a correct identification at species level following an updated taxonomy is the key to information on functional characters, e.g. useful metabo-lites and potential mycotoxins, growth conditions, resistance, etc. unfortunately, many mycological reports do not specify the taxonomy used or do not pay sufficient attention to taxonomical systems based on classification by functional characters-in contrast they are using a nucleotide sequence based phylogeny, which conveys little -if anything -about function of the organism. this situation is a major challenge for biotechnologists and mycologists in the years to come and will be highlighted by illustrative examples. the commercial interest in functional foods containing sufficient amounts of living probiotics is paralleled by the increasing scientific attention to the beneficial effect in the digestive tract. a daily intake of viable cells is proposed to ensure probiotic effect on consumer's health. one of the approaches which seems to be feasible to enhance probiotic viability and stability is to improve the fermentation conditions. during batch fermentation the viability of lactobacillus gasseri decreases after reaching a maximal value apparently indicating cell death. in this work, the apparent loss of viability can be avoided during fed-batch fermentation. a three-fold increase in viability is obtained when nutrient concentration was controlled compared with the viability reached in batch cultures. as a consequence, higher biomass concentration and lower specific lactic acid production were obtained. a mathematical model was developed to simulate and describe the effect of nutrient limitation on growth, viability, glucose consumption and lactic acid production. contribution to the metabolic adaptation to food restriction in rabbits (preliminary results) s. van harten , s. borges , p. cravo , l.a. cardoso : instituto de investigação científica tropical, cvz, lisboa, portugal; instituto de higiene e medicina tropical, lisboa, portugal. e-mail: svharten@gmail.com (s. van harten) in order to understand metabolic differences between two breeds of rabbits (halop ab and oryctolagus cuniculus algirus) during food restriction, the activities and expression of key enzymes and hormones of the rabbit were studied. animals from each breed were divided in two groups (ad libitum and restricted), revealing the results a similar difference in glycemic levels between fed and underfed rabbits, with a restriction of % of ad libitum feeding in the wild animals (decrease of % lw) and % of that ingestion in the halop breed (decrease of % lw). the activities of glutamine synthetase and glutaminase show a higher reduction of these enzymes in the wild animals superior to that of the halop breed, compromising, in this way, the ammonium detoxification and the entry of residual carbonated groups of the protein catabolism into the krebs cycle. in the latter animals, a rapid mobilization capacity of triacylglycerols (tga) appears to exist, with a rapid catabolism of fatty acids leading to their oxidation. the wild breeds' results reveal a rise of circulating tga, reflecting difficulties in the lipolysis and mobilization of nefa for oxidation. in these underfed animals, phosphoenolpyruvate and pyruvate suffered a large increase and oxaloacetate a decrease. the halop breed revealed results that indicate a diminution of glycolisis, being glucoses' energy substituted by carbonated chains of lipolysis and protein catabolism. hormone results showed a higher decrease in insulin, t and igf- in the underfed halop animals. in order to confirm the biochemical results, relative quantification of enzyme expression was studied by real time-pcr. since the introduction of genetically modified (gm) crops in , the area under their cultivation has globally increased from . million hectares in to . in . the number of countries adopting gm crops also rose from one country, the usa, in to in . despite numerous successes public opinion still questions the ecological, moral, ethical considerations and issues concerning altering the natural state of the organisms. in this study, a survey of food shoppers' knowledge, attitudes and perceptions of gm foods was carried out in food outlets in nairobi. the food outlets were determined by simple random sampling. using systematic sampling, shoppers were interviewed at targeted imported food products. focus group discussions were also conducted with farmers at city markets. the survey reflected views of a systematic sample of shoppers in seven food outlets between november and december of . it revealed knowledge at %, with positive attitudes and good perceptions towards gm foods (χ = . , d.f. , p < . ). seventy nine percent of shoppers were willing to buy and consume gm foods (χ = . , d.f. , p < . ). cross-tabulation of shopper's position on various issues raised in the survey showed a strong correlation between the respondents' respective knowledge, attitudes, and perceptions (r = . ). nineteen percent of food sampled tested positive for gms. poisson statistics were used to calculate the number of sample sequences. the statistical tools were obtained from spss version . . the results of this study will be of great interest in determining the use and adoption of gm crops in kenya. it will also guide the development of national foreign food policy on gm foods. the technology should be embraced as soon as it is acceptable to alleviate, drought, famine and hunger estimated to be affecting . million kenyans today, mostly children. consumers and gm foods: the case of turkeyÖzlenÖzgen , mustafa yildiz : department of family and consumer sciences, school of home economics, university of ankara, ankara , turkey; department of field crops, faculty of agriculture, university of ankara, ankara, turkey the future development of food biotechnology depends on consumer acceptance. scientists are aware that consumer attitudes will have a crucial impact on the process of the food biotechnology. because, food is one of the central features in human life. consumers' attitudes and trusts in the institutions will determine how gene technology will be used in food sector, in the future. recently, research concerned with consumer aspects of gm foods accelerated. but in turkey, the literature that deals with this subject is very limited and sparse. therefore, this research was carried out on the turkish consumers with the purpose of analyzing the consumers' awareness, assessments about benefits-risks, market place and labelling, and trusts in institutions, towards gm foods. this study was based on interviews with consumers who have recently purchased from major malls, during shopping hours. of the four major malls, voluntary male and female consumers were included in the research if they had main or secondary responsibility for household shopping. the questionnaire form was applied to subjects through face-to-face individual interview. the data were analyzed by using statistical methods according to explanatory variables, including age, gender and educational level. findings indicated that consumers' awareness and views about gm foods were connected to selected demographic characteristics. the results of this study can be important for consumer educators, marketing managers and policy makers. benefit-risk perceptions and moral beliefs of turkish consumers towards transgenic productsÖzlenÖzgen , haluk emiroglu , mustafa yildiz , ayşe sezen taş : department of family and consumer sciences, school of home economics, university of ankara, ankara, turkey; faculty of law, university of bilkent, ankara, turkey; department of field crops, faculty of agriculture, university of ankara, ankara, turkey the use of biotechnology in production has generated considerable debate involving the benefits-risks and moral beliefs associated with its use. consumer acceptance of genetically modified product is a critical factor will affect the future of this technology. this study was planned and conducted to determine the relationship between product/process related benefit perceptions, product/process related risk percentions and moral beliefs of consumers towards transgenic products. a total of university educated consumers, consisting of males and females, employed at the ministries selected by random sampling method in ankara, were included into study. interview techniques were used in the gathering the research materials. the interview instrument had been prepared considering previous research and literature. answers given to sentences typed likert were scored, used "varimax analysis technique" for validity. in order to test the reliability of questionnaire were calculated "cronbach alpha" as inner consistency coefficient. the t-test were performed for determining the differences dependent on gender and age variables between product related benefit perceptions, process related benefit perceptions, product related risk perceptions, process related risk perceptions and moral beliefs of consumers. the examination of relationships between product/process related benefit perceptions, product/process related risk perceptions and moral beliefs of consumers was made by correlation analysis technique. it is thought that the results of this study are important both for scientists and social scientists. the application of the dna recombinant technology for food production is generating a great debate in our society with the participation of scientists trying to explain the way of obtaining these new foods and which are their implications; environmentalist groups and anti-biotechnology associations that systematically are against to the application of this technology; legislatives bodies and the public in general, represented by consumers' organizations that expresses their right to be informed. considering that university students will be the future professionals and consumers their opinion on this topic will be decisive in its success or failure, this research is aimed to performed a global and comparative study of the agrobiotechnology perception by students from different areas of knowledge and studies. this study was carried out during the academic years - , being analyzed a total of valid surveys. the designed questionnaire included questions relatives to: evaluation of the own knowledge and interest on the topic; evaluation of the information sources mainly used by university students to obtain nutritional information; the opinion about gm food labelling; risks/benefits perception; purchasing intention and support of biotechnology. results obtained showed that % of the university students interviewed have a clear positive perception of biotechnology, mainly the students of the health sciences area. these students understand the scientific terminology and they use the university as the main source of information. they support the development of the biotechnology and they consider that in a future it will report them benefits. other group ( %) has a clear negative perception, they are mainly students from law and art history. they do not understand the scientific terminology, they consider that biotechnology will cause them risks, and as a consequence they don't have intention of buying these foods. the technology of the dna recombinant can be also used to introduce in the plants genome the gene that codes a protein of interest for their use as antigen. the application of agrobiotechnology has allowed the development of a new generation of vaccines that try to reduce or to eliminate the inconveniences of the classic ones. for the new vaccines design, the detailed knowledge of the biology of the pathogen is considered. with this knowledge the genes implied in virulence can be inactivated or modified selectively. the term "edible vaccines" it is usually applies to the use of edible parts of the plants (tubers, fruits, leaves, etc.) genetically modified with the purpose to produce specific components (antigens) of a pathogen (virus, bacteria, etc.) against which is wanted to protect a person or animal. however, oral is not the best vaccination route since the quantity of antigen for an efficient immunization it is usually high, being also needed, the co administration of an adjuvant that stimulates the immune answer. on the other hand, it is also important to highlight that the levels of antigen accumulation in transgenic plants is usually lower than the necessary ones. another problem is the irregular accumulation of the antigen in the different parts of the plants, thus difficult the appropriate control of the doses. tannin is polyphenolic component having some antioxidant properties and exists in many plants and fruits. in pomegranate juice this component causes turbidity and haze. during fruit juice clarification by conventional gelatin method, all poly phenolic substances which are responsible for antioxidant activity are removed and as a result the quality of the product is reduced. in the present study tannase enzyme (tannin acyl hydrolase; ec . . . ) was used to decompose tannin to gallic acid and glucose and as the result the amount of turbidity of the juice is decreased, however, the antioxidant properties remain unchanged since tannin is not decomposed and not separated in the juice as it occurs in the gelatin method. the amount of gallic acid in pomegranate juice samples before and after addition of tannase was measured using hplc tests and the optimum temperature, the enzyme and juice contact time, ph, and solvent concentration for clarification of pomegranate juice were obtained as • c, ph = . , h and mm citrate buffer, respectively. the potential benefits of enzymatic clarification of pomegranate juice, that is preservation of antioxidant activity and hence increasing the quality of the fruit product, in comparison to that of conventional clarification method by gelatin introduce a new technique in turbidity and haze removed in tannin containing fruit juices. the objectives of this study are to investigate the inhibitory effect of low molecular weight chitosan for its use as biopreservatives of foods. the inhibitory activity of chitosan against escherichia coli and staphylococcus aureus was investigated by determining the effect of chitosan treatment on bacterial growth during culture and its effect on the viability of non-growing cells. the activity of chitosan was decreased considerably for both strains when sucrose was added to ts broth containing chitosan. the addition of ethanol affected little on the inhibition of chitosan against s. aureus. the influence of nacl on the activity of chitosan was similar to the influence of sucrose and ethanol. however, the experiments with non-growing cells showed that the ethanol enhance drastically the antibacterial activity of chitosan on s. aureus. the results presented in this paper demonstrate that its antibacterial activity may be affected considerably by common food additives such as sucrose, ethanol and nacl. in lactococcus lactis the enzymes phosphofructokinase (pfk), pyruvate kinase (pk) and lactate dehydrogenase (ldh) are uniquely encoded in the las operon. we have applied metabolic control analysis to study the role of this organisation. earlier work showed that ldh at wildtype level has zero control on glycolysis and growth rate but high negative control on formate production (c j formate ldh = − . ). we find that pfk and pk have zero control on glycolysis and growth rate at the wildtype enzyme level but both enzymes exert strong positive control on the glycolytic flux at reduced activities. pk has high positive control on formate (c j formate pk = . − . ) and acetate production (c jacetate pk = . − . ), whereas pfk has no control on these fluxes. decreased expression of the entire las operon resulted in a strong decrease in growth rate and the glycolytic flux. increased las expression resulted in a slight decrease in the glycolytic flux. at the wildtype level the control was close to zero on both glycolysis and the pyruvate branches. the sum of control coefficients for the three enzymes individually was comparable to the control coefficient found for the entire operon at the wildtype level; the strong positive control by pk almost cancels out the negative control by ldh on formate production. the analysis suggests that co-regulation of pfk and pk provides a very efficient way to regulate glycolysis, and co-regulating pk and ldh allows the cells to maintain homolactic fermentation during regulation of glycolysis around wildtype level. bovine chymosin is used extensively in cheese production because of its specificity and low proteolytic activity. we are interested in the caprine chymosin as an alternative because in the canary islands cheese has traditionally been made using goats milk with extract from the abomasum of newborn goats as coagulating factor. we isolated and characterized the prochymosin cdna from the abomasum of milk-fed kid goats. this cdna predicts a polypeptide of amino acid residues, with a signal peptide and a proenzyme region of and amino acids, respectively. the caprine preprochymosin has % and % identity with the corresponding lamb and calf sequences. the cdna fragment encoding prochymosin was fused in frame to the killer toxin signal sequence in a constitutive vector, and to the ␣-factor signal sequence-flag in an inducible expression vector. kluyveromyces lactis pm - c, k. lactis sel , characterized by a "supersecreting" phenotype, and saccharomyces cerevisiae bj were transformed with the recombinant plasmids. activated culture supernatants of yeast transformants showed milk-clotting activity. the flag-prochymosin fusion was purified from bj culture supernatants by affinity chromatography. after activation at acid ph, proteolytic activity assayed toward casein fractions showed that the recombinant caprine chymosin specifically hydrolyzed -casein. the recombinant caprine enzyme could be an alternative milk coagulant in cheese making. lipid accumulation in schizochytrium g / s was studied under batch and continuous culture. different glucose and glutamate source concentrations were supplemented in a defined medium. during batch cultivation, lipid accumulation occurred towards the end of the growth phase but ceased when cell proliferation stopped. under continuous culture, as dilution rate decreased from . to . h − , both cell dry weight and total fatty acid content (tfa) of the cell increased. with a constant dilution rate of . h − , nitrogen limitation induced lipid synthesis ( % tfa) as described for other lipid-accumulating organisms. however, with carbon-limited conditions, some lipid accumulation was still possible, the tfa being %. finally, the batch and continuous culture methods are compared for docosahexaenoic acid ( : , n − ) production. the objectives of this study is to investigate the inhibitory effect of low molecular weight chitosan for its use as biopreservatives of foods. the inhibitory activity of chitosan against e. coli and staphylococcus aureus was investigated by determining the effect of chitosan treatment on bacterial growth during culture and its effect on the viability of non-growing cells. the activity of chitosan was decreased considerably for both strains when sucrose was added to ts broth containing chitosan. the addition of ethanol affected little on the inhibition of chitosan against s. aureus. the influence of nacl on the activity of chitosan was similar to the influence of sucrose and ethanol. however, the experiments with non-growing cells showed that the ethanol enhance drastically the antibacterial activity of chitosan on s. aureus. the results presented in this paper demonstrate that its antibacterial activity may be affected considerably by common food additives such as sucrose, ethanol and nacl. nitrite-oxidizing bacteria catalyze an essential step of nitrogen elimination in biological wastewater treatment. recently, novel and yet uncultured nitrite-oxidizing nitrospira-like bacteria were found to be abundant in municipal and industrial wastewater treatment systems where they outcompete nitrobacter, which has long been considered as the organism responsible for nitrite oxidation in bioreactors. despite the importance of nitrospira-like bacteria for wastewater treatment and for nitrogen fluxes in natural ecosystems, little is known about their ecophysiology and interactions with other organisms. cultivation-independent molecular techniques were applied to investigate the diversity, distribution, and physiological and genetic features of nitrospira-like bacteria in nitrifying activated sludge and biofilm. a surprisingly high diversity of these organisms was found to exist in these engineered and in natural habitats. moreover, significant physiological differences could be identified among various phylogenetic sublineages in the genus nitrospira. quantitative co-localization analyses performed by novel image analysis software revealed that these metabolic features are reflected by the spatial organization of nitrifiers living in biofilm and activated sludge flocs. based on an environmental genomics approach the genome of a nitrospira-like bacterium found in activated sludge is being analyzed. results obtained so far point at unexpected physiological capabilities of this organism, and allow us to propose that nitrospira-like bacteria may also play roles in the bioremediation of (per)chlorate and chlorite. the activated sludge process is the most common way to remove organic matter, nitrogen and phosphorus from wastewater by microbiologically means. knowledge about the microorganisms involved is fundamental for optimisation of existing plants and development of new plants and process designs. many of the bacteria believed to be involved in nitrification, denitrification, biological phosphorusremoval, and removal of organic matter in full scale plants are now identified by use of molecular methods. recent developments in experimental approaches have allowed the study of the ecophysiology of these uncultured and potentially important bacteria, thus providing a better understanding of their function in full-scale activated sludge ecosystems. relatively few dominant species in each functional group (e.g. denitrifiers and polyphosphate accumulating organisms) seems to be present. some species appear to be very specialized regarding nutrient requirements while others are more versatile. a new method for mercury remediation from industrial wastewater based on the enzymatic reduction of mercury by live mercury resistant bacteria immobilized on the pumice particles has been developed in gbf, germany, and implemented in the industrial scale (unknown). the experience gained during operation of this instalation led to the idea, that the process of bioremediation may be integrated in one bioreactor with the sorption of mercury from wastewater, by immobilization of the bacteria directly on the activated carbon. for this it was necessary to define several significant parameters of the activated carbon used and the sorption process itself. the paper presents results of the equilibrium and kinetics investigations of the process of mercury sorption from aqueous solutions onto seven different types of activated carbon. the effective diffusion coefficients in the particles were obtained from the transient-state experiments and the sorption isotherms, saturation capacity of the sorbents and its dependence on the temperature and ph were identified. then the hydrodynamic and sorption characteristics of the activated carbon bed in a laboratory-scale fixed-bed bioreactor were investigated in different process conditions (mercury concentration, volumetric flow rate, temperature, ph). the results (effective capacity of the bed, dispersion and diffusion coefficients, mass transfer coefficient) enable implementation of this bioreactor for modified, integrated process of mercury bioremediation from industrial wastewaters. research supported by the grant kbn t c . bacterial cr(vi) reductases convert the very mobile toxic cr(vi) to the less toxic and less mobile cr(iii). the ability to reduce cr(vi) was studied on cell extracts of ochrobactrum tritici strain bvl and microbacterium sp. strain a. both microorganisms were isolated from the same sample of chromium-contaminated sludge, taken from a wastewater treatment plant. while in the first case activity was found to be associated with the intracellular soluble extract, in the second case it was a process occurring extracellularly. cr(vi) reduction by the intracellular soluble extracts of strain bvl required the presence of nadh or nadph as electron-donor, while the extracellular fraction of strain a only used nadph. several studies were made on strain bvl intracellular soluble extracts. a k m of . m cr(vi) and a v max of . ± . nmol cr(vi) min − mg − protein were estimated from the lineaweaver-burk plot and michaelis-menten non-linear regression. the temperature and the ph optima for cr(vi) reduction were . • c and . , respectively. hyperthermus butylicus is an anaerobic hyperthermophilic crenarchaeon, isolated from the solfataric sea floor off sáo migel island, azores (zillig et al., ) . h. butylicus grows at up to • c (optimally between and • c) at ph . it can utilize peptides, polysaccharides, and other substrates, as carbon sources to produce acetate, butyrate, and n-butanol. the capability to produce enzymes (e.g. hydrolases, dna and rna polymerases, etc.) that can tolerate and function at temperatures • c higher than most other thermophilic archaea, renders h. butylicus of particular interest to the biotechnology industry. the complete genome sequence of h. butylicus was determined and it contains , , bp on a single circular chromosome. protein encoding genes were identified which use a high level of uug and gug start codons. many of these were assigned functions on the basis of sequence comparisons. our analyses revealed some unusual metabolic properties in h. butylicus. several sugar transporters were identified, although the set of genes required for glycolysis is incomplete. moreover, genes encoding enzymes converting glucose to trioses are absent and no genes encoding enzymes of the pentose phosphate cycle or the kdpg pathway were detected. the h. butylicus genome encodes many proteases and peptidases although the lon proteases, encoded in all other archaeal genomes, are absent. although it was reported that h. butylicus does not utilize free amino acids in the media, genes for amino acid transporters were identified, and several proteins involved in di-or oligo-peptide transport are encoded. genes encoding signal peptidases are absent. we will summarize gene products of special biotechnological interest. reference zillig et al., . j. bact. , - . hot genomics: insights in the thermophilic lifestyle of thermus thermophilus from its complete genome holger brüggemann , , anke henne , gerhard gottschalk : göttingen genomics laboratory, institute of microbiology and genetics, university of göttingen, germany; institut pasteur, unité de génomique des microorganismes pathogènes, paris, france thermus thermophilus is an extremely thermophilic, halotolerant bacterium, which was originally isolated from a natural thermal environment. recently completed genome sequences of two strains, hb and hb , provide a solid foundation for investigating many aspects of thermophilic lifestyle; these range from molecular stability determinants to key elements of organismic physiology. in addition, the species has considerable biotechnological potential; many thermostable proteins isolated from members of the genus thermus are indispensable in research and in industrial applications. the closely related genera thermus and deinoccoocus belong to a distinct branch of bacteria called the deinococcus-thermus group. genome comparison of t. thermophilus and d. radiodurans, a mesophilic organism, which exhibits high resistant to radiation, oxidative stress, and desiccation, is of particular interest for the identification and exploration of thermophilic determinants. a large number of orthologs with a high degree of sequence identity are shared between the two species. this opens the opportunity for comparative studies of conformational and chemical thermostability of proteins, as well as for the identification of specific traits for each organism, explaining their unique physiological properties and their intriguing differences in stress tolerance. although strains hb and hb share a highly conserved chromosome, striking differences can be found between their megaplasmids, which encode a huge proportion of genes not found in the genome of d. radiodurans. possible contributions made by the megaplasmids to a thermophilic lifestyle will be discussed. microorganisms that can live in high temperatures, extreme ph and high salt concentration are called extromophiles. extromophilic microorganisms have extended our knowledge and understanding of fundamental questions such as the origin of life. the ability to grow in extreme conditions and to produce stable proteins makes extremopliles very attractive for the researchers and also for the industry. extremozymes from extremophiles have a great economic potential in many industrial processes, including agricultural, chemical and pharmaceutical applications. concurrent development of protein engineering will increase the application of enzymes from extremophiles in industry. turkey has vast and various ecologi-cal areas, and so it has a broad microbial diversity. based on the extremophilles which defined in the scope of this project, halophilic microorganisms produced industrially important proteins were isolated from Ç amaltı saltern area in izmir, turkey. in this work, growth of isolates at different temperature, salinity and ph values were investigated to determine the effects of various growth conditions. eight isolates grow at ph between . and . and two isolates at . - . . they grow at temperature between and • c and salt concentration between % and %. the results of some phenotypic characters showed that they are gram (−) and oxidase,ürease, dnase and nitrate reduction are (−), and catalase (+). they used d(+) glucose, maltose, lactose, sucrose, l(+) arabinose, d(+) mannose, glycerol and four of isolates used d(+) xylose as a carbon source. the isolates resistant to erythromycine, ampicilin sulbactam, cefoxitin, penicillin, bacitracin, novabiocin, amikacin and sensitive to ceftazidine, ciprofloxacin, amoxycillin/clavulanic acid, imipenem, chloromphenicol, ceftazidime/clavulanic acid, aztreonam, cefepime, cefotaxime, cefoperazone amoxicillin. this project was supported by tubitak through project tbag - t . the technology of producing renewable energy sources such as ethanol, methane and hydrogen from biomass holds the potential of creating in-house energy resources while lowering the emission of greenhouse gasses as demanded by the kyoto protocol. recently, goals were defined for the european union determining that . % of the transportation fuel has to come from biofuels in year . a large-scale implementation of biofuels into the transportation sector will demand that lignocellulosic biomass, which is found in a surplus throughout the world is used as the raw material for the production process. the presentation will include a comprehensive description of the special bio-refinery concept developed in denmark for production of biofuels and other valuable products from straw. the concept includes several innovative steps such as a pre-treatment method using wet oxidation, on-site production of enzymes and a continuous fermentation process using a genetic modified thermophilic bacterium. by co-producing several biofuels in the plant optimal use of the biomass has been assured and the price of for instance of bioethanol is getting close to conventional oil-based fuels. optimizing each step in the bio-refinery, while having the full integration in mind, will be the way to make an economical viable biofuel production. in the presentation we will present our road map for achieving this goal in the nearest future. replacement of gasoline by liquid fuels produced from renewable sources is a high-priority goal in many countries worldwide. one such fuel, which has been found well suited, is ethanol. it may be produced from various lignocellulosic materials, such as forest and agricultural residues, which are fairly inexpensive. to compete with gasoline the production cost must be substantially lowered. ethanol production from lignocellulose comprises the following main steps: hydrolysis of hemicellulose, hydrolysis of cellulose, fermentation, separation of lignin, recovery and concentration of ethanol and wastewater handling. the enzymatic hydrolysis and fermentation can either be run separately (shf) or combined into a simultaneous saccharification and fermentation (ssf). the latter has been shown to result in higher ethanol yields than shf. some of the most important factors to reduce the cost are: efficient utilisation of the raw material by high ethanol yields, high productivity, high ethanol concentration in the feed to distillation and process integration in order to reduce capital cost and energy demand. in the last years we have performed several studies on the hydrolysis and fermentation of various forest and agricultural residues in a mini-pilot to improve the overall yield of ethanol and to reduce the energy demand and production cost. steam pretreatment, with small addition of acid catalyst, has resulted in sugar yields close to % of the theoretical for various types of raw materials, e.g. spruce, salix and corn stover. the ssf has been developed and optimized to give high yield of ethanol. for spruce an ethanol yield of about % of theoretical based on the composition of the raw material has so far been obtained using a two-stage steam-pretreatment of so impregnated raw material followed by ssf. improvements of the ssf step, in the form of high dry matter content, recirculation of process streams and adapted yeast have resulted in ethanol concentrations around g/l leading to substantial reduction in energy demand and production cost. these improvements have been assessed by techno-economic evaluation to determine the effect on the ethanol production cost. the process has been further optimised by process integration to further reduce the energy demand. the ethanol production cost was estimated to be around . - . euro/l ethanol assuming a yearly capacity of tonnes raw material (dry matter). production of bioethanol from spent grain, a by-product of beer production sho shindo, tadanori tachibana, akita research institute of food and brewing, akita-city, akita - , japan. e-mail: shindo@arif.pref.akita.jp (s. shindo) the breweries generate one million tons of spent grain every year, and about % of the spent grain is recycled in japan. therefore, it is environmentally and economically significant to consider the production of ethyl alcohol as biomass energy using the spent grain from the breweries industry. ethyl alcohol production from spent grain with immobilized yeast cells was investigated. spent grains were liquefied by a steam explosion treatment to obtain liquefied sugar. when kg of wet spent grain was treated under the kg/cm pressure for min using a l steam explosion reactor, g of total sugar was obtained from the liquefied spent grain. furthermore, . % (w/v) of glucose, . % (w/v) of xylose, and . % (w/v) of arabinose were produced when the liquefied spent grain was treated with glucoamylase, cellulase, and hemicellulase enzymes. ethyl alcohol production was carried out by immobilized sacchromyces cereviseae and immobilized yamadazyma stipitis simultaneously from liquefied spent grain. both yeast cells were immobilized on the glass beads carrier. xylose and arabinose were consumed after glucose was consumed completely during ethyl alcohol production. . % (v/v) ethyl alcohol was produced from liquefied spent grain that was adjusted % of initial sugar concentration after days. the vegetable oils constitute a resource of renewable potential for the production of fuels, becoming a viable alternative when compared to the diesel from petroleum. among the vegetable oils, the extracted oil of the castor plant seeds is a promising alternative source because it is constituted mainly of the ricinoleic acid ( -hydroxy- octadecenoic) that represents % of the total constitution of the oil approximately. the biodiesel obtained from castor oil can be defined chemically as being a mixture of methyl esters or ethyl esters of carboxylic acids synthesized by transesterification reaction of the existent triglyceride and an alcohol of little chain through the use of alkaline or enzymatic catalysts. in this work, we described the results found in castor oil with different degrees of purity. initially, it was made a rheological characterization followed by structural characterization (rmn c, rmn h and infrared) and thermal characterization (dtg, dta and dsc) of the crude and refined castor oil. it has been also measured the hydroxyl tenor, acidity index, saponification index and iodine index in different oils. later, these results were used to evaluate possible differences in the quality of the biodiesel (ethyl esters) produced in the enzymatic alcoholysis of the castor oil catalyzed by lipases (novozym , liposyme rm im and lipozyme tl im). the degree of substitution of castor oil derivative was performed by titration with . n hcl and confirmed by tlc analysis and the results showed conversion rates about %. has been demonstrated to have a wide range of health benefits such as prevention and therapy of various cancers, amelioration of heart disease, and prevention of renal stone formation as well as complications from diabetes. on the hand, lower phosphorylated forms of inositol, especially inositol trisphosphate (ip ) and inositol tetrakisphosphate (ip ) are important signal transduction molecules within the cells both in plants and the animal kingdom. it has been hypothesized that at least the anticancer function of ip is mediated via these lower inositol phosphates. the diversity and practical unavailability of the individual myo-inositol phosphates preclude their investigation. phytases, which catalyze the sequential hydrolysis of phytate, render production of defined myoinositol phosphates in pure form and sufficient quantities. different phytases may result in different positional isomers of myo-inositol phosphates and therefore different biochemical properties. phytases differing in ph optima, substrate specificity, and specificity of hydrolysis have been identified in plants and microorganisms. in this paper the dephosphorylation pathway of the novel phyfauia was compared to other bacterial phytate degrading enzymes. preliminary results have shown that phyfauia converted ip into ip (myoinositol , , , , -pentakisphosphates) and another isomer, which is yet to be elucidated. in a denitrifying pilot plant reactor, a new obligately anaerobic ammonium oxidation (anammox) process with great potential for nitrogen removal for high strength wastewater was discovered. after transfer of the complex microbial community to a laboratory sbr system, a highly enriched population, dominated by a single anaerobic chemolithoautotrophic bacterium related to the planctomycetes was obtained. the bacterium was purified via percoll centrifugation and characterized as 'candidatus brocadia anammoxidans'. survey of different wastewater treatment plants using anammox specific s rrna gene primers and anammox specific oligonucleotide probes revealed the presence of at least four other anammox bacteria, tentatively named 'candidatus kuenenia stuttgartiensis', 'candidatus brocadia fulgida', 'candidatus scalindua wagneri' and 'candidatus scalindua brodae'. a close relative of the two scalindua species, 'candidatus scalindua sorokinii' was found to be responsible for about % of the nitrogen conversion in the anoxic zone of the black sea and in the benguela upwelling system along the namibian coast, making anammox an important player in the global nitrogen cycle. electron microscopic studies of all five anammox bacteria showed that several prokaryotic membrane-bounded compartments are present inside the cytoplasm, which are surrounded by unique ladderane lipids. hydroxylamine oxidoreductase, a key anammox enzyme, was present exclusively inside one of these compartments, named the 'anammoxosome'. unique peptides fragments of the purified hao were used to locate the hao gene in genome assembly of 'candidatus kuenenia stuttgartiensis'. the implementation of the anammox process in the treatment of wastewater with high ammonium concentrations was started at the treatment plant in rotterdam, the netherlands, where it is combined with the partial nitrification process sharon. the estimated price for nitrogen removal with partial nitrification and anammox is about . euro/kg n. gas lift reactors could sustain the highest anammox capacity at . kg n removed/m reactor per day. an alternative configuration of anammox is the oxygen-limited canon process in which aerobic ammonium-oxidizing bacteria protect anammox bacteria from oxygen and produce the necessary nitrite. maximum nitrogen removal with canon in gas lift reactors was . kg n/m reactor per day. using several different conditions and parameters, the competition and co-existence of aerobic and anaerobic ammonium-oxidizing bacteria were modeled. in addition to ammonia, urea was also converted after a -week adaptation in the canon system. recently it was shown that anammox bacteria can use organic acids as additional energy source. murray moo-young, wa anderson department of chemical engineering, university of waterloo, waterloo, ont., canada n l g bioreactors are central to the bioremediation of contaminated environments of water, air or soil. in all three areas of application, bioreactor design is critical to the development of new or improved processes. this overview focuses on the physical limitations of bioreactors caused by biological requirements. the information is based on our own research findings. the need for more applicationsoriented bioremediation research becomes apparent. for technoeconomic reasons, the airlift type has often been the bioreactor of choice for most bioremediations. however, lack of adequate understanding of the quantitative effects of operating conditions on its performance has been an ongoing concern. these effects have been characterized for engineering implementation. to enhance productivity, innovative pretreatment techniques of the polluted sources have also been developed using photocatalytic and chemical oxidation methods. case studies on petrochemical-contaminated water and soil reveal significant enhancement potentials. other studies on microbial biofilters for air bioremediation indicate that the active mass of the biological consortia is not sufficiently understood for rational design. analysis and retrofit design of wastewater treatment facilities using process simulation tools demetri petrides, alexandros koulouris, intelligen, inc., scotch plains, nj , usa. email: dpetrides@intelligen.com (d. petrides) process simulators have been used in the petroleum and chemical industries for over four decades to facilitate the design of new processes and optimize the performance of existing ones. similar benefits can be derived from the use of such tools in the environmental arena, particularly in the field of physical and biological treatment of municipal and industrial wastewater. specifically, process simulators can be used to evaluate and improve options for: ( ) more efficient removal of nutrients (e.g., organic nitrogen and phosphorous) that cause eutrofication, ( ) estimation and control of volatile organic compound (voc) emissions from open tanks, and ( ) more efficient removal and control of hazardous compounds. the potential benefits will be illustrated with cases studies involving both municipal and industrial wastewater facilities. the microbial reduction of metals has showed recent interest as these transformations can play crucial roles in the cycling of both inorganic and organic species in a range of environments and, if harnessed, may offer the basis for a wide range of innovative biotechnological processes. under certain conditions, however, microbial metal reduction can also mobilise toxic metals with potentially calamitous effects on human health. some effluents present heavy metals as soluble compounds, several microorganisms have the capacity to precipitate these metals as insoluble compounds, and this fact allows the collection and separation of these metallic precipitates from contaminated medium. sulfate-reducing bacteria (srb), under anaerobic conditions, oxidize simple organic compounds (such as acetic acid and lactic acid) by utilizing sulfate as an electron acceptor and generate hydrogen sulfide. hydrogen sulfide reacts with heavy metal ions to form insoluble metal sulfides that can be easily separated from a solution. the purpose of this work was study the capacity of desulfovibrio sp. cultures to reduce mixtures of the heavy metals in presence or not of petroleum. for it the experimental design k (k = ) was carried out. the five studied factors were cr, cu, mn, zn and petroleum. the study was carry out with desulfovibrio sp. batch studies were performed in ml sealed bottles with different concentrations (cr(iii)- ppm, cu(ii)- ppm, mn(ii)- ppm, zn(ii)- ppm) of metal sulfate and g l − of petroleum. during batch incubation the dissolved concentration of metal studied in supernatant were decreased to undetectable levels for zn ( - %), however with cu ( - %), mn ( - %) and cr ( - %). the development of continuous process with sulfatereducing bacteria seems to be a suitable alternative to reduce metals in solution from contaminated media such as industrial or mine effluents. after these preliminary results, some experiments in course are focused to study that purpose. reduction of odour emissions from livestock buildings using a bioscrubbing system morten Øgendahl, nawaf abu-khalaf, jens jørgen lønsmann iversen department of biochemistry and molecular biology, university of southern denmark, dk- odense m, denmark. e-mail: tvede@bmb.sdu.dk (m. Øgendahl) a bioscrubbing system for reducing odour emissions from livestock buildings is presented. the bioscrubbing system consists of two separate units; an absorption column and a water purification module. the absorption column is mounted in the ventilations stacks in the livestock buildings absorbing odorants in the effluent air flow. the odorants are absorbed in a spray of droplets formed by a grid of high pressure nozzles in the inlet of the absorption column. the spray of droplets is extracted from the air flow and pumped to a centrally located water purification module, an inverse three phase fluidised bed bioreactor, where the bio-degradation of the absorbed odorants occurs. the bioreactor features a split sparging system for maximum mixing and aeration. the cleaned water is recirculated to the absorption column. an electronic tongue will quantify key odorants in the bioreactor. the absorption column is designed to be retrofitted into existing livestock building ventilation systems. the water purification module is constructed in standard size units simplifying scaling to match the requirements of individual applications. the total bioreactor volume is increased by increasing the number of standard bioreactors. this work describes a "light off" toxicity bioassay sensor based on whole cell genetically modified bioluminescent bacteria. the biosensor was constructed by mating between the environmentally isolated phenol-degrading acinetobacter sp. strain df and the plasmid putk that is an inc p␤ plasmid with the bioluminescence genes luxcdabe inserted into a genetic region involved in plasmid replication and transfer. subsequently, the bioreporter designated df /putk and used to investigate phenolics toxicity. among examined phenolics, pentachlorophenol, catechol and nitrophenol recorded the fastest effect on the bioluminescence of bioreporter df /putk over incubation period of min. the effect of various concentrations of phenol and its derivatives either in an individual, duple or triple mixture forms on the bioluminescence response of the constructed bioreporter df /putk were also examined. significant reduction of the bioluminescence was observed whenever a mixture contained pentachlorophenol, catechol and nitrophenol, respectively. to develop a system appropriate to commercialize, the constructed bioreporter df /putk was subjected for immobilization in microtiter plates using several entrapment gels. after a selection of materials was tried, lb/agar was chosen as the most suitable candidate material. characterization of key odour compounds in an air wet scrubber is presented. the key odour compounds represent five chemical groups, i.e. sulphide, alcohol, volatile fatty acids (vfas), phenol and indole. direct aqueous injection (dai) and solid phase extraction (spe) methods were used before injection of key odorants into the gas chromatography-flame ionisation detection (gc-fid). the dai and spe methods were efficient in the identification of odour compounds in the wet scrubber. the spe method had a high recovery and can be more effective in the identification of compounds at low initial concentration. however, dai showed a better linearity and a lower limit of detection (lod) than the spe method. the dai method was the method of choice for characterization, as it is cheaper, easier to handle and highly applicable. at least two odorants, phenol and -butanol, were quantified successfully using the dai method. their lod was less than their odour detection limit in the wet scrubber. dai method can be used as a reference measurement method for any further analytical application, e.g. electronic tongue. recent developments in biotechnology enabled the widespread use of microbial enzymes in textile, detergent, food and dairy industries and also in various environmental applications. microorganisms which live at extremes of temperature, ph and salinity, produce extremozymes that offer many exciting opportunities for their use in clean production. in this study, microorganisms were isolated from camaltı saltern area inİzmir, turkey. effect of medium salinity on the growth of these microorganisms was determined. seven out of isolates required salt for growth. the salinity ranges at which growth was detected were: - % for two isolates, - % for one isolate, - % for two isolates and - % for one isolate. the isolates were also screened for their capability of producing industrially important enzymes such as amylase, protease, lipase, xylanase and cellulose which are widely used not only in textile, detergent, food and dairy industries but also in various environmental applications. all of the isolates were found to be producers of both amylase and xylanase enzymes at varying salinity array within - % salt concentration range at ph . . extracellular protease activity was detected in the medium of all isolates grown at , , , and % salinity at both ph . (optimum growth ph) and ph . . out of isolates, , and were found to produce cellulase enzyme when the salt concentrations were , and %, respectively. at % salt concentration, only one isolate was found to be cellulase enzyme producer. none of the isolates were found to produce lipase enzyme at - % salt concentration range. this project was supported by tubitak through project tbag - t . chemical engineering department, middle east technical university, ankara , turkey. e-mail: ubakir@metu.edu.tr (u. bakir) glass and ceramic tiles are very widely used industrial materials. in most cases, periodical cleaning is required to maintain their optical properties such as transparency and visual aspects. because of the ever-growing demand for healthy living, there is a keen interest in materials capable of killing harmful microorganisms. the application of these tiles in care facilities to reduce the spread of infections, in public and residental places to improve hygienic conditions are of general interest. in this study the aim is developing methods to apply thin film coatings on glass tiles to make them anti-bacterial by utilizing photocatalysis and investigating their anti-bacterial properties. semiconductors because of their reasonable band gap energies find great attraction through this purpose. the photocatalytic property of semiconductors are used in this process. oxidising radicals are formed on the coated surfaces and these radicals attacks the organic pollutants and bacteria on contact with the surface. titanium dioxide (tio ) coated surfaces are considered to be very effective against organic and inorganic materials, as well as against bacteria. in the experimental procedure coating solution is prepared by sol-gel technique. after pretreatment of surfaces, the coating solution is applied on the surfaces by dip-coating method. after appropriate thermal treatments, to achieve thin, dense and strong coatings, indicator microorganism is directly applied on the coated surfaces and illuminated under solar simulater light source. finally, the number of surviving microorganisms are determined. in this study, the effects of titanium dioxide (tio ), tin oxide (sno ) solutions and metal doping to these coating solutions on anti-bacterial function were investigated. as a result of this study, the number of escherichia coli that is used as indicator microorganism, on tio and sno coated glasses with respect to the control glass reduced by - % and - %, respectively. doping with metals increased the activity of the coatings, hence the number of surviving microorganisms decreased. activity of a methanogenic ecosystem during the primary contact with a solid support s. michaud, n. bernet, p. buffière, j.p. delgenès inra-lbe, avenue des etangs, f- narbonne, france in this paper, the biological activity during the first initial contact between a methanogenic sludge and a solid support was investigated in batch experiments, at different solid concentrations, using two different granular solid materials and with glucose as the main organic substrate. in all cases, the introduction of a solid material in a methanogenic suspended biomass induced a response of the anaerobic microorganisms, after a lag phase during which biological activity was not detected. this lag phase could be the consequence of a physical stress induced by the first contact between microbial cells and the solid surface. this lag phase was not observed when the biomass used originated from a biofilm reactor, i.e. using a biomass previously exposed to a solid material. a change in the metabolism of organic matter from catabolism and methane production toward production of other compounds could be observed, characterised by a sharp decrease of the methane yield in the anaerobic system. analyses of the gas and liquid phases did not show the production of any new gaseous or soluble compound as the biological end product of this activity. this suggests the production of non-soluble compounds by an anabolic pathway, which could indicate the initiation of biofilm formation. this metabolic activity was shown to be directly correlated to the ratio between the solid surface introduced and the microorganism concentration in the anaerobic culture (m g vs − ). from kinetic observations, it could be observed that acetogenic methanogenesis recovered more rapidly than syntrophic propionate and butyrate degradation. evaluating microbial diversity of hydrocarbon degrading bacteria cleantis braithwaite, howard rosser, tawfiq al-ibrahim, hussain, al-bandi research and development center, saudi aramco, dhahran, saudi arabia the analysis of microbial diversity with molecular methods is central to isolating and identifying new and potential biocatalysts resources for research and industry. the ability to degrade hydrocarbon components of petroleum is widespread among bacteria, and is an effective method for remediation of a variety of ecosystems. due to the high carbon content of oil and the low levels of other nutrients essential for microbial growth, treatment of oil with phosphorus and nitrogen is generally required to enhance the growth of hydrocarbon-degrading bacteria and to stimulate oil sludge degradation. in this research study, three types of oily sludges from a gas plant, refinery, and terminal facilities were treated with nutrients. to assess the microbial diversity, both biolog culture method and culture independent polymerase chain reaction (pcr,) denaturing gradient gel electrophoresis (dgge) methods were used. nutrient addition significantly improved oil sludge degradation. we identified and characterized several hydrocarbon degrading bacterial strains that have the ability to convert petroleum. these bacteria included representatives both gram positive and gram-negative genera. there were slight difference in the quantity and type of hydrocarbon degrading bacteria found in the three sites. this is the first molecular analysis of hydrocarbon degrading microbial population in saudi arabian operations. mussel adhesive proteins, including the -plus variants of foot protein type (fp- ), have been suggested as potential environmentally friendly adhesives for use in aqueous conditions and in medicine. here we report the novel production of a recombinant mytilus galloprovincialis foot protein type variant a (mgfp- a) fused with a hexahistidine affinity ligand in escherichia coli, and its ∼ % purification with affinity chromatography. recombinant mgfp- a showed a superior purification yield and better apparent solubility compared to those of the previously reported recombinant m. galloprovincialis foot protein type (mgfp- ). the adsorption abilities and adhesion forces of purified recombinant mgfp- a were compared with those of cell-tak (a commercial mussel extract adhesive) and mgfp- using qcm analysis and modified afm, respectively. these assays showed that the adhesive ability of recombinant mgfp- a was comparable to that of cell-tak but lower than that of recombinant mgfp- . collectively, these results indicate that recombinant mgfp- a may be useful as a commercial bioadhesive or an adhesive ingredient in medical or underwater environments. cresol, a monomethylated phenol, is an aromatic compound. the environmental protection agency (epa) has determined the carcinogenic potential of cresol. various options are being examined for the degradation of cresol because of their unavoidable large scale production and toxicity. many aromatic hydrocarbons can be used as electron donors aerobically by species of pseudomonas, thus leading to the ring cleavage of these compounds. in the present study, pseudomonas strains were isolated from activated sludge collected from sewage treatment plant. it was repeatedly transferred onto nutrient agar plate to check the purity of the culture. the organism was grown aerobically in an inorganic medium with p-cresol as the solitary carbon source. pseudomonas was confirmed by the expression of green pigment, gram staining and biochemical tests including koh, catalase, nitrate reduction and carbohydrate fermentation reaction. inoculation status was used to determine the rate of degradation of p-cresol. the effect of temperature on p-cresol degradation was studied. moreover, the effect of different concentrations of the aromatic compounds on pseudomonas as well as substrate variability was also documented. phenolic intermediates were estimated colorimetrically using -aminoantipyrene, folin-lowry method, uv spectrophotometry and hplc. the results indicated that pseudomonas could degrade up to mg/l of p-cresol within h. pseudomonas sp. exhibited good metabolic versatility and degraded other aromatic compounds including m-cresol and p-hydroxybenzoic acid. we conclude that this strain of pseudomonas has excellent potential for bioaugmenting the degradation of p-cresol-containing waste water treatment units. a considerable amount of waste cooking oil is produced by the restaurant industry worldwide. this poses a significant environmental and economic problem, since high oil and grease concentrations in the sewage system could lead to pipes occlusion and decreased efficiency in water treatment operation plants. therefore, sending these wastes to recycling companies or hazardous waste processors is usually required. yarrowia lipolytica, a well-known lipase producer, requires the presence of lipidic compounds (i.e. vegetable oils) to boost enzyme biosynthesis. in this work, the suitability of waste cooking oil as lipase inducer in submerged cultures of this yeast has been assessed. if successful, this procedure could allow both the degradation of an abundant waste and its valorisation as a raw material for the production of a high added value product. the microorganism was grown in a liquid medium to which various amounts of waste cooking oil were added. biodegradation degrees up to % (measured as decrease in cod) were obtained after days of treatment. also, initial glucose concentration in the basal medium seemed to influence the efficiency of the process. on the other hand, addition of waste oil led to a significant increase in lipase production (more than two-fold), compared to oil-free cultures. moreover, chain-length specificity of the produced enzymes was significantly different: high activity towards medium chain length esters was found, which hinted to the occurrence of both lipases and esterases. biodesulfurization: a documental review j. ferrer, simon bolivar university, environmental engineering lab. caracas, venezuela a documental review about larger interest aspects in biodesulfurization technique is showed. especifically, the investigation is related to general framework and the justification of this technique, degradatives pathways elucidated up to now, involved microorganism, important elements in development of bacterial desulfurization and progress areas, and future tendency. in situ bioremediation of a p-nitrophenol contaminated site and assessment of its community structure debarati paul, gunjan pandey, sumeet labana, rakesh k. jain institute of microbial technology, sector a, chandigarh , india. e-mail: rkj@imtech.res.in (r.k. jain) biodegradation of p-nitrophenol (pnp), a priority pollutant, was studied as a model system for bioremediation of sites contaminated with nitroaromatic/organic compounds. bioremediation studies were carried out in pnp-spiked soil in small plots under natural field conditions using arthrobacter protophormiae rkj . role of carrier material was examined by immobilizing the bacteria on corncob powder prior to adding them to soil. these studies demonstrated successful removal of pnp by immobilized cells that were able to deplete pnp completely in days, whereas free cells were able to deplete % pnp in the same time period. monitoring the fate of released bacteria revealed fairly stable population of the cells when they were immobilized on corncob powder throughout the period of study. on the other hand, there was a decrease of . log units in colony forming units of free cells at the end of the study ( days). bacterial community structure and diversity was also studied for the pesticidecontaminated site wherein the effect of addition of an exogenous strain on the existing soil community structure and on soil functionality was determined using molecular techniques. as revealed by restriction fragment length polymorphism (rflp) studies different phylotypes could be identified on the basis of similar banding patterns. sequencing of representative clones of each phylopyte showed that the community structure of the pesticide-contaminated soil mainly constituted of proteobacteria and actinomycetes. terminal fragment length polymorphism (t-rflp) analysis showed only subtle changes in community structure during the process of bioremediation. bacteriocins encompass an array of structurally different molecules produced by a number of phylogenetically distinct bacterial groups and trigger the killing of the same or closely related species. the recombined escherichia coli strain harboring a bacterocin coding region of xanthomonas campestris pv glycines ra was disrupted to obtain cell homogenate. peptidic xanthomonas bacteriocins (pxb) were separated by lowering ph and adding salt. the resulting pxb's were partially purified using ion exchangers, gel filtration. two final active fractions, a and b, were obtained with a yield of . % and - -fold purification. the activity of pxb was stable at the ph ranging from . to . . andreja kresal, vanja kokol, vera golob textile department, university of maribor, , slovenia wastewater from textile dyeing industries is characterized by high chemical and biological oxygen demands (cod and bod) and intense color due to the extensive use of synthetic dyes. as dyes of complex aromatic structures are resistant to removal by the typical microbial population and may be toxic to the microorganisms present in the treatment plants, discharge of the wastewater to the treatment plants may lead to its failure. beside, direct discharge of these effluents into municipal wastewater plants and/or environment may cause the formation of toxic carcinogenic and/or unhealthy breakdown products. different chemical and physical methods (adsorption, coagulation-flocculation, oxidation, filtration and electrochemical treatments) for color removal have been proposed, but due capital costs and slow operating speed as well as huge amounts of sludge creation there is still a great need to develop an economic and effective method. the use of lignin degrading white-rot fungi and their enzymes (laccase, lignin peroxidase, manganese peroxidase) has attracted increasing scientific attention due their ability to oxidative degrade a wide range of recalcitrant organic compounds. in the contribution, the decolorization efficacy of different commercial textile reactive dyes (anthraquinone, azo, triphenylmethane) will be investigated after the treatment by laccase from trametes versicolor. in order to examine the reuse of enzymatically decolorized liquors, the ecological suitability and the toxicity of the degradation products after different time of enzyme exposure will be studied. this work was carried out within the scope of research project e! cawab. influence of heavy metals on growth and extracellular enzyme production of a trichoderma harzianum strain with biocontrol potential l. hatvani , l. kredics , a. szekeres , z. antal , l. manczinger , a. nagy , c. vágvölgyi : department of microbiology, university of szeged, p.o. box , h- szeged, hungary; hungarian academy of sciences, university of szeged, microbiological research group, hungary; pilze-nagy ltd. kecskemét, p.o. box , hungary. e-mail: kredics@bio.u-szeged.hu (l. kredics) trichoderma species are common soil inhabiting asexual filamentous fungi with teleomorphs belonging to the hypocreales order of the ascomycota division. besides the industrial and clinical importance of the genus, certain strains have been found to cause great losses in mushroom cultivation while other strains are well known to possess high antagonistic activity against several plant pathogenic fungi and therefore used as biocontrol agents. important mechanisms of antagonism include competition and mycoparasitism, which -among others -can be related to the fast growth of trichoderma strains and the production of several extracellular enzymes. the influence of certain, soil-occurring heavy metals on mycelial growth and the secretion of extracellular enzymes involved in competition and mycoparasitism was examined in this study regarding an effective, potential biocontrol isolate of trichoderma harzianum. the metal ions zinc, manganese, copper, iron, lead and mercury were applied at the concentrations of , , , , , and m, and dry mycelial weight as well as the activities of extracellular ß-glycosidase, cellobiohydrolase, trypsinand chymotrypsin-like protease and n-acetyl-glucosaminidase enzymes were determined. it was found that mercury totally blocked mycelial growth, while other metal ions exerted a much lower influence on growth. the presence of heavy metals did not have a significant effect on the activity of the examined extracellular enzymes with the exception of trypsin-like protease, which showed a four-to six-fold rise in activity in the presence of certain sublethal concentrations of copper. based on these results, our further aim is to develop copper-resistant derivatives by mutagenesis from trichoderma strains with biocontrol potential. since proteases play an important role in mycoparasitism, these strains could be applied within the frames of integrated pest management in combination with copper-containing fungicides, resulting in an enhanced level of crop protection even with reduced amounts of fungicides. this work was supported by grants f of the hungarian scientific research fund and grant omfb- / of the hungarian ministry of education. the significance of biocontrol agents (bcas) is that some of them possess good antagonistic abilities against plant pathogenic fungi. a significant number of the most prominent fungi for the purposes of agricultural application belong to the genus trichoderma. in previous studies, in vitro assays on agar plates were reported as the generally used method for the evaluation of antagonistic abilities, as the results of these assays are well transferable to the practical application. the aim of the present study was to develop an accurate, image analysis-based method for the evaluation of the biocontrol characters of bcas. randomly selected trichoderma isolates were tested against fusarium culmorum. in the currently developed method, the areas of the fungal colonies were calculated on petri dishes by measuring the occupied surface of the medium on digital images. the inhibition effect was recorded as the value of biocontrol index (bci), which was calculated from the ratio of the area of the trichoderma colony and the total area occupied by the colonies of trichoderma and the plant pathogen. the proposed method was tested for numerous parameters, and the results revealed that bci proves to be capable for the accurate measurement and scale of the biocontrol abilities of fungal isolates. this work was supported by grants f of the hungarian scientific research fund and grant omfb- / of the hungarian ministry of education. the effect of advanced oxidation processes and recirculation on biodegradation of leachates from aerobic landfills liliana krzystek, anna zieleniewska-jastrzębska, stanisław ledakowicz department of bioprocess engineering, technical university of lodz, - lodz, poland modern landfills are built and operated in a way which allows us to treat them as a special type of bioreactor. simulation of municipal waste biodegradation in lysimeters provides knowledge on basic processes that take place in an aerated landfill. the aim of aeration is to stabilise mainly biodegradable and nitrogen containing components and to reduce methanogenic potential. stabilised leachates from old landfills contain big quantities of refractive carbon compounds that cannot be removed by biological methods. in such case most advantageous is to apply advanced oxidation processes (aops). the objective of this study is an experimental simulation of a landfill aerobic stabilisation and the impact of aops and recirculation of leachate on the reduction of organic load. the performance of the processes was monitored by the reduction in time of basic indices of organic load (bod , cod, toc, vfa, tkn, n-nh + ) and changes in biogas composition. the simulation of aerobic landfill processes was carried out in lysimeters with a fixed bed of household solid waste stabilised during months in anaerobic conditions. leachates taken from the lysimeters were recirculated and subjected to advanced oxidation processes, i.e. ozonation and uv radiation with the addition of h o . experimental studies showed that the aerobic waste stabilisation was a very quick process. during a month the bed was stabilised, reaching a significant reduction of organic load indices. aeration of the lysimeters caused a quick reduction of mainly degradable organic substance (in terms of bod ) and n-nh + and vfa. the reduction of methanogenic potential of the landfill was even faster. the composition of gas at the outlet from the lysimeter changed and after one day already its content was similar to atmospheric air. a more frequent recirculation of leachates enhanced greatly the aerobic biodegradation. it was found that application of advanced oxidation processes (especially ozonation) contributed to a growing reduction of the organic load in the leachates from aerated lysimeters. the application of leachate ozonation resulted in a very high degree of reduction of organic compounds (up to %). the objective of the experimental study was to assess the effect of temperature on the extent of aerobic batch biodegradation of potato stillage with a mixed culture of bacteria of the genus bacillus. the experiments were performed at , , , , , , , , and • c, at ph , in five l l working volume stirred tank reactor (str) (biostat ® b, b. braun biotech international). the duration of the process was h. initial cod of the stillage amounted to . g o /l, the main carbon sources being reducing substances ( . g/l), organic acids (determined as their sum) ( . g/l) and glycerol ( g/l). at • c, no cod reduction or biomass increment was found to occur. at the other investigated temperatures, the reduction in cod measured after suspended solids (ss) separation varied from . % ( • c) to . % ( • c). without ss separation, cod reduction ranged between . % ( • c) and . % ( • c). this indicates that, in terms of the extent of cod reduction, the optimal process temperature was • c and that there was a local optimum at about • c. according to the temperature applied, the content of reducing substances decreased by . - %, that the organic acids by . - . %, and that of glycerol by . - %. the experiments also produced the following two findings: ( ) the rise in temperature brought about a decrease of biomass concentration in the str (measured as ss and bacterial number), and ( ) temperature was a factor affecting the demand for ammonia nitrogen (n-nh ), which was the highest at and • c. the high n-nh demand observed both over the higher and lover ranges of the investigated temperature should be attributed to the release of n-nh and to the large amounts of the biomass produced, respectively. the results obtained imply that the extent of potato stillage biodegradation with a mixed bacterial culture was high over a wide range of the investigated temperature. polychlorinated compounds such as tetrachloroethylene (pce) have become serious environmental pollutants. considerable attention has been paid to these organochlorine compounds. this paper describes the molecular analysis of dechlorinating gene in halorespirating bacterium and efficient bioremediation process. an anaerobic bacterium, that dechlorinates pce to tce, was isolated and identified as a species of the genus desulfitobacterium. a novel pce reductive dehalogenase (prda) gene from the desulfitobacterium sp. strain kbc- was identified. these prd genes, including membrane anchor protein, were classified as a novel type of pce reductive dehalogenase (approximately % homology with the general pce dehalogenase). according to the substrate utility of this strain kbc- and phylogenetic analysis of prda, the type of this microorganism may be expected to play the role of a primary degrader of pce in the environment. high efficient bioremediation process so called the restricted aeration system which means microaerobic/aerobic reciprocal bioremediation process was developed. strong modifications take place, as ammonia production with a subsequent rise of the ph value and a rapid heat evolution leading to temperatures of up to • c. little is known about the microbial community in the toscano cigar fermentation and its development as fermentation proceeds. the aim of this study is to investigate the microbial community composition, its dynamic and its influence on the toscano cigar production process. our results show that the fermentation could be divided into three different phases: initially yeasts are the predominant microorganisms while bacterial growth is partially inhibited; the middle phase is characterized by exponential growth of bacteria while yeasts disappear. in the final phase the microorganism population is mostly represented by sporigen microbial species. the occurrence of yeasts in the first phase could be attributed to their ability to grow at low temperature and low ph levels. the bacterial population flourishes after the yeast cells have reached a stationary phase and probably grows on residual nutrients and autolysing yeast cells. yeasts and bacteria involved in the fermentation process were isolated and characterized. the microbial community was investigated by a combination of phenotypic and molecular approaches. the phenotypic characterization was based on both colony and cell morphology. the isolates were then identified by rrna genes sequence analysis. finally, in order to clarify the role of the identified microorganisms in the production process, a preliminary biochemical characterization was carried out. biosensors have undergone rapid development over the last few years; in particular, in environmental field many biosensors using microorganisms and purified enzymes as biological component, were recently studied. benzene is present everywhere with high levels in the cities and sometimes, in petroleum processing plants. it is classified as carcinogenic compound of first class able to cause leukaemia. because the evaluation of benzene requires complex instruments and quite long analysis times, it is required to study alternative systems for benzene detection simple, fast and highly sensitive, such as biosensors. from pseudomonas putida mst, strain previously isolated in our laboratory and able to degrade benzene, we isolated genes encoding for benzene , -dioxygenase and cis- , -dihydrodiolbenzene dehydrogenase to use in the development of two different hydrocarbon biosensors based on microorganisms and on purified enzymes. the genes isolated were cloned in pvlt and we developed three microbial systems carrying: ( ) benzene dioxygenase, ( ) dihydrodiol dehydrogenase and ( ) benzene dioxygenase-dehydrogenase modified by pcr to obtain enzymes with histidine tag. the cloning was planned to construct recombinant strains able to overproduce the enzymes; the enzymatic activities will be evaluated both using whole cells and purified enzymes. study of operation condition of biofilter using fibril-form matrix for odor gas removal don-hee park, chonnam national university, this research was performed for developing of biological treatment process of odor gas such as mek, h s, and toluene, which is generated from the food waste recycling process. to establish the operational conditions of odor gas removal by small-scale biofiltration equipment, it was continuously operated by using toluene as a treating odor object. when the odor treating microorganisms were adhered to fibril form biofilter, high removal efficiency over % was obtained by biofilm formation. at ppm of inlet odor gas concentration and s of retention time, the removal efficiency was % and % in first stage reactor and second stage reactor, respectively. however, the removal efficiency remained over % at the operational conditions above s of retention time. ozonated water is produced using an ozone generator in a container filled with cold water. it is useful for sanitizing the surfaces of various products for which heat or chemical treatment is inappropriate, such as fresh food products. in this study, we investigated the antimicrobial effects of ozonated water and electrolyzed ozonated water against escherichia coli, s. aureus, bacillus subtilis and yeast, saccharomyces cerevisiae for practical use in sanitizing various products. the results demonstrated that the electrolyzed ozone water was effective for the reduction of microbial population at relatively low concentration of ozone. also, the electrolyzed and the ozonated water showed synergistic antimicrobial effects. many xenobiotics can react spontaneously with thiol moieties of glutathione (gsh), forming gsh-conjugates, or via glutathione s-transferases (gst). these enzymes participate in detoxification of potentially harmful compounds from endo or xenobiotic origin. using saccharomyces cerevisiae as experimental model, we observed that cells mutated in the gtt or gtt genes showed twice as much cadmium absorption than the control strain. we proposed that the formation of the cadmium-glutathione complex is dependent on those transferases, since it was previously demonstrated that the cytoplasmic levels of this complex affect cadmium uptake. the addition of glutathione monoethyl ester (gme), a drug that mimics glutathione (gsh), to gtt ∆ cells restored the levels of metal absorption to those of the control strain. however, with respect to gtt ∆ cells, addition of gme did not alter the capacity of removing cadmium from the medium. taken together, these results suggest that gtt p and gtt p play different roles in the mechanism of cadmium detoxification. by analyzing the toxic effects of this metal, we verified that gtt ∆ and gsh ∆ cells showed, respectively higher and lower tolerance to cadmium stress than control cells, suggesting that although gsh plays a relevant role in cell protection, formation of the gsh-cd + conjugate is deleterious to the mechanism of defense. furthermore, analyzing the harmful effects of other xenobiotic, menadione ( -methyl- , -napthoquinone), we have also observed that gtt p and gtt p isoforms play distinct functions in the process of cell protection as well as in drug remove, since both strains showed lethal phenotypes after direct exposure to mm menadione. however, after adaptive treatments (mild-heat or exposure to a lower menadione concentration), cells acquired tolerance to menadione stress, although the gtt ∆ mutant had still shown a higher sensitivity against drug toxicity. by analyzing the malondialdehyde (mda) produced in response to menadione, we observed that gtt ∆ cells exhibited increased levels of lipid peroxidation, indicating that, during menadione exposure, gsh-conjugates are formed by the same transferase isoform, gtt p, involved in cadmium stress. financial support: stint (sweden), cnpq and faperj (brazil). polycyclic aromatic hydrocarbons (pahs) are ubiquitous and persistent throughout the environment. they are generally distributed from both natural and industrial sources. many pahs can have a detrimental effect on the flora and fauna of affected habitats through uptake and accumulation in food chains, and in some instances, they induce serious health problems and/or genetic defects in humans. many research efforts have been expended to find a suitable method for remediation of soil and water environments contaminated with pahs. amongst them, the use of ligninolytic fungi is particularly suitable for the development of such processes, since they produce extracellular lignin-degrading enzymes (mnp, lip, laccase, . . .) which degrade a wide range of organic pollutants. coriolopsis rigida has been reported to produce extracellular laccase as the sole ligninolytic enzyme. this makes this fungus particularly suitable for the study of xenobiotics degradation by laccase. the purpose of this research was to obtain high laccase activities by c. rigida in solid state cultures and to determine their ability to degrade anthracene (typical pah). both in vivo and in vitro assays were performed. the former led to - % degradation in days depending on the culture conditions, whereas the latter showed a degradation percentage above % in days when low mediator concentration (hbt) was added to the reaction mixture. focus will be given on pressure-driven membrane bioreactors, gastransfer membrane bioreactors and the novel ion exchange membrane bioreactor (iemb). the latter concept has been developed and currently studied by our group. this process, based on integration of donnan dialysis with bioconversion of one or more target pollutants to harmless products, has been modeled and experimentally verified for the removal of various charged inorganic pollutants such as nitrate, perchlorate and bromate by mixed microbial cultures under anoxic conditions. tests of up to months showed a very good operational stability. the essential role of the microbial membraneattached biofilm, which develops naturally in this type of systems, will be also demonstrated and discussed. poly(lactic acid) (pla), which is one of biodegradable plastics, is depolymerized by hydrolysis and releases soluble monomer or oligomer of lactic acid. many bacteria can use the monomer and oligomer as an energy source or a carbon source. in this study, we applied pla to an electron donor for denitrification process of the previously developed bioreactor, which could remove ammonia from wastewater by simultaneously carrying out two biological processes, aerobic nitrification and anaerobic denitrification. a bench-scale bioreactor was constructed with a gel-plate containing pure-cultured cells of nitrosomonas europaea and paracoccus denitrificans and a pla-plate. the pla-plate was prepared by mixing three kinds of plas with different molecular weight and tricalcium phosphate to keep the constant release of the electron donor for a long term. batch treatment experiment with the bioreactor was repeated with an artificial wastewater containing ammonia for days. the bioreactor could remove nitrogen from the artificial wastewater at nitrogenremoval rate of approximately g n/day per square meter of gel-plate surface during the experiment period without an additional electron donor. the performance was equivalent to that obtained with our bioreactor using ethanol as electron donor for denitrification. the bioreactor using pla dose not need an additional pump for serving an electron donor (e.g., ethanol) and a hollow space for serving. therefore, the concept using solid electron donors like a pla would be effective our bioreactor to compact and simplify, and would be possible to develop a portable or disposable bioreactor. leucosporidium antarcticum as a source of enzymes for biotechnology arkadiusz wojtasik , , marianna turkiewicz , jaroslaw dziadek , pawel parniewski : centre for medical biology pas, lodowa street, - lodz, poland; faculty of biotechnology and food sciences technical university of lodz, stefanowskiego / street, - lodz, poland. e-mail: awojtasik@cbm.pan.pl (a. wojtasik) leucosporidium antarcticum is a psychrophilic yeast able to growth at low temperature. these microorganisms live in antarctic marine waters and are endemic to that cold environment. furthermore, l. antarcticum is also isolated from the digestive tract of antarctic krill euphausia superba. enzymes isolated from coldadapted microorganisms such as l. antarcticum having a specific activity at low temperatures ranging from to • c are considered for utilization at biotechnological applications such as bioremediation, production of polyunsaturated fatty acids of dietary significance and might be a source of industrially useful enzymatic proteins. the main goal of this study was to construct a cdna library of l. antarcticum. the partial cdna library was obtained and some of the clones were analysed. the sequencing analyses allowed us to find an approximately base pair nucleotide sequence which displayed a very high homology to disulfide bond chaperone belonging to the hsp family from psychrobacter sp. high similarity of that heat shock protein was found on an amino acid sequence level and was reaching nearly %. the main object of our further research is to clone hsp family protein gene and to obtain its expression in a mezophilic host strain. also, further clones will be analysed to find other interesting genes encoding the psychrophilic proteins. this work was partially funded by the kbn grant i / / . out of plant species found in the flora of turkey, about are endemic. beautiful flowering (geophytes) bulbous plants form an important part of this rich biodiversity. besides use as ornamental plants, these have great potential in perfume and pharmaceutical industry. genera of fritillaria, ornithogalum, muscari, bellevalia, tulipa, galanthus, sternbergia, crocus, arum and biarum have important and critically endangered species with high export potential that enters into this group. most of these are endangered and their collection from wild and export has been banned to conserve them. large scale production and conservation of these species could also be achieved by in vitro techniques. therefore bulb scale and immature embryo explants of sternbergia candida, s. fischeriana, muscari muscarimi, fritillaria imperialis and f. persica were cultured on different nutrient media supplemented with various concentrations of plant growth regulators using different culture applications. large numbers of bulblets were produced (over bulblets/explants) from single immature embryos on nutrient media in most species tested after months of culture initiation. regenerated bulblets were kept at • c for weeks and then transplanted to soil successfully. to our knowledge the present study is the first report for in vitro bulblet production from immature embryos of geophytes. the procedure described here provides a prolific bulblet production system that may form the basis of bioreactor culture and conservation of endemic and endangered geophytes. the commercial use of organofluorine compounds in industrial, pharmaceutical and pest-control applications has dramatically increased over the past few years, resulting in the introduction of numerous new organic compounds into the environment. organofluorine compounds are chemically very stable and are assumed to be resistant to biological degradation. given the chemical inertness of fluorinated organics, their bioactivity, and their potential for accumulation in the environment, it is important to understand their environmental fate and the mechanisms by which they might be degraded. examples of the biodegradation of fluorinated compounds in literature are scarce, being fluorobenzoic acids the most commonly reported. information on the cleavage of carbon-fluoride bonds in synthetic compounds is limited to fluoroacetate dehydrogenase. in this project we try to obtain more insight in the defluorination mechanisms by investigating the diversity of degradation routes for these compounds in several soil bacteria by making use of modern genetic tools. a gram-positive strain capable of aerobic biodegradation of -fluorophenol ( -fp) as the sole source of carbon and energy was isolated by selective enrichment from soil samples collected near an industrial site. batch cultures were set up and substrate consumption, accumulation of intermediates and product formation were monitored. the consortium was able to use -fp up to concentrations of . mg l − and was able to utilize a range of other organic compounds. stoichiometric release of fluoride ions was measured in batch cultures suggesting that there is no formation of dead-end products during -fp metabolism. biobleaching of kraft cellulose pulp by poliporus versicolor aysun ergene , nazif kolankaya : kırıkkale university, faculty of science and literature, department of biology, yahsihan, kırıkkale, turkey; hacettepe university, faculty of science, department of biology, beytepe, ankara, turkey the suitability of culture supernatant from poliporus versicolor for use in the biobleaching of kraft cellulose pulp was investigated. p. versicolor was found to grow on mycological broth ( % soytone, % d-glucose and . % cellulose pulp). maximal extracellular ligninase production was detected after days ( nkat). the optimum biobleaching conditions are • c and ph . , with days. in this condition p. versicolor decreased the kapa number from . to . and increased brightness from to . in -day treatment. boron and iron are among the microelements required for the proper development of the vegetative and generative tissues of plants. though iron is present in high amounts in almost all soil types, its bioavailability to crops is extremely reduced, hence most of the plants face an iron defficiency problem and while on one side crop productions effected, on the other hand the nutrition problems come up to human through contagious nutritional chains. boron is also among the most problematic micronutrients of the major crop plan-tation areas of turkey. both defficiency and toxicity problems exist in a total of about % of the central anatolian soil where pea is among the legumes cultivated. application of varying levels of boron and iron combinations in greenhouse and the analysis of plant acquisition via icp-aes as well as determination of the effects of the element combinations both in morphological and molecular levels are the aim of our studies. the genetic bases of the response differences of plant genotypes to b and/or fe, were investigated through the applications of molecular marker techniques. considerable growth rate and stem size differences were detected within the parents (wild-type versus cultivar) and the f plants. the presence of efficient genotypes to high micronutrient levels are expected to help us increase the cultivation of the crop in problematic areas as well as in exploring the molecular bases of the microelement uptake mechanisms. butachlor is one of the selective systemic herbicide toxins that are act by inhibition of protein synthesis. this toxin is used exclusively in the rice, barely, cotton and wheat farmlands. butachlor is belonging to chloroacetanilide herbicide group, which are consisting of butachlor, alachlor, acetochlor, metolachlor and poropachlor. in the view of bioenvironmental, butachlor is degraded in the soil by microbial activity. its stability is about - weeks. it is converted to the water-soluble derivatives in soil or water, with a slow evolution of carbon dioxide. because of butachlor is one of the herbicide toxin, it is inhibitor factor against growth of bacteria and microorganisms. microorganisms can be continuing their activities in the limited concentration of butachlor. therefore treatment of industrial wastewater consist of concentrated butachlor by the biological treatment is impossible and it is necessary chemical or physico-chemical treatment are used. in this research, biological treatment methods are used. in the biological treatment, an activated sludge system with volume of . l is used. in this method, butachlor with concentration of mg/l are treated. removal percent of butachlor for concentration of . and mg/l are calculated to . % and . %, respectively. removal percent of cod is also calculated to %. olive oil mill wastewater (omw) as the effluent of the concern of olive industry has high organic load. the conventional biological treatments despite of their simplicity and rather suitable performance are ineffective for the omw treatment since phenolics possess antimicrobial activity. in order to carry out a proper treatment on omw, use of microorganism able to degrade the phenolics thus, is necessary. the ability of phanerochaete chrysosporium immobilized purification and downstream process of xylitol obtained biotechnologically from hemicellulosic hydrolyzate of corncobs b. rivas , p. torre , j.m. domínguez , j.c. parajó , a. converti : department of chemical engineering, vigo university (campus of ourense), polytechnic building, as lagoas, ourense, spain; department of chemical and process engineering, genoa university, via opera pia , genoa, italy. e-mail: brivas@uvigo.es (b. rivas) biotechnological production of xylitol from lignocellulosic materials has been widely studied in the recent years with promising results that confirming the possible industrial application of this technology. xylitol purification from fermented broth is the limiting stage of this process. previous works suggest crystallization procedures in order to recovery xylitol from fermented synthetic solutions. the complexity of fermented hydrolyzate not allows direct crystallization. in this work, corncobs hydrolyzate obtained with autohydrolysis-posthydrolysis techniques, detoxificated with activated charcoal and concentrated was fermented to xylitol by d. hansenii. the fermented broth composition was . % of xylitol ( g/l), . % of other sugars and . % of other compounds that interferes in the crystallization process (as dry matter of the liquor). the fermented media was submitted to an absorption process with activated charcoal and concentrated until a xylitol concentration of g/l. the liquor was then submitted to a second step of precipitation with ethanol, the best results achieved in this study were obtained with an ethanol/liquor ratio of . in these conditions this treatment allows to remove a . % of the impurities. the resulting solution was evaporated and crystallized containing % of ethanol and a xylitol concentration of g/l. crystallization was performed at t = • c with slightly agitation. after h were separated xylitol crystals with a recovery yield of % and a purity degree of %. numerous publications have documented that only a minor number of the indigenous prokaryotic organisms found in complex environments such as the human intestine, biogas reactors, and soil are known, and probably only a fraction of this diversity can be accessed using traditional culturing techniques. some of the reasons for this are the lack of knowledge of specific growth conditions, specific nutrients, and obligate coculture requirements. also growth on a solid surface directly exposed to the atmosphere puts a very strong selective pressure on single cells supposed to develop into visible colonies. therefore, the knowledge of these microorganisms is scarce and generally limited to the s rrna genes that have been extracted from different environments and cloned for phylogenetic analyses. an obvious approach to circumvent these problems was the development of techniques based upon micromanipulation for isolation of single cells from complex mixtures. continuous development of modern microscopes in combination with the precision of a servo-powered micromanipulator and the development of the modern microscopic micro injectors used in ivf techniques has further aided the manipulation of single cells. this technique, however, does not solve the problems of the non-culturable cells, and other approaches are needed to gain more information about these organisms. an approach to the non-culturable cells could be genomic analysis of isolated single cells without preceding cultivation. this pcr-based technique is widely used for genetic analysis of human cells, but due to the small amounts of dna present in prokaryotic cells it has so far not been possible to produce identifiable amounts of dna from single cell amplification using conventional polymerases. a promising alternative used for amplification of small amounts of dna is the f dna polymerase operating under isothermic conditions. applying random hexamer primers, this polymerase carries out a multiple displacement amplification (mda) of high molecular weight dna template. in this study we demonstrate the successful application of mda for selective amplification of genomic dna from a single prokaryotic cell. the yield was > mg of amplified genomic dna corresponding to about a billion-fold amplification from a single cell. the technique was used to approach a large group of non-thermophilic archaea found in agricultural soil. our results show that combining mda with fluorescent in situ hybridization and cell isolation by capillary micromanipulation enables an unprecedented ability to investigate new species without cultivation. also this combination of techniques opens for studies of genetic heterogeneity within populations and processes such as horizontal gene transfer. precipitation of zn + , cu + and pb + at bench scale using biogenic hydrogen sulphide produced from the utilization of volatile fatty acids by sulphate reducing bacteria maria teresa alvarez , , carla crespo , bo mattiasson : department of biotechnology, center for chemistry and chemical engineering, lund university, p.o. box , s- lund, sweden; instituto de investigaciones fármaco bioquímicas, universidad mayor de san andrés, la paz, bolivia biological production of hydrogen sulphide (h s) from sulphate using sulphate reducing bacteria (srb) is popular within environmental biotechnology. srb require absence of oxygen, presence of nutrients required for growth and oxidizable organic substrates (to supply hydrogen atoms for reduction of sulphate). many organic wastes have been used as electron donors for the sulphate-reducers in the treatment of acid mine drainage (amd) including straw, hay, sawdust, peat, spent mushroom compost and whey, however, other wastes such as municipal organic waste can be used. the aim of this work was to study the possibility of using srb for the treatment of amd at bench-scale. this process involved three stages: the volatile fatty acid (vfa) production by hydrolytic bacteria from the degradation of vegetables and fruits, the production of h s through the utilization of the produced vfas by sulphate reducing bacteria and the precipitation of metals by using the biologically produced h s. the substrates used for vfa production consisted of tomato, papaya, apple and banana. the h s produced from the degradation of vfas was utilised for the precipitation of an artificial effluent simulating the heavy metal concentrations of a mine located at bolivian andean region, containing approximately mg/l of zn + , mg/l of cu + and mg/l of pb + . the maximum concentration of hydrogen sulphide obtained was approximately mm. removal efficiencies of %, % and % for zinc, cooper, and lead, respectively, were achieved in the present work. at • c during days. bacterial population was determined by counting in a neubauer chamber with optical microscope. sulfate concentration was measured by turbidity method and metal concentrations in the filtered supernatant were measured by icp-aes. the first part of study consists of determine the maximum concentration of each metal at which d. vulgaris and desulfovibrio sp. grow in similar way than control culture (without metal). both cultures tolerate: cr(iii) ppm, ni(ii) . ppm, zn(ii) ppm. the maximum precipitation percentages were approximately: % ( ppm cr(iii)), % ( . ppm ni(ii)) and % (for d. vulgaris- ppm zn(ii) and desulfovibrio sp.- ppm de zn(ii)). time to reach the highest precipitation was minor for mixed culture (desulfovibrio sp.) y all the cases. the next part was focused to study the precipitation percentage when metals are present in combination in the same metal levels (cr(iii)-ni(ii), cr(iii)-zn(ii), ni(ii)-zn(ii) and cr(iii)-ni(ii)-zn(ii)). the combination of metals does not affect significantly the bacterial growth and precipitation percentage of metals. this fact supposes an importance advantage so metals are commonly found together in the environment. future experiments are focused in development of this process in continuous operation mode. biosolubilisation and depolymerisation of coal has potential to produce a clean energy source or high value organic products from low rank coals such as lignite or sub-bituminous coal. these complex soluble phenolic compounds are of value as starting materials for biotransformation to value-added compounds such as antioxidants and flavourants. the bioprocess is carried out at ambient temperature and pressure and is perceived to be environmental benign. in the evaluation of coal solubilisation an important quantity for the assessment of process feasibility is the yield, i.e. the determination of the mass of product obtained per unit mass of coal solubilised. to date, results for coal biosolubilisation reported in the literature are qualitative or at best semi-quantitative, indicating trends with operating variables. the process kinetics has not been determined rigorously because measurement of fungal growth during coal solubilisation is hindered by the presence of the solid coal substrate. knowledge of the profile of biomass growth is required for the rigorous determination of the kinetic parameters necessary for process design and optimisation. in this paper, the use of an indirect method for the estimation of the growth and metabolism of fungal biomass by measuring co evolution and o consumption using an off-gas analyser is reported in the study of fungal coal solubilisation. coal determined rigorously because measurement of fungal growth during coal solubilisation is hindered by the presence of the solid coal substrate. knowledge of the profile of biomass growth is required for the rigorous determination of the kinetic parameters necessary for process design and optimisation. biosolubilisation was carried out in a stirred tank slurry bioreactor with working volume of . l. complete suspension of the coal particles of - m mean diameter was achieved at an agitation rate of rpm. growth yield coefficients based on coal and oxygen as well as maintenance coefficients were calculated from growth of the fungus under the same conditions using a non-coal carbon source such as glucose. these data were used to determine the stoichiometric coefficients for biomass growth, enabling the biomass production rate to be quantified in terms of co production rate and o consumption rate. a dna-chip platform for parallel detection of microorganisms related to biofilm in industrial systems and drinking water systems pernille skouboe, dorte lauritsen, kim holmstrøm bioneer a/s, kogle allé , dk- hørsholm, denmark. e-mail: psk@bioneer.dk (p. skouboe) an oligonucleotide microarray for simultaneous detection and identification of pathogenic bacteria related to technical water systems as well as drinking water has been developed. the approach is based on the use of a tandem hybridization technique with two ribosomal s rdna-pcr products, bp and bp long, generated from two consensus pcr reactions using conserved ribosomal primers end-labeled with cy and cy , respectively. the tandem hybridization technique implies an internally quality control for discrimination between target and non-target signals. the current prototype of the dna-chip platform includes oligonucleotide probes representing different genera (and subgroups of species), e.g. legionella, mycobacterium, aeromonas, campylobacter, vibrio and enterococcus. the platform has been used for detection and identification of species from pure cultures, and initial experiments with water samples from industrial systems have been performed. the potential as well as the limitations of using a dna-chip based detection format in its present form will be documented. particularly, its potential application as a rapid method for initial screening of environmental or food samples will be addresses. the aim is to reduce and optimize the number of samples required for traditional microbiological identification tests. in the course of a project for the development of a novel kind of a mycotoxin inactivating feed additive, the aim of this study was to isolate and characterize microorganisms with the specific ability to enzymatically break down and detoxify fumonisins, a group of structurally related fungal toxins, with fumonisin b (fb ) being the most abundant and -with respect to toxicology -also the most important representative of this group. these toxins are produced as secondary metabolites by some fusarium species such as fusarium verticillioides and f. proliferatum and are naturally occurring contaminants of cereal grains worldwide. they are found especially in maize and maize based products, and are known to be hazardous to human as well as to animal health. a natural feed additive, based on microorganisms and/or enzymes, should ensure the detoxification of fumonisins during feed uptake and digestion via microbial or enzymatic break down of these compounds, by that protecting the animal from the harmful effects of these mycotoxins. besides an extensive screening of microbial strains derived from strain collections, various different natural habitats were investigated for the presence of fb degrading microbial activity, such as intestinal contents of pigs, soil samples, and naturally fumonisin contaminated maize. while testing of nearly organisms from strain collections did not show positive results, fumonisin transforming activity could be detected in one soil sample and a number of maize samples. trials in order to isolate the respective fumonisin degrading microorganisms resulted in a number of strains, whose fb degrading activity could be proven. the most promising bacterial and yeast strains were further characterized with regard to a general taxonomic description, and to different aspects of their toxin degradation behaviour. approaching a more relevant in vivo situation, fb degradation trials in food-and feed-stuffs were conducted. further on, the applicability of the respective organisms as stabilized lyophilisates was investigated. arsenic is one of the most important global environmental pollutants and the toxicological effects are related to its chemical form and oxidation state. arsenite [as(iii)] is reported to be on average times more toxic than arsenate[as(iv)]. this work shows the ability of one strain of the species ochrobactrum tritici to grow in presence of several metals including arsenite, arsenate, selenite, selenate, tellurite and antimonite. its arsenite mic was determined as mm, whereas for arsenate, this bacterium could resist to concentrations upper than mm. we report the identification of two loci involved in high-level arsenic resistance. sequencing of the first locus identified four complete genes in the following order: arsr, arsd, arsa, arsb. the second locus containing genes for arsenic resistance was also characterized. each sequence has been compared with nucleotide and protein databank (blast programs) and significant homology with known orfs coding for arsenic resistance has been found. it is also possible that the phenomenon of high-level arsenic resistance in o. tritici could evolve other genes or loci. the ability of the ␣-proteobacterium o. tritici to tolerate high levels of arsenic in addition to other oxyanions has considerable potential for detoxification and bioremediation of contaminated environments. this work is based on the mathematical modeling of kinetics of a thermophilic bacteria cultivation system. the cultivation proceeded by way of batch and continuous on the synthetic medium with the main carbon source-lactose. this medium simulated an industrial waste approximately. mixed thermophilic aerobic bacteria popula-tion, applied to the wastewater treatment (sludge v&k bystrice pod hostynem), was used to the inoculation. the cultivation system consisted of the laboratory fermentor biostat b (b. braun biotech) with working volume l and the control unit connected with a computer. it is possible the temperature, ph, aeration, stirring and foaming regulation. physical and chemical cultivation conditions were optimized. the chemical oxygen demand (cod), generally expressive the impurity level, was selected as the main parameter for the cultivations run classification. but into the mathematical model also the kinetics of biomass growth, lactose consumption, production of choice metabolites (acetate, lactate, succinate) and dissolved oxygen concentration was included. the modeling was located to two head distinguishable growth phases of the microorganisms. an optimization and identification of mathematical model parameters was practised in the software language psi/c. the difference between simulated curves and experimental data is not statistically significant on the relevancy level . (f-test). it took place the cod degradation at . % with the average yield coefficient y chsk/x . g cod/g biomass in the batch process with the air aeration only. there is a better way to the cod elimination (> . %)-the aeration of air enriched by the pure oxygen. in experiments with the continuous system was obtained the . % cod decrease after the steady state stabilization. this work was supported by project msm . fluorescence in situ hybridization (fish) of whole cells using oligonucleotide probes was applied to study the influence of low temperature and temperature reduction on the bacterial community of biofilm reactors for the removal of chlorophenols (cps). two packed bed reactors were set up for degradation of a mixture of -cp, -cp, , -dicp, and , , -tricp as sole source of carbon and energy at • c (ra) and • c (rb) and were inoculated with bacterial consortia adapted to these respective initial temperatures. the performance of the reactors was studied under different conditions of pollutant loading, aeration rate, and hydraulic retention times over months. total chlorophenol removal capacities of and mg l − day − were achieved in the bioreactors ra and rb, respectively, under a total pollutant load of mg l − day − . the population of ␤-proteobacteria was the major bacterial community of the biofilm ( - %) followed by the ␥-proteobacteria ( - . %). two bacteria with the ability to mineralize mg chlorophenols l − were isolated from the bioreactors and characterized as ralstonia basilensis and alcaligenes sp., both belonging to ␤-proteobacteria. decreasing the temperature by • c (in two steps of • c each) resulted in an increase in the population of ␥-proteobacteria and a decrease in the population of ␤-proteobacteria in both reactors. application of genus specific probes showed an increase in the pseudomonas population from % of the ␥-proteobacteria at • c to % at • c. the pollutant removal capacity decreased to and mg l − day − in ra ( • c) and rb ( • c), respectively. the ␣and ␦-proteobacteria, cytophaga-flavobacteria and actinobacteria the survey was carried out in urban and rural areas of two cities (ankara and isparta). this paper is only analysing urban people by excluding villagers. urban sample is consisted of urban consumers and professionals. professionals were selected amongst pharmacists, doctors, agricultural engineering's and industrialists that is thought are affective in the process of developing new technologies and also the development of biotechnology in the society. basic data was gathered by a questionnaire including both structured and open-ended questions besides deep interviewed. workforce development for life sciences-the scottish experience carol booth scottish entreprise, uk the presentation will look at, the background and definition of workforce development for scottish enterprise, examine information available to support scottish enterprises economic intervention and where and when to intervene. conclusions emerging from the evidence base will be used to outline scottish enterprises approach to workforce development and look at which actions might be required to address identified issues. moving on to reasons for integrating workforce development into business development and how the life sciences cluster team at scottish enterprise, stakeholders and partners in scotland have approached their current and future contributions to workforce development for life sciences using a variety of projects. the national institute for bioprocessing research and training (nibrt) in ireland is a proposal that will be a state-of-the-art training, research and pilot plant service facility that brings together institutions with complementary expertise and state-of-the-art research technology, and industry partners. these include university college dublin, trinity college dublin, institute of technology, sligo and dublin city university. nibrt is an innovative collaboration between academic institutions at the forefront of biotechnology, cell biology, engineering and pharmacy and industry. for training, two separate training labs, for upstream and downstream training, in addition to research labs are planted. it will also include a state-of-the-art pilot plant fermentation facility for fermentation optimisation, fermentation scale-up, product separation and purification, regulatory aspects and automation. by aligning with industrial demands, the new institute will tailor its training programmes while remaining on the cutting edge of biotechnology research and technologies. the fermentation facility will offer hands-on training workshops and educational modules for outside researchers and companies. these workshops cover the fundamentals of small-scale fermenta-tion, scale-up considerations, and fermentor design and set-up. the training and educational philosophy underpinning the nibrt will focus on the needs of industry with an emphasis on providing training for accreditation of existing industry staff and prepare technicians and graduates for the technical, business, regulatory and professional aspects of the industry. the strategy is to provide specialised modules in nibrt in support of courses established in the higher education institutions, which will provide the certificates, diplomas and degrees. modules will be offered for all categories of students and will be given credits respected by other third level institutions in ireland. the role of professional graduate degrees in meeting current and future biotechnology industry workforce needs a. stephen dahms san diego state university, usa the presentation will review the status of new graduate training models designed to meet the unique needs of the biotechnology industry as it transitions to commercialization. emphasis will be on professional master's degree programs in biotechnology and their various versions, with a focus on operational and funding strategies and industry acceptance. discussion will also centre upon the creation and operation of industry-validated, specialized and highly targeted professional masters degrees in various refined aspects of the drug development process, including regulatory affairs, biomedical quality systems, clinical affairs, management of drug development, management of reimbursement affairs, bioinformatics, etc. the eurodoctorate in biotechnology, new combined mba/phd combined degrees in the molecular life sciences, the u.s. professional doctorate in chemistry and the proposed u.s. professional doctorate in biotechnology will be also discussed. data will also be presented on the current workforce and the industry's projected needs. genetic studies show that mankind is a rapidly expanded population of closely related individuals with very similar disease sensitivity. bad nutrition and infections dominate among the main health problems in the world. apart from malnutrition, the overeating habits of the developed world are now creating problems in the developing world as well. infectious diseases are also a global problem since new contagious agents like hiv, sars and avian flew do not recognize borders. thus, the global responsibilities of modern health care are obvious. many research scientists from and in developing countries find it nearly impossible to use their talents for the benefit of their own countries. some struggle to develop research and education programmes with poor facilities, some leave science completely, and others migrate to more developed countries. the talents of such people are either being wasted or lost completely to their home countries just at the time they are most needed to combat the great humanitarian challenges of hunger, illness and lack of knowledge. europe must strengthen programmes which allow third world scientists to work to their full potential in their home countries or regions. yang beijing genomics institute, chine academy of sciences, beijing, china europe has all the reasons to be proud of being the cradle of modern science and of its achievements and resources in life sciences. as a model of having solved many of the problems that many other counties are now facing, europe is expected by the whole world to make its further contribution to a better future of mankind and to play a more important role in the international community of life sciences. tropical diseases and public health basilio valladares director of the university institute of tropical diseases and, public health, university of la laguna, la laguna, tenerife, spain. e-mail: bvallada@ull.es the presentation will look at the main research interests of the university institute of tropical diseases. these are the following: . immunology and molecular biology of parasites. we express and purify recombinant proteins from leishmania sp. which have been shown to act as immunomodulators and protect against disease such as l , hsp , hsp . the study of acanthamoeba pathogenic factors has also resulted in the isolation and silencing of extracellular proteins related to their pathogenecity, which has a great potential in the development of novel chemotherapeutics. . diagnosis of parasites. the immunological diagnosis of leishmaniasis has been one of the main research interests in our laboratory for several years. as a result, we have identified peptides which could be used to develop kits for the immunological diagnosis of leishmaniasis such as hsp c-end, l n-end, etc. we have also developed some dna based methods for the identification of acanthamoeba species from biological and environmental sources. . water quality. biological parameters. our water research group has the expertise to identify and characterise bacterial, viral and parasitic indicators of faecal contamination in diverse water sources including tap water, rivers, reservoirs, sea, etc. this research area has been developed in collaboration with the local sewage treatment plant and reservoir managing authorities. currently, we are establishing a conjoined project with the center for disease control and prevention (cdc) in atlanta, usa for the identification of water-borne emerging pathogens. . development and formulation of chemotherapeutic antiparasitic agents. in this field, we evaluate the leishmanicidal activity both in vivo and in vitro of natural and synthetic drugs and synthetic peptides. in a later stage, the drugs which have shown the highest antiparasitic activity have been subjected to cytotoxicity assays and their molecular targets dissected. some of the drugs tested in the last few years have been submitted to patent due to their outstanding activity. finally, in order to allow the commercialization of these drugs, both in vivo and in vitro assays are being carried out to predict their chemical stability and degradation pathways. this will be followed by the use of liofilization and controlled crystallisation strategies for the development of efficient and safe treatments. . human and population genetics. tachykinins and their receptors in different tissues and groups of patients and their association with molecular polymorphisms is another one of our research interests. the knowledge of ligand and receptor sequences and their similarities will allow the rational development of drugs with specific activity against these receptors. furthermore, we are also interested in the interspecific variation along the evolutionary scale of these markers. . nitrate assimilation group. research in our group is focused on understanding nitrate assimilation in the yeast hansenula polymorpha. several biotechnological companies use this yeast to produce heterologous proteins (hepatitis b vaccine). genetic manipulation techniques for h. polymorpha are available in our laboratory. head of the department of biotechnology, technological institute of canary islands, pozo izquierdo santa lucía, las palmas, spain single cell analysis by flow cytometry has proved to be a tool to perform simultaneous and rapid measurements related to cell morphology and physiological state. previous studies showed the possibility of quantifying neutral and polar lipids spectrofluorometrically using a lipid specific fluorescent dye, nile red (nr), however the existence of inter and intraspecific variations in the fluorescent response had not been clearly established. in this work, two strains of marine microalgae: crypthecodinium cohnii and tetraselmis suecica, characterized both by high contents of polyunsaturated long chain fatty acids (dha and epa, respectively) and an hypersaline microalgae: dunaliella salina, characterized by a high ␤-carotene production, were grown under different conditions and collected at different growth phases to be used for in vivo lipid quantification with nr by flow cytometry. our results showed a high correlation between the mean fluorescence signal of nr stained cells and the neutral and polar lipid content measured by gravimetry for each strain. in this respect, these data make feasible the development of a rapid method for lipid quantification in monoalgal cultures. however, differences in the dye uptake related to specificity were detected. in this communication we also assess the possibility of use this cytometric technique to select microalgal strains with high lipid and polyunsaturated long chain fatty acids content from mixed samples. performance of such technique would be a good alternative to the time-consuming traditional screening protocols based on gravimetry and gas chromatography and would optimise the search of new commercial strains of microalgae. claverie-martín head of research unit, biomedical research institute, hospital universitario, de n.s. de candelaria, santa cruz de tenerife, spain our group is involved in the cloning and production of proteins of interest to the food and pharmaceutical industry. we have recently expressed in yeast the cdna that encodes the precursor of caprine chymosin. chymosin is the enzyme responsible for the coagulation of milk in the abomasum of unweaned calves. this enzyme is secreted by gastric mucosa cells as an inactive precursor, known as prochymosin. in the acidic conditions of the lumen, prochymosin is converted into the active form by autocatalytic cleavage of the n-terminal prosequence. chymosin is used extensively in cheese production because it cleaves -casein in a specific manner with low proteolytic activity. several biotechnology companies are producing the bovine recombinant enzyme for commercial use in the process of cheese making. we are interested in the caprine chymosin as an alternative because in the canary islands cheese has traditionally been made using goats milk with extract from the abomasum of newborn goats as coagulating factor. it is well known that the activity of these types of extracts varies depending on the age of the animal and the type of food ingested. these difficulties should be overcome using a recombinant caprine chymosin. the caprine mrna used for the synthesis of the cdna was obtained from the abomasum of milkfed kid goats. the cdna fragment encoding the mature portion of caprine prochymosin was fused in frame to a signal sequence in yeast expression vectors. culture supernatants of yeast cells transformed with the recombinant plasmids showed milk-clotting activity after activation at acid ph. proteolytic activity assayed toward casein fractions indicated that the recombinant caprine chymosin specifically hydrolysed -casein (patent ). the recombinant caprine chymosin could be an alternative milk coagulant in cheese making. work is underway to optimise the expression of the new recombinant prochymosin for further purification and characterization. smart molecules for health victor martín head of research, university institute of bio-organics "antonio gonzález", avda. astrofísico francisco sánchez , la laguna, tenerife, spain the instituto universitario de bio-orgánica "antonio gonzález" (iubo) is a multidisciplinary research centre that belongs to the university of la laguna. the iubo is located at the town of la laguna, inscribed on unesco's world heritage list in , and former capital of the canary island of tenerife. the geographical location in addition to its mild climate has made the canary islands to posses a variety of ecosystems with unique plants and animals. the institute was started up in the s with the need to study the natural products and secondary metabolites produced by those marine and terrestrial organisms, thus providing a new source of bioactive products. the main research lines that are being developed at iubo are summarised in the following paragraphs: anticancer agents from natural sources: several natural products and their semisynthetic derivatives are produced at the iubo in diverse joint projects for the development of new antitumour drugs with novel mechanisms of action. as an example we can mention natural products from the mevalonic, shikimic or polyketide pathways. some products have recently shown in vitro reversion of the resistance in multidrug resistant (mdr) tumour cell lines. genetic engineering: in vitro cultures of the plants atropa baetica, maytenus amazonica and m. macrocarpa are developed in order to manipulate their biosynthetic pathways and induce the production in large scale of the secondary metabolites for diverse applications, including arthritis, rheumatism, and back pain. marine organisms and toxins: dinoflagellates are marine organisms responsible for the red tides and food poisoning episodes. among others, okadaic acid and yessotoxin are the most common toxins present in european shellfish. the isolation of these products is best done from the microorganism cultures, since they are present in very low amounts in the natural sources. at the iubo we develop culture systems to provide us with amounts of toxins large enough to perform biological, metabolic and bioactive studies. insecticide and repellents: natural products are being isolated for their use against plagues, specially those affecting agriculture. these projects are run in collaboration with a number of public institutions and agrochemical companies throughout europe and latin america. fine chemicals and pharmachemicals: our institute possesses large expertise in the field of organic synthesis devoted to the synthesis of medicinal substances, with special focus on asymmetric processes. of particular interest is the development of new methodologies for the total synthesis of biologically active substances like polyether toxins, (un)natural amino acids, sphingosine analogs, alkaloids, etc. with an annual source of s of new compounds, the fine chemicals and medicinal chemistry branch at iubo have recently started and anticancer screening program in collaboration with the biomedical research unit at the hospital universitario n.s. de la candelaria. the program is committed to the discovery of novel drugs for application in cancer treatment. the outcome of this project in its first year has been outstanding, leading to the finding of several leads that form the basis for current and future projects. institute of canary islands, biotechnological department, playa de pozo izquierdo s/n, santa lucía, gran canaria, spain the presentation will look at the main research interests of the biotechnological department of the technological institute of canary islands. these are the following: nutrition and feeding in aquaculture: we conduct studies on digestion, absorption, transport, and utilization of the different nutrients applying also different techniques such as histology, enzymology, genetic or immunology among others. aquaculture feeding is the main important cost in fish farms, being higher that the prize of fries, personnel cost or energetic costs. thus, studies conducted on the improvement of diet formulation and the use of different dietary ingredients is one of our main research lines (such as vegetable oils and meals to be used as alternative to fish meal and oil, or carotenoid sources to improve fish colour). not only the use of the different ingredient is studied, but also the effect of these ingredients on fish health, flesh quality, flesh healthy aspects related with human consumption, are being studied. different formulae are being developed and patents of different diets are being obtained. studies on nutritional requirements are also conducted allowing us to patent different formulae in larvae studies, developing microdiets to substitute the high-cost processes associated with live prey in larval nutrition. development of immunostimulants, anti-stress diets, immune techniques to be applied as bio-indicators of fish health and welfare, as well as use of dietary ingredients derived from bio-reactors are other research lines in our group. genetics: genetic techniques applied to aquaculture are being an important tool. microsatellites are being used to determine genealogy of the fish, allowing to decreases important problems in aquaculture such as fish deformities. genetic techniques such as micro-arrays and gene expression are being applied to obtain indicators of stress and health in fish. these technologies also permit to make different selective breeding programs, increasing the accuracy to estimate genetic parameters and evaluating brood-stock. furthermore, this technology allows to obtain a procedure to increase the productivity and quality of fish hatcheries. new species for aquaculture. development of new culture techniques: the diversification of species cultured is one of the main objectives of european aquaculture, since nowadays only four marine species are commercially produced: gilthead sea bream. european sea bass, turbot and salmon. we have been developed rearing techniques for new species such as red porgy or canarian abalone and also we are conducting studies on different new species, such as different sparids species, octopus or yellowtail. new rearing technology: the election of adequate systems for fish growth for each species and site of production and the localization of more appropriate sites for farms, using gis technology are also of special importance in our research team. besides, new larval rearing techniques such as semi-extensive hatchery (mesocoms) were development and nowadays is being used to increase fry quality (survival, no-deformities, better growth). this technology is being exported to other countries in order to offer new technology easy to manage to be implanted in developing countries. alejandro cañeque project officer, canary islands special zone, ministry of finance, c/leon y castillo - a planta, edificio urbis, las palmas, spain. e-mail: acaneque@zec.org the canary islands special zone is the newest tax instrument within the canary islands economic and fiscal regime (ref). it offers a reduced tax rate of between and % of corporate income tax for companies setting up a business. the companies must cover the following minimum requirements: create employment and make a minimum investment. the zec offers other tax advantages such as the exemption from paying transfer tax and stamp duty and the canary islands general indirect tax (igic). this tax scheme particularly fosters the biotechnology and pharmaceutical sectors. references fahnert references bechor the antimicrobial drugs high-level production of human collagen prolyl -hydroxylase in sandwich hybridisation assay for quantitative detection of yeast rnas in crude cell lysates microbial processes and products press. biotech. bioeng. . van hijum microbial xylitol production from corn cobs using candida magnoliae the role of bacillus methanolicus citrate synthase gene, city, in regulatiing the secretion of glutamate in lysine-secreting mutants plasmid-dependent methylotrophy in thermotolerant bacillus methanolicus , -anhydrod-fructose; a versatile chiral building block: biochemistry and chemistry detailed dissection of a new mechanism for glycoside cleavage: the ␣- , -glucan lyase ␣- , -glucan lyase, a new class of starch and glycogen degrading enzyme ␣- , -glucan lyase, a new starch processing enzyme for production of , -anhydro-d-fructose efficient purification, characterization and partial amino acid sequencing of two ␣- , -glucan lyases from fungi ␣- , -glucan lyases producing , -anhydro-d-fructose from starch and glycogen have sequence similarity to alpha-glucosidases enzymatic description of the anhydrofructose pathway of glycogen degradation i. identification and purification of anhydrofructose dehydratase, ascopyrone tautomerase and ␣- , -glucan lyase in the fungus anthracobia melaloma a systems approach to dissecting immunity and inflammation integrative biological analysis of the apoe* -leiden transgenic mouse innate recognition of bacteria in human milk is mediated by a milk-derived highly expressed pattern recognition receptor, soluble cd soluble forms of toll-like receptor (tlr) capable of modulating tlr signaling are present in human plasma and breast milk proteomics of the rat gut: analysis of the myenteric plexuslongitudinal muscle preparation an integrative metabolism approach identified stearoyl-coa desaturase as a target for an arachidonate-enriched diet the challenges of modeling mammalian biocomplexity rapid enrichment of bioactive milk proteins and iterative, consolidated protein identification by mudpit technology post-genomics of lactic acid and other food-grade bacteria to discover gut functionality genetics, metabolism and application of lactic acid bacteria. fems microbiol complete genome sequence of lactobacillus plantarum wcfs functional ingredient production: application of global and metabolic models metabolic engineering of lactic acid bacteria for the production of nutraceuticals reduction in antinutritional and toxic components in plant foods by fermentation the authors gratefully acknowledge the financial supports given to this work by ankara university, biotechnology institute (project no k - ). the authors gratefully acknowledge the financial supports given to this work by ankara university, biotechnology institute (project no. k - ) and the microorganisms given to this work by prof. dr. francesco molinari at university of milano, and prof. dr. leyla aÇ ikel at gazi university. the authors gratefully acknowledge the financial supports given to this work by ankara university, biotechnology institute (project no k - ) and the microorganisms given to this work by prof. dr. francesco molinari at university of milano, and prof. dr. leyla aÇ ikel at gazi university. mrs. lynnette fernandez, johanna mäkeläinen, m.sc. and olli rämö, m.sc. are gratefully acknowledged for their help. this work was supported by the health science council, the academy of finland and the tekes-neobio program. supported by the mec of spain (ppq - -c - ) and the canary islands government. jmp thanks icic for a postdoctoral fellowship. frpc thanks cajacanarias for a fpi fellowship. tm thanks the spanish mcyt-fse for a ramón y cajal contract. this work was supported by the korean systems biology research grant from the ministry of science and technology, lg chem chair professorship, ibm sur program and bk program. this study was supported by ankara university biotechnology institute (project no: ). this work was partially supported by mec and feder (bio - ), and fundación séneca carm ( /pi/ ). this work was supported by grants f of the hungarian scientific research fund and grant omfb- / of the hungarian ministry of education. keto sugars have long been implicated as attractive intermediates or substrates for further chemical or enzymatic reactions, to generate a number of synthetic sugar derivatives and fine chemicals. the quinone-dependent pyranose dehydrogenase (pdh) purified of the basidiomycete fungus agaricus meleagris catalyzes with high specificity the oxidation of the c- of glycosidically bound d-glucose, whereas in contrast it oxidizes simultaneously the c- and c- of free d-glucose. considering the broad substrate tolerance, pdh provides a new convenient tool for high yield production of -keto- this work was partially financed by the scientific and technological research council (cicyt, spain), grant ren - , by the iii pla de recerca de catalunya (generalitat de catalunya), grant sgr- , and by the generalitat de catalunya to the "centre de referència en biotecnologia" (cerba). this work was supported by mega a.s. (czech republic) (www.mega.cz) and following vega grants: / / and / / . this work was partially supported by mec and feder (bio - ), and fundación séneca carm ( /pi/ ). financed by a marie curie re-integration grant merg-ct- - and consejería de educación y ciencia de la junta de andalucía. this project is part of the collaborative research centre sfb "development of biotechnological processes by integrating genetic and engineering methods", which is supported by the german research foundation dfg. this research was supported in part by grants from the hungarian scientific research fund (otka t , f and d ) and the hungarian-spanish intergovernmental s & t cooperation programme (omfb / ). this research was supported in part by grants from the hungarian scientific research fund (otka t , f and d ) and the hungarian-spanish intergovernmental s & t cooperation programme (omfb / ). this research was supported in part by grants from the hungarian scientific research fund (otka t , f , d ) and gvop- . . .- - - . the authors thank dr. hamid narjiss (director of morocco inra) for the instruction to identify nadorcott mandarin by molecular markers and helpful discussions regarding this paper. this work was partially funded by the program for scientific cooperation cnrst (morocco) and iccti (portugal), the international foundation for science (ifs) support in stockholm (sweden). this work has been financed by xunta de galicia (pgidt pxib pr). sevgil sadettin, gönül dönmez department of biology, faculty of science, ankara university beşevler, ankara, turkey this study was supported by ankara university biotechnology institute. demet Ç etin , sedat dönmez , gönül dönmez : department of biology, faculty of science, ankara university, beşevler, ankara, turkey; department of food engineering, faculty of engineering, ankara university, dışkapı, ankara, turkey sulfate-reducing bacteria (srb) that could grow on modified postgate c medium (pc) containing chromium(vi) were isolated from industrial wastewater and their chromium(vi) reduction capacities were investigated as a function of changes in the initial ph values, chromium, sulfate, nacl concentrations and carbon source. the optimum ph value at mg l − initial chromium(vi) concentrations was determined as . chromium(vi) reduction by srb was investigated at . - . mg l − initial chromium(vi) concentrations. at the end of the experiments, the mixed cultures of srb were found to reduce more than % of the initial chromium(vi) levels which ranged from . to . mg l − within - days period. the effects of initial - . g l − concentrations of sulfate and - % (w/v) concentrations of nacl to chromium reduction were showed that, the lowest concentrations of sulfate and nacl, were the best for chromium reduction in the pc media including mg l − chromium(vi). when the % whey was used as carbon source in the pc medium, . % of the . mg l − initial chromium(vi) concentration was reduced within this study was supported by ankara university biotechnology institute. this study was carried out as a part of the project for analyzing and controlling the mechanism of biodegrading and processing entrusted by the new energy and industrial technology development organization (nedo). this work has been financed by xunta de galicia (pgidt pxib pr). lead ions are considered a high pollutant of different waters. in our previous work were selected seven potential sorbent-strains rhodotorula mucilaginosa , rh. aurantiaca , rhodotorula sp. , williopsis californica , candida krusei t, cryptococcus sp. wt of lead ions. it was determined their stability to high concentration (up to mg/l) of lead ions in medium. the ph changes and yeast physiologies of growth were studied in medium with these heavy metal ions. the influences of environmental factors such as ph of solution, age of microbial culture, biosorbent concentration in suspension, alive or living state of biosorbent, time of contact on sorption were investigated. the levels of maximal sorption ability and biomass affinity to heavy metal ions were established by experimentally received sorption isotherms with mathematical modeling of biosorption process separately for everyone researched yeast strain. the sorption isotherms obtained in these experiments for non-living yeast biomass showed that the maximal sorption capacity was and × − mol (g sorbent) − for rh. aurantiaca and s. cerevisiae , respectively. in the case of living biomass, the high- this work has been financed by the spanish ministry of science and technology and european feder (project ctm - ). the authors wish to thank dra. m.j. martínez (cib, csic, madrid, spain) for providing coriolopsis rigida. this work was supported principally by embo and the mrc. fed-batch cultivation of haematococcus pluvialis under illumination with leds for production of astaxanthin abdolmajid lababpour, tomohisa katsuda, shigeo katoh department of molecular science and material engineering, kobe university, kobe, hyogo - , japan photosynthetic microalga haematococcus pluvialis is a most promising microorganism for production of astaxanthin, which has powerful antioxidant activity and is used both for human being as a food supplement and cosmetic; as well as animal farming such as salmon and poultry. the deficiency of nutrients in batch culture of h. pluvialis decreases the growth of cells and increases the induction of astaxanthin as an induction factor. therefore, it is impossible to reach to high cell concentrations in batch cultures. in previous experiments, the medium replacement increased the cell concentration, while accumulation of astaxanthin was not induced without other factors. in this work, the effects of fed-batch addition of culture medium on the cell growth and astaxanthin production in h. pluvialis cultures were studied. h. pluvialis was cultivated in cm culture medium containing sodium acetate, yeast extract, l-asparagines, mgcl · h o, feso · h o and cacl · h o (ph . ). light was supplied by panels of blue or red led lamps. the temperature was kept at • c and the culture was mixed with a magnetic stirrer. in fed-batch cultures of h. pluvialis, the cell concentration and production of astaxanthin increased in comparison with those in batch culture. in addition, the operation in feb-batch manner is easier than medium replacement from industrial viewpoints for production of astaxanthin. simvastatin and similar compounds are wide used as antihypercholesterolemic agents. simvastatin is obtained by c-methylation of side chain of lovastatin. this process is not perfect and some unreacted lovastatin is present in the reaction mixture. simvastatin is separated from lovastatin using fungi clonostachys compactiuscula. using of living microbials has some disadvantages such as high cost, difficult product separation from the reaction mixture. we work on overcome these difficulties by separation and immobilization of enzyme that hydrolyses lovastatin ammonium salt in presence of simvastatin ammonium salt. our results of purification and immobilization lovastatin hydrolase will be presented. investigation of peptide antibiotics produced by trichoderma strains isolated from winter wheat rhizosphere a. szekeres , l. kredics , l. hatvani , z. antal , l. manczinger , a. nagy , c. vágvölgyi : department of microbiology, university of szeged, p.o. box , h- szeged, hungry; hungarian academy of sciences, university of szeged, microbiological research group, hungry; pilze-nagy ltd. , kecskemét, p.o. box , species of the imperfect filamentous fungal genus trichoderma with teleomorphs belonging to the hypocreales order of the ascomycota division are of great economic importance as sources of enzymes, antibiotics, as plant growth promoters, decomposers of xenobiotics, and as commercial biofungicides. peptaibols and related peptaibiotics (prps) are secondary metabolites constituting a family of fungal peptide antibiotics which is constantly growing since alamethicin was isolated from cultures of trichoderma viride. these compounds are linear, amphipathic polypeptides composed of - amino acids and usually containing several non-proteinogenic amino acid residues, which are representing characteristic building blocks of the structure. one hundred and twenty trichoderma strains were isolated from roots of winter wheat grown in agricultural fields of southern hungary. the identity of species was examined based on morphological and molecular characters. the presence of prps-producing strains among the isolated trichoderma strains was detected by biological tests and the antibiotics were partially purified using a multistep chromatography procedure involving exclusion chromatography, adsorption chromatography and thin-layer chromatography. about % of the isolates proved to be able to produce prps. the antibacterial activity of the compounds was tested against staphylococcus aureus, bacillus subtilis, micrococcus luteus and escherichia coli, while the antifungal effect was recorded against fusarium oxysporum, f. culmorum, rhizoctonia solani and pythium debaryanum. ergezinger, m., bohnet, m., berensmeier, s., buchholz, k., . integrierte enzymatische synthese und adsorption von isomaltose in einem mehrphasenbioreaktionsreaktor. cit , - . glucansucrases from family of glycoside-hydrolases are transglucosidases that produce ␣-glucans from sucrose, a very cheap substrate, without any use of nucleotide activated sugars. based on sequence analyses, these enzymes have been classified in two families, the family and the family of glycoside hydrolases. among the natural diversity existing in family in which are found the glucansucrases produced by lactic acid bacteria, three enzymes have been selected for their distinctive specificities: dextransucrase from l. mesenteroides nrrl b- f (dsr-s), which catalyses almost essentially the synthesis of ␣- , -linkages, alternansucrase from l. mesenteroides nrrl b- (asr), which produces alternan polymer formed of ␣- , and ␣- , -alternated linkages and finally dextransucrase from l. mesenteroides nrrl b- (dsr-e), which is responsible for the synthesis of a branched dextran composed of about % of ␣- , -linkages in the main chain and % of ␣- , -branched linkages. for all these enzymes, the natural polymerase activity can be shifted towards oligosaccharide production or gluco-conjugate syntheses by introducing acceptors in the reaction medium. a number of sugar acceptors have been successfully glucosylated with the view of developing new functional food products. acceptor glucosylation yield as well as acceptor reaction product structures were shown to be highly dependant on the enzyme specificity. consequently, using glucansucrases of distinctive specificities and varying the acceptors give access to a large variety of applications. amylosucrase, the sole glucansucrase found in family of glycoside-hydrolases is also of great interest for functional food applications. this enzyme is able to synthesize highly resistant amylose from sucrose. again the reaction conditions can be used to modulate the yield and the size of amylose. the aim of our work is to further develop the applications of these enzymes via rational and combinatorial engineering. the most recent results obtained in this field will be discussed. novel food structure engineering concepts with enzymes johanna buchert vtt biotechnology, espoo, finland food structure is a very important quality attribute in food choice, since it affects not only the sensory perception of texture, but also release of flavour. enzymes offer specific means to engineer food structure by creating cross-links to food biopolymers, i.e. to proteins and/or carbohydrates. enzymatic cross-linking of food biopolymers can be exploited to create novel types of structures to foods without any need of added food ingredients. laccases and peroxidases can be used to crosslink ferulic acid containing carbohydrates, such as sugar beet pectin or arabinoxylan. proteins can be crosslinked by different oxidative or transferase type of enzymes. transglutaminases can crosslink protein via formation of isopeptide bond between glutamine and lysine residues. laccase and peroxidase can oxidize tyrosine residues to corresponding radicals, which in turn can further react with different groups in proteins. tyrosinases, on the other hand, oxidize tyrosine to a quinone, which can further react with aromatic ring, amine and thiol groups present in proteins. the biopolymer networks formed can be further engineered by combining adequate processing to the enzyme treatment. in this work the potential of enzymatic food structure engineering is reviewed. asparaginase-mediated reduction of acrylamide formation in baked, fried, and roasted products hanne vang hendriksen, beate kornbrust, steffen ernst, mary stringer, hans peter heldt-hansen, peter Østergaard novozymes a/s, dk- bagsvaerd, denmark. e-mail: hvhe@novozymes.com (h.v. hendriksen) in , it was discovered that acrylamide is formed in several potato and grain-based foods (e.g. chips, french fries, toasted bread, biscuits, cereals) and in coffee, all of which have been prepared at high temperatures. the level of this potential carcinogen in the final food appears to range from to ppb. later that year, the mechanism of acrylamide formation was unraveled, demonstrating that asparagine and reducing sugars are the precursors for acrylamide. this pointed to several potential enzymatic approaches to remove the root cause of the problem by degrading the precursors in situ. here, we demonstrate that asparaginase treatment leads to a more efficient reduction in acrylamide than alternative enzymatic treatments. asparaginase from aspergillus oryzae is used to reduce acrylamide formation significantly in laboratory models of a range of common food products. examples are french fries, biscuits, crisp bread, and fabricated chips. the sensory qualities appear to be constant. the implications for scaling up the processes for industrial food production are discussed. effect of cultivation conditions on folate content in yeast: exploring the potential of yeast as a bio-enrichment vehicle for folate in foods sofia hjortmo , johan patring , jelena jastrebova , thomas andlid : department of chemical and biological engineering, chalmers university of technology, po box , gothenburg, sweden; department of food science, swedish university of agricultural sciences, po box , uppsala, sweden. e-mail: sh@fsc.chalmers.se (s. hjortmo) over the past years, the interest in health benefits of the b vitamin folate has increased considerably. a good folate status may hinder progression of several diseases such as neural tube defects and downs syndrome in foetus, as well as cancer, dementia, alzheimer's disease and cardiovascular disease in adults. it is however not easy to reach the recommended intake and new strategies have to be developed to increase the folate status. in this project we explore the use folate producing microorganisms for this purpose. many yeasts have the ability to synthesise folate de novo and can thus serve as a source for humans. folate enrichment in fermented foods could be much improved by using starter cultures better at producing folate compared to traditional strains. this is, e.g. applicable to bread making fb over-expression of isoprene biosynthetic enzymes in the ␤-carotene producer zygomycete mucor circinelloides tamás mucor circinelloides has been involved to study the carotene biosynthesis genesis of fungi. this fungus is more amenable to molecular techniques than the others traditionally used in carotenogenic studies (e.g. blakeslea trispora and phycomyces blakesleeanus). moreover, mucor has a great advantage: it is a dimorphic organism. this type of morphology is preferred by the fermentation industry because yeast-like growth allows the submerged culture, when usually higher biomass production can be achieved and cells can be more easily separated from the media. β-carotene is a terpenoid-type chemical compound likewise to sterols, quinones or chlorophylls. the production can be increased by improving the non-carotene specific terpenoid biosynthesis. this can be carried out by the overexpression of the genes responsible for the ratelimiting steps of these pathways. in this study, polyethylene glycol mediated transformations of m. circinelloides protoplasts were performed with autoreplicative expression vectors containing the known terpenoid genes of m. circinelloides (e.g. isoa encoding farnesyl pyrophosphate synthase and carg encoding geranylgeranyl pyrophosphate synthase). carotene production of the transformants and the wild-type strains were analysed by high-performance liquid chromatography (hplc). transformants harbouring plasmids with isoa or carg produce about . times more carotene than the recipient strain, while carotene production increased about two times in the co-transformants containing both type of plasmids. members of the genus rhizopus are important from biotechnological aspects in consequence of their effective extracellular enzyme, alcohol and organic acid production. moreover, rhizopus strains are used for fermentation of various foods, because they are capable of transforming soybeans into edible products. the high affinity iron permease (ftr ) contains both highly conservative and variable regions applicable for phylogenetic comparisons. the aim of this study was the comparative analysis of this gene of different rhizopus species in order to elaborate a simple and fast method to identify these fungi at a species and subspecies level. conserved regions of candida albicans and rhizopus oryzae ftr genes (fu et al., ) have been analysed to design degenerate primers for polymerase chain reaction. they were used to amplify the homologous regions from different strains of r. oryzae, r. microsporus, r. stolonifer and r. niveus. isolates of the similarly thermophilic rhizomucor miehei and r. pusillus, as well as a strain of m. rouxii were involved in the study as outgroups. deduced protein sequences were aligned and phylogenetic analysis was performed. surprisingly the r. oryzae isolates formed a group completely different with a significant distance from the r. microsporus isolate. r. niveus is currently not distinguished from r. stolonifer var. stolonifer because of morphological considerations. however, phylogeny of ftr gene sequences, in agreement with earlier results based on rapd data (vágvölgyi et al., ) , raise the need to handle r. niveus as a separate species. sequences and pcr primers useful for identification of all tested rhizopus strains were elaborated. potential application in a lactose conversion process. the prebiotics market is at high demand therefore the development of the process to produce 'prebiotic' galacto-oligosacharides efficiently and inexpensively is our particular interest. citrus, particularly mandarins and clementines, are among the most economically important fruit crops in morocco. besides morphological traits multiple molecular markers have been used for the caracterisation of citrus germplasm. the main aim of this study was to evaluate the moroccan mandarin germplasm and to identify specific polymorphisms among accessions sharing identical name. eighty mandarin and two sweet orange varieties were analyzed by dna markers. issr markers were amplified using anchored primers and analysed by agarose gel electrophoresis. aflp markers analyses were performed using three primer combinations. the dice coefficient was used to estimate genetic similarities and the upgma algorithm was utilised to generate a phenogram depicting the genetic relationships among the acessions. the selection of primers out of the issr primers primarily assayed, allowed us to maximize the average number of amplified fragments analyzed per reaction ( . ), and the percentage of informative polymorphisms ( %). the three combinations of ecori/msei primers revealed reliable aflp markers, ( %) of witch were polymorphic. the range of fragment sizes varied from to bp. contrasting with the phenotypic diversity for agronomic and fruit quality traits, very low variability at the dna level has found among mandarins, which always showed a high (s > . ) coefficient of genetic similarity. the molecular marker analyses allowed the clarification of ambiguous denominations and the establishment of phenological relationships. the mandarin cultivars have been clustered into several different sub groups. this study allowed the identification of one issr marker, distinct and specific for the clementine sidi aissa and some aflp markers specific to maroc late and w. navel. many hybrids used in this study presented high coefficient of the similarity with one of their parents, such as siamelo and king of siam (s = . ), fortuna and clementine (s = . ), kara and king of siam (s = . ). this work was partially funded by the program for scientific cooperation cnrst (morocco) and iccti (portugal), the international foundation for science (ifs) support in stockholm (sweden). the new morocco mandarin variety nadorcott became very important in the international market because of high quality, good size, easy peeling and absence of seeds. also known as afourer or w. murcott, this variety was selected in - at the afourer experimental station, inra, located near to beni mellal city, as an original tree among several -year-old murcott honney (c. reticulata × c. sinensis) seedling trees. in order to shed additional light on the genetic origin of this variety, we have carried out isozyme and dna fingerprinting analyses. for better interpretation of the nadorcott molecular profiles, others mandarin cultivars, among which murcott honney, were also analyzed by molecular markers. three enzymatic systems (idh, pgm and pgi) permitted the discrimination between nadorcott and his female parent murcott honney. the molecular patterns displayed by these cultivars point out the sexual origin of nadorcott and discard the previously assumed hypothesis for its origin as a mutation of a nucellar zygote. the issr and rapd markers analyses allowed the identification of kinnow; du japon, vietnam; and swett lime as the genetically most closely related mandarins (s ∼ . ) to nadorcott. strong genetic similarity was also found with the clementine group, a possible male parent of nadorcott. the analysis by aflp markers confirmed the hybrid origin of nadorcott and the high genetic relatedness (s = . ) of this mandarin to its putative female parent murcott honey, and other cultivars as kinnow (s = . ) and clementine (s = . ). the possibility of an accurate molecular identification of nadorcott by specific molecular markers is of paramount importance for the protection and management of this original moroccan citrus variety. an effective process for the chemical-biotechnological utilization of distilled white lees was studied. a first treatment with hydrochloric acid allowed the solubilisation of tartaric acid. the influence of temperature, amount of hcl and reaction time were considered through an experimental design. under the optima conditions g/l from white distilled lees and . g/l from red distilled lees were recovered. the tartaric acid was precipitated as calcium tartrate so that it can be isolated from the rest of the raw material compounds. the solid residue was used as an economic nutrient for lactic acid production by lactobacillus pentosus using trimming wastes as substrate. the lactic acid concentrations and volumetric productivities achieved were similar to those obtained using distilled lees without tartaric acid recovery as nutrient. thermophilic cyanobacterial strains that could grow in the bg media was isolated from hot springs and their reactive dye bioaccumulation was studied under thermophilic conditions in a batch system, in order to determine the optimal conditions required for the highest dye accumulation. in the experiments performed with newly isolated synechocystis sp. and phormidium sp., the optimum ph values at about mg l − initial reactive dye concentrations was determined as . lipases are extremely versatile enzymes, that catalyze both hydrolysis and synthesis reactions. they have a wide range of industrial applications, among which the manufacture of detergents, pharmaceuticals and fine chemicals are outstanding. the fungus rhizopus oryzae has been reported to synthesize a number of commercially interesting enzymes. in this work, its ability to produce extracellular lipases when grown in solid state culture has been assessed. cultures were carried out in erlenmeyer flasks, using a complex medium and several supports, both synthetic (nylon sponge) and natural (barley bran, ground walnut or peanut). the latter appeared to be more suitable for lipase production. since the best results were initially obtained with lipid-containing supports, barley bran cultures were supplemented with a vegetable oil, in an attempt to optimise lipase production and design an efficient procedure for reusing this agroindustrial waste. surprisingly, this strategy did not improve enzyme synthesis. however, when a surfactant (triton x- ) was added to the basal medium, a dramatic increase in extracellular activity was detected (up to -fold). the results agreed with those previously obtained in submerged cultures of r. oryzae, in which addition of olive oil did not increase lipase production, while the presence of triton x- had a remarkably beneficial effect. also, enzyme concentration in solid state cultures was up to two-fold that of the submerged ones. est maximal sorption capacity was found for yeasts cryptococcus sp. wt and rh. aurantiaca . these cultures also demonstrated the high sorption affinity, which makes them especially efficient biosorbents at low concentrations of lead ions. the high efficiency of lead elution was shown with . n edta. isolation and identification of marine bacteria from deep-sea sediments els maas , cara brosnahan , vicky webb , helen neil , phil sutton : marine biotechnology, national institute for water and atmospheric research ltd., kilbirnie, wellington, new zealand; oceanography, national institute for water and atmospheric research ltd., kilbirnie, wellington, new zealand marine sediments were obtained using a piston corer with associated trigger core ( . m, . m diameter). cores were collected from depths of to m, along norfolk ridge and across challenger plateau. sediments ranged from coarse carbonate sands in the north to sandy and silty hemipelagic mud with increasing depth and latitude. all samples sites underlie subtropical surface water masses associated with, and south of, the tasman front. sediment samples were aseptically taken from the triggers cores upon recovery. samples were stored in sterile tubes at • c on board the vessel for - days. the core samples were plated on several different agar types and incubated aerobically for weeks at • c. individual colonies were sub-cultured and purified using standard microbiological techniques. morphological and molecular taxonomy revealed that the bacteria isolated from the sediments were closely related to novosphingomonas, halomonas, stappia, glaciecola, pseudoalteromonas and leeuwenhoekiella. phylogenetic trees constructed using s rrna gene sequence data showed that two other isolates were unrelated to known genera. the bacterial isolates are currently being investigated for their biotechnological potential. olive oil mill wastewater (omw) as the effluent of the concern of olive industry has high organic load. the conventional biological treatments despite of their simplicity and rather suitable performance are ineffective for the omw treatment since phenolics possess antimicrobial activity. in order to carry out a proper treatment on omw, use of microorganism able to degrade the phenolics thus, is necessary. the ability of phanerochaete chrysosporium immobilized on loofa was studied. the basal mineral salt solution along with glucose, ammonium sulfate and yeast extract were used to dilute omw properly. the fungus did not grow on the concentrated omw. therefore, omw diluted by % was used thought this study. the extent of removal in this biotreatment, of total phenolics (tp) and cod were and %, respectively. while the color and aromatocity decreased by and %, respectively. the kinetic behavior of the loofa immobilized fungus was found to follow monod equation. the maximum growth rate was . h − while the monod constant based on the consumed tp and cod (mg/l) were and , respectively. the control of water pollution has become of increasing importance in recent years. the release of dyes into the environment constitutes only a small proportion of water pollution, but dyes are visible in small quantities due to their brilliance. many dyes are difficult to decolourise due to their complex structure and synthetic origin. the adsorption of reactive dye remazol brilliant blue r (rbbr) on polyelectrolyte complex (pec) was studied in a batch systems. the adsorption parameters determined were: effect of the different values of ph on the adsorption of dye by pec, and the effect of contact time on the amount of rbbr adsorbed (in mg g − ). the data indicates that the adsorption capacity of rbbr by pec is dependent on ph. the maximum adsorption at ppm was . % equal to . mg of dye/g of polymer. the results show a tendency towards greater adsorption for reactive dyes (ph range of - ). the effect of contact time was studied at initial concentration ( ppm) of dye, the amount of rbbr adsorbed for these pec increased and reached a constant value with the increase in contact time. the increase in the extent of removal of dye after min of contact time is less and hence it is fixed as the optimum contact time. the pec show their capacity to remove rbbr to aqueous solutions by adsorption. flocculation of saccharomyces cerevisiae (diastaticus) ifo was studied. cells of ifo did not flocculate even in the stationary phase without mg + ("mg + -deficient cells") although they began to flocculate strongly h after inoculation in the presence of mg + ("complete cells"). cycloheximide completely inhibited induction of floc-forming ability of "mg + -deficient cells". co-flocculation between "complete cells" and "mg + -deficient cells" was investigated by chemical modification. treatment of "mg + -deficient cells" by proteolytic enzymes did not affect the co-flocculation with "complete cells". photo-oxidation or mercaptoethanol-reduction of "mg + -deficient cells" failed to weaken the co-flocculation with "complete cells" while treatment of "mg + -deficient cells" by periodate brought about a significant loss of the co-flocculation. on the contrary, "complete cells" deflocculated by proteolysis or chemical modification of proteinaceous component failed to co-flocculate with "mg + -deficient cells". these findings suggest that "mg + -deficient cells" are non-flocculent because of lack of proteinaceous component essential for flocculation of cells of ifo . the industrial toscano cigar production starts with the dark firecured kentucky tobacco fermentation process. during this phase the present work is a trial to study the portal serum factors which stimulate the cell proliferation of the schistosomules, aiming to find ways to block or inhibit their effects. our previous studies showed that portal serum of human and hamster (highly susceptible hosts) and a - kd fraction separated from human portal sera by ultrafiltration stimulate cell proliferation in immature schistosomules ( days old) in vitro. for further identification of the portal serum factors in the range of - kd that stimulate cell proliferation, schistosomules were incubated in vitro in medium containing % fetal calf serum, % portal human serum or % peripheral human serum or their fractions separated by native electrophoresis followed by electroelution, incubations were performed in presence of bromodeoxyuridine (brdu) in order to measure differences in cell proliferation. the results showed that human portal sera enhanced cell proliferation of schistosomules compared to the peripheral serum. this stimulatory effect was substantially reproduced by fraction separated from human portal serum with molecular weight . kd. these results may help in designing a drug or antibody therapy to block the stimulating effect of the portal serum fraction and subsequently to disturb the life cycle of the parasite at early stage of development. center of molecular biosciences, university of the ryukyus, okinawa - , japan. e-mail: naoya-s@comb.u-ryukyu.ac.jp (n. shinzato)oil strage tank sludge, mainly composed of water and solid hydrocarbons (waxes) needs to be treated when harvesting the stored oil. although the sludge treatment by microbial surfactant or microbial cracking are considered as the feasible method, microbial degradation of the waxes (ca. solid n-alkanes) have been reported in a very limited species such as acinetobacter. in addition, long-chain n-alkanes (so called paraffin waxes) are one of the major components of oil, and their resistant properties to biological attack hold up the recovery of oil-polluted environments. in this report, we have screened n-tetracosane (c ) degrading bacteria from soils in okinawan island, an unique sub-tropical area in japan, to know the bacterial diversity and their degrading mechanism. srdna phylogenetic analysis of the isolates, totally ca , showed they were not only acinetobacter and pseudomonas, but also other proteobacteria (alcaligenes), actinomycetes (gordonia, nocardia, and leifsonia), bacillus, staphylococcus, and unidentified ones. they also grew not only the solid n-alkanes but also iso-alkanes, mid-chain n-alkanes as the sole carbon source. results for the biosurfactant production will also be shown. the properties of environmental enterococci were studied. the strains were isolated mainly from surface and waste waters and several strains from sheep manure were also included. species identification was provided by combination of phenotypic (micronaut system, merlin) and molecular detection methods (automated its-pcr, ddl-pcr). several discrepancies were observed when comparing molecular and biochemical identification. six enterococcal species were overall identified; e. faecium and e. hirae were the most abundant ones, almost % of isolates belonged to these two species. the distribution of selected genes conferring virulence to enterococci (cyla, gele and esp) was investigated, the positive signal was obtained mainly for e. faecalis strains. the strains were also characterized for the possession of enterocin genes (enta, entb, entp, ent , entl ab) and high frequency of enterocins was observed. biosorption of three different dyes (reactive black , cibacron brilliant yellow, cibacron brilliant red) onto immobilized scenedesmus obliquus a microalga was investigated in a batch sys-tem. the immobilized alga exihibited the highest dye uptake capacity at the initial ph value of . for all dyes. the effect of temperature on equilibrium sorption capacity indicated that maximum was obtained at • c for rb , cby and cbr biosorption. the freundlich, and langmuir adsorption models were used for the mathematical description of the biosorption equilibrium and isotherm constants were evaluated. biocontrol properties of microbially-treated sugar beet wastes in presence of rock phosphate n. vassilev, i. nikolaeva, m. vassileva department of chemical engineering, faculty of sciences, university of granada, c/fuentenueva s/n, granada- , spain. e-mail: nbvass@yahoo.com (n. vassilev) the effect of soil application of sugar beet wastes (sb) treated with aspergillus niger in the presence of rock phosphate (rp) on the control of fusarium wilt of tomato were studied. two treatments and a control were used: inoculation with glomus intraradices (am), further inoculation with a. niger grown on sb + rp medium, and the control (c). application of the am fungus increased plant growth, p and n uptake and reduced disease caused by fusarium oxusporum f. sp. licopersici (fol) as compared to non-mycorrhizal control plants. soil amendment with sb + rp + a. niger resulted in % and % (versus c) higher plant shoot biomass in plant-soil experiments contaminated or not with fol, respectively. in this case, disease severity and number of fol cfu reached the lowest levels while soil phosphatase and beta-glucosidase activities increased compared to all other treatments. fol negatively affected plant root mycorrhization determined in the am treatment while the difference between the mycorrhization of plants grown in the presence and absence of f. oxysporum in sb + rp + a. niger-amended soil was insignificant ( % versus %, respectively). in conclusion, the fermentation mixture containing mineralized organic matter, partially solubilized rp, and a. niger biomass could be efficiently used not only in improving plant growth, nutrient uptake and properties of degraded and polluted soils, as previously reported (vassilev and vassileva, ) , but also in environmentally-mild management of fusarium wilt. vassilev, n., vassileva, m., . appl. microbiol. biotechnol. , - . biological treatment processes allow for the effective elimination of charged inorganic micropollutants, e.g. a number of oxyanions, heavy metals, etc. from contaminated drinking water supplies. however, dedicated technologies have to be implemented in order to eliminate the target pollutants without changing the quality of treated water, avoiding its secondary pollution by cells, nutrients and metabolic by-products. some innovative technologies, which combine the use of membranes with the bioconversion of charged micropollutants in order to deal with the secondary water contamination problem, will be presented and critically compared. a special on loofa was studied. the basal mineral salt solution along with glucose, ammonium sulfate and yeast extract were used to dilute omw properly. the fungus did not grow on the concentrated omw. therefore, omw diluted by % was used thought this study. the extent of removal in this biotreatment, of total phenolics (tp) and cod were and %, respectively. while the color and aromaticity decreased by and %, respectively. the kinetic behavior of the loofa immobilized fungus was found to follow monod equation. the maximum growth rate was . h − while the monod constant based on the consumed tp and cod (mg/l) were and , respectively. advanced start-up strategy of an anaerobic three-phase turbulent bed reactor treating winery wastewaters r. cresson, h. carrère, n. bernet, j.p. delgenès laboratoire de biotechnologie de l'environnement, institut national de la recherche agronomique (inra), avenue des etangs, narbonne, france. e-mail: cresson@ensam.inra.fr (r. cresson)the objective of our study was to compare two start-up strategies for an anaerobic biofilm process, to create an effective biofilm and increase the organic loading rate (olr) as quickly as possible. two methanogenic three-phase biofilm reactors have been started, using the same operational parameters (solid hold-up ratio, gas velocity of mm s − ), in order to test two different strategies:• maximal load strategy (reactor a): the olr is increased as long as the global amount of removed cod (biogas production) increased. • maximal removal strategy (reactor b): the olr is increased stepwise as soon as the cod removal rate reaches %.both reactors have been operated for days, until a volumetric olr of g cod l − j − , with more than % of carbon removal. the total amount of cod removed and methane produced were higher in reactor b ( . and . %, respectively). in both reactors, the short hydraulic retention time (hrt) applied during all the experiment caused a rapid wash-out of planktonic bacteria and an exclusive use of the substrate by the attached micro-organisms, which accelerates the biofilm growth. the lag-phase was reduced to approximately days. the reactor submitted to repetitive disturbance by the maximal removal strategy appeared to be more robust when confronted to perturbation like organic overload or nutritional deficiency. experiments have demonstrated capability and the efficiency of the aggressive strategy for controlling anaerobic bioreactor start-up. sustainability is the generally accepted paradigm for future industrial development. the re-integration of waste products into production processes is a major aspect of environmental sustainability. in this study the use of sugar cane molasses is being investigated for the production of bioplastics by mixed microbial cultures, with the added possibility of parallel biohydrogen production. polyhydroxyalkanoates (phas) are polyesters synthesized by bacteria and accumulated as granules in the cytoplasm. studies conducted by this group have shown that mixed microbial cultures subjected to dynamic feeding conditions may accumulate phas up to % cell dry weight, a value close to that obtained for pure cultures. volatile fatty acids are good substrates for the production of phas by mixed cultures. on the other hand, sugar molasses, with a very high sugar content (about % dry weight), can produce organic acids by fermentation. the two-stage process being implemented in this study includes a molasses fermentation step, in which the high sugar content of the molasses is converted into volatile fatty acids (vfas), and a pha production step, in which the vfas serve as the precursors to the formation of phas under dynamic feeding conditions. moreover, hydrogen can be produced by anaerobic bacteria from carbohydrate-rich substrates giving organic fermentation end products, h and co . to optimize the production of both high-value products, design of experiments (doe) is being used to elaborate a set of experiments to study the effect of ph, hydraulic retention time and organic loading on both the organic acids distribution (which will serve as precursors for pha production in the second step) and h production in the acidogenic fermentation reactor (a l cstr). preliminary results show that the effluent of the acidogenic reactor fed with g/l total sugars and operated at ph and d = . h − (composed mainly of acetate and propionate) can be successfully fed to a polymer-accumulating mixed culture. under these conditions, the h production yield has been estimated at . mol h /mol sucrose. vanillin is a flavour compound used in food industry, fragrances and pharmaceutical preparations, which is nowadays mainly produced by chemical synthesis. the increased demand of natural products for the food industry as well as the high cost of natural vanillin extracted from vanilla pods has recently stimulated the research for alternatives to produce this compound by a natural way. the microbial transformation of ferulic acid, a phenolic compounds from lignin degradation, is recognized as being the most interesting alternative to produce natural vanillin. the combined effects of initial ferulic acid concentration (s ) and biomass concentration (x ) on vanillin production by resting cells of escherichia coli strain were investigated using response surface methodology. e. coli jm /pbb a recombinant strain producing key enzymes of ferulate catabolic pathway from p. fluorescens bf (feruloyl-coa synthetase and feruloyl-coa hydratase/aldolase) was utilized in this work. a full-factorial design was employed for experimental design. the results shown a possible inhibition phenomena at a vanillin concentration of about . g l − leading to the accumulation in the fermentation media of secondary compounds like vanillic acid and vanillin alcohol. removal of dissolved nutrients from wastewater using a microalgae biofilter line christensen, suvina sooknandan, jens jørgen lønsmann iversen department of biochemistry and molecular biology, university of southern denmark, odense, a microalgae biofilter can be used for treatment of wastewater from landbased fish farms in order to remove excess amounts of dissolved nutrients such as nitrate, ammonium and phosphate. a bubble column bioreactor has been developed for cultivation and characterization of microalgae. this type of bioreactor is equipped with a control system that enables online determination of the photosynthetic quotient and optimization of light intensity. furthermore the bioreactor has a dualsparging system simultaneously allowing adequate mixing and high gas-liquid mass transfer coefficients. different species of microalgae have been cultivated in batch and fed batch cultures to characterize growth and ability to take up the different dissolved nutrients. the specific growth rate and substrate uptake rate have been determined to compare and select the algal species most suited for use in a biofilter. additionally the composition of lipid, protein and carbohydrates has been measured to determine the nutritional quality of the algae when used as animal feed. at present, biological nitrogen-removal is mostly carried out through several complicated steps. to simplify the present systems for nitrogen-removal, we have investigated a new nitrogenremoval bioreactor using packed gel envelopes capable of simultaneous nitrification and denitrification. the envelope consists of two plate polymeric gels with a spacer in between. ammonia oxidizer, nitrosomonas europaea and denitrifier, paracoccus denitrificans are co-immobilized in the plate gels. when the envelopes are exposed to wastewater containing ammonia, the immobilized n. europaea oxidizes ammonia to nitrite in the outer aerobic surfaces of envelopes. at the same time, as ethanol solution is injected into the internal anaerobic spaces of envelopes, the immobilized p. denitrificans reduces the nitrite to nitrogen gas using the ethanol solution as an electron donor for denitrification. in this way, the envelopes can remove ammonia from wastewater in a single step. we have already reported advantages of our bioreactor in laboratory-scale experiments. in this study, we show our large-scale bioreactor (water volume . m ) could treat three kinds of wastewater derived from coal power plants. ammoniacontaining wastewater that occurred regularly in a coal power plant was continuously treated with the bioreactor using thirty envelopes for over a year. the bioreactor could remove more than % of total nitrogen at hydraulic retention time (hrt) of h. at hrt of h, the bioreactor accomplished a maximum rate (the transformation of nh + to n ) of . g n/day m of the envelopes' surface. the performance was equivalent to that obtained in the laboratory-scale experiments. furthermore, our bioreactor showed similar nitrogen-removal performances when it treated nitrate-containing wastewater occurring regularly and condensed ammonia-containing wastewater occurring at irregular intervals in coal power plants. these results show that our bioreactor can treat various wastewater containing nitrogen in coal power plants. thus, our concept is effective to simplify the large-scale systems in coal power plants and the other plants. in order to establish an environmentally friendly process for the treatment of metal containing waste, in a portuguese refinery a process involving sulphur oxidizing acidophilic microbes is being considered. bioleaching of metal containing bottom ash, from fluidised bed incineration of sludge resulting from the refinery water treatment station, was performed using a sulphur oxidising acidophilic culture isolated from an acid pool resulting from the weathering of sulphur piles from the claus plant. this sample served as inoculum for liquid medium cultures with % sterile sulphur flowers as source of energy. application of monod kinetics to adapted culture growth of free cells presented a value of µ = . day − . yield of sulphur conversion to sulfate after days was η = %. in the presence of bottom ash from the incineration of refinery sludges µ = . day − and the yield of sulphur conversion was η = . %. a η fe = % removal of iron is obtained from the treated ash. x-ray fluorescence spectroscopy of the solid residue revealed a total removal of metals namely, v, cu, ni, zn and most of the fe after days of bioleaching. the present of heavy metals in the environment is a serious problem. they are commonly present in effluents from mining and industrial activities. usually, chemical conventional methods are very expensive and they have limitations when heavy metals are in low concentrations. at the moment the interest increases for processes that involve microorganisms as alternative method. some effluents present heavy metal sulphates which are soluble compounds. sulphate-reducing bacteria (srb), under anaerobic conditions, oxidize simple organic compounds (such as acetic acid and lactic acid) by utilizing sulphate as an electron acceptor and generate hydrogen sulphide. hydrogen sulphide reacts with heavy metal ions to form insoluble metal sulphides that can be easily separated from a solution. the purpose of this work was to evaluate the ability of srb to reduce cr(iii), ni(ii) and zn(ii) in artificial contaminated solution. desulfovibrio vulgaris and desulfovibrio sp. strains has been tested in this study. batch cultures was carried out in ml sealed bottles with different concentrations of studied metals ( - mg/l), with % of inoculum bacterial and adapted to postgate's medium c. a gaseous nitrogen current was employed to purge oxygen and obtain anaerobic conditions. the assays were incubated statically represented very low portion (less than - %) of the total bacterial community at all temperatures tested. schistosomules of schistosoma mansoni ( days old) were incubated in rpmi medium containing % fetal calf serum, % hamster portal venous or % hamster peripheral venous serum (highly susceptible host) or % rat portal venous or % rat peripheral venous serum (poorly susceptible host) in presence of bromodeoxyuridine (brdu) in order to measure differences in cell proliferation. also the rate of cell proliferation of s. mansoni were assessed in vivo in hamster to study the cell proliferation in the natural ontogeny of the organism. the rates of cell proliferation as expressed by brdu labeling indices (blis) were determined as a function of time of incubation by immunohistochemistry using monoclonal antibody to brdu. compared to schistosomules cultured in presence of rpmi plus % fetal calf serum, blis were increased by % in the presence of hamster portal, but not in peripheral serum. while in case of rat, no significant changes were observed in the blis in both portal and peripheral sera. the experiment was repeated using hamster portal and peripheral sera containing different schistosomal igg antibody titres. the results showed decreased values of blis compared to sera which did not contain the schistosomal antibody(ies). the in vivo results revealed that there was no cell proliferation of s. mansoni schistosomules ( days old) in the lungs. cell proliferation was detected in schistosomules of days old and the results revealed a significant decrement in the brdu labeling indices (blis) with the increase of the age of schistosomules in vivo. the results indicated that hamster portal venous serum (highly susceptible host) could have stimulating factor(s) for schistosomule cell proliferation which is not found in rat (poorly susceptible host) and the presence of antibody(ies) greatly inhibit the cell proliferation. this could be due to the blocking of some portal serum factors, which stimulate the cell proliferation by the antibody(ies). the release of dyes into the environment constitutes only a small proportion of water pollution, but dyes are visible in small quantities due to their brilliance. many dyes are difficult to decolourise due to their complex structure and synthetic origin. the adsorption of reactive dye remazol brilliant blue r (rbbr) on polyelectrolyte complex (pec) was studied in a batch systems. the adsorption parameters determined were: effect of the different values of ph on the adsorption of dye by pec, and the effect of contact time on the amount of rbbr adsorbed (in mg g − ). the data indicates that the adsorption capacity of rbbr by pec is dependent on ph, the maximum adsorption at ppm was . % equal to . mg dye/g of polymer. the results show a tendency towards greater adsorption for reactive dyes (ph range of - ). the effect of contact time was studied at initial concentration ( ppm) of dye, the amount of rbbr adsorbed for these pec increased and reached a constant value with the increase in contact time. the increase in the extent of removal of dye after min of contact time is less and hence it is fixed as the optimum contact time. the pec show their capacity to remove rbbr to aqueous solutions by adsorption. compared to other european nations, the austrian population shows a low level of knowledge in biosciences and a strong denial of gene technology ( ). the austrian non-profit organisation dialog<>gentechnik, a scientific society, organizes various activities to raise awareness for the "hot topics" in the life sciences. according to its principle of independence, all activities are funded publicly. projects on behalf of the austrian authorities and international cooperations demonstrate credibility and trust in dialog<>gentechnik ( ). a few examples will be presented. dialogue with the public: on the occasion of the first anniversary that the gmo labelling rules became effective, the action "gene technology on my plate" is performed austrian wide in shopping centres. here, consumers are informed about health and labelling aspects of gm food. two days of open discussions were organized in the context of the austrian genome research program gen-au ( ): topics were "gene diagnosis" ( ) and "genome research-what is in it for me?" ( ) . an open lab is currently set up in vienna to offer "hands on" experience in life sciences for everybody. motivating students: in the very successful gen-au summer school ( ), high school students spend - weeks in the lab and work with scientists. the best documentations are awarded. in an innovative project, student groups (age - ) work on the topics stem cells and cloning and develop units of an elearning course which will be accessible for all austrian schools in the near future. engaging stakeholders: dialog<>gentechnik manages interdisciplinary wor king groups that develop leaflets, brochures and questionnaires on various aspects of gene diagnosis. four products are currently distributed to the public and to health services. ( ) european commission. eurobarometer . , december ; ( ) www.dialog-gentechnik.at; ( ) www.genau.at. the aim of this paper is to compare the attitudes of the urban consumers with professionals towards to new technologies, especially to biotechnology. it was tried to find out in which area, medical, agriculture or industry, people can accept biotechnological developments and in which area not accept. key: cord- -o hvuwvq authors: harvey, david j. title: analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: an update for – date: - - journal: mass spectrom rev doi: . /mas. sha: doc_id: cord_uid: o hvuwvq this review is the sixth update of the original article published in on the application of maldi mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of . general aspects such as theory of the maldi process, matrices, derivatization, maldi imaging, arrays and fragmentation are covered in the first part of the review and applications to various structural typed constitutes the remainder. the main groups of compound that are discussed in this section are oligo and polysaccharides, glycoproteins, glycolipids, glycosides and biopharmaceuticals. many of these applications are presented in tabular form. also discussed are medical and industrial applications of the technique, studies of enzyme reactions and applications to chemical synthesis. © wiley periodicals, inc. mass spec rev : – , . this review is a continuation of the six earlier ones in this series (harvey, (harvey, , (harvey, , (harvey, , (harvey, , (harvey, , on the application of matrix-assisted laser desorption/ionization mass spectrometry (maldi) mass spectrometry (ms) to the analysis of carbohydrates and glycoconjugates. it is intended to bring the coverage of the literature to the end of . although the intention is to be a comprehensive as possible, there is an increasing tendency to publish maldi data in supplementary information. because of the ever-increasing number of papers and journals, it has not been possible to check all supplementary information and, consequently, papers that do not refer to maldi in the main text may well have been omitted. also omitted are papers that simply report the mass of glycoproteins and those concerned with nucleotides and nucleosides: the latter compounds, although containing carbohydrates are considered to be different types of compound. maldi continues to be a major technique for the analysis of carbohydrates; figure shows the year-by-year increase in papers reporting use of the technique for the period - . because the review is designed to complement the earlier work, structural formulae, etc. that were presented earlier are not repeated. however, a citation to the structure in the earlier works is indicated by its number with a prefix designating the review containing the structure (i.e., /x refers to structure x in the first review and /x to a structure in the second). a large number of books and review articles directly concerned with, or including maldi analysis of carbohydrates and glycoconjugates, have been published during the review period. those of a general nature are listed in table ; those concerned with specific carbohydrate types are listed in the appropriate sections. details of the maldi process are still not fully understood and several investigators have attempted to obtain greater understanding. although not all of these studied have involved carbohydrates, they are included here because maldi processes for different compounds are likely to be similar. one of the commonly accepted models for the formation of analyte ions in maldi-ms assumes a primary ionization of the matrix, for example, by photoionization leading, among other things, to stable protonated and deprotonated matrix ions. peptide and protein ions are then envisaged as being formed by secondary proton transfer reactions in the expanding matrix plume. this model has been checked experimentally by hillenkamp et al. ( ) by comparing the yield of positive to negative ions of three peptides (bradykinin, angiotensin i and fibrinopeptide a) and six matrices a-cyano- -hydroxycinnamic acid (chca, / ), , dihydroxybenzoic acid (dhb, / ), -azo- -thiothymine (att, / ), -nitroaniline ( -na, / ), -amino- -nitro- -picoline (anp, ) and -aminoquinoline ( -aq, ), differing in gas-phase basicity by about kj/mol. it was shown that the observed ion yields cannot be explained by any single and consistent set of parameters such as gas-phase basicity or acidity of the analyte and matrix. it was concluded that the existing simple model needs be modified to fully explain the experimental findings. liu et al. ( a) have used synchronized dual-polarity maldi ms to demonstrate incoherent production of positive and negative matrix ions. in both positive and negative ion modes, matrix ions were found to appear from thin, homoge-neous dhb matrix films at different threshold laser fluences. the presence of singly charged molecular matrix ions suggests that the existence of dhb ion-pairs may not be a prerequisite in the maldi process. photoelectrons induced by the laser excitation may assist the production of negative dhb ions, as shown in experiments conducted with stainless steel and glass substrates. at high laser fluences, the relative yield of positive and negative matrix ions remained constant when homogeneous matrix films were used, but the yield fluctuated significantly with inhomogeneous crystal morphology. this result was also inconsistent with the hypothesis that matrix ion-pairs are essential primary ions. thus, results from both low and high laser fluences suggest that the production of positive and negative matrix ions in maldi may occur via independent pathways. the same authors have examined the initial ionization reaction in maldi based on the appearance of photoelectrons. the threshold laser fluence for the ejection of photoelectrons from dhb, sinapinic acid ( / ) and , , trihydroxyacetophenone (thap, / ) on stainless steel targets was found to be . , . , and . mj/cm , respectively. these values were considerably lower than those for maldi ions, indicating that the electron detachment probably precedes other ionization reactions. the stainless steel target was thought to general review with a section on glycolipids - glycan analysis by mass spectrometry short review, maldi and esi, applications to n-linked glycans (sekiya & iida, ) deciphering carbohydrate structures by ion mobility ms short general review of glycomics and ion mobility. applications to flavonoids, gags and glycoproteins modern maldi-tof mass spectrometry development of tof mass spectrometers since the introduction of maldi (vestal, ) maldi mass spectrometry of carbohydrates short general review (in chinese) - play an insignificant role in the production of photoelectrons because suspended dhb produced a photoelectron signal similar to dhb on the surface. in addition, decreasing the dhb thickness on the target reduced the photoelectron intensity. for crystalline dhb and sinapinic acid, the photoelectron intensity was found to increase with the laser fluence (nitrogen laser at nm) in less than a second order relationship, suggesting considerable reductions of ionization potentials in comparison with free molecules. according to ab initio calculations, the ionization potential of dhb clusters was found to reduce as the cluster size increased from monomer to octamer. the paper discusses the impact of these abundant electrons on ion production in maldi. the earlier rate equation model for maldi ion formation and reaction (knochenmuss, (knochenmuss, , , has been extended to include positive and negative ions of both matrix and analyte (knochenmuss, ) . the resulting positive/negative ratios of secondary analyte ions show that a recent static equilibrium approach is not adequate for quantitative analysis of maldi experiments. although the ion ratios remain close to unity whenever the reaction free energies are at least moderately favorable, deviations from this condition result in unequal ratios of oppositely charged ions and show once again that the dynamic aspects of maldi cannot be neglected. molecular dynamics simulations of maldi have been performed to investigate laser pulse width and fluence effects on primary and secondary ionization process. at the same fluence, short ( or psec) pulses were found to give much higher initial pressures and ion concentrations than longer ones ( ns). these differences were found not to persist because the system relaxes towards local thermal equilibrium on a nanosecond timescale. higher fluences were found to accentuate the initial disparities. axial velocities of ions and neutrals were found to span a wide range and to be fluence-dependent. the total ion yield was found to be only weakly dependent on the pulse width and to be consistent with experimental estimates. secondary reactions of matrix cations with analyte neutrals were efficient even though analyte ions were ablated in clusters of matrix (knochenmuss & zhigilei, ) . lai et al. ( ) have employed transition state theory for modeling the desorption of surface ions, assuming chemical and thermal equilibrium in the solid state prior to desorption. the method was different from the use of conventional models that assume chemical equilibrium in the gas phase. this solid-state thermodynamic interpretation was used to examine the desorption of thap and of an angiotensin i/thap mixture. it successfully described the changes in ion yield with the effective temperature under various laser fluence and initial temperature conditions. the analysis also revealed the key role played by ion concentration in the modeling used to provide the best fit of the model to observations. divergence of the ion beam with laser fluence was also examined using an imaging detection method and the signal saturation normally seen at high fluence was appropriately reduced by ion focusing. simplified but deceptive theoretical interpretations were obtained when the analysis was conducted without adequate calibration of the instrument bias. the laser plume produced by several ionic liquid matrices has been studied by a post-ionization approach in which the neutrals in the ablation plume were ionized with a second laser pulse. it was found that after the initial event that produced the ions, a second, time-delayed, ablation event occurred in which the plume contained only neutral molecules. the presence of these neutral molecules was explained by a reflected-shockwave model in which the shockwave emerging from the laser ablation is reflected from the sample plate behind the sample. it was assumed that when the shockwave arrived at the sample surface it caused a second ablation of the neutral molecules (hellwig et al., ) . the nm multiphoton dissociation and ionization of , -dihydroxyacetophenone (dhap, / ) has been studied (dyakov et al., ) . the experimental results indicate that photoionization that occurs in the gas phase after dhap vaporizes from the solid phase may not play an important role in the maldi process. a combined ir-maldi ion source with an electrospray ionization (esi) emitter for post desorption ionization has been described (sampson, murray, & muddiman, ) . the source produced multiply charged ions from proteins but singly charged ions from carbohydrates (o-glycans cleaved from mucin (muc) were tested) and avoided the fragmentation produced by some other techniques. ir-maldi ms with a laser emission in the mm wavelength range, which utilized energy absorption at the co double-bond stretch region, has been investigated for analysis of several types of biomolecule. the softness of ir-maldi ms was evident in the negative ion mode where abundant [mÀh] À ions were obtained for acidic biomolecules with sulfate, phosphate, or carboxylate groups. better sensitivity was obtained than with ultraviolet (uv) maldi ms. furthermore, there was no substantial loss of sialic acid due to the prompt fragmentation from sialylated glycoconjugates such as gangliosides. such loss is a common problem with maldi analysis of sialylated carbohydrates. the technique was used in conjunction with a potent new matrix, oxamide ( ), resulting in the use of low laser fluence, and removing one of the limitations of maldi ms for biomolecular analysis of uv-maldisensitive molecules (tajiri, takeuchi, & wada, a) . tu and gross ( ) have reviewed methods for miniaturizing sample spots for maldi analysis. topics include minimizing sample dispersion by target modification, the use of hydrophobic materials as maldi-plate surfaces, the use of microdispensing devices such as piezoelectric dispensers and the use of droplet charging by induction or polarization. a simple device for maldi sample preparation, based on the spraying of matrix/sample solution through a stainless steel sieve, has been used for the preparation of maldi samples of peptides, polysaccharides (pss) and high molecular weight (mw) proteins (cristoni et al., ) . the spectra obtained by laser irradiation of the resulting microspots exhibit resolution and sensitivity higher than those achieved by the commonly employed dried droplets method. furthermore, the target surface was more homogeneous than those obtained by the dried droplet method. chca and super-dhb (dhb þ -oh, -ome-benzoic acid) (s-dhb) matrices were used and applications were to insulin and dextran oligomers. ultrasound produced by a simple piezoelectric device has been used as an alternative method for soft ionization of biomolecules. cavitation was proposed as the major mechanism producing the ions and the technique was applied to carbohydrates, proteins and fatty acids. however, although an abundant ion, said to be the [mþh] þ from the high-mannose glycan man glcnac ( / ), was obtained in the presence of dhb, thap, or sinapinic acid, neither the mass nor the stated elemental composition were consistent with this structure . the recent availability of commercial ion mobility instruments offers another dimension to carbohydrate analysis by providing the ability to separate by molecular shape and offering the possibility of rapid isomer differentiation. however, resolutions on current instruments are comparatively poor and do not match those that can be obtained with high performance liquid chromatography (hplc). a comparison of three types of ion mobility ms (field asymmetric waveform ion mobility (faims), drift tube and traveling wave ion mobility spectrometry (twims)) for separation of chiral molecules with applications to monosaccharides has been reported (enders & mclean, ) . data are best reported as rotationally averaged collisional cross-sections. these can be obtained directly with drift tubes and indirectly with the twims instruments. obtaining such measurements with faims instruments, however, is more challenging. collisional-cross sections have been measured for a large number of biologically relevant molecules including oligonucleotides (n ¼ ), carbohydrates (n ¼ ), lipids (n ¼ ), and peptides (n ¼ ). collisional cross sections increased with mass but were found to be different for each molecular type in the order oligonucleotides < carbohydrates < peptides < lipids. the specific correlations were best described by logarithmic regressions. thus, the technique was able to separate compounds of different structures but with the same or similar mws. in addition, some separation of compounds with the same mass within a particular class was possible. the latter point was demonstrated by separations of isobaric oligonucleotides, which were interpreted by molecular dynamics simulations mclean, ) . have used ion mobility to extract carbohydrate ions from incubation mixtures obtained from protein-n-glycosidase (pngase) f release of n-glycans. glycoproteins such as ribonuclease b (rnaseb) were first digested with trypsin followed by pngase f and then analyzed directly by ion mobility ms with a waters synapt instrument. the carbohydrate ions showed different mobilities from peptide and other contaminating ions allowing them to be extracted directly from the crude mixtures. both esi and maldi ion sources were used; the maldi ion source was better at ionizing the larger carbohydrates and did not suffer from the problem of producing both [mþh] þ and [mþna] þ ions that were seen in the esi source. the technique would appear to have great potential for rapid glycan analysis, particularly as the synapt instrument also offers the ability to fragment the isolated ions. hossain and limbach ( ) have reviewed matrices used for maldi analysis of several compound classed including carbohydrates. the review covers common matrices such as dhb and chca and less common systems such as liquid matrices and carbon nanotubes ( references). the use of ionic liquid matrices has also been included in a review by liu et al. ( e) although the bulk of this review discusses the use of ionic liquids for sample preparation. the thermal stability of several commonly used crystalline matrices, , -dhb, thap, chca, sinapinic acid, nor-harmane ( / ) and harmane ( / ) has been studied by heating them at their melting point and characterizing the remaining material by a variety of spectroscopic and chromatographic techniques. in general, all compounds, except for chca and sinapinic acid, remained unchanged after fusion. chca showed loss of co , yielding a trans-/cis- -hydroxyphenylacrylonitrile ( ) mixture. sinapinic acid showed mainly cis-to-trans thermal isomerization and, with very poor yield, loss of co (tarzi, nonami, & erra-balsells, ) . h-pteridine- , -dione (lumazine, ) has been described as a good matrix for phospholipids where the presence of cationcontaining compounds suppresses signals from neutral compounds. phosphatidyinositol was reported to give a signal that was an order of magnitude higher than that obtained from dhb (calvano, carulli, & palmisano, ) . a number of papers have reported the use of various nanoparticles as matrices. unlike traditional chemical matrices, these materials generally produced little or no signal in the low mass region, thus making them ideal for analysis of small carbohydrates. titanium dioxide micro-and nano-particles, prepared by hydrolysis of ti butoxide and maintained in aqueous solution, have been evaluated as matrices for the detection of several small molecules. most of the reported applications were to lipids but the nanoparticles were also applied to flavonols/anthocyanins and their glycosides in rose petals. the spectra showed ions from many more small molecules than did spectra recorded from dhb, particularly in the region of the dhb matrix ions. one advantage of the nanoparticles is that their small size (average of about nm) facilitates their penetration into tissue for in situ imaging (lorkiewicz & yappert, ) . titanium dioxide (tio ) has also be used by kawasaki, okamura, and arakawa ( ) for ionization of several types of compound, including carbohydrates, with a similar absence of low mass matrix ions. in a study of the most suitable crystalline form, the anatase-type tio , was shown to be the best. nanoparticles of diamond, tio , titanium silicon oxide, barium strontium titanium oxide, and silver have been examined for their potential as maldi matrices for direct laser desorption/ionization of carbohydrates, especially fructans, from plant tissue. two sample preparation methods including solventassisted and solvent-free (dry) deposition were compared. all examined nanoparticles except ag were found to desorb/ionize standard sucrose ( ) and fructans in both positive and in negative ion mode. in positive ion mode, sugars gave [mþna] þ and/or [mþk] þ ions depending on the ionic composition of the sample spot, and [mÀh] À ions in negative mode. ag nanoparticles yielded good signals only for non-salt doped samples that were measured in the negative ion mode. when used to study fructans in plant tissues all the nanoparticles except ag could desorb/ionize carbohydrates in both ion modes. nanoparticles gave similar limit of detection (lod) for standard fructan triose ( -kestose, ) in the positive ion mode and better lods in the negative ion mode than those given by the common crystalline organic maldi matrices such as dhb, nor-harmane or carbon nanotubes. although lower signal-to-noise ratio (s/n) signals were obtained from the tissues with solvent-free matrix deposition than when solvent was used, the reproducibility averaged over all sample was more uniform (gholipour et al., ) . maldi detection at the level of several hundred zmoles has been achieved by the addition of gold nanoparticles (aunps) with a diameter of several tens of nanometers into a sample solution (shibamoto et al., ) . n-acetyl-tetraose fmol/ml gave a strong signal in the presence of nm aunp ( .  particles). the mechanism appears to be related to surface plasmon (sp) excitation of the aunps. citrate-capped aunps have been shown to act as matrices for the determination of several types of biomolecule, including carbohydrates such as starch, dextrins, and glycosphingolipids (gsls) in the presence of high concentrations of salt (wuhrer, koeleman, & deelder, b) . with the gsls, however, some loss of sialic acid ( / ) was found. a combination of immobilized silica and dhb on iron oxide magnetic nanoparticles has been shown to give a clean background and to be appropriate for the analysis of a range of small molecules, including glycolipids . the ratio between sio and dhb was found to affect the surface immobilization of dhb on the nanoparticle, critically controlling the ionization efficiency and background. enhancements of molecular ion signal over those produced from dhb alone were noted and high quality product-ion spectra were obtained. fullerene ( )-derivatized silica (pore size nm) also appears to be good for small molecules including monosaccharides (szabo et al., b) . multifunctional zro nanoparticles and zro -sio nanorods have been successfully used as matrices for cyclodextrins in atmospheric pressure and vacuum maldi. the lod for cyclodextrins was found to be . - fmol and the matrices were used to analyze cyclodextrins in urine samples (kailasa & wu, ) . manganese dioxide nanoparticles have been used to ionize ginsenosides with the advantage that the spectra lacked matrix ions in the low mass region allowing low-mass postsource decay (psd) ions to be clearly visible. the technique was named nanoparticle-assisted laser desorption/ionization (nano-paldi) (sahashi, osaka, & taira, ) . single-crystalline euf hexagonal microdisks with hollow interiors have been prepared as background free matrices. the long-lived excited state of europium ions is capable of transferring energy to the molecules allowing the microdisks to act as matrices. they were successfully used for analysis of small peptides, amino acids and hydroxypropyl b-cyclodextrin ( ) (chen et al., d) . another new matrix with low background is mesoporous silica, sba- , functionalized with quinoline . the material was obtained by using calcined . compared with dhb and sba- itself, the modified material demonstrated several advantages in the analysis of small molecules such as less background interference ions, high homogeneity, and better reproducibility. detection limits in the region of pmol were reported. in a more traditional approach for reducing low-mass matrix ions, fujita et al. ( ) have used b-cyclodextrin ( / ) mixed with thap or , -dhap. the latter compounds, in particular, formed inclusion complexes with the cyclodextrin as demonstrated by the lack of a similar effect when the corresponding linear carbohydrate, maltoheptaose ( ) was used as a co-matrix. a method for recording negative ion spectra that is suitable for small molecules in that it produces no matrix-related ions in the low mass region uses the proton sponge, , -bis(dimethylamino)naphthalene (dman, ) as a solution in ethanol as the matrix. this compound has very high proton affinity and can take up protons from even weak acids to form deprotonated anions. moreover, dman in solution has a strong uv absorption band in the region - nm, fully compliant with frequencies of nitrogen and neodymium-doped yttrium aluminium garnet (laser) (nd:yag) uv lasers. the matrix was found to be suitable for a range of small molecules including fatty acids, carbohydrates and prostaglandins (shroff & svato, ; shroff & svatos, ; . the authors proposed to name the technique as matrix-assisted ionization/laser desorption, abbreviated as maild, ms . n(ch ) (ch ) n , -bis(dimethylamino)naphthalene, zhang et al. ( i) have reported a new method for the analysis of small molecules by using matrices such as metalphthalocyanines (mpc, ) which form matrix-analyte adducts and shift the molecular ions into a high and interference-free mass range. the mass of the target analyte was calculated by subtracting the mass of mpc from the mass of the mpc-analyte adduct. the mpcs themselves were also detectable and could serve as internal standards. various mpcs with aromatic or aliphatic groups and different metal centers were explored. aluminum-phthalocyanines (alpcs), gallium-phthalocyanines (gapcs), and indiumphthalocyanines (inpcs) were found to be efficient matrices for a large number of compound types and formed mpc-analyte adducts in either the positive or negative ion mode. the detection limits varied from to fmol, depending on analyte. mass spectrometry reviews doi . /mas graphene has also been reported to be a good matrix for low mw compounds with essentially no ions from the matrix (dong et al., ) . colloidal graphite has also been used for atmospheric pressure maldi and has been shown capable of producing both [mþh] þ and [mÀh] À from many types of small molecules, including glycosylated flavonoids (perdian, schieffer, & houk, ) . two reviews on ionic liquids, including their use as maldi matrices have been published (soukup-hein, warnke, & armstrong, ; sun & armstrong, ) in an extensive study to find new liquid matrices matrices were tested and new ionic liquids were prepared (crank & armstrong, ) . both the anionic and cationic moieties were varied systematically to find a matrix with the best physical properties, analyte signal intensity and widest mass detection range. the developed matrices showed a wide mass detection range (< , da to > , da) for proteins and peptides with greater s/n ratios than solid matrices and could effectively ionize proteins of large mass without disrupting noncovalent interactions between monomers. it was found that both the proton affinity and pk a of the cation have a large effect on the ability of the matrices ionize the analyte. the matrices could be used to detect carbohydrates with fewer degradation products than solid matrices. n,n-diisopropylethylammonium a-cyano- -hydroxycinnamate and n-isopropyl-nmethyl-t-butylammonium a-cyano- -hydroxycinnamate were the best matrices for proteins and peptides, while n,n-diisopropylethylammonium a-cyano- -hydroxycinnamate and n,ndiisopropylethylammonium ferulate were found to be the best matrices for carbohydrates. another novel ionic liquid matrix has been made from the , , , -tetramethylguanidinium ( ) salt of thap and found to be well suited for the maldi analysis of glycopeptides and glycans, particularly as it appeared to overcome the well-known ionization suppression of carbohydrates in the presence of peptides. for example, even glycopeptides containing short peptide backbones and large carbohydrate moieties gave high signal intensities when using this matrix even though they did not produce spectra directly from thap. in a second experiment, glycans were released with pngase f from total tryptic digests derived from glycoproteins and analyzed by maldiquadrupole ion trap (qit)-time-of-flight (tof). when using the liquid matrix, it was possible to detect the glycans with high intensities in the presence of the tryptic peptides, whereas, once again, glycan ionization was completely suppressed when measured with thap alone. the extent of metastable decay of glycopeptides was also found to be reduced when using the liquid matrix (ullmer & rizzi, ) . ionic liquid matrices have shown considerable advantages over conventional matrices for maldi-ms analysis of polysulfated carbohydrates such as heparin and heparin sulfate. these compounds are not easily analyzed by uv-maldi ms analysis because of the thermal lability of their o-and n-so moieties. two new ionic liquid matrices based on the combination of -( -hydroxyphenylazo)benzoic acid (haba, / ) with , , , -tetramethylguanidine or spermine ( / ) have been successfully applied to the analysis of these compounds (przybylski et al., b) . these matrices gave improved signalto-noise ratios as well as a decrease of fragmentation and desulfation. sulfated oligosaccharides were detected with higher sensitivity than with the usual crystalline matrices, and their intact sulfated ions [mna] À were easily observed. maldi-ms characterization of challenging analytes such as heparin octasaccharide carrying eight o and four n-sulfo groups, and heparin octadecasulfated dodecasaccharide was also successfully analyzed. in a paper published in , gomenez et al. ( ) reported that dhb, with vacuum drying to improve target spot homogeneity, was a better matrix than sinapinic acid for obtaining reliable molecular mass values of intact glycoproteins because it prevented sugar fragmentation. in a follow-up to this work, the group have investigated the use of liquid matrices prepared from dhb and sinapinic acid with the aim of avoiding the vacuum drying step (giménez et al., ) . the best results were obtained with a variety of glycoproteins such as bovine a acid glycoprotein, bovine fetuin and human transferrin (tf) from matrices prepared by adding an equimolar amount of butylamine ( . ml) to , ml of a mm solution of sinapinic acid or dhb in meoh. the mixture was vortexed and sonicated for min, evaporated to approximately ml with air, and finally reconstituted with ml of etoh or mecn, for dhb and sinapinic acid mixtures, respectively. furthermore, it was noted that both matrices gave the same masses for the tested glycoproteins that agreed with literature values, suggesting that no fragmentation of the carbohydrate moieties had occurred. a matrix consisting of chca and aniline ( ) has also proved to be successful for a number of different compounds, including raffinose ( ) (calvano, carulli, & palmisano, ) . although it was noted that this matrix gave a stronger signal than chca alone, the latter matrix is not very efficient in ionizing carbohydrates. a comparative investigation of several matrices for analysis of the small carbohydrates, glucose (glc, / ) and sucrose ( ) has been performed by yang et al. ( c) . of the matrices studied, sodiated dhb, carbon nanotubes, an ionic liquid matrix of dhb-pyridine, a binary matrix of dhb-aminopyrazine ( / ), zinc oxide nanoparticles and aunps, the best sensitivity was provided by the carbon nanotubes. the detection limit was pmol. both carbon nanotubes and the ionic liquid matrix exhibited the highest reproducibility. tzeng, zhu, and chang ( ) have investigated doping various maldi matrices with alkali metal hydroxides. it was found that for neutral underivatized oligosaccharides, the addition of % naoh to dhb caused partial alkaline degradation by glycosidic cleavages upon laser desorption. the effect intensified when nonacidic compounds such as thap or -amino- -mercapto- , , -thiadiazole (amt, / ) were used as the matrix. the degradation allowed identification of the reducing end residue of the analyte and facilitated its structural characterization by psd tof-ms. use of matrices consisting of lioh and thap or amt led to the production of singly as well as multiple lithiated ions. multiple lithiation appeared to occur with carbohydrates containing free -oh groups. up to li atoms were found to be incorporated for maltoheptaose, b-cyclodextrin, and dextran . such a "lithium tagging" technique makes it possible to differentiate positional isomers of milk-neutral oligosaccharides, lacto-n-difucohexaose i and ii (lndfh-i ( ) and lndfh-ii, ( )), without the need of chemical derivatization or tandem ms analysis. lndfh-ii, = glcnac, = galactose, = fucose, = glucose (for details, see (harvey, et al., c) choi and lee ( ) have studied the ionization efficiencies of maltooligosaccharides (degree of polymerization, dp - ) with the cations na þ , li þ , and k þ in salts containing tfa À , cl À , and oh À with dhb as the matrix. the signal strength rose with the number of glucose units with sodium consistently giving the most intense signals. the nature of the anion also affected the ionization with the tfa À salts generally being the most effective. a sample preparation method developed by nishikaze and amano ( ) has been compared with the conventional drieddroplet or ethanol (etoh) recrystallization methods and reported to give superior results in terms of ion yield and signalto-noise ratio. the method, named the "reverse thin layer method" consists of first, complete drying of the oligosaccharide solution on the maldi target plate and then deposition of the matrix dissolved in a small amount of meoh. using this method, a relatively homogeneous matrix crystal was generated and higher yields of both positive and negative ions were obtained. the authors comment that the method could be applied to various other matrices including both solid and liquid matrices. a method for direct archaebacterial glycolipid and lipid analysis by maldi ms in intact membranes, without prior extraction/separation steps, has been developed by angelini et al. ( ) . the purple membrane isolated from the extremely halophilic archaeon halobacterium salinarum was used as a model, lyophilized and ground with -aminoacridine ( -aa, / ). the mixture was crushed in a mechanical die press to form a thin pellet, small pieces of which were attached directly to the maldi target. in parallel, solution spectra of individual phospholipids and glycolipids, were analyzed by maldi-tof/ ms using -aa as the matrix. results show that -aa is a suitable matrix for the conventional maldi-tof/ms analysis of lipid extracts from archaeal microorganisms, as well as for fast and reliable direct dry lipid analysis of lyophilized membranes. a new technique termed matrix-free material-enhanced laser desorption/ionization mass spectrometry (mf-meldi-ms) has been described which employs a single compound prepared by immobilizing bradykinin on silica gel coupled to -( triethoxysilylpropylureido)azobenzene ( ) for both the maldi matrix and a stationary phase for thin-layer chromatography (tlc). the technique was applied to the analysis of carbohydrate reference standards such as glucose, sucrose, raffinose and plant extracts from quercus robur (oak). carbohydrates formed [mþna] þ and abundant [mþk] þ ions. the meldi material adsorbs the laser energy sufficiently for desorption and ionization and delivered excellent results in respect to signal-to-noise (s/n) ratio (s/n ratio: > / ) and sensitivity with a lod in the low ng/ml range. for preparation of the tlc plates, the modified silica gel was suspended in methanol, acetone, acetonitrile or acetonitrile/water ( : ) for min. about - ml of the suspension was applied in narrow channels cut into a stainless steel target in the form of a thin layer and dried at room temperature. the sample was placed on this layer for subsequent tlc in n-buoh/acetone/acetic acid/ h o ( : : : ) as mobile phase (qureshi et al., ) maldi imaging has seen many developments during the review period and several reviews and related articles have been published; details are in table . a mass spectrometer with ion mobility separation capability (waters synapt) has been used by snel and fuller ( ) to produce high spatial resolution images of glucosylceramide ( ) in the spleens of mouse models of gaucher disease. the matrix (chca) was applied to the tissue sections with an airbrush. for data acquisition, the laser was continually fired at one position until no more ions were observed and then the sample was moved by mm (laser diameter about mm). ions were generated from only the un-irradiated surface at each of these positions achieving an effective spacing of mm. at this spacing, it was possible to visualize macrophages enriched in glucosylceramide which could be distinguished from other cell types in the spleen. current maldi mass spectrometric imaging (msi) spatial resolution is typically limited by the diameter of the laser spot-size, which is usually between and mm, covering an area equivalent to tens of mammalian cells. perdian and have addressed the problem of acquiring high resolution imaging with high resolution ms on an orbitrap mass spectrometer. at a spatial resolution of mm, the orbitrap mass analyzer is able to image a ,  , mm sample area in - min with one scan per step, depending on the resolution. if the spatial resolution is increased to mm, the same size sample area will take more than - hr to complete the experiment, a time that is not practical in the normal laboratory. however, because the orbitrap also has a linear ion trap (it) analyzer, this was utilized, together with a two-motion plate movement to reduce the time while maintaining the resolution. thus, for every pixel position on the target, the laser spot was moved in a spiral fashion such that both orbitrap and ms/ms data were acquired. with a fast nd:yag laser the data acquisition time was decreased by - % compared to that from orbitrap-only scans; however, % or more time could be saved for higher mass resolution and with a higher repetition rate laser. using this approach, a high spatial resolution of mm was maintained at it imaging, while orbitrap spectra were acquired at a lower spatial resolution, - mm, all with far less data acquisition time. furthermore, various ms imaging methods were developed by interspersing ms/ms and ms fragmentation n times (ms n ) it scans during orbitrap scans to provide more analytical information. the method was applied to several flavonol glycosides from an arabidopsis flower petal in which ms/ms, ms n , it, and orbitrap images were all acquired in a single data acquisition. spectra were acquired in negative mode and no matrix was required. for uv-absorbing compounds such as flavonoids and their glycosides, a matrix is not necessary for imaging as demonstrated by imaging at the single cell level of secondary metabolites in arabidopsis thaliana and hypericum species (hölscher et al., ) . the highest spatial resolution achieved, mm, was much higher than that achieved by commonly used maldi) imaging protocols. a new matrix-free technique called nanostructure-initiator mass spectrometry (nims) has been developed for the analysis e c n e r e f e r s n o i t a t i c s t n e t n o c t c e j b u s general review short article, mainly applications (murayama et al., ) general review methods and applications to peptides and lipids (including glycolipids) general review short general review (award lecture) general review from origins to state of the art (francese et al., a) concise review of mass spectrometry imaging (sugiura et al., b) and tissue imaging of carbohydrates and steroids (patti et al., ) . analysis was accomplished by spray depositing nacl or agno with a fused-silica picotip emitter onto a porous silicon surface to provide a uniform layer rich with cationization agents prior to desorption of the fluorinated polymer initiator. upon laser irradiation, carbohydrates produced [mþna] þ ions whereas steroids formed [mþag] þ . for glucose, a plot of concentration against the c/ c ratio was linear with a correlation coefficient r of . over the range - mm. carbohydrates and steroids could be detected down to the -amol and -fmol levels, respectively. the ability of the method to perform tissue imaging was demonstrated by imaging the distribution of sucrose in a gerbera jamesonii flower stem and the distribution of cholesterol ( ) in a mouse brain. the flower stem and brain sections were placed directly on the ion-coated nims surface without further preparation and analyzed directly. no deposition of a matrix onto the sample surface was needed. a similar matrix-free method, termed nano-assisted laser desorption-ionization (naldi) ms for tissue imaging has been demonstrated by vidová et al. ( ) . commercially available nano-structured surfaces were used as substrates for imprinting tissue sections. the lithographic transfers were washed and the lipids were imaged by laser desorption mass spectrometry (ldms). the naldi imaging of lipid transfers was compared with standard maldi imaging of matrix-coated (chca) tissue sections and the resulting images were found to be of the same quality with no spatial information being lost due to the imprinting process. naldi imaging was reported to be faster due to the absence of the time-consuming matrix deposition step, and the naldi mass spectra were less complex and easier to interpret than maldi spectra. the method was applied to the analysis of phospholipids, gsls and glycerophospholipids in mouse kidney slices. naldi ms was able to identify the same lipid species as maldi and was reported to provide better distinction between kidney and adrenal gland tissues based on the lipid analysis. miura et al. ( a) have developed a maldi imaging system that is claimed to be able to image from single cells in thin tissue sections. an indium tin oxide-coated glass slide marked with a mm-wide mesh work to enabled matching of optical and ms images was used for mounting tissue sections. a suspension of hela cells in culture medium was mounted onto the slide and incubated for hr at ˚c. cells were then washed with phosphate-buffered saline (pbs) and the appearance of single cells adhering to the glass was observed by optical imaging. the single cell-adhered glass slide was then mounted onto maldi sample plate with a parallel experiment involving brain tissue slices and the matrix, -aa, was applied with an airbrush. several metabolite peaks were detected at the position of the single cell. adenosine triphosphate (atp, ; m/z . , identified by comparison with a standard sample) was identified with a good signal-to-noise ratio. peaks were also obtained from other metabolites such as fructose- , -bisphosphate ( ) a major hurdle for imaging gangliosides in tissue using ms is that sialic acid residues can be dissociated in the ionization process. chan et al. ( a) have investigated the ionic liquid matrix chca/ -methylimidazol ( / ) ( : w/w) and have found that it produces excellent sensitivity for ganglioside detection without significant loss of sialic acid residues. the matrix was applied to the tissue samples with an airbrush; the method best adapted to handling a mixed matrix whose components have different volatilities. image analyses of mouse brain tissue sections demonstrated that the n-fatty acyl chains of gangliosides were differentially distributed in mouse hippocampal regions. gangliosides with n-c acyl chains were enriched in the ca region, while gangliosides with n-c acyl chain were enriched in dentate gyrus. colsch and woods ( ) have developed a method for imaging sialylated gsls in mouse brain. the total glycolipid profile was obtained by maldi-tof following solvent extraction and then individual species were mapped from frozen brain slices by maldi using a linear tof/tof system in negative ion mode. the matrix, which consisted of saturated , dihydroxyacetophenone (dhap, / ) dissolved in % ethanol with the addition of ammonium sulfate ( mm) and . % of heptafluorobutyric acid (hfba) was applied with a chip- chemical inkjet printer with a piezoelectric head. twenty-eight nanoliters of matrix were deposited per spot with the distance between two spots of mm. the ammonium sulfate in the matrix mixture limited the formation of salt adducts, while the addition of hfba increased the stability of dhap in the vacuum and reduced its rapid sublimation. some sialic acid loss was noted, particularly with gd and gt , which were detected as the ganglioside ( the results showed differences in gsl localization in several brain regions depending on the sialic acids and the ceramide (cer). imaging of lipids, including gsls, has been reported by landgraf et al. ( ) using a hybrid linear it/orbitrap mass spectrometer that allowed the acquisition of ms/ms spectra. a dramatic improvement in mass spectral resolution and a decrease in background were observed from lipids distributed within nerve tissue when compared with results obtained from fragmentation within the linear it. the dhb matrix was applied to the dried tissue samples with an epson inkjet printer and the maldi ion source was operated at a pressure of about mtorr. the technique was used to image lipids from rat spinal cord sections. goto-inoue et al. ( ) have imaged the glycolipid, seminolipid ( ) in mouse testis. this sulfated glycolipid comprises more than % of the glycolipid in mammalian testis and disruption of the gene catalyzing transfer of galactose (gal) results in male infertility due to the arrest of spermatogenesis. different molecular species are defined by fatty acid composition. tissue imaging was performed from thaw-mounted tissue slices that had been sprayed with dhb with an airbrush. it was found that the major molecule (c : -alkyl-c : -acyl) was expressed throughout the tubules: some (c : -alkyl-c : acyl and c : -alkyl-c : -acyl) were predominantly expressed in spermatocytes and the other (c : -alkyl-c : acyl) was specifically expressed in spermatids and spermatozoa. this is the first report to show the cell-specific localization of each molecular species of seminolipid during testicular maturation. experimental details for performing imaging of glycolipids using the airbrush application method for applying dhb, as used by this group, has been published separately (goto-inoue, hayasaka, & setou, a) . the distribution and localization of gsls present in mouse brain sections have also been investigated using nanoparticleassisted laser desorption/ionization imaging ms. aunps modified with alkylamine were used as a new matrix to maximize the detection of gsls. the mouse brain was frozen in liquid nitrogen, and serial sections were thaw-mounted onto indiumtin oxide (ito)-coated glass slides. a thin layer of aunps or dhb matrix was applied to the surface with an airbrush. ims analyses were performed by raster-scanning in the x-axis with a scan pitch of mm in the y-axis. sulfatides and gangliosides were detected in mouse brain sections with the new matrix whereas it was difficult to detect them using dhb (goto-inoue et al., c ). an oscillating capillary nebulizer (ocn) was also used by chen et al. ( g) for analysis of sphingolipids in tissue slices in tay-sachs/sandhoff disease. in addition to the above-mentioned matrix-free methods, jung et al. ( ) have reported imaging of cellulose in poplar (populus deltoids) stem using more traditional techniques. analysis of microcrystalline cellulose was performed first to determine the best matrix. dhb gave much stronger signals than matrices such as chca or sinapinic acid and was applied to poplar cellulose with an ocn. ions at m/z , , , , and , (dp , , and ) were monitored with a voyager de str instrument and produced images that closely resembled the optical image. taira et al. ( a) , on the other hand used an airbrush with chca to image ginsenosides in cross-sections of panax ginseng root and used ms/ms to obtain detailed structural information. ginsenosides were found to be located more in the cortex and periderm than in the medulla and that they were at greater concentration in the root tip than in the center of the root. several carbohydrates including hexoses (hex) and d-fructose -phosphate have been imaged in eggplant (solanum melongena, also known as aubergine) fruits from dhb although the paper was mainly concerned with imaging of g-aminobutyric acid (gaba, ) (goto-inoue, setou, & zaima, b) . li, bohn, and sweedler ( h) have compared two ms imaging methods, maldi and sims, for glycan detection in the stems of the perennial grass miscanthus  giganteus. several methods of sample preparation were investigated for maldi. a thin ( nm) gold coating provided high quality signals of oligosaccharide-related ions and dhb also showed high efficiency for ion production. on the other hand, chca produced only very weak spectra, consistent with its use as a stand-alone matrix for carbohydrates. direct laser ablation of untreated sections gave high resolution images, although coating the sections with a nanometer thick layer of gold greatly enhanced the quality of the sims images. two reviews describing derivatization reactions have been published (busch, ; ruhaak et al., b) , the second ( references) is more comprehensive and deals specifically with n-linked carbohydrates. these derivatives are most often used for introducing fluorescent tags for chromatographic detection but also find use in ms. -aminobenzamide ( -ab, / ) and -aminopyridine ( -ap, / ), favored by japanese investigators, are the derivatives most frequently encountered; use of the latter derivatives has been reviewed by hase ( ) . several new derivatives (or labels) have been reported. thus, -amino- -naphthalenesulfonic acid (ansa, ) has been used to derivatize n-glycans by reductive amination for capillary electrophoresis (ce), hplc, and maldi-tof analysis (briggs et al., ) . the derivative was reported to give superior resolution in both ce and hplc analysis to the wellused -aminopyrene- , , -trisulphonic acid (apts) derivatives and in maldi-tof analysis, the negative charge enabled both neutral and acidic glycans to be examined simultaneously. -amino- -ethylcarbazole ( / ) (mou et al., ) , another new derivatizing agent, has been reported to increase sensitivity of ms detection. -picoline-borane ( ) has been proposed as an alternative to toxic sodium cyanoborohydride for the reductive amination reaction (ruhaak et al., a) . pabst et al. ( ) have compared the preparation and performance of different labels for n-glycan analysis. several cleanup procedures were developed for cleaning these derivatives before analysis, of which hydrophilic interaction solidphase extraction (spe) appeared to be the most widely used. however, the cleanup was laborious and a better method was sought. simple addition of acetone resulted in the formation of a precipitate, which turned out to be the labeled oligosaccharide. a single addition and removal of acetone reduced the amount of reagent to approximately . % (measured with -ab). two further extractions of the pellet with acetone reduced the excess of amine reagent by at least as much as clean-up with a cyano-spe cartridge. in addition, reduction of the schiff base of -aplabeled glycans proceeded faster and/or required less reagent when the samples were pre-purified before the addition of reducing agent. acetone extraction was successfully applied to many other labels such as -ab and -aminobenzoic acid ( -aa) ( / ). with respect to the performance of the individual labels, procainamide ( ) emerged as more sensitive than -aa for normal-phase hplc, but its chromatographic performance was not convincing. -ap gave the lowest retention on reversedphase and graphitic carbon columns and, thus, appeared to be most suitable for glycan fractionation by multidimensional hplc. most glycan derivatives performed better than native carbohydrates in maldi and esi ms, but the sensitivity gain was small and hardly sufficient to compensate for sample loss during preparation. amano et al. ( a) have labeled oligosaccharides with a pyrene derivative in order to acquire negative ion maldi-qit-tofms n spectra. the oligosaccharides were reacted with pyrene butanoic acid hydrazine ( / ) and then reduced with nabh and cleaned with a small c column. the derivatives gave intense and stable molecular ions in both positive and negative ([mÀh] À ions) modes and as little as fmol of pyrene-labeled oligosaccharides, such as monofucosyllacto-nhexaose ( ) gave sufficient signals for analysis. although some fucose loss by in-and post-source occurred in positive ion mode, this loss was significantly less in negative mode. a method, termed glycan reductive isotope labeling (gril) has been introduced for glycan quantitation. free glycans or those released from glycoproteins, were derivatized by reductive amination with either [ c ]aniline or [ c ] aniline. these dual-labeled aniline-tagged glycans could be recovered by reversed-phase chromatography and could be quantified by uv absorbance or ms. one labeled variety was used as the reference standard against which the test glycan, labeled with the other isotope was measured. unlike previously reported isotopically coded reagents for glycans, the derivatives did not contain deuterium, which can sometimes be chromatographically resolved. the technique allowed linear relative quantitation of glycans over a -fold concentration range and could accurately quantify sub-picomole levels of released glycans (xia et al., ) . on-target derivatization with the matrix -aminoquinoline ( / ) has yielded schiff bases which could be measured directly in positive and negative ion mode from one single spot. the optimal conditions were mg/ml of -aq in a mecnwater mixture ( : v/v) with . % of an inorganic acid to give a ph of . in negative ion mode, spectra from chloride adducts of the derivatives were acquired from fmol of oligosaccharide. furthermore, psd fragmentation in positive and negative ion mode was enhanced, providing information on oligosaccharide sequence, linkage, and branching. the method was applied to trifucosyllacto-n-hexaose ( ) and trifucosyl-para-lacto-n-hexaose ( ), two isomers occurring in human breast milk samples, which were easily identified and distinguished (rohmer et al., ) . tfplnh, (symbols as defined for structures and . anomericity not specified) reductive amination derivatization has also been exploited in other areas, combined with their use in ms. binding of sugar chains to proteins, viruses and cells is conveniently monitored by the surface plasmon resonance (spr). key to this method is the use of linker compounds for immobilization of the sugar chains to the gold-coated spr chip. sato et al. ( a) have developed a novel linker molecule, named "f-mono," which combines a linker with a fluorescent moiety to allow high sensitivity monitoring of the glycans. since the molecule ( ) contains a , -diaminopyridyl group and a thioctic acid group, conjugation with sugar chains can be achieved by the reductive amination reaction. the mass spectra of thee compounds contained a peak da higher than the molecular ion due to the reduction of the thioctic acid moiety providing a convenient method for identifying the glycans even using unfractionated crude samples. immobilization of the derivatives onto gold-coated chips, and their subsequent spr analyses were successively accomplished. use of hydrazone formation removes the need for a reductive step to stabilize the derivative as required by schiff base formation from primary amines. experimental details for synthesis of the phenylhydrazone derivatives discussed in earlier reviews (lattova & perreault, a,b; lattova, perreault, & krokhin, ) have recently been reported in an edition of methods in molecular biology devoted to glycomics . small oligosaccharides and n-glycans from chicken ovalbumin have been converted into their biotin derivatives by incubating them with biotinamidocaproyl hydrazide (bach, ) (kapková, ) . the derivatives imparted improved mass spectral sensitivity over that of the free glycans and, because the labeling reagent contained a biotin handle, it allowed the interaction between lectins and biotin-derivatized oligosaccharides to be investigated. fragmentation of the n-linked glycans was dominated by y and b/y-type glycosidic ions. han et al. ( a) have used a new substituted hydrazine, -( -cyanophenyl)- -piperidinecarbohydrazide ( ) and produced a derivative that increased detection sensitivity by about -fold over that from the underivatized glycan. the observation of [mþna] þ ions rather than the expected [mþh] þ species suggested that the sensitivity increase was the result of increased hydrophobicity. maldi analysis employed dhb and super-dhb; recrystallization of the super-dhb sample with acetonitrile substantially improved the signal-to-noise ratio and reproducibility. girard's t reagent ( / ), with its constitutive cationic charge, has been used in quantitative measurements, as described below and for measuring a-galactose-containing n-glycans in porcine pig corneal endothelial cells and keratocytes (kim et al., c) . because of the in-built charge, signal strengths from glycans of different structures were thought to be equal. this is not necessarily the case for formation of [mþna] þ ions. rather than reacting the reducing-terminal aldehydes of carbohydrates with amines or hydrazines, the reverse reaction can be used if the glycan is first converted into its glycosylamine ( ). in fact, this type of reaction can be used directly on pngase f-released n-glycans because these are released in this form. chemical formation of glycosylamines generally utilizes the kochetkov reaction in which the glycan is treated with an excess of ammonium carbonate. unfortunately, this reaction is slow and can take up to five days for completion. to overcome this problem, liu, salas-solano, and gennaro ( h) have used microwave assistance to speed the reaction up to as little as min. following amidation the glycans were reacted with tris-( , , -trimethoxyphenyl)phosphonium acetic acid n-hydroxysuccinimide ester ( ) to introduce a permanent charge on the glycan and the investigators were able to detect derivatized maltoheptaose at fmol/ml by maldi-tof ms using dhb and chca matrices. glycans from rnaseb, chicken ovalbumin and asialofetuin were also detected at high sensitivity. a potential problem arises from possible cleavage of the reducingterminal n-acetylglucosamine (glcnac) residue as such a reaction has recently been reported as a by-product of the kochetkov reaction when, for example, ammonium bicarbonate at ˚c was used (murase & kajihara, several carbohydrates, including maltoheptose, blood type b antigen, pullulan and the glucan of ganoderma lucidum have been converted into their naphthimidazole (naim) derivatives ( ) in high yields by the iodine-promoted oxidative condensation (scheme ; lin et al., b) . the reaction took about hr to go to completion giving derivatized carbohydrates that gave enhanced maldi signals ([mþh] þ ions) compared with those from the free carbohydrates or their -ab derivatives with dhb or thap as matrices. less than pmol of carbohydrate could be detected. furthermore, the derivatives could easily be hydrolyzed to the parent glycans. a further series of such derivatives has been synthesized by condensation with diamines such as substituted benzene- , -diamine ( ) in order to increase hydrophobicity and detection sensitivity (lin et al., c) . using maltotriose ( , n ¼ ), derivatives with pyrimidine- , diamine ( ), pyridine- , -diamine ( ) and , , -oxadiazole- , -diamine ( ) b. derivatives of other sites permethylation has long been used in carbohydrate analysis, most frequently for linkage determination by gas chromatography/mass spectrometry (gc/ms) following hydrolysis and acetylation (permethylated alditols acetates). however, there appears to be an increasing trend to employ the reaction for maldi analysis. the advantage of permethylation is that it increases sensitivity and several investigators employing its use appear to detect larger glycans, particularly n-glycans, than by the use of underivatized glycans. however, sample clean-up of the highly basic reaction mixtures can be a problem with, in some cases, losses offsetting any gain in sensitivity. introduction of solid-phase permethylation has improved the situation. experimental details of the solid phase permethylation method (methyl iodide on sodium hydroxide beads) (kang, mechref, & novotny, ; kang et al., ) that is capable of permethylating very small amounts of carbohydrate have been published in methods in molecular biology (mechref, kang, & novotny, a) . on the negative side, it has been reported that permethylation can lead to loss of o-linked acetyl groups from certain sialic acids (liu & afonso, ). an extension of the solid-phase method to allow sulfated glycans to be analyzed by maldi-tof ms has been developed (lei, mechref, & novotny, ) . sulfated glycan samples were permethylated followed by methanolytic cleavage of the sulfate groups. the desulfated, permethylated glycans were then subjected to another permethylation step using deuteromethyl iodide to label the hydroxyl groups that were exposed by removal of the sulfates. the number of attached sulfate groups could be calculated from the mass-shift caused by the presence of the deuterium label and the position of the sulfate substitution could be determined by collision-induced dissociation (decomposition) (cid). the method was validated with linear standard glycans and used to identify sulfated n-glycans released from bovine thyroid-stimulating hormone (btsh). yu et al. ( c) have shown that it is possible to permethylate sulfated glycans with methyl iodide and sodium hydroxide (ciucanu and kerek method) (ciucanu & kerek, ) , without loss of the sulfated glycans. the trick is to avoid the normal chloroform/water partition stage, which results in much of the sulfated material (unmethylated) partitioning into the aqueous phase. instead, clean-up employed c reversedphase spe cartridges and microtips self-packed with nh -beads. the methylation reaction was capable of methylating phosphates but not sulfates, allowing them to be readily identified. formation of methyl esters by reaction of the sodium salt with methyl iodide has frequently been used to stabilize sialic acids to maldi analysis by eliminating the labile proton on the acid group. an alternative procedure for methyl ester formation that provides information on the sialic acid linkage directly from the maldi spectrum has been published by wheeler, domann, and harvey ( ) (fig. ) . the sugars were desalted, dried, dissolved in methanol and treated with -( , -dimethoxy- , , triazin- -yl)- -ethylmorpholinium chloride (dmt-mm, / ). after removal of the solvent, the products were transferred directly to the maldi target and examined from dhb. however, for the analysis of small amounts of n-glycans derived from biological sources, the method benefited from an additional clean-up stage involving the use of a nafion membrane. unlike the reaction with methyl iodide with the sodium salt that produced a single peak from each sialylated glycan, irrespective of the linkage, the reaction with dmt-mm produced different derivatives from a ! -and a ! -linked sialic acids. the a ! -linked sialic acids produced only methyl esters whereas a ! -linked sialic acids were converted into their lactones providing a da difference in mass, thus enabling the linkage to be determined directly from the maldi-tof spectrum (fig. ) . negative ion cid mass spectra of these neutralized glycans provided information, in many cases, on the antenna of n-linked glycans to which the variously linked sialic acids were attached. in an application of the method to the glycoprotein carcinoembryonic antigen-related cell adhesion molecule (ceacam ), it was shown that both a- ! -and a- ! linked sialic acids were present (harvey, baruah, & scanlan, a) . -( , -dimethoxy- , , -triazil- -yl)- -methylmorpholinium chloride in the presence of ammonium chloride, converts sialylated glycans into amides with the same linkage-specific reactivity difference. thus, the a ! -linked sialylated glycans yield lactones, as above, whereas the a ! -linked compounds form amides. have used this reaction to examine blood serum glycoproteins but their technique differed from the above methyl ester formation in that the glycans were permethylated after reaction with dmt-mm. this reaction formed the methyl ester from the a ! -linked compounds as the result of the opening of the lactone ring, whereas the amide that was derived from the a ! -linked compounds was converted into the corresponding dimethylamide with a da increase in mass over that of the methyl ester. -( , -dimethoxy- , , -triazil- -yl)- -methylmorpholinium chloride can also be used to form substituted amides directly and has been used by endo et al. ( ) to link the fluorescent derivative -( -pyridylamino)ethylamine (paea, ) to the carboxy group of sialic acids in sialo-oligosaccharides and gangliosides. the derivative gave good hplc and tlc sensitivity and possessed the following advantages for maldi analysis: suppression of preferential cleavage of neu ac; enhancement of molecular-related ion intensities; simplification of ms spectra; and finally, since paea-amidation did not cleave the linkage between sugar and aglycon, allowed maldi-tof-ms and ms/ ms analyses to reveal the complete structure of the molecule. -( , -dimethoxy- , , -triazil- -yl)- -methylmorpholinium chloride has also been used to form amides from hexuronic acids (hexas) in an investigation of the n-linked glycosylation of structural subunit rvh of rapana venosa hemocyanin . as well as containing the rather unsusal (for n-glycans) hexuronic acid, the glycans also contained naturally methylated hexoses and internal fucose residues. gil et al. ( ) have stabilized sialic acids by formation of amides with acetohydrazide under mild acidic conditions in the presence of -ethyl- -( -dimethylaminopropyl) carbodiimide hydrochloride (edc, ) . glycoproteins were first reduced and alkylated and then the sialic acids were amidated. glycans were released with pngase f and a permanent charge was attached to the reducing terminus by further reaction with girard's t reagent. alternatively, derivatization with -aa was used and the products were examined both by hplc and maldi-tof ms. the amidation reaction was performed on the glycoprotein because acetohydrazide would also have reacted with the aldehyde function of the released glycan, precluding derivatization with an amine more suited to the detection method used for monitoring the glycans. the method was applied to the analysis of n-glycans from transgenic pig-derived human factor ix. another method for synthesis of methylamides has been reported by liu et al. ( i) . sialylated glycans were reacted with methylamine in the presence of ( -azabenzotriazol- yloxy)trispyrrolidinophosphonium hexafluorophosphate (pyaop, ) and n-methylmorpholine ( ) for min at room temperature. after methylamidation, sialylated glycans were analyzed by maldi-tof and esi ms without loss of the sialic acid moiety. both a ! -and a ! -linked sialic acids were quantitatively converted to their methylamides. this method was validated with n-glycans released from the well-characterized glycoproteins, fetuin, human a -acid glycoprotein, and bovine a -acid glycoprotein and was used to study n-glycans from serum glycoproteins from human, mouse, and rat. both neu ac and oacetylated analogues were stable under maldi conditions. glycopeptides tend to produce weaker signals than nonglycosylated peptides and it is frequently difficult to observe their molecular ions in samples rich in unglycosylated peptides. have developed a highly sensitive on-plate pyrene derivatization method using -pyrenyldiazomethane ( ) for acquisition of maldi ms n spectra of glycopeptides in amounts of less than fmol. unusually, the pyrene groups were easily released from glycopeptides during ionization when dhb was used as a matrix. thus, most ions were observed in their underivatized form. at the same time, pyrene derivatization was found to reduce the ionization of peptides and to produce signals from the glycopeptides that were strong enough for acquisition of ms n spectra that contained ions from both glycan and peptide. when the liquid matrix, aq-chca, was used, the sialic acid linkages of the pyrene sialylated glycopeptides were found to be stable because of inefficient release of the pyrene group allowing ms n spectra of the intact glycans to be obtained. the method was used to examine glycopeptides from ng of prostate specific antigen. ohara et al. ( ) have developed a method for analysis of sulfated carbohydrates by forming complexes with -(pyren- ylmethyl)guanidine (pmg, ) from a matrix consisting of this compound and p-nitroaniline ( / ). two types of sulfated carbohydrate were examined, chondroitin sulfate ( ) and carrageenan ( ). the pmg complexed with the sulfate group and was eliminated under maldi conditions such that the molecules produced a ladder of peaks separated by masses corresponding to losses of each complexed sulfate (mass difference u). in positive ion mode, the molecular ions from the chondroitin sulfates contained one more pmg molecule than the number of sulfate groups. one pmg group was presumed to bind to a carboxylate group. in negative mode, one less pmg molecule was bound. carrageenans showed a slightly different pattern in that the number of pmg molecules found in the positive ion spectra equaled the number of sulfates. sialic acids are classically analyzed by hplc after formation of fluorescent , -diamino- , -methylenedioxybenzene (dmb, ) derivatives. these derivatives require an a-keto acid group in the sialic acid. galuska et al. ( b) have developed a method for specifically measuring nucleotide-activated sialic acids in the presence of unactivated acids by first reducing the keto group that is present only in the non-activated acids. under the conditions of the derivatization reaction, the nucleotide group was removed leaving, in the case of the activated acids, only, an intact a-keto acid group that reacted with the dmb reagent. subsequent analysis was by hplc and maldi-tof. maldi ms is used extensively in analyses with glycan arrays as summarized in recent reviews (see table ). table lists glycans that have been used in array construction. most of the above work has been with carbohydrate preparations prior to array construction. however, maldi ms has also been used to interrogate arrays directly. as an example, a new type of glycan array covalently or non-covalently attached to aluminium oxide-coated glass slides has been developed for studies of enzymatic reactions and protein binding . the array was prepared by tagging glycans with a polyfluorinated hydrocarbon ( ) tail and spotting them robotically onto the aluminium oxide-coated slide surface which contained a layer of polyfluorinated hydrocarbon terminated with phosphonate. after incubation and washing, the noncovalent array was characterized by maldi-tof at a low laser energy without addition of matrix. a cellotetraose (d-glc-(b-( ! )-d-glc) -b-( ! )-d-glc) array was developed to study the activity and specificity of different cellulases and to differentiate the exo-and endoglucanase activities. compared to the preparation of glycan arrays on glass slides and other surfaces, this method using phosphonic acid reacting with aluminium oxide-coated was said to be more direct, convenient and effective and represented a new platform for the highthroughput analysis of protein-glycan interactions. polyfluorinated hydrocarbon tag, in another application, a ligand affinity capture (lac) method for detection of biotinylated biomolecules has been developed by jørgensen, juul-madsen, and stagsted ( ) . metal-coated glass slides were treated with amino-silane and derivatized with biotin followed by avidin. biotinylated biomolecules could then be captured and detected in the low femtomole to low picomole range by maldi-tof ms using chca in a dried droplet method. the technique was used to detect biotinylated lipopolysaccharide (lps) and its binding to the antagonist polymyxin b. the a-mannose-specific lectin concanavalin a (con a) has been bound to polydopamine-modified gold, indium, and iridium surfaces and its activity was demonstrated with rnase b using spr spectroscopy. surface-maldi-tof ms experiments revealed that the affinity of the immobilized con a depended on the oligosaccharide structure and composition. thus con a was found to bind certain man -( / ), man -( / ), and man -glcnac rnase b glycoforms more strongly than man -and man -glcnac glycoforms (morris, peterson, & tarlov, ) . concern has frequently been expressed about the ability of maldi-tof ms to provide quantitative information. fortunately, this concern appears to be unjustified as two recent studies have shown. thus, a systematic study of widely different glycopeptides was performed by thaysen-andersen, mysling, and højrup ( ) to determine the relationship between the relative abundances of the individual glycoforms and the maldi-tof ms signal strength. glycopeptides derived from glycoproteins containing neutral glycans (rnaseb, igg, and ovalbumin) were profiled and yielded excellent and reproducible quantitation (correlation coefficient r ¼ . , n ¼ ) when evaluated against a normal-phase hplc -ab glycan profile. similarly, precise quantitation was observed for various forms of n-glycans (free, permethylated, and fluorescence-labeled) using ms. in addition, three different sialo-glycopeptides from fetuin were site-specifically profiled, and good correlation between peak intensities and relative abundances was found with only a minor loss of sialic acids (r ¼ . , n ¼ ). for glycopeptide purification, a range of hydrophilic and graphite materials packed in microcolumn format was evaluated and proved capable of desalting without loss of quantitative information. thus, maldi-tof ms signal strength of glycopeptides has been found to accurately reflect the relative quantities of glycoforms, enabling rapid and sensitive site-specific glycoprofiling of n-glycan populations. the second study relates to the concern that has often been expressed over possible losses of fucose residues when glycans are ionized by maldi. tajiri, kadoya, and wada ( b) have recently assessed possible fucose loss and found it to be insignificant. fucose (fuc) is known, however, to migrate within [mþh] þ ions particularly when these are derivatized by reductive amination. experiments on fucose loss were conducted with fucosylated glycopeptides from tf and haptoglobin. studies with increasing collision energy on the [mþh] þ ions showed that the major fragmentation was cleavage at glcnac residues. biantennary glycans containing a , / -antenna fucose or a , -core fucose showed different fragmentation behaviors in experiments. stability was dependent on peptide backbone sequences. cleavage of the glcnacb ! man linkage occurred at a slightly lower activation energy than for the core fucosylated (cf) species, while the linkage of a , core fucose was more stable than that of antenna a , / fucose. however, these fragmentations only occurred at relatively high collision energy. consequently, the authors concluded that quantitation of fucosylated glycans by ms of glycopeptides, without collisional activation, was justified. the fucosylation levels calculated from the signal intensities in nanospray ionization and uv maldi mass spectra were essentially the same. the mass spectrometric profiling of glycopeptides from tf from patients with congenital disorders of glycosylation (cdg-ia and cdg-iic) demonstrated that the elevation or reduction of fucosylation in pathological conditions can be reliably determined by ms of glycopeptides. in spite of these reassurancies, it is possible that for mixtures of compounds, complex suppression effects may degrade quantitative results. consequently some investigators prefer lc/ms methods because the lc step removes, or minimizes, the effect that other compounds in a mixture have on the ionization of the target compounds. instability of sialylated glycans under maldi conditions complicates quantification and errors can possibly also be introduced by unequal ionization of glycans in mixtures. to overcome these problems with n-glycans from a therapeutic glycoprotein from a chinese hamster ovary (cho) cell line, jang et al. ( a) first formed methyl esters of the sialic acids to stabilize them and then converted the glycans to their girard's t derivatives. these derivatives have a constitutive positive charge, thus overcoming the problem of unequal ionization. percentages of sialylated glycans were measured at . and . % in two cell lines. the results were comparable to those obtained by np-hplc combined with fluorescence detection using -ab or -ap derivatization. girard's t derivatives have also been used by kim et al. ( a) to quantify glycans released from gsls originating from miniature pig endothelia and islet cells. a method using a deoxyribonucleic acid (dna) sequencer has been described for the quantitative analysis of plant nglycans released with pngase a or f and derivatized with apts ( / ). maldi-tof analysis was used to confirm structures with the aid of exoglycosidase digestions (lee et al., i) . a method for the quantification of fructo-oligosaccharides using maldi tof ms with dhb as the matrix, has been developed with the fructan, raftilose, a partially hydrolyzed inulin with a degree of polymeration - as the test compound (onofrejová, farková, & preisler, ) . nystose ( / ), which is chemically identical to the raftilose tetramer, was used as the internal standard. two mathematical approaches, conventional calculations and artificial neural networks (ann), were compared for data processing. the conventional method relied on the assumption that a constant oligomer dispersion profile will change after the addition of the internal standard. on the other hand, ann was found to compensate for a non-linear maldi response and variations in the oligomer dispersion profile with raftilose concentration. as a result, the application of ann led to lower quantification errors and excellent day-to-day repeatability compared to the conventional data analysis. this reproducibility was satisfactory for ms quantification of raftilose in the range of - pg with errors below %. the method was applied to measurements of the content of raftilose in dietary cream and it was stressed that no special optimization of the maldi process was carried out. maldi-tof ms with dhb, thap or p-nitroaniline ( / ) has been used to determine the concentrations of the unsaturated disaccharide from chondroitin sulfate ( ) obtained by enzymatic digestion of native chondroitin sulfate with chondroitin abc lyase. the signal-to-noise (s/n) ratio in the spectrum was used as a quantitative measure: amounts of chondroitin sulfate (measured as the disaccharide) down to at least fmol could be detected and there was a direct correlation between the s/n ratio and the amount of chondroitin sulfate between about and pmol although the curve was sigmoidal. the influence of different parameters such as the matrix, the applied laser intensity and different methods of data analysis were also tested. advantages and drawbacks of this approach are critically (song et al., d) bacillus anthracis tetrasaccharide with thiol linker maldi for attachment to a maleimide functionalized microarray to study of carbohydrate-antibody interactions (oberli et al., ) glycodendrimers with n group terminating in α-man, β-glcnac or β-gal tof immobilized on an acetylenyl-terminated gold substrate via click chemistry high-mannose glycans -oxime linked tof used to probe binding to malectin muc glycopeptides tof synthesis on an amine-reactive hydrogelcoated microarray glass surface. to detect autoantibodies in breast cancer correction: (blixt et al., ) n-glycan-asn-fmoc conjugates from chicken ovalbumin, bovine fetuin, and horseradish peroxidase (hrp) tof/tof printed onto commercially available nhydroxysuccinimide (nhs) -activated glass slides after deprotection. glycans interrogated using plant lectins and antibodies in sera from mice infected with schistosoma mansoni (song et al., e) n-glycan clusters tof (dhb) biantennary and high-mannose n-glycans linked to non-reducing terminus of man glcnac core, plus biotin (song et al., f) thiol-terminated nonamannoside tof coupling of a thiol-terminated mannoside to maleimide-functionalized glass surfaces derived from γ -aminopropyl silane slides (dietrich et al., ) various oligosaccharides derivatized with -( aminoethyl)aniline by reductive amination reagent has amine groups at both ends allowing the modified carbohydrates to be covalently attached to an amino-reactive nhs-activated glass surface by formation of stable amide bonds (seo et al., ) discussed in the paper. finally, the method was validated by the determination of the chondroitin sulfate content in samples of known concentration as well as in enzymatically digested bovine nasal cartilage and compared with two further established methods of chondroitin sulfate determination (the carbazole and alcian blue methods) . positive ion fragmentation of carbohydrates is fairly well understood with two types of cleavage, glycosidic cleavage that occurs between the sugar rings and involve hydrogen migration and cross-ring cleavages that involve the rupture of two of the bonds forming the rings. glycosidic cleavages revealing sequence information predominate in positive ion spectra whereas negative ion spectra frequently contain very abundant cross-ring product ions that provide information on linkage. the nomenclature introduced by domon and costello ( ) is universally used to describe the ions. the stabilities of glycosyl bond linkages in various carbohydrates have been investigated by computational calculations to find general rules of fragmentation of sodiated oligosaccharides . the calculations revealed that a-glucose, a-galactose, b-mannose, a-fucose, b-glcnac and b-galnac linkages were cleaved more easily than b-glucose, b-galactose, and a-mannose linkages because the transition states of the former were stabilized by the anomeric effect. the - linkage was more stable than the others whereas the sialyl bond was the most labile of all the linkages investigated. comparison of activation energies and binding affinities to the sodium cation revealed an increase in activation energy in proportion to the increment in binding affinity. the calculated stabilities of glycosyl bonds were: a-man (mana ! , or man ) > a-neunac (neunaca ! or ); this result was close to the experimentally deduced trend. in-source decay frequently accompanies ionization of permethylated glycans. although the presence of fragments can lead to complex spectra, they can also be used to obtain pseudo-ms spectra. smargiasso and de pauw ( ) have investigated matrices and conditions for optimal production of such ions and have concluded that dhb was the most versatile matrix; the presence or absence of isd fragments could be controlled, depending on the location of the laser shots. spectra obtained from the center of dhb targets produced mainly [mþna] þ ions that did not yield isd fragments, whereas spectra from the crystals surrounding the target yielded mainly [mþh] þ ions that fragmented readily. -aa ( / ), on the other hand, formed homogeneous matrix spots and did not induce isd. soltwisch and dreisewerd ( ) have noted that collisional cooling in an orthogonal tof mass spectrometer stabilizes fragment ions that are generated in-source and that by varying the buffer gas pressure, production of isd and post-source (psd)-type ions could be varied. under high-pressure conditions, isd-type fragments of o-linked glycosylated peptides were generated that retain the glycan. psd fragments, on the other hand, showed partial loss of the glycan from y-type peptide fragments. the detailed positive ion psd and isd fragmentation of deprotonated d-ribose ( / ) and d-fructose ( / ) has been studied with the aid of the isotopically labeled analogues, [ - c]-d-ribose, [ - c]-d-ribose and [c- - h]-ribose (bald et al., ) . the fragmentation was compared with fragmentation through dissociative electron attachment (dea). fragmentations of deprotonated monosaccharides formed in the maldi process, as well as their transient molecular anions formed upon electron attachment, were characterized by loss of different numbers of h o and ch o units. two different fragmentation pathways leading to cross-ring cleavage were identified. metastable decay of deprotonated d-ribose proceeded either via an x-type cleavage yielding fragment anions at m/z ¼ , , and (dominant ion, c h o ), or via an a-type cleavage resulting in m/z ¼ , and . this result is in contrast to previous cid studies where only a-type cross-ring cleavage was proposed. it was found that the heavier fragment anions at m/z ¼ and m/z ¼ generated via metastable decay exclusively arise from an x-type cleavage whereas the smaller fragment anions at m/z ¼ and arise predominantly from an a-type cleavage. a fast and early metastable cross-ring cleavage of deprotonated d-ribose observed in isd was dominated by x-type cleavage leading mainly to m/z ¼ and ; the latter ion is not the same as that found in psd. for dea of d-ribose, a sequential dissociation was identified that included metastable decay of the dehydrogenated molecular anion leading to m/z ¼ . the most dominant fragment ions in dea were due to faster decompositions occurring within several hundred nanoseconds (as in isd) and, thus, sequential reactions including an initial dehydrogenation could be excluded. several oligosaccharides (aldoses) and oligosaccharide alditols derived from agaroses, kand i-carrageenans obtained by hydrolysis of agaroses and carrageenans, two important types of red seaweed polysaccharides, have been used as model compounds to study prompt (isd) and psd fragmentation. sulfated alditols gave [mÀh] À ions in negative-ion mode together with prompt fragment ions produced mainly by desulfation. sulfated aldoses gave mainly prompt fragmentation ions (c-cleavages and desulfation). non-sulfated aldoses and alditols only gave ions ([mþna] þ ) in positive ion mode with no prompt fragmentation. the aldoses yielded cross-ring fragmentation in the psd mode. several different matrices (dhb, norharmane ( / ), ferulic acid ( / ) and the ionic liquid matrices dhb/acid-n-butylamine and ferulic acid/n-butylamine) were investigated; the best results were obtained with dhb and nor-harmane (fatema et al., ) . cid on tof/tof and magnetic sector instruments have been compared with several types of biomolecule . the sector instruments produce high-energy collisions ( - kev) yielding more structural information from many compounds than instruments producing only low energy ( kev) collisions. the case with different tof/tof instruments is less clear-cut because the collision energy is spread over a wide range. fewer differences were seen with carbohydrates than with some other compound types because most fragmentations (except formation of x-type cross-ring cleavages) occur at low energies. high-energy fragmentation in positive ion mode generally enhanced the relative abundance of cross-ring cleavage fragments, particularly x-type ions and has been used with hplc (offline) to provide a powerful method for glycoprotein analysis . full experimental details are given in the paper. experimental details for obtaining infrared multiphoton dissociation (irmpd) spectra from carbohydrates have been described . the technique offers the advantage that, because both parent and product ions can absorb ir photons, the spectra can be richer in information than those obtained by cid. in the same publication, the authors discuss sustained off-resonance irradiation-collision-induced dissociation (sori-cid) implemented with a maldi-ft-icr mass spectrometer which produced similar spectra to irmpd. experimental details are described. sori-cid with maldi-ft-icr has also been applied to muc-type o-glycans . the nm photodissociation of n-linked glycopeptides has been investigated in a modified maldi tof/fof instrument by irradiating the ions within the collision cell. singly charged glycopeptide ions from horseradish peroxidase (hrp) yielded abundant peptide and glycan fragments. the peptide fragments included a series of x-, y-, v-, and w-ions with the glycan remaining intact. these ions provide information about the peptide sequence and the glycosylation site. the glycan fragmented to give both glycosidic fragments and abundant cross-ring fragments that were not observed in low-energy cid spectra. doubly charged glycopeptides generated by nanospray in a linear it mass spectrometer also yielded peptide and glycan fragments. however, the former were dominated by low-energy fragments such as b-and y-type ions while the glycan was primarily cleaved at glycosidic bonds (zhang & reilly, ). maldi-lift-tof-ms/ms and esi tandem mass spectrometry (esi-it-ms/ms n ) have been used for the characterization of free oligosialic acids and those derivatized with dmb, as well as partially o-acetylated derivatives. electrospray required the acids to be lactonized but the larger lactones could only be detected by maldi-tof. the fragmentation spectra were dominated by simultaneous cleavage of glycosidic linkages and the corresponding lactone ring, whereas classical cross-ring fragments were of minor abundance. however, the combined use of the two different types of fragmentation analysis allowed a sensitive and detailed characterization of both short-and longchained species. furthermore, oxidation of the nonreducing end sugar moiety enabled sequence determination and localization of acetylated and nonacetylated sialic acid residues (galuska et al., a) . the effect of the pressure of cooling gas in the ion source of an orthogonal-tof ms has a strong influence on the extent of analyte ion fragmentation. soltwisch et al. ( ) have investi-gated the effect of this parameter on peptide and oligosaccharide fragmentation using substance p and the milk sugar, lnfp-ii ( ), respectively in both uv-and/or ir-maldi. a range of pressures, from . to . mbar was used in combination with seven different buffer gases (he, ne, ar, n , co , ch , and isobutane). the influence of the ion extraction voltage on the analyte fragmentation was also investigated for a selected set of gas parameters. fragment ions exhibited characteristic fragment yield-pressure dependencies that were classified into three groups. for lnfp-ii, the yield of molecular ions rose with pressure until at the higher pressures, it was similar to that from substance p. the authors speculated that the consistently lower ion yields reported from oligosaccharides could be attributed to the generally low pressures used when recording their spectra. with respect to fragmentation, glycosidic fragment ions (termed type i) ions dominated the spectra at low pressure but their relative abundance fell dramatically as the pressure rose. the ions resembled species that are also found in maldi-psd ms. the abundance of type ii ions, which resembled typical isd fragments, and consisted mainly of cross-ring products, rose with pressure, probably because of a reduction in the secondary fragmentation process that resulted in loss of fucose. a few other ions, termed type iii ions did not show such dramatic changes with pressure. comparing the yields of fragmentation for the different buffer gases revealed a correlation between their internal degrees of freedom and their collisional cooling efficiency. changing the buffer gas pressure and/or extraction field provided an easy means to influence analyte ion fragmentation and to switch from the primary production of one type of fragment species to another. hexose rearrangements of protonated molecules of n-glycopeptides and reductively aminated n-glycans have been observed by maldi-tof/tof-ms/ms and esi-it-ms/ms (wuhrer, koeleman, & deelder, b) . fragmentation of proton adducts of -ab and -aa-labeled high-mannose n-glycans from rnaseb resulted in transfer of one to five hex residues to the fluorescently labeled innermost glcnac residue. glycopeptides from various biological sources containing high-mannose glycans were likewise shown to undergo hex rearrangement reactions, resulting in migration of one or two hex moieties to the chitobiose core. tryptic fc-glycopeptides from igg peptides containing biantennary n-glycans were also shown to undergo hex rearrangements. such rearrangement products can cause major problems with the interpretation of unknown glycans but, fortunately, do not appear to occur from [mþna] þ ions, the major ions in maldi spectra of most neutral carbohydrates. the use of computer software for analyzing carbohydrate spectra is not as advanced as that for proteins; nevertheless, many investigators have developed software for spectral interpretation, composition calculations, and have constructed databases, usually for specific glycan types. much of this software is applicable to spectra generated from the majority of common ion sources. a good source of information is the book bioinformatics for glycobiology and glycomics: an introduction by the late claus-wilhelm von der lieth, luetteke, and frank ( ) . in addition, various tools for glycomics and available databases are covered in a comprehensive review by frank and schloissnig ( ) . the simplest of these algorithms calculates compositions from m/z values. although such software is extremely valuable, a composition is not a structure and such programs should not be used as the basis of labeling spectra unless the proposed structures are confirmed by suitable techniques. one such tool reported in the review period is called lipid and calculates molecular compositions for glycerophospholipids, gsls, fatty acids and small oligosaccharides from m/z values entered as single values or as mass lists. the user-extendable software is a microsoft excel add-in developed using visual basic for applications and is compatible with all versions of ms excel since ms excel (hübner, crone, & lindner, ) . kronewitter et al. ( ) have constructed a library of possible n-glycan masses by successive dismantling of tetraantennary hybrid and high-mannose glycans. these calculations gave the possible masses that would be expected in a glycan mixture. three hundred thirty one distinct neutral compositions were obtained but many of these will represent several isomeric glycans. the smallest mass difference that was observed was . da. however, many of the masses coincided with isotope peaks from ions of different compositions meaning that, without deisotoping, a resolution of at least , would be needed to resolve all peaks. the theoretical masses were matched against measured masses from n-glycans released from human serum glycoproteins and discrete compositions were detected. in a similar way, an in silico glycan database of possible nglycan compositions has been constructed by addition of known monosaccharide residues, such as those in a neu ac-gal-glcnac antenna, to the common trimannosyl chitobiose core. the derived masses were then matched to the experimental mass and the software, named glyquest, predicted compositions and possible structures. next, it calculated possible glycosidic fragments from the proposed structures, matched these to the experimental mass spectrum and constructed a spectrum labeled with the proposed structures (gao, ) . the software could also be applied to glycans containing fluorescent labels such as -ab but was not applicable to glycopeptides with unknown modifications. however, as with much of the software developed for this work, the detailed structural details such as the linkage of each monosaccharide are not available and the software must be regarded as a guide to the total structure. a similar "branchand-bound" algorithm developed by peltoniemi, joenväärä, and renkonen ( ) starts with the trimannosyl-chitobiose core and then constructs n-type glycans in an iterative process until the target carbohydrate composition is reached. the algorithm identified several glycans from tf and human serum samples. glycospectrumscan is a web-based tool that identifies the glycoheterogeneity on a peptide from mass spectrometric data. two experimental data sets are required as inputs: ( ) oligosaccharide compositions of the n-and/or o-linked glycans present in the sample and ( ) in silico derived peptide masses of proteolytically digested proteins with a potential number of nand/or o-glycosylation sites. glycospectrumscan uses ms rather than ms/ms data, to identify glycopeptides and determine the relative distribution of n-and o-glycoforms at each site. it can be used to assign monosaccharide compositions on glycopeptides with either single or multiple glycosylation sites. the algorithm allows the input of raw mass data, including multiply charged ions, making it applicable for both esi and maldi data from all mass spectrometer platforms. low resolution data from, for example, its are heavily smoothed to yield the average mass whereas data from high resolution instruments receive a milder smooth and deisotoping to give the monoisotopic mass. the software was used to characterize the n-and o-linked glycopeptides from human secretory iga (siga), consisting of secretory component ( n-linked sites), iga ( n-linked, o-linked sites), iga ( n-linked sites) and the j-chain ( n-linked site). glycospectrumscan is freely available at www.glycospectrumscan.org (deshpande et al., ) . prediction of glycosylation sites is another area of software development. a program that predicts n-and o-glycosylation sites based on local information, general protein information, sub-cellular localization and binding specificity of glycosyltransferases has been developed and was claimed to be about % accurate (sasaki, nagamine, & sakakibara, a) . however, as with all predictive programs that are not % accurate, results should only be taken as a guide for designing appropriate location experiments. software that attempts to predict structures from spectra is possibly the most active area in computer applications. simglycan is one such tool (apte & meitei, ) . the software accepts raw or standard experimental ms data files, matches them with its own database of theoretical fragments and generates a list of probable candidate structures. each structure is scored to reflect how closely it matches the experimental data. the software also predicts novel glycan structures by drawing a glycan and mapping it onto the experimental spectrum. other biological information is also available for easy reference. the program can be downloaded from http://www.premierbiosoft. com/glycan/index.html. another software platform for carbohydrate assignment is sysbioware, developed by vakhrushev, dadimov, and peter-katalinić ( ) and designed to work directly from raw ms data. the data are first imported into the spectrum browser, baseline corrected and denoized. peak detection is based on shape matching and the software detects monoisotopic m/z values and charge states. a biological filter is used during compositional analysis of the monoisotopic ions. the software was successfully tested with human urine. sysbioware, a software platform developed for ms data evaluation in glycomics, has been applied to the interpretation of spectra from human serum gsls. the masses of predicted ions arising from cleavages in the glycan and the ceramide moieties were calculated, thus enabling structural characterization of both entities. the calculated masses were then used to match with those in the spectra for structural identification . böcker, kehr, and rasche böcker ( ) have presented an algorithm for calculating glycan structures from tandem mass spectra. twenty-four spectra (of [mþh] þ ions) of -ap-labeled n-glycans obtained from batroxobin (from bothrops moojeni venom) were used as test compounds, the spectra were measured with a tof/tof instrument with a maldi ion source and the algorithm rapidly predicted possible topologies. biological restraints needed to be used to limit the predictions to reasonable structures. goldberg et al. ( ) have compared three algorithms, "max subgraph," "parsimony," and "randomwalk" that make inferences about glycan synthesis from biological knowledge for their ability to assign structures from single-ms spectra from a variety of tissues and organisms, containing more than , manually annotated peaks. max subgraph behaved better than the other two but only uniquely assigned the correct structure to about half of the peaks in out of the spectra. a computer model that predicts n-linked glycan profiles based on cellular enzyme activities has been developed (krambeck et al., ) . the paper describes the expansion of a previously developed detailed model for n-linked glycosylation (krambeck & betenbaugh, ) with the further application to analyze maldi-tof mass spectra of human n-glycans. the glycosylation reaction network is automatically generated by the model, based on the reaction specificities of the glycosylation enzymes and allows prediction of the complete glycan profile and its abundances for any set of assumed enzyme concentrations and reaction rate parameters. a predicted mass spectrum of model-calculated glycan profiles is obtained and enzyme concentrations are adjusted to bring the theoretically calculated mass spectrum into agreement with that obtained experimentally. the result is a complete characterization of a measured glycan mass spectrum containing hundreds of masses in terms of the activities of enzymes. in addition, a complete annotation of the mass spectrum in terms of glycan structure is produced, including the proportions of isomers within each peak. the method was applied to mass spectrometric data obtained from normal human monocytes and monocytic leukemia (thp ) cells. a kinetic model originally developed for the prediction of peptide cid spectra has been extended to predict the cid spectra of n-glycopeptides. the model was trained with cid spectra obtained with an ion trap and was able to predict cid spectra with excellent accuracy in ion intensities for n-glycopeptides up to , da in mass. the program is said to be capable of predicting up to common glycoforms including high-mannose, hybrid and complex n-glycans with two to four antennae (zhang & shah, ) . spencer et al. ( ) have devised a computational approach to predict the fine structure and patterns of domain organization of heparan sulfate (hs). analysis uses chemical composition data obtained after complete and partial enzymatic digestion of mixtures of hs chains and produces populations of theoretical hs chains with structures that meet both biosynthesis and enzyme degradation rules. the program was used to analyze hs from various cell types and good agreement was found between experimental data and computer predictions. glycoviewer (http://www.systemsbiology.org.au/glycoviewer) is a web-based tool that can visualize, summarize, and compare sets of glycan structures. it takes as its input a list of glycan structures in international union of pure and applied chemistry (iupac) format or glycan structures constructed with a sugar structure builder. the output is a graphic, which summarizes all salient features of the glycans according to features such as the nature and length of any chains and the types of terminal epitopes. the tool can summarize several hundred glycan structures in a single figure. the report contains an example of use of the tool for analysis of normal and disease associated glycans from the human glycoproteome (joshi et al., ) . several glycan databases are available. the kyoto encyclopedia of genes and genomes (kegg) glycan databases contain useful information on glycan structures and metabolic pathways (hashimoto & kanehisa, ) and data mining the protein data bank for glycol-related data using the glycosciences. de internet portal has been discussed in methods in molecular biology (lütteke & von der lieth, ) . ito et al. ( a) have synthesized n-and o-linked glycan libraries (named glycan mass spectral database, gmdb) and constructed a library of their positive ion ms , ms and ms fragmentation spectra. n-glycans were in the form of their -ap derivatives whereas oglycans that were released by b-elimination were not. the library was said to be accessible on-line at http://riodbdev.ibase. aist.go.jp/rcmg/glycodb/ms (however, attempts by the reviewer to connect to the site have failed.) it can be searched either by mw of glycan composition in terms of isobaric monosaccharides and instructions on how to use the software are given in the paper. although such databases and tools for glycomics are readily available on the web, these have, until now, been isolated. this unfavorable situation has been discussed (von der lieth, ) and has been largely overcome by ranzinger et al. ( ) who have developed glycomedb, a meta-database for public carbohydrate sequences. at the time of publication ( ) it contained , unique structures in glycoct (www. glycome-db.org) encoding, referencing more than , external records from , different taxonomic sources. a user-friendly, web-based graphical interface has been developed which allows taxonomic and structural data to be entered and searched. the structural search possibilities include substructure search, similarity search, and maximum common substructure. a novel search refinement mechanism allows the assembly of complex queries. with glycomedb, it is now possible to use a single portal to access all digitally encoded, public structural data in glycomics and to perform complex queries with the help of a web-based user interface. a list of databases is given in aoki-kinoshita ( ). n-and o-glycan structures are usually depicted with small cartoons in which each constituent monosaccharide is shown by a symbol. unfortunately, there is no consensus on which symbol to use for any particular monosaccharide with most investigators preferring to develop their own system. several years ago, the consortium for functional glycomics (cfg) attempted to redress the problem with the introduction of a system that has since been adopted by several laboratories. unfortunately, this system has several major drawbacks: (a) it does not diagrammatically show linkage or anomericity and (b) it uses color to differentiate hexoses, thus causing problems when structures are printed in black and white and making the system difficult to use with pen or pencil on paper. a new system that overcomes these problems has recently been introduced (harvey et al., c) . monosaccharides are shown as shapes with various additions to indicate functional groups (e.g. an inclusive dot to indicate a deoxy-sugar and a filled shape to code for an n-acetyl sugar). linkage is shown by the angle of the lines linking the sugar symbols and anomericity is shown by the type of line (full for a b-bond and broken for an a-link). examples can be seen below. although color is not used to define monosaccharides, the cfg colors can be used with the oxford symbols. unfortunately, color was omitted from the table of symbols in the original article but was published later as an erratum (harvey et al., b) . the article received comments from the authors of the cfg system (varki et al., ) and discussion is continuing. this scheme and others are compared in a review by frank and schloissnig ( ) . two tools for displaying n-glycans found in the mammalian cho cell line have been developed (mcdonald et al., ) . both take as input the -digit identifier devised by krambeck and betenbaugh ( ) that uniquely defines each structure assuming the existence of the trimannosyl-chitobiose core. the first of these tools, glycoform, is designed to display a single structure from an identifier entered by the user. the display is updated in real time, using symbols for the sugar residues, or in text-only form. the two symbol sets discussed above are used, the symbols and layout devised by the cfg http://www. functionalglycomics.org/static/consortium/nomenclature.shtml or the alternative "oxford" system used by glycobase, a relation database of -ab labeled n-glycans (campbell et al., ) . however, although glycoform can display structures using the oxford system, unfortunately, it does not display the correct linkage information that is inherent to the full oxford system. in addition, glycoform can display the name of the glycan as used by glycobase. glycobase formalism does not yet handle nacetyllactosamine (naclac, ) repeating units and is, therefore, currently limited to structures with one gal residue per branch. structures can be added to a library, which is recorded in a preference file and loaded automatically. individual structures can be saved as image files either portable network graphics (png), jpeg or windows bitmap (bmp) formats. the second program, glycologue, reads a file containing columnar data of nine-digit codes, which can be displayed on-screen and printed at high resolution. both programs, for windows, mac os x and linux x gtk can be downloaded from http://www.boxer.tcd. ie/gf. a major problem with the analysis of monosaccharides or small oligosaccharides by maldi is the presence of very abundant ions from matrices such as dhb in the low mass region of the spectra. various methods for overcoming this disadvantage are discussed above. nevertheless, maldi with conventional matrices has produced results and the technique works well for the larger oligosaccharides. thus, oligosaccharides from dextran, alginate, hyaluronan and chondroitin sulfate have been characterized by maldi-tof ms directly from a tlc plate after soaking it in the dhb matrix. the plate had a metal backing to ensure electrical contact. the tlc solvent system was n-butanol/formic acid/water ( : : , v/v/v). it was found that the high content of formic acid caused few problems but was responsible for partial formylation of glycosaminoglycans (gags) and minor n-acetyl loss from hyaluronan and chondroitin sulfate . a comparative study of maldi and a new technique, electrospray droplet impact secondary ion mass spectrometry (edi/sims) has been applied directly to fruits such as bananas, apples, grapes and strawberries. the major constituents, fructose ( / ), glucose ( / ), sucrose ( ) and organic acids gave abundant [m þ k] þ ions positive mode and cf coo À adducts in negative mode (the cf coo À ions came from cf cooh in the esi spray. these negative ion spectra were almost free of background ions. maldi from dhb, on the other hand, although producing positive ions gave virtually no ionization in negative ion mode (asakawa and hiraoka, ) . reviews of the analysis of polysaccharides are listed in table . large polysaccharides need to be hydrolyzed, often enzymatically but sometimes chemically, to smaller fragments before they are amenable to maldi analysis. one chemical method involves the selective cleavage of the rhap-( ! )-a-galap linkage in rhamnogalacturonans. enzymic cleavage of this linkage is often ineffective, especially in highly branched rhamnogalacturonans but deng et al. ( ) have developed an improved chemical fragmentation method based on belimination of esterified -linked galacturonic acid (galpa, ) residues that overcomes the problem. at least % of the carboxyl groups of the galpa residues in a. thaliana seed mucilage were converted to methyl or hydroxypropyl esters and b-elimination was found to be more extensive with hydroxypropyl-esterified than with methyl-esterified rhamnogalacturonans. the non-reducing -deoxy-b-l-threo-hex- -enepyranosyluronic acid ( ) residue formed by the b-elimination reaction was removed by treatment with aqueous n-bromosuccinimide. this method was used to fragment the branched rhamnogalacturonan from peppergrass seed mucilage with product characterization by maldi-tof ms, glycosyl residue composition analysis, and and d nmr spectroscopy. the results showed that the most abundant low-mw fragments contained a backbone rhamnose ( / ) residue substituted at o- with a single side-chain, and suggest that peppergrass seed mucilage rhamnogalacturonans is composed mainly of the another chemical method for analysis of rhamnogalacturonan ii makes use of mild acid hydrolysis to hydrolyze the acidlabile monosaccharides -deoxy-d-manno-oct- -ulosonic acid (kdo, / ), -deoxy-d-lyxo- -heptulosonic acid (dha) and apiose (api, ) at the branchpoint between the sidechains and the oligogalacturonide backbone to release short polysaccharide chains that were analyzed by esi and maldi-tof ms (séveno et al., ) . the method was optimized using citrus pectin and then applied to other plant species. experimental details for examination of extracellular polysaccharides (epss) from plants following digestion with a variety of endoglycosiodases and maldi-tof analysis from dhb has been reported by günl, gille, and pauly ( ) . other examples are listed in tables (plants) and (bacteria). ionization efficiencies of cyclodextrins and corresponding linear compounds (maltohexaose and maltoheptaose) have been compared together with differences in the ionization efficiencies of aand b-cyclodextrins ( / ) . alkali metal salts of li þ , na þ , k þ , and cs þ were used as the cationizing agents to enhance the ionization efficiency. relative ion intensities of the cyclodextrins were much larger than those of the linear carbohydrates and the difference showed an increasing trend with the size of the alkali metal cation. b-cyclodextrin had higher ionization efficiency than a-cyclodextrin ( / ) and the difference increased with increasing size of the alkali metal cation. the ionization efficiency was also found to be affected by the counter anions. the higher ionization efficiencies of cyclodextrins were explained with the number of coordination sites and the binding energies. analysis of milk oligosaccharides appears to be receiving increasing attention. reviews of mass spectrometric methods for their analysis have been published by niñonuevo and lebrilla ( ) , kolarich and packer ( ) and urashima et al. ( ) . wu et al. ( b) have developed an annotated library of neutral human milk oligosaccharides with characterization by hplc, maldi-ft-icr ms and exoglycosidase digestion. pyrene labeling (amano et al., a) has been used by amano et al. ( b) to enable neutral carbohydrates from human milk to be observed by negative ion maldi-tof ms n . the neutral oligosaccharides from the milk of a woman (blood type a, le bþ ) were obtained by anion-exchange column chromatography after the removal of lipids and proteins. further fractionation was performed by means of aleuria aurantia lectin-sepharose column chromatography and reversed-phase hplc after labeling. twenty-two oligosaccharides with decaose cores were identified and, of these had novel structures. locascio et al. ( ) have published a method for measuring the consumption of human milk oligosaccharides by strains of bifidobacteria. oligosaccharides were quantified with deuterated and reduced oligosaccharide standards that were added after bacterial growth and results were processed with inhouse software called glycolyzer after removal of contributions from c isotopes. high growth was found for bifidobacterium longum biovar infantis strains, which consumed nearly all available substrates, while other bifidobacterial strains showed low or only moderate growth ability. other examples of the use of maldi analysis of milk glycans are listed in table . glycoproteins and their attached n-and o-glycans is possibly the largest group of compounds that have been analyzed by maldi, catalyzed largely by developments in the biotechnology industry. analysis of these compounds has been reviewed by many authors (table ) . n-glycans are normally attached to an asparagine residue in a asn-(xxx)ser-(thr) consensus sequence where xxx is any amino acid (xxx) except proline however, asn-linked n-glycans have recently been found at the . - . % level on a non-consensus amino acid sequence (tvswn sgal) in the c h domain of human antibodies and on igg (valliere-douglass et al., b). many investigators have published methods for glycoprotein enrichment. solid-phase glycan/glycoprotein capturing methods have become popular in recent years and some of these have been highlighted in an article by . (kooy et al., ) upis ceramboides (alaskan beetle) xylomannan first identification of a nonprotein anti-freeze compound (walters et al., ) commercial chitosan oligosaccharides analysis of commercial samples and preparation of sample with glcnac - not stated but has been found in cryptococcus neoformans human macrophage activation shown to be triggered by chitotriosidase-mediated chitin and chitosan degradation (gorzelanny et al., ) format (not all items present): maldi method (matrix), compounds run (derivative), other methods. enrichment strategies for glycopeptides based on lectinaffinity chromatography and polysaccharide hydrophilic affinity physicochemical chromatography have been discussed by ito, hayama, and hirabayashi ( b) . the combined use of these techniques effectively removes non-glycosylated peptides. the ability of boronic acids to form cyclic derivatives with the cis-dihydroxy groups present in most glycans has been extensively used. thus, -aminophenylboronic (apb) acid ( / )functionalized beads, mesoporous silica, and nanodiamonds have been developed to enrich glycosylated peptides and proteins but the direct immobilization of the apb group was found to be insufficient to suppress nonspecific adsorption/ adhesion. consequently jang et al. ( b) have designed a selfassembled monolayer (sam)-based plate, which contained a spacer group such as oligo(ethylene glycol) to reduce the nonspecific adsorption/adhesion, for direct detection of glycoproteins after affinity-capture (or enrichment) on the plate. the utility of the plate was demonstrated with model glycoproteins such as ribonuclease g and tf. a two-stage glycopeptide enrichment technique using boronate-functionalized beads has been developed by chen et al. ( f) . samples were incubated with the functionalized magnetic beads in slightly alkaline conditions at room temperature for about hr with gentle shaking. the beads were washed and the enriched glycoproteins/peptides were eluted under acid conditions and dried in a speed-vac evaporator. the glycoproteins were then either dissolved in mm ammonium bicarbonate and digested by lys-c overnight or separated by sodium dodecyl sulfate (sds)-polyacrylamide gel electrophoresis (page) and the resulting gel-bands were digested in-gel by lys-c overnight. the compounds were then ready for a second enrichment. alternatively, digestions could be carried out with trypsin. analysis was by maldi-tof. boronic acid functionalized nanoparticles have also been used to concentrate antibodies by capturing the carbohydrates attached to the fc region of igg . chalagalla and sun ( ) have prepared a boronic acid-containing polymer capped with biotin ( ) for linkage to a magnetic bead and used the product for glycan capture and lin et al. ( f) have constructed magnetic nanoparticles with immobilized apb acid for glycoprotein capture. "sno @poly(hema-co-st-co-vpba)" core-shell nanoparticles containing boronic acid groups have been prepared by of copolymerization between hydroxyethyl methacrylate grafted ( ) on sno nanoparticles, styrene, and -vinylphenylboronic acid (vpba, ). they have been used to extract tryptic peptides from hrp, bovine asilotf and human serum glycoproteins. analysis was by maldi-ms/ ms using an axima qit instrument (sheng, xia, & yan, survey epo and of several analytical methods including maldi, chromatography (reichel & gmeiner, ) application of proteomics in biomarker discovery mainly proteins and glycoproteins. a novel boronic acid functionalized mesoporous silica, which holds the attractive features of high surface area and large porosity has also been used to concentrate glycopeptides. in comparison to direct (traditional) analysis, this method was stated to enabled two orders of magnitude improvement in the detection limit of glycopeptides irrespective of the nature of the attached glycans . the same group has also synthesized boronic acid functionalized core-satellite composite nanoparticles that possess both the superparamagnetic properties of magnetic nanoparticles and the surface chemistry of aunps. glycoproteins or glycopeptides could be obtained in high yield by use of a magnet. the composite nanoparticles were used to enrich glycosylated proteins from human colorectal cancer tissues for identification of n-glycosylation sites. in all, unique glycosylation sites mapped to different glycoproteins were identified, of which sites ( . %) were new. boronic acid functionalized gold-coated si wafers have been used as maldi plates to isolate and enrich glycopeptides . this method was claimed to be beneficial for several reasons. thus, solution transfer and eluting steps required in conventional enrichment strategies were not needed, thereby reducing sample loss. secondly, the lower limits of detection of glycopeptides were said to have been increased by two orders of magnitude. thirdly, non-specific bindings were not detected even when non-glycopeptides were times more concentrated than glycopeptides. furthermore, glycopeptides could be detected in the presence of mm ammonium bicarbonate or the physiological buffer, pbs. in a related method (tang et al., a; yao et al., ) , aunps were first spotted and sintered onto a stainless steel plate, then modified with -mercaptophenylboronic acid ( ) to provide a porous substrate with a large surface for capturing glycopeptides from peptide mixtures. the captured peptides were then analyzed by maldi-tof ms simply by deposition of a dhb matrix. the technique enabled sample enrichment, washing and detection steps to be fulfilled on a single maldi target plate. well-characterized glycoproteins, such as hrp and asialofetuin, were employed as standards to investigate the enrichment efficiency. fe o @c@au magnetic microspheres functionalized with -mercaptophenylboronic acid have been synthesized by the same group (qi et al., ) and successfully used for enrichment of glycoproteins and glycopeptides. a polyfunctional device has been constructed from macroporous silica foam (mosf) containing boronic acid (bmosf) or amino groups (nh -mosf) and used to immobilize enzymes such as trypsin and selectively enrich glycopeptides. use of the device considerably speeded up hydrolysis times as demonstrated with glycopeptides from hrp (qian et al., ) . tang et al. ( a) have immobilized the lectin con a on apb acid-functionalized magnetic nanoparticles using methyl a-d-mannopyranoside as a linker. the selective capturing ability of the con a-modified nanoparticles was tested using standard glycoproteins and cell lysate of human hepatocelluar carcinoma cell line , . regeneration of the protein-immobilized nanoparticles could easily be performed by utilizing the reversible binding between the boronic acid and the sugar. cona has also been used in conjunction with hollow fiber flow field-flow fractionation (hf ) to preconcentrate high mannose type nlinked glycoproteins from bacterial lysates as exemplified by glycoproteins from streptococcus pyogenes . the specificity of datura stramonium agglutinin (dsa) for triand tetra-antennary glycans has been utilized to enrich human liver glycoproteins containing these larger glycans which were then separated and identified by sds-page followed by maldi-tof analysis (sun et al., b) . the performance of chromatographic columns consisting of agarose-bead-bound -aminophenyl boronic acid, agarosebound wheat-germ agglutinin (wga) or a mixture of both compounds (boronic acid lectin affinity chromatography, blac) has been evaluated for glycoprotein enrichment using the model proteins of rnaseb and trypsin inhibitor in the presence of the non-glycosylated proteins, myoglobin (neutral) and lysozyme (basic) over a wide temperature range ( - ˚c). the results showed that glycoaffinity micropartitioning at ˚c provided the highest recovery rate for glycoprotein enrichment. a large amount of lysozyme was present in the elution fractions of the -aminophenyl boronic acid-containing micropartitioning columns due to an ion-exchange mechanism occurring between the positively charged protein and the negatively charged stationary phase. at ˚c, nonspecific interactions with the agarose carrier prevailed, evidenced by the presence of myoglobin in the eluate (olajos et al., ) . a novel boronate affinity monolith, poly-( -acrylamidophenylboronic acid-co-ethylene dimethacrylate) ( ) has been prepared in mm capillaries by a one-step in situ polymerization procedure . the monolith was used to separate glycopeptides from peptides produced from hrp and to separate this glycosylated protein from non-glycosylated bovine serum albumin (bsa). the maldi-tof spectrum of the hrp peptides showed little evidence of the presence of glycopeptides before passage through the capillary but revealed abundant glycopeptide ions after treatment. mass spectrometry reviews doi . /mas b. other solid-phase methods titanium dioxide (tio ) microspheres, synthesized by a sol-gel method, have a high affinity for the acid groups of sialic acids and peptides. they have successfully been used for simultaneous enrichment of glycopeptides and phosphopeptides from, for example bovine rnaseb and human igg . detection was by esi but the method would be equally applicable to maldi-tof analysis. mysling et al. ( ) have used zic-hilic in a microcolumn format for spe and glycoprotein enrichment involving trifluoroacetic acid (tfa) ion pairing to increase the hydrophilicity difference between glycopeptides and non-glycosylated peptides. three mobile phases were investigated: % formic acid, . % tfa and % tfa all containing % acetonitrile and experiments were conducted on single glycoproteins, a five-glycoprotein mixture and depleted plasma. the presence of tfa, particularly at the % level, in the mobile phase significantly improved the glycopeptide enrichment ( . -fold) as evaluated by maldi-tof ms and rp-lc-esi-ms/ms. four types of hydrazine functionalized carboxyl and epoxysilanized magnetic particles (hfmp) have been developed by sun et al. ( a) for isolation of glycopeptides. particles prepared by adipic dihydrazide functionalization from carboxylsilanized magnetic particles yielded the maximum capture capacity. the method was verified by successful isolation of all formerly glycosylated peptides from standard glycoproteins (fetuin, rnaseb, and human serum albumin (hsa)) and by identification of their glycosylation sites. c. other techniques maldi-tof ms has been used by carvalho et al. ( ) to characterize the aand b-subunit of recombinant and pituitary glycoprotein hormones that have been separated by a new method of incubating the glycoproteins overnight with acetic acid ( . - . m) at ˚c. a. use of mass spectrometry to detect glycosylation of proteins a simple method to detect glycosylation is to measure the mass of a glycoprotein and then to repeat the measurement after incubation with pngase f to remove the n-glycans. the method has been used to detect the presence of n-glycans in recombinant bovine cd expressed in pichia pastoris (muller-steffner et al., ) . the molar masses of non-glycosylated ( , da) and penta-glycosylated thermomyces lanuginosus lipase ( , ) as measured by maldi-tof ms have confirmed glycosylation and shown that each glycan-moiety adds approximately , da to the molar masses (pinholt et al., ) . in another example, cu/zn superoxide dismutase monomer was determined to have a mass of , da before deglycosylation and , da afterwards giving a mass for the glycan of about , (nedeva et al., ) . the difference between the sequence mass ( , da) and measured mass of about , da of the peroxidase from royal palm tree (roystonea regia) together with the fact that the amino-acid sequence includes possible n-glycosylation sites, suggests heavy glycosylation. glycosylation sites were identified, in this case, by n-terminal sequencing and maldi-tof-ms analysis of tryptic peptides . b. mass spectrometric detection of glycoforms of intact glycoproteins although resolution of glycoforms by maldi-tof ms is generally inferior to that obtained by esi, glycoproteins with masses in the region of kda, containing only a limited number of glycoforms have been resolved successfully as illustrated by the resolution of four glycoforms of antithrombin in a study of altered glycosylation causing antithrombin deficiency . glycans appeared to be sialylated biantennary (residue mass , da). ogawa et al. ( ) have observed resolution of glycoforms of stereum purpureum endopolygalacturonase i produced in p. pastoris ( . kda). three main ion peaks corresponding to the protein with the high-mannose glycans man - glcnac together with some minor unresolved ions were observed. c. detection of glycosylation sites and site occupancy the most common method for detecting site occupancy is to utilize the conversion of the asn to which the n-glycans are attached to aspartic acid (asp) when the glycans are released with pngase f. the increase by one mass unit is readily detected by ms. the method has been used, for example to confirm occupancy of six of the seven potential n-linked glycosylation sites in the envelope glycoprotein gp and three of the four potential sites in the gp protein of the yellow head virus from the penaeus monodon shrimp (soowannayan et al., ) . periodate oxidation and hydrazide capture on a solid support have been used by lewandrowski and sickmann ( ) to study glycosylation sites in human platelet proteins. the bound glycoproteins were sufficiently stable to allow washing, following which the proteins were hydrolyzed. glycopeptides remained bound to the solid support through the glycan moiety from where they were released with pngase f and the glycosylation site was identified by means of the asn to asp conversion. a new method using tandem o stable isotope labeling (tosil) to quantify the n-glycosylation site occupancy has been reported (liu et al., k) . glycoproteins were digested with trypsin and pngase f in the presence of either h o or h o. three o atoms were introduced into n-glycosylated peptides, two at the carboxyl terminus of all peptides and the third at the n-glycosylation site. the samples were mixed to give pairs of ions in the resulting maldi or esi spectra. a unique mass shift of da was produced by n-glycosylated peptide with a single glycosylation site, whereas non-glycosylated peptides produced an ion pair spaced by only da. intensity ratios could be used to monitor site occupancy in various physiological and disease conditions. the method yielded good linearity within a -fold dynamic range with the correlation coefficient r > . . the standard deviation (sd) ranged from . to . , for four glycopeptides from two model glycoproteins. the method was used to monitor glycoproteins in the sera from a patient with ovarian cancer and healthy individuals. eighty-six n-glycosylation sites were quantified and n-glycosylation levels of glycopeptides showed significant changes. a similar o-labeling technique was used by alvarez-manilla ( b) to identify n-glycosylation sites in con-a-extracted glycopeptides from pluripotent murine embryonic stem cells. glycopeptides rather than glycoproteins were extracted from tryptic digests to avoid false positives produced from non-glycosylated proteins that were bound to extracted glycoproteins if no digestion had been performed. segu et al. ( b) have published a method for detecting sites occupied by glycans carrying a fucose residue attached to the core. the method made use of the endoglycosidase m which, like pngase f has a broad spectrum of activity with the exceptions that (a) it is not active on core-fucosylated glycans and (b) it shows reduced activity with larger glycans. the second exception was overcome by conducting incubations in the presence of sialidase, b-galactosidase and b-n-acetylhexosaminidase, which reduced the size of the glycans. then, the results were compared with the products of digestions performed with pngase f allowing the core-fucosylated sites to be determined. analyses were by lc/ms but the technique would be equally applicable to maldi-tof analysis. d. analysis of glycopeptides because many glycoproteins are too large and heavily glycosylated for direct analysis by ms, much work is performed on derived glycopeptides, most commonly tryptic glycopeptides. tryptic cleavage of glycoproteins is frequently hindered by steric hindrance imposed by the glycans but improvements can be made by the use of heat to increase the rate of proteolysis. segu, hammad, and mechref ( a) have used microwaveassisted enzymatic digestion to achieve higher sequence coverage of several model glycoproteins such as fetuin, tf, and fibrinogen. efficient digestion was achieved in min at an optimum temperature of ˚c; there was no apparent loss or partial cleavage of the glycans. signals from glycopeptides are often weak or absent from the spectra of mixed peptides and glycopeptides, a situation that can be improved by fractionation of the two compound classes. wohlgemuth et al. ( ) have investigated several techniques and have shown that hydrophilic interaction chromatography (hilic) chromatography with zic-hilic and tskgel amide- are very specific at capturing glycopeptides from mixtures. sialylated glycopeptides could also be enriched with tio . capture using a hydrazide column resulted in lower recovery and involved a more complex enrichment scheme. a new material for glycopeptide concentration, termed "click maltose," has been synthesized by linking the alkynyl-derivatized maltose chain to the azide derivatized silica gel through click chemistry. unlike the rigid structure of sepharose, the saccharide chain of click maltose exhibits a certain amount of flexibility, which provides a sufficient number of hydroxyl groups for the effective formation of hydrogen bonds with the glycans attached to glycopeptides. the material was used to isolate glycopeptides from igg, rnaseb, and agp . cellulose columns have also been used for concentration of glycopeptides (snovida et al., ) . a method for detecting core-fucosylated (cf) glycoproteins for screening purposes has been reported by jia et al. ( ) . after igg depletion, fucosylated plasma proteins were enriched by use of lens culinaris lectin and the bound glycoproteins were digested by trypsin. these compounds were enriched by use of a , da cut-off filter, a procedure that also combines de-salting and concentration. the recovered glycopeptides were then treated with endoglycosidase f , which specifically cleaves the glycosidic bond between the two proximal (core) glcnac residues and leaves the fucosyl-glcnac residues attached to the peptides. four standard glycoproteins, apo-tf, fetuin, rhepo, and rnaseb, was used to illustrate the method. in addition, a part of the untreated tryptic peptides was treated with pngase f in order to locate the glycosylation site by the asn to asp conversion. products were detected by maldi-tof. in a related method using an ion trap, a neutral loss scan for fucose ( da) was also used to detect fucosylation. the methods were applied to the detection of fucosylated glycoproteins in the plasma of healthy subjects and subjects with hepatocellular carcinoma. over fucosylated glycoproteins and attachment sites were identified, and over , mass spectra of cf glycopeptide were analyzed. a method termed the sulfate emerging method has been described for specifically extracting sulfated glycopeptides (toyoda, narimatsu, & kameyama, ) from mixtures. the method overcomes the often negative contribution from other charged groups in the molecules. to accentuate the negative charge on the sulfate group, basic amino acids were eliminated and carboxylic acids were neutralized as follows: the protein was first hydrolyzed with trypsin and then the positively charged c-terminal lysines (lyss) and arginines (args) were eliminated by incubation with carboxypeptidase b. the negative charges of the carboxylic acid groups on the peptides were then neutralized by chemical modification with acetohydrazide using the recently reported quantitative modification of carboxyl groups in sialic acid using this reagent and -ethyl- -( -dimethylaminopropyl) carbodiimide hydrochloride ( ). the sulfated glycopeptides in the mixture were then captured by anion exchange resin with a basic buffer (ph . ) in which protonation of histidine residues was suppressed. finally, the sulfated glycopeptides were eluted from the anion exchange resin by increasing the ionic strength of the elution buffer for analysis by ms. rather than trypsin, pronase has been used as a nonselective enzyme to reduce the protein to a single amino acid (asn) or short peptide attached to n-glycans. these compounds are generally smaller than those obtained from o-linked glycopeptides, probably because o-glycans lie closer to the peptide backbone than n-glycans and protect the polypeptide from enzymatic digestion. n-glycans usually rise above the peptide backbone exposing the polypeptide to enzymatic digestion. dodds et al. ( ) have described immobilized pronase which retains its activity after repeated use for at least weeks. use of negative ion detection has been reported by nwosu et al. ( ) as providing distinct advantages over detection in positive ion mode for the detection of glycopeptides produced by pronase digestion. analysis in positive ion mode, although most commonly used for glycopeptide characterization, is hampered by potential charge-induced fragmentation of the glycopeptides and poor detection of the glycopeptides carrying sialic acids. furthermore, cid spectra of glycopeptides in the positive ion mode predominantly yields glycan fragments with minimal information on the peptide moiety. in the study by nwosu et al., which employed bovine lactoferrin for detection of n-glycosylation and k-casein for o-glycosylation, potential n-linked glycopeptides were detected in the positive ion mode whereas potential n-linked glycopeptides were detected in negative ion mode. analysis of k-casein, which contained mainly sialylated glycans, yielded improved results in negative mode. experimental details for peptide mass fingerprinting and identification of glycosylation sites have been published by wilson, simpson, and cooper-liddell ( ) . unlike the case with released glycans, where a relatively low mass accuracy measurement is usually sufficient to determine the composition in terms of the constituent monosaccharides, the situation is very different for measurements of glycopeptides. desaire and hua ( ) have examined the accuracy required and have concluded that in only a few cases can the mass accuracy provided by most commercial instruments be sufficient to unambiguously assign compositions to all glycopeptides in a mixture. e. n-glycan release once glycosylation sites have been determined, detailed structural analysis of the attached glycans is more conveniently carried out after releasing the glycans from the protein or peptide. both chemical and enzymatic methods are available but although, in the past, chemical release with hydrazine was popular, most investigators now prefer enzymatic methods. i. enzymatic release pngase f. peptide n-glycosidase f (pngase f), an amidase, is the most popular enzyme for cleaving n-glycans from their asn linkage site. it shows a broad range of substrate specificity with the exception that it does not release glycans bearing a fucose residue attached to position of the reducingterminal glcnac; in these cases, pngase a is appropriate. pngase f cleaves the entire glycans, which are released as the corresponding glycosylamines. these compounds rapidly hydrolyze to the glycan with retention of the reducing terminus. this method is, thus, distinctly advantageous to techniques such as b-elimination, popular with o-glycans (see below) because this site can conveniently be used to attached tags for fluorescence or other detection methods. wang et al. ( i) have recently found that the activity of the enzyme towards denatured glycoproteins can be enhanced by removal of the nterminal h helix from the enzyme. bereman et al. ( b) have studied methods for optimizing the release of glycans with this enzyme. dialysis of plasma prior to incubation was found to have little or no effect. however, microwave-assisted glycan release was found to be beneficial; min at ˚c with approximately w was found to give optimal results. surprisingly, no protease digestion was required as needed with standard incubation methods, and it was found that an -hr incubation with no detergent (np ) led to the greatest ion abundance of glycans from plasma glycoproteins. data could be obtained in less than day from raw plasma samples utilizing microwave irradiation. pngase f-glycan release from human serum glycoproteins has been achieved in min by using a constant microwave power of w, giving a temperature of ˚c (kronewitter et al., ) . in this study, the glycans were recovered by spe using a robotic liquid handler and examined by maldi with an ft-icr instrument from dhb. replicate analysis gave coefficients of variation of less than . . the standard protocol for n-glycan release from glycoproteins requires relatively long deglycosylation times (from several hours to, usually, overnight) and relatively high enzyme concentration (from : to : enzyme/substrate ratio). szabo, guttman, and karger ( a) have used a high-pressure method, both to reduce the reaction time and the amount of enzyme required. thus, a pressure-cycling device was use to cycle the pressure from atmospheric to as high as kpsi. greater than % release of the asn-linked glycans from bovine rnaseb, human tf, and polyclonal human immunoglobulin was achieved in only a few minutes using as low as : , enzyme: substrate molar ratio. a reactor with immobilized pngase f on a monolithic polymer support in a capillary has been developed that allows fast and efficient release of n-linked glycans. performance was determined with rnaseb, chicken ovalbumin, and human igg with detection by maldi-tof ms. the optimized reactor was integrated into a multidimensional system comprising on-line glycan release and hydrophilic interaction liquid chromatography (lc) followed by esi-tof ms detection. using this system, human igg was deglycosylated at room temperature in . min to an extent similar to that achieved with the soluble enzyme after hr at ˚c (krenkova, lacher, & svec, ) . immobilization of pngase f on detonation nanodiamonds has resulted in glycan release from glycoproteins in less than min . the method, using trypsin immobilization, also gave good results and proved to be better for proteolysis than the use of commercial immobilized trypsin beads. artefacts associated with pngase release of n-glycans. for solution release of glycans, the glycoprotein is usually denatured by reduction and alkylation or by use of detergents or other compounds such as urea. in general, low concentrations of urea (< m) do not usually cause irreversible protein denaturation. indeed, pngasef itself is stable in . m urea at ˚c for hr and still possesses about % activity in m urea. however, other glycoproteins appear more susceptible. for example, analysis by sds-page and maldi-tof ms have revealed that additional . kda of glycans can be released by pngase f if the deglycosylation is conducted in m urea suggesting that urea treatment exposes a glycosylation site that was previously inaccessible to pngasef . use of urea, however, can cause problems with the released glycans because it has been reported to compete with water for hydrolysis of the initially formed glycosylamines with the formation of a urea complex (omtvedt et al., ) . another artefact that has been found in glycans released with pngase f involves the reaction of the glycosylamines with h s to form the glycan-sh analogue. the h s arises from dithiothreitol (dtt, / ), a reagent used for protein alkylation and present in some commercial preparations of the reagent. the consequence of this reaction is an increase in da giving the impression, from a simple mass measurement, that the glycan has an additional oxygen atom. addition of da can also be observed in maldi spectra as a consequence of the formation of [mþk] þ rather than [mþna] þ ions, but this possibility can be excluded by formation of [mþcs] þ ions whereupon the da mass increase will still be present. the reaction with h s was first noted with glycans from human igg and negative ion ms/ms of the artefactual products located the da to the reducing-terminal glcnac residue. the negative ion fragmentation pattern was the same as that expected for a glycan with a hexose attached to the -position of the residue rather than fucose (deoxy-hex) that was actually the case. this result emphasizes how careful one must be, not only in deducing compositions from glycan masses but also in interpreting their ms/ms spectra . another artefact of the pngase f release step, detectable by ce but not by ms has been identified as the product of epimerization of the terminal glcnac residue to n-acetylmannosamine (mannac, ) under the slightly basic conditions usually employed in the release reaction. reducing the ph to . effectively removed the by-product (liu, salas-solano, & gennaro, h) . other endoglycosidases. another popular enzyme for n-glycan release is endo h which cleaves the chitobiose core of high-mannose and hybrid glycans but not complex ones, leaving a glcnac residue, with any linked fucose, attached to the protein. pace et al. ( ) have made use of this property to release and identify minor glycans in igg without interference from the more abundant complex glycans. endo f has a similar specificity and has been used by, for example, voutilainen et al. ( ) to detect glycosylation in talaromyces emersonii cellobiohydrolase cel a produced in the yeast saccharomyces cerevisiae. ammonium hydroxide/carbonate-based chemical deglycosylation and pngase a enzymatic release have been compared for glycan release from a plantibody produced in tobacco plants (triguero et al., ) . although both methods gave similar profiles as evaluated by hplc of -ab derivatives, the main drawback of the chemical release method was that it induced degradation of a , -fucosylated n-glycans. ii. extraction and purification of released glycans. cleanup of samples prior to ms is crucial to obtaining good spectra. many methods are in use; porous graphatized carbon (buser et al., ) is popular and we have found nafion membranes (börnsen, mohr, & widmer, ) to be convenient at removing both salts and some hydrophobic compounds. avoiding the introduction of contaminants is also important. disposable plasticware such as plastic test tubes that are normally used to process samples have been shown to be a major source of contamination. the contaminants, which produce ions, mainly prompt fragments across the entire mass range to about m/z , , originate from polymers that are used to protect the plastic against oxygen or uv light degradation. such compounds are hindered amine light stabilizers (halss) used in modern polyolefin (polypropylene, polyethylene) stabilization. the polymeric agent: poly-(n-b-hydroxyethyl- , , , -tetramethyl- -hydroxy-piperidinyl succinate, ), known as tinuvin- or lowilite , has been found to leach from laboratory polypropylene or polyethylene plastic test tubes into solvents used for sample preparation. . ml plastic tubes were found to be the major source of the contamination but the authors of the paper found that this could be minimized by using large solvent volumes of, for example, matrix solution (sachon et al., ) . amano and nishimura ( ) have used two kinds of hydrazide-functionalized glycobeads, termed glycoblot h and glycoblot abc of which the latter carries an additional fluorescent probe, to extract pngase f-released n-glycans from solution. sialic acids were then converted into methyl esters using the -methyl- -p-tolyltriazene (mtt, / ) reagent de-scribed by miura et al. ( ) and the products were examined by maldi-tof ms after release of the glycans from the beads by mild acid hydrolysis. the method was used to examine human serum glycoproteins for cancer biomarkers. full experimental details of the extraction and derivatization procedure are given in the paper. enrichment of serum and cellular glycoproteins with glycoblot h beads has also been used for o-glycan analysis . glycans were released from human milk osteopontin and urinary muc glycoproteins with ammonium carbamate and the method was proposed as ideal for identification of biomarkers. a method for sequentially enriching sulfated glycans by strong anion-exchange chromatography according to their degree of sulfation has been described by lei, novotny, and mechref ( ) . the method is based on modifying the binding ability of strong anion-exchange material with different sodium acetate concentrations, thus enabling selective binding and a subsequent elution of different glycans according to their degree of sulfation. before this enrichment, the negative charge on any sialic acid was eliminated by permethylation. the method was initially optimized using sulfated oligosaccharide standards and then used to examine the sulfated n-glycans from btsh, a glycoprotein possessing mono-and disulfated n-glycans. f. analysis of released glycans ahn et al. ( ) have obtained good resolution of n-glycans as their -ab derivatives using hilic columns packed with . mm sorbent. glycans were released from rnase b with pngase f and extracted using a microelution hilic spe well plate. the labeled glycans were also extracted from the preparative reagents using the same plate and their integrity was checked by maldi-tof ms. in another technique, guillard et al. ( ) have experimented with optimizing a linear ion trap instrument for automated measurement of permethylated n-glycans in serum. glycans were released with pngase f, cleaned with graphitized carbon and permethylated with the sodium hydroxide method. dhb, although the favored matrix for carbohydrates, failed to give the necessary reproducibility because of the large crystal size. chca, on the other hand, proved to be satisfactory. full experimental details for analysis of o-and n-linked glycans have been published by several authors (azadi & heiss, ; morelle et al., a; north et al., b) . g. total methods for glycoprotein structure there have been many reports of methods for total glycoprotein analysis of which the following are representative. a small-scale method for n-glycan release and analysis from plants used to produce recombinant glycoproteins has been described . concentration, protease digestion and deglycosylation are carried out in a single concentrator unit mass spectrometry reviews doi . /mas without the need for intermittent purification. this approach minimized adsorptive losses and facilitated handling. the plant protein was concentrated in a unit with a kda cutoff and after buffer exchange, pepsin digestion was carried out in the concentrator overnight. deglycosylation was carried out with pngase a for hr. released n-glycans were purified using reversed-phase and cation exchange chromatography in micro-columns and analyzed by maldi-tof ms without derivatization. a chip-based reversed-phase lc/ms method for n-glycan analysis suitable for biomarker discovery has been developed by . n-glycans were released from bovine fetuin as a model glycoprotein and human serum glycoproteins with pngase f and reduced to alditols with an ammonia-borane complex. the glycans were then permethylated in dimethylformamide to avoid artefacts in ms measurements and their structures were checked by maldi-tof measurements. reversed-phase microfluidic lc of the permethylated n-linked oligosaccharide alditols was then performed and was shown to resolve some closely related structures. optimized lc gradients, together with nanospray ms were then used with human serum samples to distinguish breast cancer patients from control individuals. a previously established two-dimensional hplc technique has been adapted as a hplc-maldi ms method for n-glycan analysis by gillmeister et al. ( ) . glycans were released from glycoproteins with pngase f purified with graphitized carbon and fluorescently labeled with -ap. the labeled glycans were analyzed on a -mm reversed phase (rp) hplc column and spotted onto a maldi-tof ms plate together with the dhb matrix using an automated plate spotter. the method gave a -fold reduction in the required amounts of starting protein compared with the earlier procedure. the entire process could be carried out in - days for a large number of samples as compared to - weeks per sample for previous two-dimensional hplc methods. the modified method was verified by identifying n-glycans from an igg antibody from human sera samples and applied to analysis of tissue plasminogen activator (tpa) from cho cell cultures under varying culture conditions. kim et al. ( b) have released glycans on a polyvinylidine difluoride (pvdf) membrane with pngase f and cleaned them with graphitized carbon contained in a -well plate before converting them into girard's t derivatives to introduce a constitutive cationic charge for quantification. analysis was by maldi-tof ms and the robust method was used to profile n-glycans from ovarian cancer patients using as little as ml of serum. a high-throughput method for the analysis of human plasma glycomes using a -channel multiplexed capillary gel electrophoresis (cge) dna sequencer with laser-induced fluorescence detection (cge-lif) system has been described with maldi-tof ms used to provide structural information (ruhaak et al., c) . glycans were released from plasma glycoproteins in a -well plate using pngase f and converted into apts derivatives with the help of -picoline borane ( ) as the reducing agent. analysis by cge-lif using the dna sequencer allowed samples to be analyzed in only . hr (the experimental time was longer because of two overnight incubations). the method was applied to a study of glycosylation patterns during first, second, and third trimesters of pregnancy, as well as weeks, months, and months postpartum. although analysis of glycoproteins carrying neutral glycans is now routine, analysis of glycoproteins with sialylated glycans is more difficult. hao, ren, and xie ( ) have approached the problem by first performing a tryptic digestion to give peptides and glycopeptides. peptide mass fingerprinting was performed on the peptides in order to identify the protein. the glycopeptides, separated by hilic chromatography, were examined by maldi-tof ms and ms/ms and treated with pngase f to release the glycans, which, together with the resulting peptides were again examined by ms. the asn to asp conversion in the peptide fraction enabled the glycosylation site to be identified. finally, the glycans were desialylated with dilute hcl and again analyzed by ms. the technique was applied to glycoproteins from human serum separated by -d electrophoresis and the differences in n-glycosylation were successfully determined for a -antitrypsin between different gel spots. in another method, sialylated glycoproteins have been selectively periodate-oxidized, captured on hydrazide beads, trypsinized and released by acid hydrolysis of the sialic acid glycosidic bonds. mass spectrometric fragment analysis allowed identification of glycan structures and additional fragmentation of deglycosylated ions yielded peptide sequence information which allowed glycan attachment sits to be identified together with identification of the protein. using this method, the investigators identified n-linked and o-linked glycosylation sites on glycoproteins from human cerebrospinal fluid . related to this method is one developed by klement et al. ( ) for enrichment of o-glcnac-modified proteins. glycoproteins were again oxidized with periodate and captured by hydrazide resin capture. rather than release of the peptide enzymatically, the glycopeptide was released by hydroxylamine treatment which also converted the aldehyde groups of the oxidized glycan to oximes. the open nature of the carbohydrate ring, following oxidation, lead to the production of characteristic fragment ions facilitating both glycopeptide identification and site attachment. the method was applied to analysis of a-crystallin a and the drosophila proteaosome. h. comparisons of methods for n-glycan analysis a comparative study of three techniques, maldi-tof, sds-page and cge-on-a-chip, for measuring the mws of large glycoproteins has been reported by müller et al. ( b) . it was found that all three techniques were capable of determining the mw of all high mw (glyco)proteins tested. the non-commercial cge-on-a-chip assay allowed electrophoretic separation of proteins in the mw range from kda to mda. mw assignment was limited to kda in the case of sds-page but with the proper matrix (thap for most glycoproteins, sinapinic acid for a -macroglobulin) and sample preparation, analysis with a standard maldi-tof-ms provided accurate mws for all high mw proteins up to mda. three methods for n-glycan characterization, namely maldi-ms of glycopeptides from tryptic digestion, negativeion esi-ms/ms of released n-glycans, and normal-phase hplc of fluorescently labeled glycans, in combination with exoglycosidase sequencing, have been evaluated for glycan identification using monoclonal antibodies expressed in tobacco plants as model compounds (triguero et al., ) . the ms methods identified the major glycans, but the hplc method was found to be the best for identification and relative quantitation. negative-mode esi-ms/ms easily provided direct identification of features such as the linkage position of the fucose residue linked to the inner core glcnac residue. grey et al. ( ) have developed a high-performance ion exchange chromatographic method for n-glycan analysis and have shown that it gives very similar results to analysis by maldi-tof. a series of standard glycans was examined and the method was extended to the analysis of n-glycans released from igg . an inter-laboratory study involving eleven uk laboratories using their routine glycan analysis procedures looked at reproducibility on glycan profiling from n-glycans released before the study from four glycoproteins, human and bovine agp, bovine pancreatic rnaseb and human serum immunoglobulin g (higg). data interpretation focused on the relative amounts of different glycan structures present, the degree of sialylation, galactosylation profiles, fucosylation, and bisecting glcnac content and the number of glycan components identified. all laboratories found high levels of sialylation for human and bovine agp, but varying amounts of di-, tri-, and tetra-antennary glycans. values obtained from mass spectrometric and chromatographic methods clustered separately. the proportion of the major man glcnac from rnaseb was between % and %. proportions of fucosylated and bisected glcnac glycans from higg were between % and % and % and %, respectively. mass spectrometric approaches consistently identified more glycan species, especially when both n-glycoylneuraminic acid (neu gc) and neu ac were present (thobhani et al., ) . a recent test of the ability of several laboratories to identify n-glycans released with pngase f from a mixture of four glycoproteins, asialo-fetuin, chicken ovalbumin and both human and bovine agp has also yielded some alarming results (orlando, leymarie, & keck, ) . although of the laboratories detected the presence of fucosylated complex nglycans, of them incorrectly located the fucose to the core glcnac of human agp rather than to an antenna. all nine of the labs using ms (not ms ) misidentified the site and all five of laboratories relying on software to identify the site also reported it incorrectly. although the ionic charge was not specified, it is assumed to be positive because it would be impossible to make this mistake with negative ion fragmentation. features such as the position of fucose residues produce diagnostic cross-ring fragments whose mass depends on the location of the fucose residue . such mis-identifications are particularly worrying because serum agp is elevated in inflammatory disease and is of potential use as a biomarker. many laboratories using this potential biomarker in serum also report the structure incorrectly. another problem in the survey arose with n-glycoylneuraminic acid ( / ), present in the bovine version of a glycoprotein. this carbohydrate is antigenic and is of concern to pharmaceutical companies producing pharmaceuticals in organisms that utilize this sialic acid. in the survey, eight of the laboratories, including seven of the eight participating industrial laboratories failed to detect its presence. all of the four laboratories that used a fluorescence tag, failed to detect this sialic acid. slightly better results were obtained by laboratories using maldi; of ten labs that used this technique, only two failed to detect this sialic acid. most laboratories that successfully detected this carbohydrate, permethylated their samples, which, of course, would stabilize it to maldi conditions. with mixtures containing different quantities of glycans, no lab correctly detected either three or four of the changes, one lab identified two of the four changes, seven labs identified one change but the other labs failed to identify any changes correctly. clearly, current analytical methods leave much to be desired. i. applications of maldi to the detailed structural determination of n-linked glycans a large number of reports have appeared on the applications of the above techniques to analysis of n-glycans from specific glycoproteins. these are summarized in tables and . other examples can be found in the tables on medical applications of maldi ms (table ) and biopharmaceuticals (table ) . some of the more unusual structures to be discovered are tetraantennary glycans with poly-n-lactosamine extensions with up to nine fucose residues in human seminal plasma , a man glcnac glycan with a bisecting glcnac residue ( ) (buser et al., ) ; a man glcnac -type glycan with two bisecting glcnacs ( ) proposed from dictyostelium discoideum feasley et al., ) and an unusual glycan with internal fucose and glucuronic acid (glca) from rapana venosa hemocyanin (velkova et al., ) . however, in the latter case, the structure was based on evidence from only one positive ion ms/ms spectrum and is open to alternative interpretations. symbols as defined for structures and plus = mannose j. miscellaneous studies among other studies, maldi-tof ms has been used to confirm the glycan compositions of several well-known glycoproteins in a study showing that glycosylation protects proteins against free radicals generated from toxic xenobiotics (martínek et al., ) the rice-derived recombinant human transferrin (rhtf) has been shown to be non-n-glycosylated by maldi and pngase f enzyme digestion . structural determination (highmannose, with bisect on branching mannose and antenna) (continued) (continued) sodium hydroxide followed by reduction to prevent a peeling reaction is the most common method but has the disadvantage of eliminating the reducing terminus, thus preventing tagging with fluorescent or other tags. some investigators have, thus, investigated the use of milder bases with the aim of avoiding the reductive stage and retaining the reducing terminus. zheng, guo, and cai ( ) have compared ammonia, methylamine and dimethylamine at ˚c for hr for release of n-acetylgalactosamine (galnac) from a small glycopeptide. b-elimination with dimethylamine and methylamine resulted in the conversion of the glycopeptide to . % of the dimethylamine derivative at m/z . and . % of the methylamine derivative at m/z . , respectively. however, the incubation of the glycopeptide with ammonia only resulted in % production of the product. the authors concluded that elimination with dimethylamine was the most efficient for release the o-linked glycans. release with methylamine was used by sun et al. ( b) to determine the glycosylation sites in human protein c inhibitor by the . mass increment introduced by the reaction. release with ammonia has been investigated in detail by yu et al. ( a) for o-glycan chains with b , -linked cores. in contrast to b , -linkages of the n-glycan-type, which were shown to be stable under the ammonium-based alkaline conditions, the b , -linkage was found to be labile and to give considerable peeling . the results indicated that complete prevention of peeling under nonreducing alkalicatalyzed hydrolysis conditions remains difficult. the yields of o-and n-glycans from bovine fetuin released by conventional means (pngase f and reductive b-elimination with naoh) were found to be greater. it was concluded that great care should be taken when employing such non-reducing alkaline conditions in glycomic analysis and in obtaining some o-glycans for functional studies. because the hydroxide ion appears to cause the unfavorable peeling reactions, miura et al. ( b) have investigated the use of the ammonium salt, ammonium carbamate for glycan release. the efficiency of release with ammonium carbamate was compared with a common conventional procedure, namely saturated ammonium carbonate/aqueous ammonia with bovine submaxillary mucin (bsm) as the test compound. release with ammonium carbamate did not exhibit significant loss of glcnac-b , (neu aca , )galnac or glcnacb , (neu gca , )galnac structural determination (highmannose, bisected high-mannose glycans). development associated with alteration of fucosylated glycans structural determination (highmannose, hybrid, complex glycans). poly-fucosylation human (ht- m colon cancer cells) pngase f, tof, glycans (per-me) pattern of n-glycosylation serves as a recognition signal for galectin- . high-man, hybrid, bi-, tri-tetra-antennary. (stechly et al., ) human (cytolytic t lymphocytes) pngase f, tof/tof (dhb), glycans (per-me) loss of effector function shown to be accompanied by major changes in nand o-glycosylation (antonopoulos et al., ) marmota monax (woodchuck with liver cancer), gal-α-( → )-gal in mouse epidermis but galnac-β-( → )-glcnac in human. high-mannose, hybrid complex (uematsu et al., ) pngase a pngase f, tof/tof (dhb/ % plants engineered to produce lewis x epitopes. (paucimannosidic glycans) mass spectrometry reviews doi . /mas and the profile of the major o-glycans was similar to that obtained following conventional reductive amination with naoh/nabh . on the other hand, glycans obtained by treating bsm with ammonium carbonate/ % aqueous ammonia and analyzed by maldi-tof ms showed a significant increase of the disaccharide components, neu aca , galnac and neu -gca , galnac, suggesting the presence of a peeling reaction. use of ammonium carbamate, thus, appears to produce efficient release without concomitant peeling. the release was performed by addition of powdered ammonium carbamate and incubation for hr at ˚c. yamada et al. ( ) have used a recently developed automated release apparatus using lithium hydroxide to obtain o-glycans from leukemia and epithelial cancer cells. because these cells usually contain free glycans, the investigators first reduced these with sodium borohydride and then labeled the released glycans with -aa in order to avoid interference. a new release method reported by goetz, novotny, and mechref ( a) combined enzymatic and chemical techniques and used b-elimination to cleave glycans from serine (ser) and threonine (thr) but not asn. the method involved first a nonspecific proteolysis with pronase, followed by solid-phase hydrazine, tof (dhb), glycans ( -ap) structural determination. high-man, hybrid, bi-, tri-, tetra-antennary complex (sumiyoshi et al., ) rabbit and chicken (erythrocytes) tof, glycans ( -ap) to study alterations in receptor-binding properties of h -type swine influenza viruses in embryonated chicken eggs. high-man, hybrid, bi-, tri-antennary (takemae et al., ) rat ( structural determination (highmannose, hybrid, complex glycans) girard's t for quantitation (kim, et al., c) sus domestica (respiratory epithelial cells) pngase f, tof, tof/tof (dhb), glycans (per-me) influenza virus shown to utilise α → linked neu ac to infect cells permethylation with sodium hydroxide. the basic sodium hydroxide caused the glycans to be released by b-elimination and these were immediately permethylated. this combination of the enzymatic and chemical procedures was reported to give a substantial improvement in sensitivity and analytical reproducibility over existing methods by minimizing sample losses. moreover, the approach was reported to extend the cleavage protocols to large glycoproteins where small oligosaccharides were not accessible to conventional chemical treatment. the method was developed with fetuin and used to identify new o-glycans from bile salt-stimulated lipase (bssl). maniatis, zhou, and reinhold ( ) have released o-glycans with aqueous dimethylamine in the presence of sodium borohydride by use of a microwave oven. the release was performed at ˚c and, for a heptapeptide carrying a glcnac group attached to thr, was complete in min. the reaction also labeled the site of detachment with a dimethylamino group. use of a : mixture of dimethylamine and [ h ] nh produced doublets in the peptide mass spectrum separated by six units, allowing the glycosylation sites to be readily identified. n-glycans are frequently removed from glycoproteins before o-glycan removal by b-elimination. however stone et al. ( ) have reported improved recovery of o-glycans from murine tissues without prior n-glycan removal. kbh and koh were used to remove the o-glycans and possible low levels of concomitantly released n-glycans were tolerated. ii. use of hydrazine. hydrazine has also been used to release these glycans with a new gas-phase method using anhydrous hydrazine being evaluated by goso, tsubokawa, and ishihara ( ) with muc-type oligosaccharides from porcine gastric mucin (pgm) and bovine fetuin. released glycans were examined by hplc and maldi-tof as -aa derivatives. glycans obtained by the treatment with hydrazine at ˚c for hr resembled those obtained by b-elimination, except for the additional disaccharide fractions derived from the core side of the oligosaccharides by further degradation. the other degraded products derived from the core side could not be derivatized by -aa, therefore, were not visible by fluorescence detection. release of the glycans was incomplete after hr but almost complete liberation was achieved by extending the treatment to hr. however, degradation increased. in this case, the addition of barium oxide to the reaction vessel decreased the degree of further degradation. results similar to pgm were obtained from bovine fetuin, but with less degradation. application of this method to the analysis of rat gastric mucin (rgm) showed that rgm has a large oligosaccharide portion on the core side. iii. other methods. a new method for o-glycan removal for study of the residual deglycosylated protein has been reported . desialylated glycoproteins whose sugar chains consisted of gal-galnac, were immobilized on alkali-stable, reversed-phase poros beads and treated with periodate to oxidize the cis-glycol groups in the gal residue. the resulting aldehydes were then susceptible to b-elimination under mild (nh ) basic conditions. the remaining galnac residue, which now contained a cis-glycol group, was oxidized with further treatment with periodate and removed with base. although the number of cycles required depended on the number of gal-galnac repeats, large core -type glycans that usually only have gal attached to the -position of the core galnac, could be deglycosylated in only two steps. o-linked glycosylation often occurs in muc-type domains that are heavily and heterogeneously glycosylated. several strategies to determine the heterogeneity of these domains have recently been investigated with four glucanases secreted in large quantities from trichoderma reesei, all of which contained heavily o-glycosylated muc-like linker regions, being used as models. the strategies involved monosaccharide compositional analysis and identification of the released glycans by hpaecpulsed amperometric detection (pad) and carbon-lc esi-ms/ ms. glycosylated peptides were generated by different protease digestions (trypsin, papain, asp-n, pretaq) and enriched by hilic microcolumns. the complex o-glycan heterogeneity was determined by maldi-ms and esi-ms, but the dense o-glycosylation in the muc-type domains conferred high resistance to protease cleavage. etd-ms/ms of the glycopeptide-enriched protease digests was unsuccessful for the assignment of o-glycosylation at individual sites within the muctype domains but allowed several previously unknown o-linked sites outside the defined linker region to be found on two of the four glucanases (christiansen et al., ) . iv. comparison of methods. three samples of iga isolated from the serum of patients with multiple myeloma have been distributed on behalf of the human proteome organization human disease glycomics/proteome initiative to laboratories for comparative analysis of their o-glycans. a range of techniques was used; the two strategies that yielded the best data were direct positive ion ms analysis of permethylated glycans and lc-ms analysis of native reduced glycans in negative ion mode. the studies reinforced the pre-eminent performance of ms techniques for o-glycan profiling (wada et al., b) . several reviews and general analytical methods have been reported; these are listed in table . loss of sulfate is a major problem in the maldi analysis of these compounds but this can be avoided by use of desorption esi (przybylski et al., a) . little use appears to have been made of the peptide binding method for stabilization of sulfate groups in these compounds as reported by juhasz and biemann ( ) . one report concerns the identification of a pentasulfated hexasaccharide responsible for binding to the growth factor pleiotrophin . the compound was complexed with (arg-gly) and identified by maldi-tof from dhb. in another study , a pentasulfated hexasaccharide with a novel structure (d , hexaa - galnac( s)b - idoa( -s)a - galnac( s)b - idoa( s)a - galnac( s)) has been isolated from the chondroitinase ac-i digest of shark skin. again, (arg-gly) was used as the complexing agent. bultel et al. ( ) have developed a method for analysis of heparin oligosaccharides by using controlled nitrous acid degradation followed by high-performance anion exchange chromatography (hpaec) separation and uv-maldi-tof analysis. the use of three different matrices, dhb, chca, and nor-harmane were investigated but only dhb and nor-harmane were needed to assign the position of sulfate groups. dhb allowed the molecular ion to be detected in nearly all cases and gave fragments arising from the loss of sulfate groups. nor-harmane, in contrast, produced mainly fragments. in all cases, ions retaining the sulfate groups were observed making these fragments essential for assigning the sulfated positions of each residue. while nor-harmane was not able to produce enough analyte desorption/ionization, fragments useful for structural assignment were produced by the addition of butylammonium formate to the dhb matrix. a method for analysis of gpi anchors on the "proteomic" scale has been described . partially purified proteins were separated by sds-page and then blotted onto a pvdf membrane. following identification of the protein, the gpi anchor was analyzed by three methods. first, the compound was hydrolyzed with hcl in the presence of [ , , , , , - h ]myo-inositol and the hydrolysate was analyzed as trimethylsilyl (tms) derivatives by gc/ms. next, the phosphate bonds were cleaved and the carbohydrate structure was elucidated by electrospray or maldi-tof ms. finally, the diacylglycerol-attached myo-inositol moiety was detached by nitrous acid deamination and analyzed by negative ion electrospray. bütikofer et al. ( ) have studied lipid remodeling of gpi glycoconjugates in procyclic-form trypanosomes and shown that, in trypanosoma congolense, the steady-state lipids consist of lyso-(acyl)phosphatidylinositol (pi, ), deacyl-pi and deacyl-(acyl)pi species, where (acyl) indicates an acyl group attached to the inositol moiety. maldi-qit-tof in negative ion mode from thap was used to analyze the pi species after deamination with nitrous acid. structural determination (core , core ). samples from geographically remote labs. similar glycans (babu, et al., ) human, (enterocytelike ht- cells) β-elimination, tof, glycans (per-me), gc/ms structural determination (core ) (morelle, et al., b) human ( absence of fucose on core glycans impairs baba-mediated adhesion to gastric mucosa (magalhães et al., ) (continued) although many bacteria produce s-layer proteins with glycosylation, qazi et al. ( ) have used maldi-tof ms to show that those from clostridium difficile are not glycosylated. this topic has been reviewed by zhang et al. ( f) and capote and sanchez ( ) . maldi is used mainly to determine the extent of glycation of intact proteins or to determine the sites of attachment of the covalent glycans. a. specific methods for glycated peptides amadori peptides have been enriched with boronate affinity tips for measurement by maldi-tof/ms. the tips showed the highest binding efficiency for glucose at ph . employing ammonium chloride/ammonia buffer with ionic strength of mm. the bound constituents were released by sorbitol ( / ) or formic acid. using sorbitol for elution required desalting prior to analysis. of three different sorbents tested: fullerenederivatized silica, ziptips (c ), and c silica, fullerenederivatized silica and ziptips showed the same performance with respect to the numbers of glycated peptides and gave better performance than c silica. fewer glycated peptides were detected by lc-ms/ms than by maldi . a novel fullerene(c )-derivatized silica material has been compared with octadecyl(c ) and triaconthyl(c )-silicas for their ability to recover peptides from digests of hsa and fibrinogen. c -and particularly the c ( nm)-spe materials were found to be the two most effective. after glycation the digests of fibrinogen and hsa were also separated. this new method made the detection of a considerably higher number of glycated peptides possible compared to the unfractionated digests and the use of boronate affinity chromatography in the case of fibrinogen. for hsa, new sites of glycation at lys and arg residues were found . a mass spectrometric method for screening large tandem mass spectrometric (ms/ms) datasets for protein glycation with glucose ( / ), lactose ( ) and maltose ( ) has been developed (montgomery, tanaka, & belgacem, ) . control experiments using a standard peptide containing a single glycation site led to the discovery of characteristic neutral loss fragmentation patterns in ms/ms analysis for glucose, lactose and maltose condensed with peptide. for glucose glycation, neutral losses of , , and da were observed in accordance with previously published reports. the neutral loss patterns for lactose and maltose were found to be identical, with characteristic losses of , , , and da. these signature losses were observed irrespective of the maldi mass spectrometer used and were valid in both tof-tof and qit-tof instruments. these neutral loss signatures were then applied to elucidation of modified peptides from a complex hsa digest glycated with each of the proposed sugars. screening of these large datasets o-labeled digests indicated that lyss and also had significant degrees of modification. the modifications that occurred at these sites were variations of fructosyl-lys and advanced glycation end products (ages) which included -alkyl- formyl- , -glycoyl-pyrole ( / ) and pyrraline ( / ). the fragmentation behavior of the peptide ac-paapaa-papaektpv-oh (human histone h . , positions - ) glycated via its lys residues to adp-ribose has been studied by fedorova, frolov, and hoffmann ( a) . under maldi conditions, the adp-ribosyl group was cleaved, almost completely at the pyrophosphate bond by isd and psd. however, this cleavage was very weak in esi-ms. the remaining phospho-ribosyl group was stable, providing a direct and reliable identification of the glycation site via the b-and yion series. as well as being associated with health problems, in for example, diabetes, protein glycation is important in the food industry in, for example, the browning of food during cooking. the reaction is also being used to attach carbohydrates to proteins to improve technological and biological functionalities. in relation to this latter use, corzo-martnez et al. ( ) have used maldi-tof ms to study the reaction between b-lactoglobulin and the sugars galactose and tagatose ( ) and found that the reaction can be competitively moderated with pyridoxamine ( ). other reports of the use of maldi to study glycation of specific proteins are summarized in table . typical structures consist of glcnac-murnac ( ) disaccharides cross-linked by short peptides. they are usually analyzed as muropeptides following enzymatic digestion. reports of the use of maldi to study peptidoglycans and muropeptides are summarized in table . this is a very large group of compounds but most work involving maldi has been concentrated on the lps from bacteria and the gsls. work, mainly involving structural determination, on the many other types of glycolipids found in bacteria and similar organisms, is summarized in table . a general review on analysis of microbial glycopolymers has been published by , and fuchs and schiller ( ) phenol/chloroform/petroleum ether and the molecules are frequently split into their component parts by mild acid hydrolysis for further structural analysis. dephosphorylation and deacylation are also common. reviews on the structural investigation of bacterial lps by ms and ms/ms have been published by banoub et al. ( ) and by grice and wilson ( ) . lipid a from coxiella burnetii, the causative agent of q fever has been discussed by toman, skultety, and ihnatko ( ) and a more general review of the core region and lipid a of lps has been published by holst and molinaro ( ) . the first structures of lps to be elucidated from cyanobacteria has been reported by snyder et al. ( ) . two strains of marine water-soluble proteins glucose l-tof ( , -dhap) use of maldi-tof to study glycation during malting synechococcus, wh and cc , were used and were shown to have very simple structures without the complex o-chain found in most proteobacteria. the lps ( ) of these cyanobacteria did not contain phosphate, heptose (hep) or kdo ( / ) but instead possessed -linked glucose as their main saccharide component, with low levels of glcn and galacturonic acid (gala). maldi-tof ms of the intact minimal core lps revealed triacylated and tetraacylated structures with a heterogeneous mixture of both hydroxylated and nonhydroxylated fatty acids connected to the di-glcn backbone. in contrast to enteric lipid a, which can be liberated from lps by mild acid hydrolysis, lipid a from these organisms could be produced only by two novel procedures: triethylamine-assisted periodate oxidation and acetolysis. unique caryophyllose (a- , -dideoxy- -c-(d-altro- , , , tetrahydroxyhexyl)-d-xylo-hexopyranose, )-containing cell wall glycolipids have been identified in lps from mycobacterium marinum (rombouts et al., ) . maldi-tof spectra were obtained from dhb and esi-ms/ms was used to elucidate the glycan sequence. b. lipid a sample preparation has been shown to be critical for assessing the true composition of these compounds. in a comparative study, kawasaki ( ) have shown that maldi-tof ms analysis of lipid a prepared using a commercial "tri-reagent"based procedure with a -chloro- -mercaptobenzothiazole (cmbt) ( / ) matrix gave the best results for compounds with a phosphoethanolamine (petn) modification. in contrast, the analysis of lipid a prepared using an lps extraction kit-based procedure with dhb was preferable for the detection of an aminoarabinose modification. for isolation of lipid a from yersinia enterocolitica, pérez-gutiérrez et al. ( ) used an ammonium hydroxide-isobutyric acid method. lyophilized crude cells were incubated with isobutyric acid and ammonium hydroxide ( : , v/v) at ˚c for hr, washed twice with methanol and the insoluble lipid a was solubilized in chloroform-methanol-water ( : . : . , v/v/v). analysis was by maldi-tof ms from dhb in negative ion mode because of the presence of two phosphate groups. the lipid as a contained both four or six fatty acyl chains whose (boniface et al., ) ratio changed with growth temperature. a similar method has been used by march et al. ( ) to study lipid a from acinetobacter baumannii. the molecules were found to have from four to seven acyl groups. a microwave-assisted method for obtaining lipid a from helicobacter pylori has been developed by zhou et al. ( a) . lyophilized cells were suspended in sodium acetate buffer (ph . ) containing proteinase k and subjected to microwave irritation at w for min at ˚c and then kept for hr at c . after centrifugation and washing, the dried supernatant was examined by maldi-tof/tof from cmbt. the reliability of the technique was demonstrated by analysis of the lipid a from bacterial cells of different h. pylori strains. the phosphorylation and acylation patterns could be elucidated using material from a single colony. furthermore, the investigators found unusual heptaacyl lipid a species present in low abundance in h. pylori mutant that have not been previously reported. the study was claimed to provide the first characterization by ms of the lipid a component from a single bacterial colony. the mass spectrometric behavior of lipid a is highly dependent on both the matrix and phosphorylation patterns. zhou et al. ( a) have investigated the effects of different matrices and co-matrices using lipid a from escherichia coli o as a model system. good results were obtained with cmbt with added edta ( / ) ammonium salt as the matrix. this matrix system was found to enhance the sensitivity of the detection of diphosphorylated lipid a by more than -fold and, in addition, provided tolerance to high concentrations of sds and to both sodium and calcium chlorides at mm concentrations. the method was evaluated for analysis of lipid a with different phosphorylation patterns and from different bacteria, including h. pylori, salmonella enterica serovar riogrande, and francisella novicida. an lc/ms-based assay has been developed for the quantitation of aminosugars, including glcn ( ), galactosamine (galn, ) and aminoarabinose (aran, ) together with ethanolamine (etn), present in lipid a and has been applied to the analysis of lipid a isolated from several biosynthetic and regulatory mutants of s. enterica serovar typhimurium and francisella tularensis subspecies novicida characterized by maldi-tof. lipid a was treated with tfa to liberate and deacetylate individual aminosugars and mass tagged with -aminoquinolyl-n-hydroxysuccinimidyl carbamate ( ), which reacts with primary and secondary amines. the derivatives were separated using rp-chromatography and analyzed with a quadrupole ms to detect quantities as small as fmol. galn was detected only in francisella and aran only in salmonella, while glcn was detected in lipid a samples from both species (kalhorn et al., ) use of grazing-incidence x-ray scattering and monte carlo simulation to investigate physics of bacterial survival against cationic antimicrobial peptides . (continued) c. core oligosaccharide during an analysis of the permethylated derivative of the core oligosaccharide from aeromonas hydrophila, prepared by the hakamori method, ions appearing at mass units higher than the [mþh] þ ions were observed. these ions were determined to be dimethylsulfoxide (dmso) covalent addition products resulting from the michael addition of the dimsyl anion on the c- -c- double bond of a , -anhydro kdo ( / ) residue followed by an addition of a proton on the double bond. corresponding ions were also seen in methylations performed using the naoh technique and represent the first characterization of these addition products (sioud et al., ) . typical pre-maldi techniques for the analysis of these compounds include separation by tlc and cleavage of the cer portion so that the glycan can be analyzed without the heterogeniety produced by the lipid. reviews on the analysis of gsls are summarised in table . a. analysis of intact compounds stübiger et al. ( ) have separated lipids, including gsls by high-performance tlc, stained them with coomassie blue and analyzed them either directly from the tlc plates or, after their removal, with an appropriate solvent. thap ( / ) was used as the matrix with acetone as the solvent for the on-plate analysis because its high volatility minimized sample spreading. a method for structural profiling of individual gsls in a single thin-layer chromatogram by multiple sequential immunodetection has been developed by souady et al. ( ) . structures of the antibody-detected gsls were determined by direct coupling of tlc with ir-maldi after treatment of the tlc plate with glycerol. this combined technique was used to demonstrate structural gsl profiling of crude lipid extracts from human hepatocellular cancer. a new method for analysis of gsls involves selective ozonolysis of the c-c double bond in the ceramide moiety of biological samples and subsequent enrichment of the generated gsl aldehydes by chemical ligation using aminooxy-functionalized gold nanoparticles (aognp). the gsl-bound nanoparticles were removed by ultrafiltration and the gsls were analyzed by maldi-tof and -tof/tof ms from dhb. the method was used for structural profiling of mouse brain gangliosides such as gm , gd a/gd b, and gt b for adult or gd in the case for the embryonic mouse. because the saturated acyl groups remained intact, the spectra provided information on both the carbohydrate and fatty acyl moieties (nagahori, abe, & nishimura, ) . the direct structural characterization of microbial gsl receptors by use of the tlc overlay assay combined with ir-maldi-o-tof-ms has been described (müsken et al., ) . glycan mixtures were separated by tlc in three parallel lanes. one lane was stained with orcinol and a second was overlayed with gsl-specific bacteria. the bound microbes were detected with primary antibodies against bacterial surface proteins and the relevant gsls were detected in the third lane by ir-maldi-tof. the combined method worked on the microgram scale of gsl mixtures and was successfully applied to the compositional analysis of globo-series neutral gsls recognized by p-fimbriated e. coli bacteria, used as model microorganisms for infection of the human urinary tract. (niedziela et al., b) incubation of botulinum neurotoxin serotype d with the gsl, gt b has produced a complex (mw , ) that was detected intact by maldi-tof ms from sinapinic acid and provided evidence that the toxin attacks neurons in a ganglioside-dependent manner (strotmeier et al., ) . a method for generation of novel fluorocarbon derivatives from gsls has been described by li et al. ( f) . the derivatives had high affinity for fluorocarbon phases allowing them to be recovered from biological matrices by fluorous solid phase extraction (f-spe). sphingolipid ceramide n-deacylase was used to remove the fatty acid from the ceramide moiety, after which the fluorocarbon-rich substituent (f-tag, ) was linked to the free amine. finally, the molecules were permethylated for ms analysis and the method was used to examine a crude ganglioside mixture extracted from bovine brain. in addition, the flourous tag was used in a microarray format to fix f-tagged gm ganglioside to a fluorous glass surface, with the glycan intact and available for interaction with a fluorescent derivative of cholera toxin b chain. cheng et al. ( a) have used -aa ( / ) as a matrix for quantitative analysis of sulfatides in biological samples. the matrix was said to promote selective ionization of sulfatides in negative ion mode with a detection limit in the high attomole range. experimental details have been published for highperformance tlc separation of glycolipids followed by blotting to a pvdf membrane in a technique termed far-eastern blot with analysis by maldi-tof ms (taki et al., ) . b. studies on the glycan moiety following removal of the ceramide for studies of the carbohydrate portion of these molecules, lipid heterogeniety is frequently reduced by removing the ceramide with enzymes such as rhodococcal endoglycoceramidase or leech ceramide glycanase. li et al. ( e) have described the preparation of the intact oligosaccharides from gm (neuacggose cer) and gbose cer as examples to show the use of ceramide glycanase and have optimized the specificity and detergent requirements of rhodococcal endoglycoceramidase for the release of glycan chains from various gsls. a novel method of detecting -gala series gsls (those possessing an r-galb - galb - -cer, group) has been reported (ishibashi et al., ) . the method used the specificity of endogalactosylceramidase, an enzyme that is capable of hydrolyzing -gala series gsls to produce intact oligosaccharides and ceramides but which also catalyzes transglycosylation reactions. in the latter reaction, the enzyme transferred oligosaccharides from the gsls to acceptors such as fluorescent -alkanols. in this application, -nitro- , , -benzoxadiazole pentanol (nbd-pentanol, ) was used as an acceptor. the fluorescent products, nbd-pentanol-conjugated- -gala oligosaccharides, were separated and detected by tlc or hplc with a fluorescent detector and characterized by maldi-tof ms. the method could also be applied to glycoglycerolipids and digalactosyldiacylglycerol and was successfully applied to detect -gala series gsls in the fungus, rhizopus oryzae and the parasite, taenia crassiceps. other applications of maldi to the analysis of these compounds is summarised in table . rhamnolipids are glycolipids of the type produced by pseudomonas spp. maldi-tof ms approaches have been developed for high-throughput screening of naturally occurring (fungus), mycelia tof (chca), psd ident. of novel neogala-series glycosphingolipids with terminal man and glc (tani et al., ) human (mesenchymal stem cells from bone marrow) macrobdella decora endoglyco-ceramidase, r-tof/tof, ft-icr (dhb), glycans (per-me) structural determination (heiskanen, et al., ) human (colonic adenocarcinoma cell lines) ceramide glycanase, tof/tof, q-tof (dhb), glycans (per-me) enhanced expression of β -gal-t activity induces in vivo synthesis of extended type chains on lactosyl-cer human (umbilical vein endothelial cells) tof (chca) acyl mainly : . may act as biomarker for inflammation, gsl = gb (okuda et al., ) human (umbilical vein endothelial cells) tof (chca) structural determination and dynamics of globotetraosylceramide under tnf-α stimulation (okuda, et al., ) human (kidney and colon) tof identification of gsls that bind shiga toxin from e. coli human (embryonic stem cells) switching of the core structures of gsls from globo-and lacto-to ganglio-series on cell differentiation miniature pig (endothelial and islet cells) ceramide glycanase, r-tof (dhb), esi (-ve), glycans (per-me and girard's t) structural determination. identification of neu gc epitopes ) (krambeck, et al., ) mouse (thymus) tof/tof (dhb), esi structural determination and natural killer t cell development (li et al., f) mouse (liver and serum) tof (-ve) acute kidney injury down-regulates gene expression of hepatic cerebroside sulfotransferase. quant. by maldi-tof. (zhang et al., h) mouse ( use of -aminoacridine ( -aa) as matrix, structural determination -phospholipids, gal-cer-sulfate, gb trichoderma viride (fungus) l-tof ( -nh - -mecoumarin) ident. of phosphocholine-containing glycosyl inositol phosphoceramides respectively. the method was validated by compositional analysis using gc/ms, fractionation by rp-hplc and analysis by and d nmr applications of maldi to the analysis of other glycolipids are listed in table . although much work has been published on glycosides during the review period, maldi appears to occupy a relatively minor position with fast atom bombardment (fab) and, particularly esi being the preferred techniques. most work has been on the identification of glycosides from various plant sources using a variety of techniques such as nuclear magnetic resonance (nmr), uv and ir spectrometry. applications of maldi to the analysis of glycosides and other natural products are summarised in table . several investigators have reported that the main fragmentation pathways of flavonoids are apparently independent of the ionization mode (esi, atmospheric pressure chemical ionization (apci), or maldi) or the types of analyzers used to acquire the spectra (triq, it, or qtof) as reported in a review of the structural characterization of flavonoid glycosides by multistage ms (vukics and guttman, ) . maldi-tof (dhb) was used by bankefors, nord, and kenne ( ) to examine saponins from quillaja saponaria bark extracts in connection with the development of a multidimensional method using hplc and esi-it ms n for profiling complex mixtures of natural products. increasing use of maldi has been made in the characterization and detection of disease and in the identification of biomarkers. some of the glycan biomarkers reported for, for example, cancer, however, appear to be more associated with glycopro-teins involved in inflammation and are, thus, secondary to this disease. blomme et al. ( ) have noted that "although individual liver diseases have their own specific markers, the same modifications, hyperfucosylation, increased branching and a bisecting glcnac, seem to continuously reappear in all liver diseases." increases in fucosylated triantennary glycans from agp is a case in point. several reviews have appeared and are summarised in table . practical details for the characterization by maldi-tof and esi-ms of n-linked glycosylation on recombinant glycoproteins produced in p. pastoris and for detecting potential cancer biomarkers in various cell lines and sera from patients have been published. bereman, williams, and muddiman ( a) have developed a nano-lc linear trap quadrupole (ltq) orbitrap method for analysis of released n-glycans and compared the spectra with those obtained by maldi-ft-icr. whereas the maldi spectra showed much loss of sialic acids from the sialylated glycans, the orbitrap spectra showed no decomposition. the method was applied to glycans released from plasma glycoproteins in benign gynecologic tumors and from epithelial ovarian cancer patients. one of the biantennary glycans found to be down-regulated in the cancer patients was a fucosylated biantennary glycan in which the fucose was unusually shown attached to a gal residue rather than to the core as determined by ms/ms. the compounds were ionized as [mþh] þ species suggesting that this might be an erroneous structure and the result of an internal rearrangement that is known to occur under these conditions. a detailed statistical analysis has been performed on eight data sets of n-glycans released from serum glycoproteins from prostate, breast and ovarian cancer patients (barkauskas et al., ) and measured by maldi-ft-icr ms. significant differences between control and cancer groups were found in all eight datasets. two structurally related compounds were found to be significantly different between control and cancer groups in all three types of cancer. these compounds had compositions of hex -hexnac -fuc and hex -hexnac -fuc and were probably from igg rather than being produced by the cancer cells. narimatsu et al. ( a) have developed a high-throughput method for detecting cancer biomarkers in early stages of the disease. briefly, the method consisted of the extraction of tissue mrnas and measurement of the expression by quantitative realtime polymerase chain reaction (pcr). the results suggested that different glycan structures were synthesized in different cell lines. secreted proteins from the same cancer cells were collected from serum-free culture and then applied to a lectin microarray to select lectin(s) that showed differential binding to glycoproteins secreted from each cancer cell line. after selection of a specific lectin, isotope-coded glycosylation sitespecific tagging (igot) was used to identify core proteins that carry an epitope bound by a specific lectin. each candidate biomarker was immunoprecipitated from serum using commercially available antibodies and their glycan structures were profiled by lectin microarray, and finally determined by ms n technology with measurements by maldi-qit-tof ms. other applications are listed in table . α-glc( - )-α-hep-( - )-α-kdo p-( - )-β-glcn p-( - )-α-glcn p arthrobacter globiformis and a. scleromae diglycosyl glycerol ft-icr (dhb), nmr structural determination. mannose and galactose (paściak et al., b) aspergillus fumigatus glycoinositolphospho-ceramides tof the mita gene shown to be required for mannosylation (kotz et al., ) hymenobacter sp. carotenoids maldi identification of '-methyl and '-xylosyl derivatives of 'hydroxyflexixanthin (klassen et al., ) leishmania major glycoinositolphospholipids tof (chca), esi-ms n demonstration of a udpglucose independent udpgalactose salvage pathway (lamerz et al., ) lipoteichoic acid tof (dhb) identification of two enzyme systems involved in biosynthesis phosphatidyl-myoinositol mannosides (pims) glycopeptidolipids shown to mask pims in cell wall (rhoades et al., ) there is currently great interest in the production of therapeutic antibodies and maldi-tof ms is frequently used in the analysis of their attached glycans. reviews have been published on methods for the production and ms analysis of igg (huhn et al., ) therapeutic antibodies (beck et al., ; del val, kontoravdi, & nagy, ; higgins, ; zhang, pan, & chen, i ) and on the humanization of recombinant glycoproteins expressed in insect cells (tomiya, ) . practical details for characterization of antibody glycans have been published by several investigators janin-bussat et al., a ,bjanin-bussat et al., a . a discussion, with experimental details, of methods based on blot detection with glycan-specific probes, ms of released glycans and lc/ms detection of glycopeptides with the aim of determining whether, how and where plant-derived biopharmaceuticals are glycosylated has also been published . several new methods have been reported. thus: a highthroughput method for monitoring igg glycosylation using a well plate format with iggs purified from ml of human plasma has been developed by selman et al. ( ) . iggs were extracted using immobilized protein a, cleaved with trypsin and the resulting glycopeptides were purified by reversed-phase or hydrophilic interaction spe. glycopeptides were analyzed by intermediate pressure maldi-fticr-ms using both dhb and chca, both of which produced signals from sialylated as well as nonsialylated glycopeptides. the method showed an interday variation of below % for the six major glycoforms of both igg and igg and was found to be suitable for isotype-specific high throughput igg glycosylation profiling from human plasma. the method was applied to the igg glycosylation of human samples. two lectin-affinity chromatography techniques, con a and lens culinaris agglutinin, have been used to enrich, by removal of high-mannose glycans, the nonfucosylated n-glycans from igg with product detection by maldi-tof following pngase f digestion (tojo et al., ) . prien et al. ( ) have used a stable isotopically labeled derivative for rapid glycan screening of biotherapeutics. glycans were labeled with either [ c ]-or c ]- -aa for both maldi-tof or lc-ms analysis. the -aa label provided high sensitivity detection in negative ion mode and the mass separation of six units between the isotopically labeled variants eliminated problems arising from isotopic overlap. pegylation of proteins is frequently used to prolong the serum half-life time of recombinant proteins but their very high mws put many of them outside the mass range of commercial maldi-tof systems using conventional secondary electron multiplier (sem) detectors. seyfried et al. ( ) have investigated the use of a high mass (hm) detector combined with a maldi linear tof ms system for the detection of pegylated (glyco)proteins in the range of - kda. the system consisted of a shimadzu axima cfrþ instrument equipped with both a conventional detector and additionally, with an inline moveable hm ion conversion detector (icd hm , from covalx). spectra were run from sinapinic acid in the linear positive ion mode and were obtained from small (interferon a a), middle (hsa) and high (coagulation factor viii and von willebrand factor (vwf), both heavily glycosylated proteins) molecular mass proteins. the particular challenge was the heterogeneity of the (glyco)proteins in the high mw range in combination with heterogeneity added by the pegylation, nevertheless, the performance of maldi linear tof ms was found to be superior to that of other methods. although the sem was able to obtain information about protein pegylation in the mass range up to kda (e.g., pegylated hsa), the hm system was crucial for detection of ions from the larger compounds, the masses of which sometimes exceeded . mda. detection of these compounds was impossible with the standard sem. the particular challenge for the analysis was the heterogeneity of the (glyco)proteins in the high mw range in combination with additional pegylation, which even introduced more heterogeneity and was more challenging for interpretation. nevertheless, the performance of maldi linear tof ms with both detector systems in terms mw and heterogeneity determination depending on the m/z range was superior to the other methods. isolation and characterization of rhamnolipid-producing bacterial strains from a biodiesel facility (rooney et al., ) proposed as a member of a new genus and species (golyshina et al., ) aplysia kurodai (sea hare) glycosaminoglycans tof, ms/ms structural determination (yoon et al., a) arthrobacter crystallopoietes n- α ,α -trehalose tof, esi, nmr first report that trehalose can be produced from maltose in this bacterium beverage from fermented plant (gülcemal et al., ) radix puerariae (kudzu) tof identification from cultivated kudzu root (nguyen et al., b) red wine procyanidins and anthocyanins tof (indole- -acrylic acid) phenolic extracts shown to induce nitric oxide (no)mediated vasoprotectivity (auger et al., ) salmo salar ( a novel method for the determination of aminoglycoside antibiotics used surface-assisted laser desorption/ionization mass spectrometry (saldi ms) with the aid of silver-coated gold nanoparticles (au@agnps) capped by anionic citrate. these nanoparticles were used both as concentrating agents and as matrices in saldi ms. the lods at signal-to-noise ratio of were , , , , and nm for paromomycin, kanamycin a, neomycin, gentamicin and apramycin respectively. the lods of the first four of these antibiotics in plasma samples were , , , and nm respectively. recoveries of the antibiotics from plasma were about % (wang et al., h) . further examples of the use of maldi ms in the analysis of therapeutics are given in table . applications in this section mainly involve the use of maldi to investigate products of newly isolated enzymes. these are summarised in table . other studies are aimed at elucidating enzyme activity as illustrated by the development of a method to determine the cleavage site in small oligomannoses that has been developed by hekmat et al. ( ) . enzymatic cleavages were performed in o-labeled water, conditions that introduced o into the anomeric position of the cleaved glycans. thus, measurements by maldi-tof could determine if a product arose from the non-reducing end of the original oligomannose by its incorporation of the label. the addition of a small amount of various ionic liquids has been found to modify the activity and regioselectivity of different immobilized preparations of rhizomucor miehei lipase that catalyzes the hydrolysis of hexa-o-acetyl lactal in aqueous media (filice, guisan, & palomo, ) . the activity of the enzyme glcnac-transferase vb, which transfers glcnac to the -position of the -antenna in n-glycans has been compared with that of gnt-v. one unusual product found after hr was the addition of glcnac to the -position of both antennae (alvarez-manilla et al., a). relevant reviews on carbohydrate synthesis are summarised in table principal component analysis, shows glycobiological differences between normal and cancer. hybrid, complex glycans (goetz et al., b) breast cancer cd hc clycoprotein pngase f, tof/tof (dhb), complex glycans (per-me) identification of the glycoprotein that binds to galmbp (fragment of mannose-binding protein) (powlesland et al., ) breast cancer serum trypsin, tof/tof (dhb), glycopeptides expression of helix pomatia lectin binding glycoproteins in women with breast cancer in relationship to their blood group (welinder et al., ) breast cancer cells β-elimination (nh carbamate), r-tof/tof (dhb), glycans, ms/ms use of new release method using glycoblotting and ammonium carbamate for β-elimination, core o-glycans breast cancer serum set of glycans identified that can be used as biomarkers (hammoud et al., ) (continued) changes in glycans . three n-glycans sufficient for detection with % accuracy hepatocellular carcinoma serum glycoproteins tof/tof (dhb), glycans (per-me) analysis using hierarchical clustering analysis. potential glycan markers identified. man glcnac , bi-, tri-antennary (tang et al., c) hepatocellular carcinoma serum and liver glycoproteins of rats pngase f, r-tof (dhb), glycans (per-me) increase in core-α- , fucosylated glycoproteins. possible biomarker hepatocellular carcinoma suggests shiga toxin as potential therapeutic agent (distler et al., ) enhanced expression of monosialylated triantennary glycans in cancer (yoon et al., b) stomach cancer mkn cells, serum automated β-elim. (matsuno et al., ) , tof (dhb), glycans (phenylhydrazones) mkn cells found to express characteristic trisialopolylactosamine-type glycans. reduced triantennary complex and increased biantennary glycans at asn- in patients table and general reactions in table . methods to change the glycosylation of a glycoprotein are common for recombinant antibiotic production as outlined above. a general method for producing homogeneous glycoproteins with eukaryotic n-glycosylation has been reported and involves the transfer of the campylobacter jejuni glycosylation machinery to e. coli and production of glycosylated proteins with the key glcnac-asn linkage. the bacterial glycans were then trimmed and remodeled in vitro by enzymatic transglycosylation to give a eukaryotic-type n-glycosylation (schwarz et al., ) . a method for immobilization of unstable membrane-bound enzymes to a commercially available sepharose support for glycan synthesis has been published ito et al. ( c) . it involves modification of the protein c-terminus and a transpeptidase reaction by staphylococcus aureus sortase a (srta) has been developed. recombinant human b , -galactosyltranseferase or recombinant h. pylori a , -fucosyltransferases were bound with simple aliphatic amino groups displayed on the surface of the solid support and were shown to have the required glycosyltransferase activity. as with previous reviews in this series, two types of compound appear to be particularly suited to maldi-tof analysis; namely glycodendrimers and carbohydrate/protein complexes. lysosomal proteins in mouse model in contrast to previous assumptions, cho cells shown to be capable of adding antigenic α-gal residues to n-glycans (bosques et al., ) α -antitrypsin (in human age hn cells) tof to characterize novel human cell line (northoff et al., ) α -proteinase inhibitor (in aspergillus niger) trypsin, pngase f, βelimination, tof/tof (dhb), glycans (per-me), gc/ms production, purification, and characterization, glycans = high-mannose (large antennae) (chill et al., ) anti-cd igg s, anti-cd igg rituximab mutant (s d/s a i e), (human) removal of fucosylated antibody ingredients from therapeutics shown to elicit high antibody-dependent cellular cytotoxicity in blood by two mechanisms anti-egfrxanti-cd bispecific igg ( model for comparison of release and glycan analysis methods (triguero, et al., ) centellosides (in centella asiatica plant cell cultures) tof to obtain a more efficient production system. α-amyrin, converted into centellosides by c. asiatica cells introduction of bisecting glcnac suppresses → -fucosylation and xylose attachment to form paucimannosidic glycans (karg, et al., ) α-l-iduronidase (human in seeds of brassica napus and nicotiana tabacum) tof (sinapinic), glycoprotein. gc/ms of monosaccharides attached carboxy-terminal er-retention motif, sekdel, reduces xyl and fuc in n-glycans but has little effect on enzyme activity (galpin et al., ) iga and iga (in murine melanoma cells) pngase f, l-, r-tof (dhb, thap), glycans structural determination. hybrid, bi-, tri-, tetra-antennary complex. igg ( use of glycosidase inhibitor to produce igg with man glcnac . effect on xtal structure and effector functions (crispin et al., a) igg (mouse in tobacco by cells) hydrazine, tof (dhb), glycans ( -ap) use of an er retention signal (kdel) increased high-mannose glycans but did not eliminate paucimannosidic glycans igg (human ij in silkworm larva hemolymph) pngase f, tof, glycans ( -ap) produced mg per kg of larvae with recovery of . %. paucimannosidic glycans igg (in cho cells) pngase f, tof, glycans production of non-fucosylated glycans by mutation (glycans -high-man, biantennary complex) (von horsten et al., ) igg (in cho cells) pngase f, tof chromatographic method to enrich nonfucosylated glycans (tojo, et al., ) igg ( effect of culture medium components on product yield. glycans: g f, g f (paul et al., b) a. synthesis of multivalent carbohydrates, dendrimers, and glycoclusters an extensive review of dendrimers (astruc, # ) , with a section on glycodendrimers, illustrates the breadth of this subject. masses of the larger compounds are frequently in the range - kda. the largest compound, based on a polyglycerol scaffold contained an estimated mannose residues but the authors had difficulty obtaining a maldi spectrum because of the high mw. syntheses frequently involve huisgen-type click chemistry because high-yield reactions are essential when so many carbohydrate molecules have to be attached. table lists papers reporting syntheses of glycodendrimers and similar compounds. among the largest of these compounds to have been reported during the review period has involved conjugation of lpsderived oligosaccharides to diphtheria toxin crm protein in an attempt to develop a vaccine against invasive meningococcal disease. conjugates with a mass of kda were analyzed by maldi-tof from sinapinic acid, the most widely used matrix for compounds of this type. conjugates can be characterized by maldi-tof ms but for large molecules the resolving power of most instruments is insufficient to distinguish each product and only a broad peak is observed. whereas the center of the peak represents the mean copy number of ligands per protein, information on the dispersity of the sample is usually neglected. patel et al. ( ) have produced a mathematical approach for calculating dispersity. by simply measuring the width at half maximum of the broad peaks that usually arise from carbohydrate-protein complexes and from the unmodified proteins, they were able to calculate the product distribution variance. furthermore, since the area between ae s equates to % of the total, m ae s represents the range within which % of adducts exist, this gives a direct measure of dispersity. as one example of the type of work involved with this type of compound, the glycosylation sites of o-specific polysaccharide of vibrio cholerae o , serotype ogawa linked to bsa via squaric acid chemistry have been determined by maldi-tof/ tof ms (from chca). the spectra showed the presence of hapten-bsa neoglycoconjugates with different hapten-bsa ratios ( . , . , and . ). sites of glycation were determined by comparison of the masses of the peptides resulting from the digestion of the bsa glycoconjugates and bsa itself using tandem ms/ms with a high-collision energy cell. the spectra improved secretion of molecular chaperone-assisted igg. no alterations in n-glycans (dojima, et al., ) interferon-γ, (human in cho cells) tof, glycans (per-me) study of intracellular glycosylation activities. effects of nucleotide sugar precursor feeding (wong et al., b) interferon ( enzyme, which usually fucosylates core of n-glycans, shown to fucosylated chitooligosaccharides (ihara et al., a) galactofuranosyltransferase glft (recombinant in m. tuberculosis h rv) l-, r-tof (chca) polymer length shown to be controlled by a template-independent polymerase (may et al., ) β-d-galactoside-α , -gal-transferase and β-d-galactoside-β , -galtransferase from pigeon two novel enzymes catalyzing the formation of galα → galβ →-galβ - glcnac galnac transferase (human) tof/tof (dhb) adds galnac to several unnatural peptides (yoshimura et al., ) galnac transferase (human) tof (dhb) enzyme found to have a unique galnac-o-ser/thr-binding site (perrine et al., ) galnac transferase (human) tof found in testis and brain (peng et identification of gal-transferase adding gal to core fuc. (titz et al., ) gnt-i fuzed to maltose binding protein from nicotiana tabacum tof/tof recombinant expression and characterization (dohi et al., ) gnt-v and gnt-vb (recombinant human. incubations in t cells) maldi enzymes transfer glcnac to position of -antennae in n-glycans. comparisons of enzymes (alvarez-manilla, et al., a) gpi-modifying β - -glcnac transferase from trypanosoma brucei enzyme characterization (izquierdo et al., ) glycosidases penicillium canescens tof (dhb) isolation and biochemical characterization, (burtseva et al., ) cellulase cel a from trichoderma reesei tof/tof (dhb, thap), esi-ms/ms enzyme shown to catalyze hydrolysis of glucosidic bonds adjacent to mono-substituted anhydroglucose units (enebro et al., b) chitinase-a from cycas revolute (cycad) tof (dhb) biochemical characterization, cdna isolation, and posttranslational modification (taira et al., b) chitinase-a and b from serratia marcescens strain bjl expressed in e. coli top effects on inhibition efficacy of allosamidin, a general competitive inhibitor of family chitinases. (zakariassen et al., ) chitinase and n-acetylhexosaminidase from verticillium fungicola tof (dhb) effect of ph on the production of enzymes in submerged cultures cellulomonas fimi tof (dhb) use of mutations to affect mannose binding (hekmat, et al., ) exocellulase cel b from thermobifida fusca tof proposal of a novel hydrolysis mechanism involving proton-transfer er glucosidase ii from rat liver tof found to be a broad specificity hexosidase (miyagawa et al., ) family gh α-mannosidase from streptococcus pyogenes tof, high-man glycans (per-me) characterization. α → mannosidase (suits et al., ) β-galactosidases from arabidopsis subfamily a tof comparative characterization (gantulga et al., ) (continued) β-galactosidases and from tomato tof enzymatic activity and substrate specifcity (ishimaru et al., ) β-galactosidases (bgac protein) from streptococcus pneumoniae specific for galβ - glcnac moiety (jeong et al., ) α-glucosidase from aspergillus niger r-tof (dhb) monitoring hydrolysis and transglycosylation activity (shimba et al., ) α-glucosidase from aspergillus niger tof (thap) activity towards dextrin and starch (ota et al., ) β-glucuronidase from aspergillus niger tof isolation and substrate specificity for transglycosylation (kiryu et al., ) shown to be involved in the maintenance of chronic infections (domenech et al., ) cholinephospho-transferase from mycoplasma fermentans tof/tof (dhb) molecular cloning and expression of enzyme involved in glycol-glycerophospholipid biosynthesis hemicellulase from chrysosporium lucknowense c tof investigation of the fungus for hemicellulase production (hinz et al., ) showed the presence of three conjugation sites on lys residues , , and , assumed to be the most accessible. the identification of y-series product ions was found to be useful for sequencing of various peptides and the a-and b-product ions confirmed the sequence of the conjugated peptides . other examples are listed in table . maldi-tof analysis, combined with ir spectroscopy has demonstrated covalent modifications of chitin with silk-like proteins in the formation of shells in the mollusc mytilus galloprovincialis (weiss et al., ) . maldi-tof ms has also been used as a standard against which to evaluate and optimize a fluorophore-assisted carbohydrate electrophoresis (face) method for analysis of pectic oligosaccharides (sun et al., a) (article in chinese). koroleva et al. ( ) have used maldi-tof ms to identify all the major glucosinolates that accumulate in s-cells of arabidopsis leaves and flower stalks. cell sap was diluted in ml methanol and mixed : with chca before being spotted onto the maldi target plate. the analysis was performed in negative-ion mode using an ultraflex tof/tof instrument. finally, mun, rho, and kim ( ) have used maldi to confirm the molecular sizes of commercial cycloamyloses and patsos et al. ( ) have used it to detect aryl glycans in cells treated with inhibitors of o-glycan processing. maldi continues to be a major technique for carbohydrate analysis although electrospray is more widely used. a major advantage of maldi is that is gives a cleaner profile of glycan mixtures because of the absence of multiple charging. new techniques, such as ion mobility have emerged to complement both maldi and electrospray but there is still much scope for improvement in carbohydrate analysis. surveys during the review period have highlighted the fact that many laboratories still make mistakes when assigning structures. much of this can be attributed to assumptions made between a simple mass (urch et al., ) s-adenosyl-l-methionine dependent methyltransferase from haloferax volcanii tof (chca) enzyme shown to methylate n-linked pentasaccharide tlmk from strepto-alloteichus hindustanus ft-icr functional characterization in tallysomycin biosynthetic pathway (wang et al., g) trehalose synthase from thermotoga maritima dsm tof molecular cloning and characterization (ryu et al., ) udp-arabinopyranose mutase from rice tof (sinapinic), glycoprotein arg residue shown to be reversibly glycosylated with single glycosyl residue; residue needed for activity (konishi et al., b) (gao et al., b) aminated xyloglucan (from tamarind) tof (dhb) physico-chemical properties of aminated xyloglucan extracted from tamarind seed (simi & abraham, c) bridged c-furanosides tof intramolecular nucleophilic attack of bzo group in a triflated cyclooctenol (jürs & thiem, ) -deoxynojirimycins with dansyl capped n-substituents tof as probes for morbus gaucher affected cell lines (fröhlich et al., ) , -dibromo- -methyl- phenylphospholane -oxide tof as novel anticancer agent (yamada et al., b) synthesis of polysaccharide intracellular adhesins using an acid reversion reaction of n-acetylglucosamine in hf-pyridine (leung et al., ) amino-bridged oligosaccharide mimetics r-tof (dhb), fab synthesis using reductive amination. glycomimetic target structures as potential ligands for the receptor protein nkr p of natural killer cells (neumann & thiem, ) amylose chains tof from starch by action of phosphorylase. preparation of a-type crystals (montesanti et al., ) bi-fluorescently-labeled maltooligosaccharides tof for amylase assays (oka et al., a) bis-hydrazides tof for conjugation with proteins etc. (adak et al., ) blood group tetrasaccharides b (types , and ) tof -aminopropyl glycosides of tetrasaccharides synthesised using acetylated galα( → )-(fucα( → ))gal trichloroacetimidate as a glycosyl donor (korchagina et al., ) , -branched arabinogalactan-type tetraand hexa-saccharides tof for investigation of monoclonal antibodies raised against arabinogalactan proteins from pressed juice of echinacea purpurea. carboxymethylated cyclosophoraose tof as a novel chiral additive for the stereoisomeric separation of flavonoids by ce (jeon et al., ) chitooligosaccharides tof by the enzymatic hydrolysis of chitosan ) chitooligosaccharides tof by the enzymatic hydrolysis of chitosan (xu et al., a) chitooligosaccharides tof/tof (dhb) glycans (amac) characterization of family chitosanase from streptomyces coelicolor a ( ) and use for degradation of chitosans chitooligosaccharides tof to study the antibacterial activity against bifidobacteria (Šimůnek et al., ) chitosan and chitooligosaccharides adsorption properties for uranium (paper in chinese) (jiang et al., b) chitosans tof synthesis from lobster chitin by naoh deacetylation and enzymatic hydrolysis. to protect crops from main pathogens (falcón et al., ) chito-tetrasaccharide β- , -glcnac-β- , -glcn repeat tof (dhb) by condensation of two disaccharides deacetylated chitohexaose tof hydrolysis of chitosan. could limit cell proliferation of breast cancer cells (xiong et al., ) -deoxy cyclic and linear oligosaccharides tof (dhb) synthesis by oligomerization of monomers (paul et al., a) β-d-fructopyranosyl-( → )-d-glucopyranose tof synthesis from d-glucose and d-fructose by thermal treatment (yamamori et al., ) galactofuranose oligomers tof/tof (chca) to probe mechanism by which polymer length is controlled in mycobacteria (splain & kiessling, ) galactomanno oligosaccharides tof from hydrolysis of guar gum by βmannosidase (paper in chinese) (zhao, et al., b) galactooligosaccharides ft-ms, gc/ms synthesis by acid hydrolysis of the polysaccharides from nerium indicum mill α-glucan pentasaccharide from aconitum carmichaeli use of chiral-auxiliary-mediated , -cisglycosylations for the solid-supported synthesis (boltje et al., ) glucan with α -( → ) linkages and α -( → ) and α -( → ) branch points produced from glucan of leuconostoc mesenteroides nrrl b- by microwave assisted hydrolysis (majumder et al., ) glucosylated raffinose tof (dhb) use of glucansucrase (alternansucrase) for synthesis glc-glc, gal-gal, gal-glc, gal-gal disaccharides tof (dhb) -member library containing all regioisomers. solid-phase synthesis (Ágoston et al., b) linear isomaltooligosaccharides (dp - ) tof one-step synthesis using synthetic fusion enzyme of dextransucrase and dextranase (kim et al., e) macrocyclic oligosaccharides tof copper(i)-catalyzed huisgen's , -dipolar cycloaddition of alkyne and azide provides size-defined macrocyclic carbohydrates (muthana et al., ) mannose-capped disaccharide with a thiol terminus to provide a tethered sugar for attaching to gold nanoparticles to mimic carbohydrateinvolved cell-surface interactions (continued) chemical stereochemically controlled synthesis (pastore et al., a) nigerose-containing oligosaccharide tof/tof (chca) escherichia coli non-glycosidically linked pseudodisaccharides tof (thap) thioethers, sulfoxides, sulfones, ethers, selenoethers, and their binding to lectins oligosaccharides from red seaweed polysaccharides tof (chca), esi efficient conversion of galactans into cglycosyl aldehydes (ducatti et al., ) oligosaccharide mimics of sialyl lewis a trisaccharide. neu ac and fuc replaced with hso and d-ara respectivly (jakab et al., ) pentasaccharide anticoagulant (idraparinux) maldi compound is fully o-sulfated, o-me mimic of antithrombin iii binding domain of heparin ) n-quaternary chitosan derivatives tof synthesis, characterization and antibacterial activity (rúnarsson et al., ) sialylated lactosides tof (dhb), per-me for coupling to bsa by huisgen reaction. as glycocongugate antigen (mosley et al., ) sialyllacto-n-tetraose and sialyllacto-n-neotetraose tof use of α - and α - -sialylated lactosaminide precursors obtained by enzymatic procedures with glycosylations employing triflic acid/n-iodosuccinimide (schmidt & thiem, ) sucrose-based guanidinecontaining g molecular transporters tof show different patterns of intracellular localization depending on the nature of the linker chains as well as the fluorescent dyes (lee et al., l) sulfated oligofucosides maldi synthesis of sulfated octyl tetra-to octaoligofucosides with different sulfation patterns (zong et al., a) α to achieve steric instead of electrostatic stabilization. two-step synthesis (kloser & gray, ) cyclic β-glucan r-tof/tof (chca) synthesis using laminarinase a glycosynthase mutant from the basidiomycete phanerochaete chrysosporium (vasur et al., ) dextrin-hydroxyethylmethacrylate and dextrinvinyl acrylate hydrogels tof/tof (dhb) for the determination of biocompatibility and biodegradability in mice (moreira et al., ) epoxy-starch derivatives tof (dhb) synthesis by epoxidation of allylated starch (huijbrechts et al., ) poly-n-acetyllactosamine tof (dhb, chca) chemo-enzymatic synthesis. characterization for cgl -galectin-mediated binding of ecm glycoproteins to biomaterial surfaces (sauerzapfe et al., ) triblock co-oligomers of tri-o-methylated and unmodified cello-oligosaccharides: tof (dhb) synthesis and structure-solubility relationships (kamitakahara & nakatsubo, ) xylan-based polysaccharides tof (dhb) amino-modified low mw xylan reacted with unmodified xylan and cellodextrins ) glycosaminoglycans and related compounds n-acetyl-heparosan oligosaccharides tof digestion of n-acetyl-heparosan with heparitinase. for study of enzymology (sugiura et al., a) , -desulfated heparin maldi for control of inflammation by inhibition of e-selectin (lakshmi et al., ) heparan sulfate oligosaccharides tof/tof (dhb), esi use of modular building blocks for preparation of a library of oligosaccharides used to probe the structural features of hs for inhibiting the protease, bace- (arungundram et al., ) heparan sulfate oligosaccharides tof synthesis and inhibition of endothelial cell functions essential for angiogenesis (cole et al., ) hyaluronan tof for nmr study of chemical proton exchange over the β( → ) glycosidic linkage (nestor et al., ) hyaluronic acid decasaccharide maldi chemical synthesis using preactivation-based chemoselective glycosylation strategy hyaluronic acid subunit and fully protected oligomers maldi tetra-, hexa-and octa-saccharides. multigram synthesis (virlouvet et al., ) isosteric sulfonate analogues of at-iii binding domain of heparin tof (thap) d-glca-and l-idoa-containing disaccharide. related to antithrombin-binding pentasaccharide of heparin. one sulfate ester replaced by na sulfonato-me moiety bisected octasaccharide maldi chemical synthesis (wang et al., e ) galβ( → )[neuacα( → )] glcnacβ( → )man motif tof chemical synthesis as molecular probe (bao et al., ) glucuronyl oligosaccharides tof, glycans ( -ap) synthesis of glucuronyl and sulfoglucuronyl oligosaccharides from hnk- glycoprotein (yagi et al., ) high-mannose glycans tof (dhb) related to hiv gp . one-pot catalytic glycosidation/fmoc removal (pastore et al., b) high-mannose glycans with glc units tof (dhb) study of the conformational properties of the glc man unit (mackeen et al., ) high-mannose glycansmethotrexate derivatives tof identification of the recognition motif of the glycoprotein-folding sensor enzyme, udp-glc: glycoprotein glucosyltransferase biantennary and high-mannose n-glycans linked to non-reducing terminus of man glcnac core, plus biotin for arrays n-glycan library tof (dhb), glycans ( -ap) enzymatic construction of library for building ms database phosphorylated highmannose glycans tof high-mannose glycans from ribonuclease. incubation with recombinant glcnac phosphotransferase. (bohnsack et al., ) (continued) (song, et al., f) sialic acid containing complex-type n-glycan tof new solid-phase synthesis. stereoselective βmannosylation, microfluidic α-sialylation and glycosylation of n-phf acetimidate on jandajel resin thiol-terminated nonamannoside tof for synthesis of microarrays (dietrich, et al., ) various complex glycans tof development of hek t expression system for human glycosyltransferases enzymatic construction of library for building ms database glycosylated amino acid derived from psgl- tof use of trichoroacetimidate donors and thioglycosyl acceptors (vohra et al., ) kl- antigen tof/tof (dhb) library of glycans to determine binding to anti-muc antibody aglycoristocetin derivatives containing hydrophobic side chain-substituted cyclobutenedione. synthesis and anti-influenza activity androgenic gland hormone of the woodlouse, (armadillidium vulgare) tof with various glycans. showed that thermodynamically most stable form not most active: result of disulfide pairing (katayama et al., b) antifreeze glycopeptide analogues tof (dhb, chca) microwave-enhanced synthesis and functional studies (heggemann et al., ) β-hfsh with chitobiose units at the natural linkage sites tof use of the sinaÿ radical glycosidation for simultaneous installation of biantennary chains and glycal chemistry to construct the tetrasaccharide core (nagorny et al., ) β-sheet-rich protein plus glcnac at various positions tof effect of glcnac position on protein folding kinetics and thermodynamics (price et al., a) biantennary n-glycans plus peptide tof/tof (dhb) solid-phase peptide synthesis. glycans linked -nh-co-ch -s-peptide (murase et al., ) bivalent glycopeptide tof mannosides linked with squaric acid (lindhorst et al., a) cd glycopeptide antigens tof (dhb, chca) chemo-enzymatic synthesis of glycopeptide with n-and o-linked glycans (huang et al., c ) c-glycosyl β -and β/β peptides maldi solution-phase synthesis. - amino acids (inaba et al., ) c-linked antifreeze glycoprotein analogues tof (dhb) effect of the length of the amide-containing side chain between the carbohydrate moiety and the polypeptide backbone influences ice recrystallization inhibition (tam et al., ) c-mannosylated peptides tof (dhb, chca) peptides shown to interact with hsc to modulate its signaling in raw . cells ) cyclic antifreeze glycopeptides tof exhibited antifreeze activity by forming hexagonal-bipyramidal ice crystals (hachisu et al., ) cyclic neoglycopeptides maldi (chca) for binding to adjacent protein binding sites in wheat germ agglutinin (schwefel et al., ) dihydrofolate reductase tof glycans = glcnac, lactose, maltotriose analysis as tryptic peptides. for study of effects of glycosylation on stability (tey et al., ) mass spectrometry reviews doi . /mas microwave-assisted synthesis. ( )-carboxyfluorescein shown to be stable fmoc-threonine bearing a core- glycan tof as building block for psgl- via fmocassisted solid-phase peptide synthesis (krishnamurthy et al., ) o-fucosylated epidermal growth factor-like repeat of mouse notch- receptor chemical synthesis and studies on folding (hiruma-shimizu et al., ) galactosylated naringinase tof (sinapinic) modification of glycosylation to effect deglycosylation of rhamnosylated drugs (garnier et al., ) gal-β- galnac-α-lys tof immunogen design for tumor t antigen immuno-targeting (sendra et al., ) glycopolypeptide-based cholera toxin inhibitors maldi effect of peptide charge and glycan linker length on activity (maheshwari et al., ) glycopeptide carrying tetra-n-ac-lactosamine containing core decasaccharide tof (dhb) solid-phase synthesis (ueki et al., ) glycosaminoglycan-protein linkage tetraosyl peptide moieties of betaglycan tof to investigate structures that best serve as a hexosamine acceptor for enzymatic glycosyl transfer (tamura et al., ) glycosulfopeptides from nterminus of human endoglycan tof/tof (thap) containing tyrosine sulfate residues and sialyl lewis x in core o-glycans and bind to human l-selectin (leppänen et al., ) glycosylated peptoids tof by on-resin click (huisgen reaction) glycoconjugation (norgren et al., ) glycosylated cell-penetrating peptide (r /w ): ac-rrwwrrwrr-nh tof one, two, or three galactose(s), with or without a spacer introduced via a triazole link (dutot et al., ) grgdy grafted to chitosan tof (chca) linked with sulfosuccinimidyl- -[ '-azido- 'nitropheny-lamino]hexanoate as drug carrier n-glycosylated insulin l-tof (dhb) addition of three glcnac residues at nh groups on peptide. use of endo m to transfer glycan to one of them. (tomabechi et al., a) n-glycoproteins carrying intact natural n-glycans tof/tof (dhb) enzymatic synthesis of biantennary glycans (huang et al., b) glycosylated analogues of glucagon-like peptide tof, lc-ms to improve proteolytic stability and blood glucose-lowering activity. sugars = glcnac, lacnac, sialyl lacnac synth. by chemoenzymatic approaches (ueda et al., b) glycosylated neurotensin analogues tof containing o-linked β-melibiose and α-tf antigen or β-lactose units linked by a peg spacer. exhibit subpicomolar anticonvulsant potency in model of epilepsy (lee et al., f) glycosylated tetracontapeptide with acidlabile sialyl-t n antigens maldi (dhb) -residue glycopeptide-α-thioester and residue glycopeptide with cys at n-terminal. solid phase synthesis; coupled by cys ncl ser . o-glycopeptide mimetics tof (dhb, chca) aziridine ring opening as regio-and stereoselective access to o-glycosyl amino acids and their transformation into oglycopeptide mimetics (schäfer et al., ) heptasaccharide from campylobacter jejuni plus acra - tof/tof (chca) development of nmr method for d structural determination of glycoproteins using enzymatically synthesised glycoprotein (slynko et al., ) (continued) -oh-proline oligomers q-tof (dhb) compounds form very stable polyproline ii helices (owens et al., ) iga-hinge peptide tof to study effect of glycosylation on cis/trans isomerization of prolines (by nmr) man - glcnac -asn-n- c r-tof (dhb) for micro-method for determining precise oligosaccharidic specificity of mannosebinding lectins (debray et al., ) mannosylated lysine derivative tof bivalent carbohydrate branching unit. suitable for solid-phase peptide synthesis chemical synthesis, for enzymatic study in dictyostelium (see table ,o-glycans) (wang, et al., k) s-linked glycopeptides maldi thioglycosylated building blocks prepared from per-ac sugars via glycosyl iodides in one-pot fashion and used in sub-monomer solid phase strategy (comegna & de riccardis, ) synthesis using using the newly developed n-troc-protected gm and galn intermediates (komori et al., ) anchor with n-terminal cys maldi general method for producing proteins containing a natural gpi anchor using expressed protein ligation (schumacher et al., ) anchor with unsaturated lipid chains tof to investigate mechanism of gpi anchoring (swarts & guo, bearing pyrazolines, isoxazolines, and dihydropyrimidine- ( h)-thiones. as antibiotics (el-sayed et al., ) hydroquinone fructoside l-tof synthesis using leuconostoc mesenteroides levansucrase hydroquinone glucoside l-tof (dhb) synthesis using leuconostoc mesenteroides levansucrase lobatoside e-related triterpene glycoside maldi gold(i)-catalyzed glycosylation with glycosyl ortho-alkynylbenzoates as donors man-α-( → )-man-o-octyl analogues maldi synthesis and evaluation as potential substrates and inhibitors of a ppm-dependent α-( → )-mannosyltransferase involved in lam/lm biosynthesis (tam & lowary, ) mannosylated pyreneperfluoroalkyl lipid maldi to study multivalent binding on the lateral phase separation of adhesive lipids (liem et al., ) mannosyl glycolipids with perfluoroalkyl membrane anchors maldi to assess the cluster glycoside effect during the binding of concanavalin a to mannosylated artificial lipid rafts (noble et al., ) mesogenic azobenzenes tethered to sugar alcohols (gretskaya & mikhalyov, ) cholesterol plus linear glucose tof (dhb) synthesis and gelling properties , -dipalmitoyl- -(npalmitoyl- '-amino- '-deoxyα-d-glucosyl)-sn-glycerol tof glycoglycerolipid of a marine alga with a high inhibitor activity against human myt kinase. synthesis starting from α-me-glcp enzymatic synthesis from , dipalmitoylglycerol in one pot reaction hydroquinone galactosides l-tof (dhb) a potential skin whitening agent. synthesis by reaction of lactase with lactose as donor. fluorescently labelled synthetic ionophore synthesises in steps with % yield (coppola et al., ) phosphatidyl-myo-inositol mannosides tof to study mannosyltransferase corynebacterium glutamicum pimb' (batt et al., ) puerarin-cycloamylose inclusion complex l-tof (dhb) enzymatic synthesis using -αglucanotransferase and maltogenic amylase (choi et al., ) sialic cinnamoyl-α-cyclodextrin tof initiates polymerization of δ-valerolactone (osaki et al., ) cinnamoyl α-cyclodextrin tof cds self-organize to give different supramolecular complexes in aqueous solutions (tomimasu et al., ) (continued) synthesized via hydrosilylation reaction under thermal or uv-activated polymerization (tian et al., ) cd-based telluronic acid plus mn(iii)meso-tetra[ -( adamantyl methyl ketone)- pyridyl] porphyrin maldi as artificial enzyme with superoxide dismutase and glutathione peroxidase activities (yu et al., d) cyclodextrin dimers tof one homo-dimer of β-cd and two heterodimers of α-cd and β-cd as hydrolases (ikeda et al., ) cyclodextrin dimers and trimers tof/tof, esi bridged cds with links at different positions formed supramolecular adducts with shapespecific ligands (aime et al., ) β-cyclodextrin cyclic-nitrone conjugate maldi with superoxide radical anion and dodecyl chain for membrane insertion α-, βand γ-cyclodextrinesters (acrylate, pent- -enoate and undec- -enoate) synthesis with nitrophenol esters (nielsen et al., a) β-cd plus hydroquinone-αglycoside tof synthesis and doxorubicin-inclusion abilities (oda et al., ) β-cd monoalkyn tof (dhb) for conjugation to long-chain thiols for selfassembled monolayer prep. (dubacheva et al., ) cyclodextrin-polyester polymers tof ring-opening polymerization -heating cyclic esters and cds (harada, ) γ-cds possessing an azido group and a triisopropylbenzenesulfonyl group as useful synthetic and authentic intermediates for unsymmetrically functionalized derivatives (himeno et al., ) β-cd-ended linear poly(nisopropylacrylamide) (β-cd-pnipam) tof (thap, dctb) self-assembly of pnipam-based amphiphiles formed by inclusion complexation (zou et al., ) β-cd with octadecyl-linked perylene bisimide maldi self-assembled amphiphilic perylene-cd conjugate for vapor sensing of organic amines (jiang et al., a) [ -deoxy- -( -h- , , triazol- -yl)(me) -( -omebi-ph- '-yloxy) hexanoyl]-βcyclodextrin tof (dhb) synthesis, liquid-crystalline properties, and supramolecular organization for controlled release application. (adrian et al., ) inclusion complexes of β-cd with bipyridine guests tof with , '-vinyl-enedipyridine, , 'vinylenedipyridine, -( -pyridyl)- -( pyridyl)ethylene, , '-ethylene-dipyridine, , '-dithiodipyridine, and , 'dithiodipyridine insulated molecular wire with highly conductive πconjugated polymer core tof rod-like -ph-c=c-ph-core coated with α-cds (terao et al., b) ionic-liquid-functionalized βcyclodextrin tof as bonded chiral stationary phases for hplc (zhou et al., b ) linear α-cyclodextrin oligomers tof controlled synthesis using copper-catalyzed huisgen , -dipolar cycloaddition (rawal et al., ) oligothiophene derivatives bearing β-cyclodextrin tof (dhb) t-β-cd and t-β-cd , with bithiophene and terthiophene with β-cd at each end form supramolecular assemblies in aqueous solns. (sakamoto et al., b) perylene bisimide-bridged bis-(permethyl-β-cds) ft-icr as solid-state fluorescence sensor for vapor detection perylene-bridged bis(βcyclodextrin) ft-icr (kitano et al., ) glycodynamers maldi dynamic polymers bearing oligosaccharide residues such as glc obtained from α-cd (ruff et al., ) membranes (cross-linked) tof (dhb) based on acrylated cyclodextrins and polyethylene glycol dimethacrylates (rölling et al., ) multivalent galactotrehaloses tof α,β-gt isomers converted into vinyl monomers then radical copoly-merization with -acrylamidophemyl-β-glc or β-glcnac) with acrylamide (miyachi et al., b) pentafluorostyrene copolymers with glucose tof synthesis by nitroxide-mediated polymerization and "click" chemistry (becer et al., ) polydioxanone with a protected monosaccharide end-group l-tof (dithranol) ring opening polymerization of p-dioxanone using a protected monosaccharide ( , ; , -di-o-isopropylidene-α-d-galactopyranose) /al(oipr) initiator system (sugih et al., ) polyfluorene derivative with mol% , , benzothiadiazole plus α-man on an amine-reactive hydrogel-coated microarray glass surface. to detect autoantibodies in breast cancer correction: (blixt, et al., ) nanofibres tof nanofibers formed through π· · ·π stacking of the complexes of glucosyl-c -salicyl-imine and phenylalanine (continued) for study of carbohydrate-protein interaction oligosaccharides from plants tof (dhb) for construction of microarray to screen for plant transglycosidases activity (kosík, et al., ) -sulfo-n-acetyl-dglucosamine containing, on gold tof to study mechanism of amyloidosis of amyloid β peptides (fukuda et al., b) miscellaneous β-d-allopyranoside-grafted ru(ii) complex tof synthesis and both acid-base and dnabinding properties azo-sugar nucleotides plus many alkynes tof (dhb) click chemistry (huisgen reaction) to produce inhibitors of glycosyltransferases -deoxy- -nitropiperidinoses maldi synthesis from a protected galactooctopyranose (collin & vasella, ) , -di-f- , -di-me- -bora- a, a-diaza-sindacene- propionic acid derivative of man glcnac . (bodipy) for development of fluorescence assay (haga et al., ) carbohydrate-functionalized salphen-metal complexes tof complexes with peripheral glucose and galactose substituents. self-assembled supramolecular structures were produced (hui et al., ) dioxolane-type ( 'anthracenyl)methylene acetals tof synthesis, regioselective hydrogenolysis, partial hydrogenation, conformational study (jakab et al., ) (kim et al., f) amino-sugars tof (chca), esi deprotection method for the , , trichloroethoxycarbonyl (troc) group using tetrabutylammonium fluoride , -anhydrosaccharides tof use of mol % of aubr to catalyse glycosylations of -oh-propargyl/me mono-, diand tri-saccharides (thadke & hotha, ) colored carbohydrates tof addition of a fmoc analogue protecting group based on guaiazulene (aumüller & lindhorst, ) (bindschädler et al., ) glycoamino acid building blocks tof use of staudinger ligation glycopeptides tof use of pyruvoyl as a novel protecting group for solid-phase synthesis (katayam et al., ) gpi-anchored proteins tof sortase a-catalyzed transpeptidation of gpi derivatives for chemoenzymatic synthesis (wu et al., f) heptasaccharide asparagine building block tof (dhb) use of one-pot catalytic glycosidation/fmoc removal (mezzato & unverzagt, ) homolinear α( → )-linked octamannosyl thioglycosides maldi imidazolium cation-tagged mannosyl fluoride and thiomannoside using block couplings. efficient alternate approach for oligosaccharide synthesis (yerneni et al., ) homooligosaccharides tof (dhb) (per-ac, per-me) base-promoted glycosylation of unprotected glycosyl fluorides (steinmann et al., ) lipophilic thioglycosides tof use of heavy lipophilic tag to assist biphasic liquid-liquid separation (encinas & chiara, ) macrocyclic neoglycoconjugates tof (chca) macrocyclization of linear d-galacto- heptulopyranose-containing oligoketosides by intramolecular glycosidation and ring-closing metathesis mucin-type oglycopeptides and oligosaccharides tof (dhb, thap, chca) using transglycosylation and reverse-hydrolysis activities of bifidobacterium endo-α-nacetylgalactosaminidase neu gc-containing glycans tof sialylation with n-glycolylneuraminyl phosphite (hanashima et al., ) n-linked glycopeptides tof condensation of glycosylamines with asn. demonstrated with man glcnac . (chen & tolbert, ) measurement and structure, particularly when structures are selected directly from databases. at present, no one mass spectrometric technique can identify all structural features of carbohydrates. sialylated and sulfated glycans still remain a problem although sialylated glycans can be handled after suitable derivatization, particularly by permethylation or simply by methyl ester formation. permethylation appears to be becoming more popular, particularly for quantification and maldi. the next few years are expected to bring many developments, particularly in instrumentation and ionization techniques, that will possibly address some of the problems with carbohydrate analysis highlighted above. -ab -aminobenzamide -aa -aminobenzoic acid -aa -aminoacridine aboe aminobenzoic acid octyl ester aeab -amino-n-( -aminoethyl)-benzamide age advanced glycation end products agp a -acid glycoprotein alpcs aluminum-phthalocyanines a-tf a-thomsen-freidenreich antigen all allose amac aminoacridone amt -amino- -mercapto- , , -thiadiazole ann artificial neural networks anp -amino- -nitro- -picoline ansa -amino- -naphthalenesulfonic acid -ap -aminopyridine apb aminophenylboronic apci atmospheric pressure chemical ionization api apiose apts -aminopyrene- , , -trisulphonic acid aq aminoquinoline ( -or -) use of genetic engineering to produce glycoproteins in e. coli which are then enzymatically remodelled (see text) (schwarz, et al., ) o-sulfated trisaccharyl glycopeptide tof (dhb) solid-phase synthesis. use of new benzyl protection method. (kawahira et al., ) oligosaccharides tof (dhb) rate acceleration on stereoselectivity and velocity of o-glycosylation reactions (ishiwata et al., ) pseudooligosaccharides maldi use of cross-metathesis reaction between sugarolefins, followed by intramolecular cyclization (ronchi et al., ) s-linked glycoconjugates tof, esi by-thiyl glycosylation of olefinic proteins (floyd et al., ) sialylated glycans tof use of koenigs-knorr reaction (with ag co ) (pazynina et al., ) thioglycosides r-tof/tof solvent-free synthesis by use of ball-milling (patil & kartha, ) trisaccharide libraries tof use of linker-tagged building blocks imobilized on a soluble polymeric support (elsayed, ) acceptor specificity in transglycosylation reactions using endo-m l-tof (dhb), esi (tomabechi et al., b ) acetolysis of -deoxyhexosemethyl glycosides. role of sugar configuration tof (dhb) (cirillo et al., ) acid-catalyzed hydrolysis of β- , -glucan, including cellobiose and crystalline cellulose with so h-bearing amorphous carbon tof (suganuma et al., ) allyloxycarbonyl group removal provides a practical orthogonal protective strategy for carbohydrates tof (zong et al., ) amadori ketoses synthesis in microwave field via mo vi -catalyzed stereospecific isomerization of -c-branched sugars bearing azido function tof (hricovíniová, (camponovo et al., ) benzhydrylamine-lysine capped with to mono-, di-and tri-α-d-manp residues tof reactive n-oh-succinimide esters to ensure complete reaction of dendrimer amines (g mass = , da) (greatrex et al., ) , -bis( , -bis( -( -( azidoethoxy)-ethoxy)ethyl)fluorenyl)benzo-thiadiazole with mannose residues tof (dhb) as an intelligent energy transfer pair for label-free visual detection of concanavalin a boltorn h (commercial polymer) with gal-cer groups tof binds hiv- gp optically pure fullerodendron formed by diastereoselective diels-alder reaction calixarene-based glycocluster oligonucleotide with galactose residues tof click chemistry. triazole-tethered glycoclusters with arrangements. affinities towards pa-il and rca with dna-based glycoarray. (moni et al., ) carbosilane with , or sialyl-α-( → )-lactose residues tof for anti-influenza properties carbosilane with , , or neu ac residues tof, fab, esi influenza neuraminidase inhibitors. dendrimers uniformly functionalized with thioglycoside-type sialic acid moieties resistant to neuraminidases (sakamoto et al., a) carbosilane with or mannose residues tof (dithranol) hydrosilylation of allyl tetra-ac-man with carbosilane dendrimers containing monohydrosilane end groups and the subsequent deacetylation (ortega et al., ) carbosilane with sialyl-nacetyllactosamine groups tof combined chemical and enzymatic synthesis (matsuoka et al., ) cyclic α-( → )-octaglucoside with from - mannose residues tof oxidation of vic-diols, reductive amination with -aminoethylmannoside β-cyclodextrin with mannose or glucose residues tof (dhb) heteroglycoclusters. antennae each with two mannose and one glucose. for lectin binding (gómez-garcía et al., ) β-cyclodextrin with dextran tof (chca) synthesis by click chemistry using huisgen reaction (nielsen et al., b) cyclopeptide with mannose residues tof synthesis by click chemistry and molecular recognition study by surface plasmon resonance (chen et al., c) dihydroxy-benzamide based with - mannose, galactose, lactose, glucose or glcnac groups tof (chca) octa-dendrimers. for screening of lectins for multivalency effects. click chemistry (pera et al., b) diphenyldisulfide with glucose residues and others without sugars tof, gc/ms use as catalyst to convert allyl alcohols into carbonyl compounds (tsuboi et al., ) cysteine with or (from dicysteine) mannose residues tof as inhibitors of type fimbriae mediated bacterial adhesion (schierholt et al., ) ethylene glycols with , or galactose,lactose, maltose or lacnac groups ferrocene with one or two glucose, mannose or lactose residues tof/tof as electrochemical probes for molecular recognition studies (casas-solvas et al., b) polyglycerol substituted phenylboronic acid tof dendrimer formed adducts with, fru, d-(+)-gal, d-(+)-glc, d-(+)-man, and me-α-d-man, by removal of four h o (hashidzume & zimmerman, ) fullerene. sugar balls with iminoglucose residues tof synthesis by click chemistry. glucosidase inhibition shown with resulting iminosugar balls (compain et al., ) fullerene. sugar balls with glucose or galactose residues tof, esi, fab residues on arms, synthesis by click chemistry gd-diethylenetri-aminepentaacetic acid with glucose residues tof synthesis, in vitro and in vivo studies of gd-dtpa-xda-d -glc(oh) complex as a new potential mri contrast agent (ozaki et al., a) hydroxy benzenes and naphthalenes with , or mannose residues tof synthesis by click chemistry (rajakumar et al., ) l-lysine plus gd chelates and galactose residues tof/tof as liver imaging probes (luo et al., ) tetramer of glycodecapeptide from muc with sialyl t n antigen as vaccine candidate (keil et al., ) peptide/tris-oh-methylmethylamine. galactose or mannose residues attached to β-cd containing doxorubicin tof for uptake studies in the human hepatocellular carcinoma cell line hepg (bernardes et al., ) peptide/tris-oh-me-methylamine/ [ru(bipy) ] + with galactose or mannose residues maldi to study lectin interactions by monitoring change in fluorescence quantum yield of ru(ii). (kikkeri et al., a) phloroglucinol, triazyne, tetrachlorosilane, pentaerythritol, myo-inositol with , , or clinked sialic acids tof synthesis by huisgen cycloaddition of azide and alkyne (click chemistry). to explore sialic acid binding to cell surfaces (papin et al., ) phthalocyanine with α-galp residues tof potential application as photosensitizers in photodynamic therapy (soares et al., ) pentaerythritol, methyl α-dglucopyranoside, d-glucose, and dmannitol with , or mannose tof (dhb) synthesis by click chemistry, measurement of binding affinities (sattin et al., ) poly-glycerol with mannose residues tof maldi poor because of high mw (up to kda) (kizhakkedathu et al., ) poly-lysine with biantennary nglycans tof synthesis by click chemistry. effect of sialic acid linkage on in vivo dynamics. mass around kda (tanaka et al., b) poly-lysine on tris-( -ethylamino)amine with aulfated cellobiose groups tof (sdhb) synthesized by sulfation of polylysinedendritic cellobiose (prepared from cellobiose and polylysine dendrimer generation ) (han et al., b) porphyrin with lactose glycans tof synthesis by huisgen click cycloaddition of azide and alkyne (okada et al., b) porphyrin with galactose or lactose maldi synthesis by click chemistry. activity on carcinogenic hep cells activators of natural killer lymphocytes (renaudet et al., ) ruthenium(ii) bipyridine with or mannose or galactose residues tof as probes to study lectin-carbohydrate interactions and to measure mono and oligo-saccharide concentrations electrochemically (kikkeri et al., b) ruthenium porphyrins with glucose residues tof water-soluble catalyst for carbenoid transfer reactions tetrabenzo-porphyrins with glucose residues tof to improve targeting of cancer cells (ménard et al., ) tetraphenyl-ethylene with lactose or sialyl-lactose residues tof/tof synthesis by click chemistry. as fluorescent probes for detection of influenza virus tetraphenyl-ethylene with or mannose residues tof (dhb) for fluorescence turn-on sensing of lectins based on aggregation-induced emission (sanji et al., ) dihydroxybenzamide base with galabiose residues tof (chca) for isolation of pathogenic streptococcus suis bacteria (pera et al., a) , , -tris-( -propynyloxy)-benzene with β-cyclodextrin rings tof (dhb) synthesis by microwave-assisted click chemistry as fluorescent tripod detection system for pesticides (mallard-favier et al., ) trimesic acid and others with , or phosphocholine residue related to glycol-sphingolipid from earthworm pheretima hilgendorfi tof for enhanced immune responses when compared to their monovalent counterparts various amino-alkyl with , or lactose residues tof (chca) synthesis from carbamate-linked lactose ) zinc(ii) phthalocyanines with glc, gal or cellobiose residues tof (dhb) chemical synthesis as photosensitizer in photodynamic therapy (iqbal et al., a ) zinc(ii) phthalocyanines with glc, gal, cellobiose or maltose residues tof (dhb, chca) eight residues. for photodynamic therapy (iqbal et al., ) zinc(ii) naphthalocyanines with glucose residues tof as photosensitizer in photodynamic therapy (iqbal et al., b ) zinc(ii) naphthalocyanine with glucose or galactose residues tof, esi ex post glycoconjugation (berthold et al., ) free amino group on lps used for site-specific conjugation. towards bivalent immunogens (grandjean et al., ) -amino- -deoxy-larabinose (ara n) maleimideactivated bsa potent immunogen (müller et al., a) n-acyl-modified sialylated glycans hsa tof squaric acid chemistry. as inhibitors of adenoviruses causing epidemic keratoconjunctivitis biantennary gal, -keto-gal + fluorescent probe single-chain antibody tof method for drug targeting using antibodies galactose bsa tof new photoinduced thiol-ene coupling galactose hsa tof two step synthesis of gal as optical imaging agent for peritoneal carcinomatosis (regino et al., ) ginsenoside rg bsa tof (sinapinic) generation and characterization of monoclonal antibody to ginsenoside rg (joo et al., ) α-glc, α-man, β-gal, α- to study role on antigen in immune system interaction (tefsen et al., ) heparin tetra-saccharide complement factor h tof (sinapinic, chca) study of interaction between protein and tetrasaccharide by cross-linking (blaum et al., ) (hirata et al., ) α-mannosides (bi-and penta-) bsa tof immunogenicity and induction of candidacidal activity methyl glyoxal (advanced glycation end product, polysialic acid insulin maldi shown to increase insulin lifetime in vivo (bezuglov et al., ) serogroup pneumococcal oligosaccharides bsa seldi-tof, fab synthetic carbohydrate conjugates express epitopes found in native capsular polysaccharides (parameswar et al., ) tn antigen hsa maldi to study effects of hapten density on induced antibody repertoire tumor-associated muc glycopeptides to study multivalent binding to human α-defensin (hd ) (lehrer et al., ) faims field asymmetric waveform ion mobility spectrometry fc fragment (crystallisable) region of igg fmoc -fluorenylmethoxycarbonyl fru fructose f-spe fluorous solid phase extraction ft fourier transform fuc fucose g (g , g ) biantennary glycans with , ( or ) galactose residues g ca coumaric , , , ,-tetra-methylguanidine gaba gamma-aminobutyric acid gags glycosaminoglycans gal galactose gala galacturonic acid galn galactosamine galnac n-acetylgalactosamine g ca coumaric , , , ,-tetra-methylguanidine (liquid matrix) gapcs gallium-phthalocyanines gc/ms gas chromatography/mass spectrometry production of chitooligosaccharides and their potential applications in medicine synthesis of biotinylated sialoside to probe cd -ligand interactions nanofibers formed through p…p stacking of the complexes of glucosyl-c -salicyl-imine and phenylalanine: characterization by microscopy, modeling by molecular mechanics, and interaction by a-helical and b-sheet proteins bishydrazide glycoconjugates for lectin recognition and capture of bacterial pathogens the applicability of enzymes in cellulose ether analysis electron impact ion fragmentation pathways of peracetylated c-glycoside ketones derived from cyclic , -diketones inclusion complexes of gcyclodextrin and carboxyl-modified g-cyclodextrin with c : synthesis, characterization and controlled release application via microgels biochemical characterization of two xylanases from yeast pseudozyma hubeiensis producing only xylooligosaccharides synthesis of -o-glycosylated , -anhydro-d-fructose and of , -anhydro-d-tagatose from a common intermediate , -o-isopropylidene-d-fructose solid-phase random glycosylation comparative solution and solid-phase glycosylations toward a disaccharide library mutational and functional analysis of large in a novel cho glycosylation mutant separation of -aminobenzamide labeled glycans using hydrophilic interaction chromatography columns packed with . mm sorbent depolymerization of sodium alginate under hydrothermal conditions cell wall b-( , )-glucan of saccharomyces cerevisiae. structural characterization and in situ synthesis new cyclodextrin dimers and trimers capable of forming supramolecular adducts with shape-specific ligands development of a mouse monoclonal antibody against the chondroitin sulfate-protein linkage region derived from shark cartilage reflection colour changes in cholesteric liquid crystals after the addition and photochemical isomerization of mesogenic azobenzenes tethered to sugar alcohols preparative enzymatic synthesis of polyprenyl-pyrophosphoryl-n-acetylglucosamine, an essential lipid intermediate for the biosynthesis of various bacterial cell envelope polymers plant cell wall proteomics: mass spectrometry data, a trove for research on protein structure/function relationships introducing capillary electrophoresis with laser-induced fluorescence detection (ce-lif) for the characterization of konjac glucomannan oligosaccharides and their in vitro fermentation behavior introducing capillary electrophoresis with laser-induced fluorescence (ce-lif) as a potential analysis and quantification tool for galactooligosaccharides extracted from complex food matrices nmr spectral mapping of lipid a molecular patterns affected by interaction with the innate immune receptor cd bioconjugation of d-glucuronic acid sodium salt to well-defined primary amine-containing homopolymers and block copolymers glycomic analysis of sialic acid linkages in glycans derived from blood serum glycoproteins chip-based reversed-phase liquid chromatography-mass spectrometry of permethylated n-linked glycans: a potential methodology for cancerbiomarker discovery plasticity of xyloglucan composition in bean (phaseolus vulgaris)-cultured cells during habituation and dehabituation to lethal concentrations of dichlobenil comparison of the substrate specificities and catalytic properties of the sister n-acetylglucosaminyltransferases, gnt-vand gnt-vb (ix) glycoproteomic analysis of embryonic stem cells: identification of potential glycobiomarkers using lectin affinity chromatography of glycopeptides negative-ion maldi-qit-tofms n for structural determination of fucosylated and sialylated oligosaccharides labeled with a pyrene derivative structural determination by negative-ion maldi-qit-tofms n after pyrene derivatization of variously fucosylated oligosaccharides with branched decaose cores from human milk derivatization with -pyrenyldiazomethane enhances ionization of glycopeptides but not peptides in matrix-assisted laser desorption/ionization mass spectrometry large-scale glycomics for discovering cancer-associated n-glycans by integrating glycoblotting and mass spectrometry a concise review of mass spectrometry imaging a glycomics approach to the discovery of potential cancer biomarkers determination of glycosylation sites and site-specific heterogeneity in glycoproteins glycomics and disease markers structural analysis of a fucoidan from the brown alga fucus evanescens by maldi-tof and tandem esi mass spectrometry structural analysis of a highly sulfated fucan from the brown alga laminaria cichorioides by tandem maldi and esi mass spectrometry n-glycan targeted gene delivery to the dendritic cell sign receptor oxazole-modified glycopeptides that target arthritis-associated class ii mhc aq and dr proteins glycocluster design for improved avidity and selectivity in blocking human lectin/plant toxin binding to glycoproteins and cells carbamate-linked lactose: design of clusters and evidence for selectivity to block binding of human lectins to (neo)glycoproteins with increasing degree of branching and to tumor cells maldi-tof/ms analysis of archaebacterial lipids in lyophilized membranes dry-mixed with -aminoacridine structural characterization of cell wall polysaccharides from two plant species endemic to central africa, fleurya aestuans and phragmenthera capitata disease-associated glycosylated molecular variants of human c-reactive protein activate complement-mediated hemolysis of erythrocytes in tuberculosis and indian visceral leishmaniasis the n domain of human angiotensin-i-converting enzyme: the role of n-glycosylation and the crystal structure in complex with an n domain-specific phosphinic inhibitor, rxp loss of effector function of human cytolytic t lymphocytes is accompanied by major alterations in n-and oglycosylation glycome informatics: methods and applications a-carba-glycopyranoside primers: potential building blocks for biocombinatorial synthesis of glycosphingolipid analogues identification, characterization and immunogenicity of an o-antigen capsular polysaccharide of francisella tularensis characterization of oligomeric xylan structures from corn fiber resistant to pretreatment and simultaneous saccharification and fermentation dense-shell glycodendrimers: uv/vis and electron, paramagnetic resonance study of metal ion complexation bioinformatics in glycomics: glycan characterization with mass spectrometric data using simglycan tm functional identification of the proteus mirabilis core lipopolysaccharide biosynthesis genes three enzymatic steps required for the galactosamine incorporation into core lipopolysaccharide redox proteomics of fat globules unveils broad protein lactosylation and compositional changes in milk samples subjected to various technological procedures lactosomes: structural and compositional classification of unique nanometer-sized protein lipid particles of human milk the pleurotus ostreatus hydrophobin vmh and its interaction with glucans comparison of the water-soluble carbohydrate composition and fructan structures of agave tequilana plants of different ages synthesis, conformation, and biological characterization of a sugar derivative of morphine that is a potent, long-lasting, and nontolerant antinociceptive modular synthesis of heparan sulfate oligosaccharides for structure-activity relationship studies direct profiling of saccharides, organic acids and anthocyanins in fruits using electrospray droplet impact/secondary ion mass spectrometry syntheses of mucin-type o-glycopeptides and oligosaccharides using transglycosylation and reverse-hydrolysis activities of bifidobacterium endo-a-nacetylgalactosaminidase recovery of water-soluble compounds from ganoderma lucidum by hydrothermal treatment binding and cellular activation studies reveal that toll-like receptor can differentially recognize peptidoglycan from gram-positive and gram-negative bacteria grafting of aminated oligogalacturonans onto douglas fir barks. a new analysis of carbohydrates and glycoconjugates & route for the enhancement of their lead (ii) binding capacities defining criteria for oligomannose immunogens for hiv using icosahedral virus capsid scaffolds dendrimers designed for functions: from physical, photophysical, and supramolecular properties to applications in sensing, catalysis, molecular electronics, photonics, and nanomedicine the red wine extract-induced activation of endothelial nitric oxide synthase is mediated by a great variety of polyphenolic compounds coloring carbohydrates: investigation of azulene derivatives as blue protecting groups differences in the sialylation patterns of membrane stress proteins in chemical carcinogen-induced tumors developed in balb/c and il- a deficient mice mass spectrometry of n-linked glycans structural characterisation of neutrophil glycans by ultra sensitive mass spectrometric glycomics methodology structural characterization of non-reducing oligosaccharide produced by arthrobacter crystallopoietes n- fragmentation of deprotonated d-ribose and d-fructose in maldi-comparison with dissociative electron attachment multidimensional profiling of components in complex mixtures of natural products for metabolic analysis, proof of concept: application to quillaja saponins structural investigation of bacterial lipopolysaccharides by mass spectrometry and tandem mass spectrometry probe design and synthesis of galb( ! )[neuaca( ! )]glcnacb( ! )man motif of n-glycan glycoprofiling bifidobacterial consumption of galacto-oligosaccharides by mass spectrometry reveals strain-specific, preferential consumption of glycans n-glycosylation of plant recombinant pharmaceuticals synthesis of thiourea-tethered cglycosyl amino acids via isothiocyanate-amine coupling permeate from cheese whey ultrafiltration is a source of milk oligosaccharides detecting glycan cancer biomarkers in serum samples using maldi ft-icr mass spectrometry data quantitative analysis of glycation sites on human serum albumin using o/ olabeling and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry characterization of wrss and wrss , new second-generation virg(icsa)-based shigella sonnei vaccine candidates with the potential for reduced reactogenicity mass spectrometry in the characterization of human genetic n-glycosylation defects combined treatment of human mcf- breast carcinoma with antibody, cationic lipid and hyaluronic acid using ex vivo assays ex vivo assays of cem cells cultured and treated in the three dimensional cultures glycan analysis and influenza a virus infection of primary swine respiratory epithelial cells: the importance of neuaca - glycans acceptor substrate discrimination in phosphatidyl-myo-inositol mannoside synthesis. structural and mutational analysis of mannosyl transferase corynebacterium glutamicum pimb synthesis and antimicrobial evaluation of amphiphilic neamine derivatives clicking pentafluorostyrene copolymers: synthesis, nanoprecipitation, and glycosylation trends in glycosylation, glycoanalysis and glycoengineering of therapeutic antibodies and fc-fusion proteins chemical structure of bacteriovorax stolpii lipid a core chirality based tailoring of the liquid crystalline properties of supermolecular tetrapedes development of a nanolc ltq orbitrap mass spectrometric method for profiling glycans derived from plasma from healthy, benign tumor control, and epithelial ovarian cancer patients development of a robust and high throughput method for profiling n-linked glycans derived from plasma glycoproteins by nanolc-fticr mass spectrometry genomic and biochemical analysis of n-glycosylation in the mushroom-forming basidiomycete schizophyllum commune the structurally similar, penta-acylated lipopolysaccharides of porphyromonas gingivalis and bacteroides elicit strikingly different innate immune responses design, synthesis and biological evaluation of carbohydrate-functionalized cyclodextrins and liposomes for hepatocyte-specific targeting ex post glycoconjugation of phthalocyanines modification of recombinant proteins by covalent polysialation illustrated with the example of human insulin influence of protein molecular mass on the glycation historical overview of glycoanalysis synthesis of differentially protected glucosamine building blocks and their evaluation as glycosylating agents de novo synthesis of differentially protected l-iduronic acid glycosylating agents chemical analysis of flavonoid constituents of the seagrass halophila stipulacea: first finding of malonylated derivatives in marine phanerogams substantial spatial flexibility and hydrogen bonding within the catalysis exerted by cyclodextrin artificial glycosidases lysine and arginine side chains in glycosaminoglycanprotein complexes investigated by nmr, cross-linking, and mass spectrometry: a case study of the factor h-heparin interaction glycomic analysis of n-linked carbohydrate epitopes from cd of mouse brain o-glycosylation pattern of cd from mouse brain a high-throughput oglycopeptide discovery platform for seromic profiling a high-throughput oglycopeptide discovery platform for seromic profiling-correction alteration of protein glycosylation in liver diseases investigating the molecular basis for the virulence of escherichia coli k by nuclear magnetic resonance analysis of the capsule polysaccharide monitoring of malting process by characterization of glycation of barley protein z determination of glycan structure from tandem mass spectra use of fullerene-, octadecyl-, and triaconthyl silica for solid phase extraction of tryptic peptides obtained from unmodified and in vitro glycated human serum albumin and fibrinogen site specific conjugation of fluoroprobes to the remodeled fc n-glycans of monoclonal antibodies using mutant glycosyltransferases: application for cell surface antigen detection innovative approach for producing injectable, biodegradable materials using chitooligosaccharides and green chemistry reciprocal principle of molecular recognition in supramolecular chromatography-highly selective analytical separation of cyclodextrin congeners on a silica-bonded [ ]fullerene stationary phase cation-independent mannose -phosphate receptor. a composite of distinct phosphomannosyl binding sites synthesis of lacdinac-terminated glycoconjugates by mutant galactosyltransferase-a way to new glycodrugs and materials new , -difluoro- a, a-diaza-sindacene (bodipy)-labeled sphingolipids for membrane studies solid-phase synthesis of a pentavalent galnac-containing glycopeptide (tn antigen) representing the nephropathy-associated iga hinge region chiral-auxiliary-mediated , -cis-glycosylations for the solid-supported synthesis of a biologically important branched a-glucan multimeric bivalent immunogens from recombinant tetanus toxin hc fragment, synthetic hexasaccharides, and a glycopeptide adjuvant the elucidation of the structure of thermotoga maritima peptidoglycan reveals two novel types of cross-link a m b family metallopeptidase of helicobacter pylori required for cell shape, pole formation and virulence lysosomal storage of oligosaccharide and glycosphingolipid in imino sugar treated cells multivalent dendrimeric compounds containing carbohydrates expressed on immune cells inhibit infection by primary isolates of hiv- ion exchange and purification of carbohydrates on a nafion(r) membrane as a new sample pretreatment for matrix-assisted laser desorption-ionization mass spectrometry chinese hamster ovary cells can produce galactose-a- , -galactose antigens on proteins characterization of atll, a bifunctional autolysin of staphylococcus lugdunensis with nacetylglucosaminidase and n-acetylmuramoyl-l-alanine amidase activities dimeric architecture of the hendra virus attachment glycoprotein: evidence for a conserved mode of assembly unusual molecular architecture of the machupo virus attachment glycoprotein synthesis of polymerizable cyclodextrin derivatives for use in adhesion-promoting monomer formulations physicochemical properties of microbial glycopolymers an analytical system for the characterization of highly heterogeneous mixtures of n-linked oligosaccharides site directed processing: role of amino acid sequences and glycosylation of acceptor glycopeptides in the assembly of extended mucin type o-glycan core high content phenotypic cell-based visual screen identifies mycobacterium tuberculosis acyltrehalose-containing glycolipids involved in phagosome remodeling strategies for analysis of the glycosylation of proteins: current status and future perspectives characterization of irx and irx -like reveals an essential role in glucuronoxylan biosynthesis in arabidopsis uv-maldi-tof mass spectrometry analysis of heparin oligosaccharides obtained by nitrous acid controlled degradation and high performance anion exchange chromatography distribution of o-glycosylhydrolases in marine fungi of the sea of japan and the sea of okhotsk: characterization of exocellular n-acetyl-b-d-glucosaminidase of the marine fungus penicillium canescens derivatization in mass spectrometry identification, characterization, and biosynthesis of a novel n-glycan modification in the fruiting body of the basidiomycete coprinopsis cinerea lipid remodelling of glycosylphosphatidylinositol (gpi) glycoconjugates in procyclic form trypanosomes: biosynthesis and processing of gpis revisited caenorhabditis elegans n-glycan core b-galactoside confers sensitivity towards nematotoxic fungal galectin cgl novel mannosidase inhibitors probe glycoprotein degradation pathways in cells chitosan oligosaccharides modulate the supramolecular conformation and the biological activity of oligogalacturonides in arabidopsis efficient synthesis of a -deoxytalose tetrasaccharide related to the antigenic o-polysaccharide produced by aggregatibacter actinomycetemcomitans serotype c aniline/a-cyano- -hydroxycinnamic acid is a highly versatile ionic liquid for matrix-assisted laser desorption/ionization mass spectrometry h-pteridine- , -dione (lumazine): a new maldi matrix for complex (phospho)lipid mixtures analysis characterization of acp, a peptidoglycan hydrolase of clostridium perfringens with nacetylglucosaminidase activity that is implicated in cell separation and stress-induced autolysis glycobase and autogu: tools for hplc-based glycan analysis carrageenans: biological properties, chemical modifications and structural analysis-a review click" glycodendrimers containing , and modified xylopyranoside termini the plasma von willebrand factor o-glycome comprises a surprising variety of structures including abh antigens and disialosyl motifs analytical progress for protein glycosylation in china strategies for proteomic analysis of nonenzymatically glycated proteins preparation of saturated and unsaturated fatty acid hydrazides and long chain c-glycoside ketohydrazones glycoproteome study in myocardial lesions serum by integrated mass spectrometry approach: preliminary insights changes of serum-associated fucosylated glycoproteins and changes in glycosylation of iga in human cirrhosis efficient isolation of the subunits of recombinant and pituitary glycoprotein hormones ferrocene-cyclodextrin conjugates: synthesis, supramolecular behavior, and use as electrochemical sensors ferrocene-carbohydrate conjugates as electrochemical probes for molecular recognition studies characterization of main anthocyanins extracted from pericarp blue corn by maldi-tof ms highly glycosylated human alpha interferon: an insight into a new therapeutic candidate the protease-associated domain and c-terminal extension are required for zymogen processing, sorting within the secretory pathway, and activity of tomato subtilase (slsbt ) design and creativity in synthesis of multivalent neoglycoconjugates -deoxy-dmanno-octulosonic acid (kdo) hydrolase identified in francisella tularensis, helicobacter pylori, and legionella pneumophila synthesis and characterization of biotin chainend functionalized boronic acid-containing polymer (boropolymer) as functional glyco-affinity macroligand maldi mass spectrometry imaging of gangliosides in mouse brain using ionic liquid matrix das inhibits h n influenza virus infection of human lung tissues synthesis, characterization, and self-assembled nanofibers of carbohydrate-functionalized mono-and di( , : , -terpyridinyl)arenes developmental regulation of oligosialylation in zebrafish glycan array on aluminum oxide-coated glass slides through phosphonate chemistry mannose receptor interacts with fc receptors and is critical for the development of crescentic glomerulonephritis in mice efficient synthesis of idraparinux, the anticoagulant pentasaccharide the efficient total synthesis of bisglycosyl apigenin from naringenin: a greener way a mixed cyclodextrinbiphenyl thermotropic liquid crystal: synthesis, liquid-crystalline properties, and supramolecular organization boronate affinity monolith for highly selective enrichment of glycopeptides and glycoproteins application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof-ms) in preparation of chitosan oligosaccharides (cos) with degree of polymerization (dp) - containing well-distributed acetyl groups study of on-resin convergent synthesis of n-linked glycopeptides containing a large high mannose n-linked oligosaccharide facile synthesis of cyclopeptide-centered multivalent glycoclusters with 'click chemistry' and molecular recognition study by surface plasmon resonance glycosite analysis in glycoproteomics by mass spectrometry combination of matrix-assisted laser desorption ionization and electrospray ionization mass spectrometry for the analysis of intact glycopeptides from horseradish peroxidase an introduction to sphingolipid metabolism and analysis by new technologies one-pipeline approach achieving glycoprotein identification and obtaining intact glycopeptide information by tandem mass spectrometry imaging maldi mass spectrometry of sphingolipids using an oscillating capillary nebulizer matrix application system single-crystalline euf hollow hexagonal microdisks: synthesis and application as a background-free matrix for maldi-tof-ms analysis of small molecules and polyethylene glycols selective desorption/ ionization of sulfatides by maldi-ms facilitated using -aminoacridine as matrix tethered derivatives of d-glucose and pentacyclic triterpenes for homo/heterobivalent inhibition of glycogen phosphorylase total synthesis of a furostan saponin, timosaponin bii subcompartment localization of the side chain xyloglucan-synthesizing enzymes within golgi stacks of tobacco suspension-cultured cells carbohydrates as recognition receptors in biosensing applications expression system for human glycosyltransferases and its application production, purification, and characterization of human a proteinase inhibitor from aspergillus niger direct amidation of aldoses and decarboxylative amidation of a-keto acids: an efficient conjugation method for unprotected carbohydrate molecules novel succinylated and large-sized osmoregulated periplasmic glucans of pseudomonas syringae pv. syringae chiral separation of hesperetin and hesperetin-o-glycoside in capillary electrophoresis using microbial b- , -glucans structural modification and characterization of rice starch treated by thermus aquaticus -a-glucanotransferase enzymatic biosynthesis of a puerarincycloamylose inclusion complex by -aglucanotransferase and maltogenic amylase analysis of mixture of maltooligoses using maldi-tofms: influence of cationizing agent types comparison of ionization behaviors of ring and linear carbohydrates in maldi-tofms challenges of determining o-glycopeptide heterogeneity: a fungal glucanase model system mutational analysis of bacillus megaterium qm b cortex-lytic enzymes mass spectrometric imaging for biomedical tissue analysis synthesis of muc peptide and glycopeptide dendrimers synthesis and biological evaluation of multivalent carbohydrate ligands obtained by click assembly of pseudo-rotaxanes pseudomonas aeruginosa exploits lipid a and muropeptides modification as a strategy to lower innate immunity during cystic fibrosis lung infection straightforward synthesis of novel akt inhibitors based on a glucose scaffold the role of sugar configuration in the acetolysis of -deoxyhexose methyl glycosides synthesis of a b-glcn-( ! )-murnac building block en route to n-deacetylated peptidoglycan fragments a urea-linked glucosamine dimer as a building block for the synthesis of linear and cyclic neosaccharides a simple and rapid method for the permethylation of carbohydrates trypanosoma brucei amp-activated kinase subunit homologs influence surface molecule expression porphyromonas gingivalis resistance to polymyxin b is determined by the lipid a -phosphatase, pgn_ a new archaeal b-glycosidase from sulfolobus solfataricus. seeding a novel retaining b-glycan-specific glycoside hydrolase family along with the human non-lysosomal glucosylceramidase cba b-glycosyl azides as substrates for a-glycosynthases: preparation of efficient a-l-fucosynthases synthetic heparan sulfate oligosaccharides inhibit endothelial cell functions essential for angiogenesis towards the synthesis of -deoxy- -nitropiperidinoses synthesis and evaluation of s-and c( )-substituted analogues of lincomycin localization and imaging of sialylated glycosphingolipids in brain tissue sections by maldi mass spectrometry an efficient modular approach for the assembly of s-linked glycopeptoids glycosidase inhibition with fullerene iminosugar balls: a dramatic multivalent effect modification of gastric mucin oligosaccharide expression in rhesus macaques after infection with helicobacter pylori identification of novel contributions to high-affinity glycoprotein-receptor interactions using engineered ligands synthesis and conformational analysis of a novel carbohydrate-fused bis-crown ether: crown-cyplos design, synthesis and characterisation of a fluorescently labelled cyplos ionophore application of liquid chromatography-tandem mass spectrometry for the characterization of galactosylated and tagatosylated b-lactoglobulin peptides derived from in vitro gastrointestinal digestion effect of glycation on the gastrointestinal digestibility and immunoreactivity of bovine b-lactoglobulin role of pyridoxamine in the formation of the amadori/heyns compounds and aggregates during the glycation of b-lactoglobulin with galactose and tagatose acceptor products of alternansucrase with gentiobiose. production of novel oligosaccharides for food and feed and elimination of bitterness glucosylation of raffinose via alternansucrase acceptor reactions investigating the candidacy of lps-based glycoconjugates to prevent invasive meningococcal disease: chemical strategies to prepare glycoconjugates with good carbohydrate loading investigating the candidacy of lps-based glycoconjugates to prevent invasive meningococcal disease: immunology of glycoconjugates with high carbohydrate loading towards a second generation of ionic liquid matrices (ilms) for maldi-ms of peptides, proteins, and carbohydrates sinorhizobium fredii hh cgs mutants are unable to nodulate determinate-and indeterminate nodule-forming legumes and overproduce an altered eps carbohydrate and domain architecture of an immature antibody glycoform exhibiting enhanced effector functions a human embryonic kidney t cell line mutated at the golgi a-mannosidase ii locus sieve-based device for maldi sample preparation. ii. instrumental parameterization a link between the assembly of flagella and lipooligosaccharide of the gram-negative bacterium campylobacter jejuni nonglycosidically linked pseudodisaccharides: thioethers, sulfoxides, sulfones, ethers, selenoethers, and their binding to lectins stable expression of a human-like sialylated recombinant thyrotropin in a chinese hamster ovary cell line expressing a , -sialyltransferase impaired lysosomal trimming of n-linked oligosaccharides leads to hyperglycosylation of native lysosomal proteins in mice with a-mannosidosis biochemical and immunocytological characterizations of arabidopsis thaliana pollen tube cell wall engineering of n. benthamiana l. plants for production of n-acetylgalactosamine-glycosylated proteins-towards development of a plant-based platform for production of protein therapeutics with mucin type o-glycosylation xylan-based nanoparticles: prodrugs for ibuprofen release towards unnatural xylan based polysaccharides: reductive amination as a tool to access highly engineered carbohydrates a simple micromethod for determining precise oligosaccharidic specificity of mannose-binding lectins functional and morphological adaptation to peptidoglycan precursor alteration in lactococcus lactis towards the implementation of quality by design to the production of therapeutic monoclonal antibodies with desired glycosylation patterns molecular characterization of a putative sucrose: fructan -fructosyltransferase ( -sft) of the cold-resistant patagonian grass bromus pictus associated with fructan accumulation under low temperatures biomarkers and diagnosis of congenital disorders of glycosylation improved procedures for the selective chemical fragmentation of rhamnogalacturonans when can glycopeptides be assigned based solely on high-resolution mass spectrometry data? mmps promotes glycopeptidolipids biosynthesis and export in mycobacterium smegmatis glyco-spectrum-scan: fishing glycopeptides from ms spectra of protease digests of human colostrum siga common sialylated glycan in actinobacillus suis the major surface carbohydrates of the echinococcus granulosus cyst: mucin-type o-glycans decorated by novel galactosebased structures surface analytical characterization of carbohydrate microarrays shiga toxin receptor gb cer/ cd : tumor-association and promising therapeutic target in pancreas and colon cancer synthesis of some quaternary n-( , -anhydro- -deoxy-d,l-ribitol- -yl)ammonium salts a barley cellulose synthase-like cslh gene mediates ( , ; , )-b-d-glucan synthesis in transgenic arabidopsis analytical performance of immobilized pronase for glycopeptide footprinting and implications for surpassing reductionist glycoproteomics recombinant expression and characterization of n-acetylglucosaminyltransferase i derived from nicotiana tabacum comparison of the n-linked glycosylation of human b , -n-acetylglucosaminyltransferase expressed in insect cells and silkworm larvae improved secretion of molecular chaperone-assisted human igg in silkworm, and no alterations in their n-linked glycan structures structure of glycosylated cu/znsuperoxide dismutase from kluyveromyces yeast nbimcc glycan structures and antiviral effect of the structural subunit rvh of rapana hemocyanin baca, an abc transporter involved in maintenance of chronic murine infections with mycobacterium tuberculosis a systematic nomenclature for carbohydrate fragmentations in fab-ms/ms spectra of glycoconjugates transformation of linear oligoketosides into macrocyclic neoglycoconjugates a new ligation strategy for peptide and protein glycosylation: photoinduced thiol-ene coupling graphene as a novel matrix for the analysis of small molecules by maldi-tof ms envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens clinical collection and protein properties of expressed prostatic secretions as a source for biomarkers of prostatic disease mycobacterium marinum mmar- , a predicted transmembrane acyltransferase, is essential for the presence of the mannose cap on lipoarabinomannan electrochemically controlled adsorption of fc-functionalized polymers on b-cd-modified selfassembled monolayers production of carbohydrate building blocks from red seaweed polysaccharides. efficient conversion of galactans into c-glycosyl aldehydes polysaccharide mimicry of the epitope of the broadly neutralizing anti-hiv antibody, g , induces enhanced antibody responses to self oligomannose glycans glycosylated cell-penetrating peptides and their conjugates to a proapoptotic peptide: preparation by click chemistry and cell viability studies nm multiphoton dissociation and ionization of , -dihydroxyacetophenone lack of complex n-glycans on hiv- envelope glycoproteins preserves protein conformation and entry function prebiotic oligosaccharides: in vitro evidence for gastrointestinal epithelial transfer and immunomodulatory properties c-furyl glycosides, ii: synthesis and antimicrobial evaluation of c-furyl glycosides bearing pyrazolines, isoxazolines, and , -dihydropyrimidine- ( h)-thiones structural characterization of bordetella parapertussis lipid a polysaccharide pharmacokinetics: amphotericin b arabinogalactan conjugate -a drug delivery system or a new pharmaceutical entity hexosamine template. a platform for modulating gene expression and for sugar-based drug discovery combinatorial syntheses of trisaccharide libraries on a soluble polymeric support lipophilic thioglycosides for the solution-phase synthesis of oligosaccharides using biphasic liquid-liquid separation chiral and structural analysis of biomolecules using mass spectrometry and ion mobility-mass spectrometry fluorescent labeling of a carboxyl group of sialic acid for maldi-ms analysis of sialyloligosaccharides and ganglioside liquid chromatography combined with mass spectrometry for the investigation of endoglucanase selectivity on carboxymethyl cellulose investigation of endoglucanase selectivity on carboxymethyl cellulose by mass spectrometric techniques cellulosic graft copolymer: poly(methyl methacrylate) with cellulose side chains radially oriented cellulose triacetate chains on gold nanoparticles amphiphilic carbohydrate-phthalocyanine conjugates obtained by glycosylation or by azide-alkyne click reaction different and new nod factors produced by rhizobium tropici ciat following na þ stress identification of the polyketide synthase involved in the biosynthesis of the surface-exposed lipooligosaccharides in mycobacteria bioanalysis of recombinant proteins and antibodies by mass spectrometry lectin and carbohydrate microarrays: new high-throughput methods for glycoprotein, carbohydrate-binding protein and carbohydrate-active enzyme analysis chitosans as bioactive macromolecules to protect economically relevant crops from their main pathogens serum n-glycome biomarker for monitoring development of dena-induced hepatocellular carcinoma in rat secondary cell wall formation in cryptococcus neoformans as a rescue mechanism against acid-induced autolysis identification and quantification of protein posttranslational modifications matrixassisted laser desorption/ionization time-of-flight (maldi-tof) mass spectrometry analysis of oligosaccharides and oligosaccharide alditols obtained by hydrolysis of agaroses and carrageenans, two important types of red seaweed polysaccharides glycopeptidome of a heavily n-glycosylated cell surface glycoprotein of dictyostelium implicated in cell adhesion fragmentation behavior of amadori-peptides obtained by non-enzymatic glycosylation of lysine residues with adp-ribose in tandem mass spectrometry effect of , ; , -b-d-glucan and the products of its enzymatic transformation on the formation of germs of buckwheat fagopyrum esculentum mönch synthesis of , -branched arabinogalactan-type tetra-and hexasaccharides for characterization of monoclonal antibodies phosphoglucomutase of yersinia pestis is required for autoaggregation and polymyxin b resistance mass spectrometric investigation of molecular variability of grass pollen group allergens cyclodextrin aldehydes are oxidase mimics simultaneous glycoproteomics on the basis of structure using ion mobility-mass spectrometry characterizing ion mobility-mass spectrometry conformation space for the analysis of complex biological samples effect of ionic liquids as additives in the catalytic properties of different immobilized preparations of rhizomucor miehei lipase in the hydrolysis of peracetylated lactal synthetic pseudopterosin analogues: a novel class of antiinflammatory drug candidates purification, characterization and in vivo studies of salmon heparin thieme chemistry journal awardees-where are they now? synthesis of the marine glycolipid dioctadecanoyl discoside thiyl glycosylation of olefinic proteins: s-linked glycoconjugate synthesis synthesis of the lewis b pentasaccharide and a hsa-conjugate thereof maldi mass spectrometry imaging, from its origins up to today: the state of the art detection of honeybee venom in envenomed tissues by direct maldi msi bioinformatics and molecular modeling in glycobiology expression of rat b( , )-n-acetylglucosaminyltransferase iii in nicotiana tabacum remodels the plantspecific n-glycosylation glycoprotein expression in human milk during lactation -deoxynojirimycins with dansyl capped n-substituents as probes for morbus gaucher affected cell lines new secoiridoid glucosides from ligustrum lucidum induce erk and creb phosphorylation in cultured cortical neurons application of maldi-tof mass spectrometry in lipidomics an update of maldi-tof mass spectrometry in lipid research maldi-tof-ms directly combined with tlc: a review of the current state isolation and structural analysis in vivo of newly synthesized fructooligosaccharides in onion bulbs tissues (allium cepa l.) during storage maldi mass spectrometry using , , -trihydroxyacetophenone and , -dihydroxyacetophenone with cyclodextrins: suppression of matrix-related ions in low-molecular-weight region change in glycosylation pattern with extension of endoplasmic reticulum retention signal sequence of mouse antibody produced by suspensioncultured tobacco by cells chemical characterization of the oligosaccharides in bactrian camel (camelus bactrianus) milk and colostrum dendritic sugar-microarrays by click chemistry aggregation of alzheimer amyloid b peptide ( - ) on the multivalent sulfonated sugar interface scope and limitations of imidazolium-based ionic liquids as room temperature glycosylation promoters the carboxy-terminal erretention motif, sekdel, influences the n-linked glycosylation of recombinant human a-l-iduronidase but has little effect on enzyme activity in seeds of brassica napus and nicotiana tabacum mass spectrometric fragmentation analysis of oligosialic and polysialic acids quantification of nucleotide-activated sialic acids by a combination of reduction and fluorescent labeling synaptic cell adhesion molecule syncam is a target for polysialylation in postnatal mouse brain comparative characterization of the arabidopsis subfamily a b-galactosidases preparation and gelling properties of sugar-contained low-molecular-mass gelators: combination of cholesterol and linear glucose generation of asparagine-linked glycan structure databases and their use the first total synthesis of -o-b-dglucopyranosyl- -o-a-l-rhamnopyranosyl apigenin via a hexanoyl ester-based protection strategy efficient synthesis of -amido-n -acetyl- -deoxyneuraminic acid and its application to the c- modification of sialic acids endothelial galectin- binds to specific glycans on nipah virus fusion protein and inhibits maturation, mobility, and function to block syncytia formation lectin-directed enzyme activated prodrug therapy (leapt): synthesis and evaluation of rhamnosecapped prodrugs characterization of a new b( - )-glucan branching activity of aspergillus fumigatus enhanced production and partial characterization of an extracellular polysaccharide from newly isolated azotobacter sp. ssb phase diagrams of monoacylated amide-linked disaccharide glycolipids diamond, titanium dioxide, titanium silicon oxide, and barium strontium titanium oxide nanoparticles as matrixes for direct matrix-assisted laser desorption/ionization mass spectrometry analysis of carbohydrates in plant tissues synthesis of a tetrasaccharide corresponding to the teichoic acid from the cell wall of streptomyces sp. vkm ac- synthesis of the hexasaccharide repeating unit corresponding to the cell wall lipopolysaccharide of azospirillum irakense kbc sialic acids in different leishmania sp., its correlation with nitric oxide resistance and host responses -o-acetylated sialic acids enhance entry of virulent leishmania donovani promastigotes into macrophages high throughput quantification of n-glycans using one-pot sialic acid modification and matrix assisted laser desorption ionization time-offlight mass spectrometry gdp-d-mannose , -epimerase (gme) plays a key role at the intersection of ascorbate and non-cellulosic cell-wall biosynthesis in tomato an hplc-maldi ms method for n-glycan analyses using smaller size samples: application to monitor glycan modulation by medium conditions towards a reliable molecular mass determination of intact glycoproteins by matrixassisted laser desorption/ionization time-of-flight mass spectrometry ionic liquid matrices for maldi-tof-ms analysis of intact glycoproteins synthesis of pegylated lactose analogs for inhibition studies on t. cruzi trans-sialidase glycosylation site-specific analysis of clade c hiv- envelope proteins enzymatic/chemical release of o-glycans allowing ms analysis at high sensitivity glycomic profiling of invasive and non-invasive breast cancer cells assessment of chemoselective neoglycosylation methods using chlorambucil as a model glycan family analysis for deducing n-glycan topology from single ms detection of hepatocellular carcinoma using glycomic analysis discovery of rifampicin as a new anti-glycating compound by matrix-assisted laser desorption/ionization mass spectrometry-based insulin glycation assay first total synthesis of , -dipalmitoyl- -(n-palmitoyl- -amino- -deoxy-a-dglucosyl)-sn-glycerol-a glycoglycerolipid of a marine alga with a high inhibitor activity against human myt -kinase acidiplasma aeolicum gen. nov., sp. nov., a euryarchaeon of the family ferroplasmaceae isolated from a hydrothermal pool, and transfer of ferroplasma cupricumulans to acidiplasma cupricumulans comb. nov synthesis of polyhydroxy [n]-polyurethanes derived from a carbohydrate precursor characterization of n-linked glycosylation on recombinant glycoproteins produced in pichia pastoris using esi-ms and maldi-tof dna-templated homo-and heterodimerization of peptide nucleic acid encoded oligosaccharides that mimick the carbohydrate epitope of hiv human macrophage activation triggered by chitotriosidase-mediated chitin and chitosan degradation evaluation of conditions for release of mucin-type oligosaccharides from glycoproteins by hydrazine gas treatment imaging mass spectrometry of glycolipids visualization of spatial distribution of g-aminobutyric acid in eggplant (solanum melongena) by matrix-assisted laser desorption/ionization imaging mass spectrometry the specific localization of seminolipid molecular species on mouse testis during testicular maturation revealed by imaging mass spectrometry the detection of glycosphingolipids in brain tissue sections by imaging mass spectrometry using gold nanoparticles practical heavy fluorous tag for carbohydrate synthesis with minimal chromatographic purification production and nglycosylation of recombinant human cell adhesion molecule l from insect cells using the stable expression system. effect of dimethyl sulfoxide glyco-sams by 'dual click': thiourea-bridged glyco-oeg azides for cycloaddition on surfaces mass spectrometry in the elucidation of the glycoproteome of bacterial pathogens investigation towards bivalent chemically defined glycoconjugate immunogens prepared from acid-detoxified lipopolysaccharide of vibrio cholerae o , serotype inaba the synthesis and immune stimulating action of mannose-capped lysine-based dendrimers some patterns in dimer ii formation in bodipy-fl-labeled lipids bodipy-labeled ganglioside probes for membrane and biological studies development of a high performance anion exchange chromatography analysis for mapping of oligosaccharides analytical approaches towards the structural characterization of microbial wall glycopolymers differential effect of plasma or erythrocyte age-ligands of rage on expression of transcripts for receptor isoforms biosynthesis of a new udpsugar, udp- -acetamido- -deoxyxylose, in the human pathogen bacillus cereus subspecies cytotoxis nvh - effective enlargement of fluorescence resonance energy transfer of poly-porphyrin mediated by b-cyclodextrin dimers total synthesis of the heptasaccharide repeating unit of the iron-binding exopolysaccharide secreted by klebsiella oxytoca bas- mass spectrometry characterization of the glycation sites of bovine insulin by tandem mass spectrometry oxidative modifications in glycated insulin new insights into the early steps of phosphatidylinositol mannoside biosynthesis in mycobacteria. pimb' is an essential enzyme of mycobacterium smegmatis fatty acyl structures of mycobacterium tuberculosis sulfoglycolipid govern t cell response automated measurement of permethylated serum n-glycans by maldi-linear ion trap mass spectrometry monoterpenoid glucoindole alkaloids and iridoids from pterocephalus pinardii production, refining, structural characterization and fermentability of rice husk xylooligosaccharides oligo mass profiling (olimp) of extracellular polysaccharides facile synthesis of three bidesmosidic oleanolic acid saponins with strong inhibitory activity on pancreatic lipase sortase-catalyzed peptide-glycosylphosphatidylinositol analogue ligation one-pot synthesis of cyclic antifreeze glycopeptides syntheses of glycoclusters containing a phosphocholine residue related to a glycosphingolipid from the earthworm pheretima hilgendorfi establishment of a real-time analytical method for free oligosaccharide transport from the er to the cytosol purification, characterization and molecular cloning of a novel endo-b-n-acetylglucosaminidase from the basidiomycete, flammulina velutipes comparative glycomic profiling in esophageal adenocarcinoma derivatization and analysis of oligosaccharides by matrix-assisted laser desorption/ionization time of-flight mass spectrometry sulfated oligosaccharide cluster with polylysine core scaffold as a new anti-hiv dendrimer lipophilic b-cyclodextrin cyclic-nitrone conjugate: synthesis and spin trapping studies sialylation using n-glycolylneuraminyl phosphite donors to synthesize neu gc-containing glycans chemical de-oglycosylation of glycoproteins for application in lc-based proteomics secondary acylation of vibrio cholerae lipopolysaccharide requires phosphorylation of kdo rapid characterization of n-linked glycans from secreted and gel-purified monoclonal antibodies using maldi-tof mass spectrometry synthesis of porphyrin-carbohydrate conjugates using "click" chemistry and their preliminary evaluation in human hep cells an improved protocol for n-glycosylation analysis of gel-separated sialylated glycoproteins by maldi-tof/ tof artificial polymerases and molecular chaperones interaction of nectarin with a fungal protein triggers a microbial surveillance and defense mechanism in nectar n-glycans on the link domain of human hare/stabilin- are needed for hyaluronan binding to purified ecto-domain, but not for cellular endocytosis of hyaluronan analytical characterization of monoclonal antibodies: linking structure to function identification of rhodococcus equi lipids recognized by host cytotoxic t lymphocytes matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates analysis of carbohydrates and glycoconjugates by matrixassisted laser desorption/ionization mass spectrometry: an update covering the period - analysis of carbohydrates and glycoconjugates by matrixassisted laser desorption/ionization mass spectrometry: an update covering the period analysis of carbohydrates and glycoconjugates by matrixassisted laser desorption/ionization mass spectrometry: an update for electrospray and maldi mass spectrometry: fundamentals, instrumentation, practicalities, and biological applications analysis of carbohydrates and glycoconjugates by matrixassisted laser desorption/ionization mass spectrometry: an update for the period analysis of carbohydrates and glycoconjugates by matrixassisted laser desorption/ionization mass spectrometry: an update for identification of by-products formed during the release of n-glycans with protein n-glycosidase f in the presence of dithiothreitol application of negative ion ms/ ms to the identification of n-glycans released from carcinoembryonic antigen cell adhesion molecule (ceacam ) structural and quantitative analysis of n-linked glycans by maldi and negative ion nanospray mass spectrometry proposal for a standard system for drawing structural diagrams of n-and o-linked carbohydrates and related compounds fragmentation of negative ions from n-linked carbohydrates, part . fragmentation of complex glycans lacking substitution on the -antenna identification of high-mannose and multiantennary complextype n-linked glycans containing a-galactose epitopes from nurse shark igm heavy chain o-glycoside biomarker of apolipoprotein c : responsiveness to obesity, bariatric surgery, and therapy with metformin, to chronic or severe liver disease and to mortality in severe sepsis and graft vs host disease pyridylamination as a means of analyzing complex sugar chains switching the selectivity of a polyglycerol dendrimer monomolecularly imprinted with d-(À)-fructose kegg glycan for integrated analysis of pathways, genes and glycan structures sphingolipidomics: methods for the comprehensive analysis of sphingolipids antifreeze glycopeptide analogues: microwaveenhanced synthesis and functional studies degradation of chitosans with a family chitosanase from streptomyces coelicolor a ( ) glycomics of bone marrow-derived mesenchymal stem cells can be used to evaluate their cellular differentiation stage rational engineering of mannosyl binding in the distal glycone subsites of cellulomonas fimi endo-b- , -mannanase: mannosyl binding promoted at subsite - and demoted at subsite - uv-laser ablation of ionic liquid matrices structure analysis of n-glycoproteins toward synthesis of the isosteric sulfonate analogues of the at-iii binding domain of heparin plant immunity induced by oligogalacturonides alters root growth in a process involving flavonoid accumulation in arabidopsis thaliana conversion of a-amyrin into centellosides by plant cell cultures of centella asiatica carbohydrate analysis throughout the development of a protein therapeutic positive and negative analyte ion yield in matrix-assisted laser desorption/ionization revisited g-cyclodextrins possessing an azido group and a triisopropylbenzenesulfonyl group as useful synthetic and authentic intermediates for unsymmetrically functionalized derivatives hemicellulase production in chrysosporium lucknowense c genetically engineered mannosylated-human serum albumin as a versatile carrier for liverselective therapeutics free oligosaccharides to monitor glycoprotein endoplasmic reticulumassociated degradation in saccharomyces cerevisiae chemical synthesis, folding, and structural insights into o-fucosylated epidermal growth factor-like repeat of mouse notch- receptor a water-soluble ruthenium glycosylated porphyrin catalyst for carbenoid transfer reactions in aqueous media with applications in bioconjugation reactions synthesis and self-assembled nanostructures of novel chiral amphiphilic liquid crystals containing b-d-galactopyranoside end-groups synthetic antitumor vaccines from tetanus toxoid conjugates of muc glycopeptides with the thomsen-friedenreich antigen and a fluorine-substituted analogue matrix-free uv-laser desorption/ionization (ldi) mass spectrometric imaging at the singlecell level: distribution of secondary metabolites of arabidopsis thaliana and hypericum species core region and lipid a components of lipopolysaccharides matrix-assisted laser desorption/ionization mass spectrometry (maldi-ms) application in carbohydrate analysis glycoproteomic analysis of wgabound glycoprotein biomarkers in sera from patients with lung adenocarcinoma synthesis, characterization and in vitro pharmacology of novel pregabalin derivatives an efficient approach to the discovery of potent inhibitors against glycosyltransferases electrospray and maldi mass spectrometry: fundamentals, instrumentation, practicalities, and biological applications design, synthesis, and immunochemical characterization of a chimeric glycopeptide corresponding to the shigella flexneri y opolysaccharide and its peptide mimic mdwnmhaa antiproliferative cardenolide glycosides of elaeodendron alluaudianum from the madagascar rainforest the influence of microwave irradiation on stereospecific mo(vi)-catalyzed transformation of deoxysugars a new approach to amadori ketoses via mo vicatalyzed stereospecific isomerization of -c-branched sugars bearing azido function in a microwave field substrate specificity and catalytic mechanism of a xyloglucan xyloglucosyl transferase hvxet from barley (hordeum vulgare l.) determination of n-glycosylation site and glycan structures of pectin methylesterase in jelly fig (ficus awkeotsang) achenes the composition of polysaccharides in primary walls of litchi chinensis sonn xyloglucans of monocotyledons have diverse structures the effect of acid dextrinisation on enzyme-resistant starch content in extruded maize starch new oligosaccharides prepared by acid hydrolysis of the polysaccharides from nerium indicum mill and their anti-angiogenesis activities lectin microarray widely applicable deprotection method of , , -trichloroethoxycarbonyl (troc) group using tetrabutylammonium fluoride hydrolytically stable bioactive synthetic glycopeptide homo-and copolymers by combination of nca polymerization and click reaction chemoenzymatic synthesis and lectin array characterization of a class of n-glycan clusters expeditious chemoenzymatic synthesis of cd glycopeptide antigens arthrobacter endo-b-n-acetylglucosaminidase shows transglycosylation activity on complex-type n-glycan oxazolines: one-pot conversion of ribonuclease b to sialylated ribonuclease c glycosynthases enable a highly efficient chemoenzymatic synthesis of n-glycoproteins carrying intact natural n-glycans synthesis of oleanolic acid saponins mimicking components of chinese folk medicine di wu lipid-a software tool for automated assignment of lipids in mass spectra a lipid profile typifies the beijing strains of mycobacterium tuberculosis. identification of a mutation responsible for a modification of the structures of phthiocerol dimycocerosates and phenolic glycolipids igg glycosylation analysis supramolecular assembly of carbohydrate-functionalized salphen-metal complexes synthesis and application of epoxy starch derivatives an echinococcus multilocularis coproantigen is a surface glycoprotein with unique o-gycosylation glycoproteomics in neurodegenerative diseases structural elucidation of a novel b. cenocepacia et- lipooligosaccharide isolated from a cystic fibrosis patient after lung transplantation against the rules: a marine bacterium, loktanella rosea, possesses a unique lipopolysaccharide first structural characterization of burkholderia vietnamiensis lipooligosaccharide from cystic fibrosis-associated lung transplantation strains fucosylation of chitooligosaccharides by human a , -fucosyltransferase requires a nonreducing terminal chitotriose unit as a minimal structure c-mannosylated peptides derived from the thrombospondin type repeat interact with hsc to modulate its signaling in raw . cells two mechanisms of the enhanced antibody-dependent cellular cytotoxicity (adcc) efficacy of non-fucosylated therapeutic antibodies in human blood production of a recombinant mouse monoclonal antibody in transgenic silkworm cocoons homotropic cooperativity of cyclodextrin dimer as an artificial hydrolase glycomimicry: display of the gm sugar epitope on escherichia coli and salmonella enterica sv typhimurium enzymatic conversion of diacetylated analysis of carbohydrates and glycoconjugates & sophoroselipid into acetylated glucoselipid: surface-active properties of novel bolaform biosurfactants preparation and conformational analysis of c-glycosyl b -and b/b -peptides the multiplicity of n-glycan structures of novine milk kda lactophorin (milk glycam- ) synthesis of an octasubstituted galactose zinc(ii) phthalocyanine anomerically glycosylated zinc(ii) naphthalocyanines synthesis of octaglycosylated zinc(ii) phthalocyanines. synthesis sesquiterpene coumarins from ferula gumosa transglycosylation-based fluorescent labeling of -gala series glycolipids by endogalactosylceramidase molecular cloning and expression of a novel cholinephosphotransferase involved in glycoglycerophospholipid biosynthesis of mycoplasma fermentans enzymatic synthesis of mycoplasma fermentans specific glycoglycerophospholipid from , -dipalmitoylglycerol isolation and structural determination of reducing fructooligosaccharides newly produced in stored edible burdock antiallergic potential of oligomannose-coated liposome-entrapped cry j as immunotherapy for japanese cedar pollinosis in mice enzymatic activity and substrate specifcity of recombinant tomato b-galactosidases and development of highly efficient and stereocontrolled o-glycosylation methodologies and its application to the construction of bacterial glycans effects of frozen conditions on stereoselectivity and velocity of o-glycosylation reactions specificity analysis of lectins and antibodies using remodeled glycoproteins production, purification and structural characterization of an exopolysaccharide produced by a probiotic lactobacillus plantarum mtcc in vitro and in vivo enzymatic syntheses and mass spectrometric database for nglycans and o-glycans strategy for glycoproteomics: identification of glyco-alteration using multiple glycan profiling tools enrichment strategies for glycopeptides photooxidative mineralization of microorganisms-produced glycolipid biosurfactants by a titania-mediated advanced oxidation process highly oriented recombinant glycosyltransferases: sitespecific immobilization of unstable membrane proteins by using staphylococcus aureus sortase a a first total synthesis of ganglioside hlg- identification of a glycosylphosphatidylinositol anchormodifying b - n-acetylglucosaminyl transferase in trypanosoma brucei direct profiling of tissue lipids by maldi-tofms glycation sites in neoglycoglycoconjugates from the terminal monosaccharide antigen of the o-ps of vibrio cholerae o , serotype ogawa, and bsa revealed by matrix-assisted laser desorption-ionization tandem mass spectrometry contribution of porphyromonas gingivalis lipopolysaccharide to periodontitis synthesis of new sulfonic acid-containing oligosaccharide mimetics of sialyl lewis a synthesis, regioselective hydrogenolysis, partial hydrogenation, and conformational study of dioxane and dioxolanetype ( -anthracenyl)methylene acetals of sugars mass spectrometric quantification of neutral and sialylated n-glycans from a recombinant therapeutic glycoprotein produced in the two chinese hamster ovary cell lines method development for direct detection of glycoproteins on aminophenylboronic acid functionalized self-assembled monolayers by matrix-assisted laser desorption/ionization mass spectrometry antibody glycans characterization structural characterization of antibodies by mass spectrometry mucin-lectin interactions assessed by flow cytometry mucin-type o-glycosylation-putting the pieces together carboxymethylated cyclosophoraose as a novel chiral additive for the stereoisomeric separation of some flavonoids by capillary electrophoresis characterization of the streptococcus pneumoniae bgac protein as a novel surface b-galactosidase with specific hydrolysis activity for the galb - glcnac moiety of oligosaccharides characterization of n-linked protein glycosylation in helicobacter pullorum a strategy for precise and large scale identification of core fucosylated glycoproteins self-assembly of amphiphilic perylenecyclodextrin conjugate and vapor sensing for organic amines structures of chitosan and chitooligosaccharides and their adsorption toward radionuclide uranium a high-throughput purification of monoclonal antibodies from glycoengineered pichia pastoris a bifunctional enzyme in a single gene catalyzes the incorporation of glcn into the aeromonas core lipopolysaccharide genetics and proteomics of aeromonas salmonicida lipopolysaccharide core biosynthesis selective monitoring of rutin and quercetin based on a novel multi-wall carbon nanotube-coated glassy carbon electrode modified with microbial carbohydrates a-cyclosophorohexadecaose and succinoglycan monomer m utilizing the o-antigen lipopolysaccharide biosynthesis pathway in escherichia coli to interrogate the substrate specificities of exogenous glycosyltransferase genes in a combinatorial approach design, synthesis, and evaluation of n-acyl modified sialic acids as inhibitors of adenoviruses causing epidemic keratoconjunctivitis natural phosphoryl and acyl variants of lipid a from neisseria meningitidis strain i differentially induce tumor necrosis factor-a in human monocytes profiles of structural heterogeneity in native lipooligosaccharides of neisseria and cytokine induction golgi targeting of drosophila melanogaster b galnactb requires a dhhc protein family-related protein as a pilot strong igg antibody responses to borrelia burgdorferi glycolipids in patients with lyme arthritis, a late manifestation of the infection generation and characterization of monoclonal antibody to ginsenoside rg a novel, simple and sensitive ligand affinity capture method for detecting molecular interactions by maldi mass spectrometry glycoviewer: a tool for visual summary and comparative analysis of the glycome mass spectrometric molecular-weight determination of highly acidic compounds of biological significance via their complexes with basic polypeptides enzymatic synthesis of salicin glycosides through transglycosylation catalyzed by amylosucrases from deinococcus geothermalis and neisseria polysaccharea direct analysis of cellulose in poplar stem by matrix-assisted laser desorption/ionization imaging mass spectrometry superior in vivo efficacy of afucosylated trastuzumab in the treatment of her -amplified breast cancer benzyl ethers as nucleophiles: from hydroxy cyclooctanes toward bridged c-furanosides multifunctional zro nanoparticles and zro -sio nanorods for improved maldi-ms analysis of cyclodextrins, peptides, and phosphoproteins a synthetic vaccine consisting of a tumor-associated sialyl-tn-muc tandem-repeat glycopeptide and tetanus toxoid: induction of a strong and highly selective immune response fully synthetic vaccines consisting of tumor-associated muc glycopeptides and a lipopeptide ligand of the toll-like receptor ghrelin-like peptide with fatty acid modification and o-glycosylation in the red stingray, dasyatis akajei synthesis of glycopeptides reactivity of rare sugar d-allose during glycation of human serum albumin arabidopsis thaliana alg mutant synthesizes immature oligosaccharides in the er and accumulates unique n-glycans two arabidopsis thaliana golgi a-mannosidase i enzymes are responsible for plant n-glycan maturation a sensitive liquid chromatography/mass spectrometry-based assay for quantitation of amino-containing moieties in lipid a selfpoisoning of mycobacterium tuberculosis by targeting glge in an aglucan pathway definitive evidence that a single n-glycan among three glycans on inducible costimulator is required for proper protein trafficking and ligand binding aglj adds the first sugar of the nlinked pentasaccharide decorating the haloferax volcanii s-layer glycoprotein aba-and bab-triblock cooligomers of tri-o-methylated and unmodified cello-oligosaccharides: syntheses and structure-solubility relationship lectin-based enrichment method for glycoproteomics using hollow fiber flow field-flow fractionation: application to streptococcus pyogenes synthesis and characterization of hydroquinone fructoside using leuconostoc mesenteroides levansucrase high-throughput solid-phase permethylation of glycans prior to mass spectrometry solid-phase permethylation of glycans for mass spectrometric analysis antibacterial activity of a disaccharide isolated from streptomyces sp. strain jj against xanthomonas sp insight into the regulation of glycan synthesis in drosophila chaoptin based on mass spectrometry a tandem mass spectrometric approach to the identification of o-glycosylated glargine glycoforms in active pharmaceutical ingredient expressed in pichia pastoris mass spectrometric analysis of carbohydrates labeled with a biotinylated tag localization of the attachment site of oligoglucans to mesorhizobium loti hambi murein synthesis of a heptasaccharide fragment of the mannan from candida guilliermondii cell wall and its conjugate with bsa synthesis of , -branched oligomannoside fragments of the mannan from candida albicans cell wall corresponding to the antigenic factor reduction of n-linked xylose and fucose by expression of rat b , -n-acetylglucosaminyltransferase iii in tobacco by- cells depends on golgi enzyme localization domain and genetic elements used for expression a small-scale method for the preparation of plant n-linked glycans from soluble proteins for analysis by maldi-tof mass spectrometry analyses of biologically active steroids: antitumor active osw- and cardiotonic marinobufotoxin, by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight tandem mass spectrometry structural analogues of diosgenyl saponins: synthesis and anticancer activity pyruvoyl, a novel amino protecting group on the solid phase peptide synthesis and the peptide condensation reaction chemical synthesis of mouse pro-opiomelanocortin( - ) by azido-protected glycopeptide ligation via the thioester method correct disulfide pairing is required for the biological activity of crustacean androgenic gland hormone (agh): synthetic studies of agh loss of udp-galnac: polypeptide n-acetylgalactosaminyltransferase and reduced oglycosylation in colon carcinoma cells selected for hepatic metastasis development of tetraphenylethylene-based fluorescent oligosaccharide probes for detection of influenza virus further structural study of the xyloglucanase-derived eggplant xyloglucan oligo-saccharides initiation of methylglucose lipopolysaccharide biosynthesis in mycobacteria synthesis of a chito-tetrasaccharide b- , -glcnac-b- , -glcn repeating unit a novel polycondensation method for the synthesis of a two-faced b- , -glucan solid-phase synthesis of o-sulfated glycopeptide by the benzylprotected glycan strategy salmonella enterica serovar typhimurium lipopolysaccharide deacylation enhances its intracellular growth within macrophages influence of crystalline forms of titania on desorption/ionization efficiency in titania-based surfaceassisted laser desorption/ionization mass spectrometry alternative procedures for analysis of lipid a modification with phosphoethanolamine or aminoarabinose recognition of endogenous ligands by ctype lectins: interaction of serum mannan-binding protein with tumorassociated oligosaccharide epitopes highly fucosylated n-glycan ligands for mannan-binding protein expressed specifically on cd (dppvi) isolated from a human colorectal carcinoma cell line structural analysis of two trisaccharides isolated from fermented beverage of plant extract phosphorylase-catalyzed n-formyl-a-glucosaminylation of maltooligosaccharides gold-catalyzed glycosidations: unusual cleavage of the interglycosidic bond while studying the armed/ disarmed effect of propargyl glycosides dendrimers of vaccines consisting of tumor-associated glycopeptide antigens and t cell epitope peptides bacillus anthracis surfacelayer proteins assemble by binding to the secondary cell wall polysaccharide in a manner that requires csab and tago sialic acids acquired by pseudomonas aeruginosa are involved in reduced complement deposition and siglec mediated hostcell recognition mass spectrometric analysis of sulfated n-and oglycans steroidal monoglycosides from the far eastern starfish hippasteria kurilensis and hypothetic pathways of polyhydroxysteroid biosynthesis in starfish two new steroid glycosides from the far east starfish hippasteria kurilensis lectin biosensing using digital analysis of ru(ii)-glycodendrimers ru(ii) glycodendrimers as probes to study lectin-carbohydrate interactions and electrochemically measure monosaccharide and oligosaccharide concentrations enzymatic synthesis and characterization of hydroquinone galactoside using kluyveromyces lactis lactase click synthesis of estradiol-cyclodextrin conjugates as cell compartment selective estrogens grandidentatin isomer from bark of suwon poplar (populus alba l.  populus glandulosa uyeki) biotransformation of mulberroside a from morus alba results in enhancement of tyrosinase inhibition glycosylation pattern of humanized igg-like bispecific antibody produced by recombinant cho cells mass spectrometric analysis of the glycosphingolipid-derived glycans from miniature pig endothelial cells and islets: identification of neugc epitope in pig islets rapid and high-throughput analysis of n-glycans from ovarian cancer serum using a -well plate platform identification of a-gal and non-gal epitopes in pig corneal endothelial cells and keratocytes by using mass spectrometry qualitative and quantitative comparison of n-glycans between pig endothelial and islet cells by highperformance liquid chromatography and mass spectrometry-based strategy construction of a fusion enzyme of dextransucrase and dextranase: application for onestep synthesis of isomalto-oligosaccharides enzymatic synthesis of alkyl glucosides using leuconostoc mesenteroides dextransucrase alteration in the glycan pattern of pilin in a nonmotile mutant of synechocystis sp. pcc suppression of b-n-acetylglucosaminidase in the n-glycosylation pathway for complex glycoprotein formation in drosophila s cells carbohydrate moieties contribute significantly to the physicochemical properties of french bean s globulin phaseolin structural characterization of multibranched oligosaccharides from seal milk by a combination of off-line high-performance liquid chromatographymatrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and sequential exoglycosidase digestion phenylethyl glycosides from digitalis lanata acceptor and substrate specificity of b-glucuronidase with transglycosylation activity from aspergillus niger kinetic study on the binding of lectin to mannose residues in a polymer brush high molecular weight polyglycerol-based multivalent mannose conjugates -methyl and -xylosyl derivatives of -hydroxyflexixanthin are major carotenoids of hymenobacter species modifications of human total serum n-glycome during liver fibrosis-cirrhosis, is it all about immunoglobulins? immunoglobulins are the major glycoproteins involved in the modifications of total serum n-glycome in cirrhotic patients enrichment of o-glcnac modified proteins by the periodate oxidation-hydrazide resin capture approach surface grafting of cellulose nanocrystals with poly(ethylene oxide) in aqueous media a quantitative model of ultraviolet matrix-assisted laser desorption/ionization a quantitative model of ultraviolet matrix-assisted laser desorption/ionization including analyte ion generation a bipolar rate equation model of maldi primary and secondary ionization processes, with application to positive/ negative analyte ion ratios and suppression effects electrospray and maldi mass spectrometry: fundamentals, instrumemtation, practialities and biological applications molecular dynamics simulations of maldi: laser fluence and pulse width dependence of plume characteristics and consequences for matrix and analyte ionization enzymatic polymer synthesis: an opportunity for green polymer chemistry synthetic studies on the carbohydrate moiety of the antigen from the parasite echinococcus multilocularis mass spectrometry for the analysis of milk oligosaccharides amphiphilic block copolymers based on cyclodextrin host-guest complexes via raftpolymerization in aqueous solution synthesis and conformational analysis of (a-d-galactosyl)phenylmethane and a-,b-difluoromethane analogues: interactions with the plant lectin viscumin structures of aldouronic acids liberated from kenaf xylan by endoxylanases from streptomyces sp study on systematizing the synthesis of the a-series ganglioside glycans gt a, gd a, and gm using the newly developed n-troc-protected gm and galn intermediates characterization of sialylated and fucosylated glycopeptides of b -glycoprotein i by a combination of hilic lc and maldi ms/ms glycopeptide profiling of beta- -glycoprotein i by mass spectrometry reveals attenuated sialylation in patients with antiphospholipid syndrome application of deep sea yeast-production of biosurfactants biosurfactant-producing yeast isolated from calyptogena soyoae (deep-sea cold-seep clam) in the deep sea an arginyl residue in rice udp-arabinopyranose mutase is required for catalytic activity and autoglycosylation structural identification of the main ellagitannins of a boysenberry (rubus loganbaccus  baileyanus britt.) extract by lc-esi-ms/ms, maldi-tof-ms and nmr spectroscopy quantification and characterization of enzymatically produced hyaluronan with fluorophore-assisted carbohydrate electrophoresis block synthesis of blood group tetrasaccharides b (types , and ) advances in the separation, sensitive detection, and characterization of heparin and heparan sulfate glucosinolateaccumulating s-cells in arabidopsis leaves and flower stalks undergo programmed cell death at early stages of differentiation polysaccharide microarrays for high-throughput screening of transglycosylase activities in plant extracts synthesis of a neoglycoconjugate containing a chlamydophila psittaci-specific branched kdo trisaccharide epitope the mita gene of aspergillus fumigatus is required for mannosylation of inositol-phosphorylceramide, but is dispensable for pathogenicity sulfated oligosaccharides: new targets for drug development stability of ( )-carboxyfluorescein in microwave-assisted synthesis of fluoresceinlabelled o-dimannosylated peptides triterpene glycosides from agrostemma gracilis porphyrin-cyclodextrin conjugates as a nanosystem for versatile drug delivery and multimodal cancer therapy a mathematical model of n-linked glycosylation a mathematical model to derive n-glycan structures and cellular enzyme activities from mass spectrometric data glycosyl azides: novel substrates for enzymatic transglycosylations multidimensional system enabling deglycosylation of proteins using a capillary reactor with peptide-nglycosidase f immobilized on a porous polymer monolith and hydrophilic interaction liquid chromatography-mass spectrometry of glycans glycomic analysis: an array of technologies synthesis of an fmoc-threonine bearing core- glycan: a building block for psgl- via fmoc-assisted solid-phase peptide synthesis the development of retrosynthetic glycan libraries to profile and classify the human serum n-linked glycome human serum processing and analysis methods for rapid and reproducible n-glycan mass profiling shigella sonnei oligosaccharide-protein conjugates immunochemical studies of shigella flexneri a and , and shigella dysenteriae type o-specific polysaccharide-core fragments and their protein conjugates as vaccine candidates a,a-trehalose-based polyacetals and macrocyclic acetals application of spectroscopic methods for structural analysis of chitin and chitosan the molecular basis of inhibition of golgi a-mannosidase ii by mannostatin a glycomics and proteomics analyses of mouse uterine luminal fluid revealed a predominance of lewis y and x epitopes on specific protein carriers productionof sophorolipid biosurfactants by multiple species of the starmerella (candida) bombicola yeast clade four new triterpenes from anchusa azurea var. azurea sialyltransferases of marine bacteria efficiently utilize glycosphingolipid substrates dethreading of deoxyribonucleotides through a-cyclodextrin efficient guest inclusion by b-cyclodextrin attached to the ends of dna oligomers upon hybridization to various dna conjugates determination of sialic acid and gangliosides in biological samples and dairy products: a review solid-phase thermodynamic interpretation of ion desorption in matrix-assisted laser desorption/ionization controlled release of , -desulfated heparin exerts its anti-inflammatory activity by effectively inhibiting e-selectin deletion of udp-glucose pyrophosphorylase reveals a udp-glucose independent udp-galactose salvage pathway in leishmania major imaging of lipids in spinal cord using intermediate pressure matrix-assisted laser desorption-linear ion trap/orbitrap ms utilization of the linear mode of maldi-tof mass spectrometry in the study of glycation during the malting process profiling of n-linked oligosaccharides using phenylhydrazine derivatization and mass spectrometry labelling saccharides with phenylhydrazine for electrospray and matrix-assisted laser desorption-ionization mass spectrometry matrix-assisted laser desorption/ ionization tandem mass spectrometry and post-source decay fragmentation study of phenylhydrazones of n-linked oligosaccharides from ovalbumin method for investigation of oligosaccharides using phenylhydrazine derivatization n-glycomic changes in human breast carcinoma mcf- and t-lymphoblastoid cells after treatment with herceptin and herceptin/lipoplex mass spectrometric study of n-glycans from serum of woodchucks with liver cancer chemical characterization, antiproliferative and antiadhesive properties of polysaccharides extracted from pleurotus pulmonarius mycelium and fruiting bodies polymerizable vancomycin derivatives for bactericidal biomaterial surface modification: structure-function evaluation the chap domain of cse functions as an endopeptidase that acts at mature septa to promote streptococcus thermophilus cell separation biorecognition of chemically modified bovine serum albumin with lactose prepared under different conditions conjugates of bovine serum albumin with chitin oligosaccharides prepared through the maillard reaction characterization of -a-glucanotransferase from synechocystis sp. pcc and its application to various corn starches effects of differential glycosylation of glycodelins on lymphocyte survival targeted molecular imaging of vegf receptors overexpressed in ischemic microvasculature using chitosan-dc conjugates the poplar gt e and gt f glycosyltransferases are functional orthologs of arabidopsis parvus involved in glucuronoxylan biosynthesis the f h glycosyltransferase is a functional paralog of fra involved in glucuronoxylan biosynthesis in arabidopsis down-regulation of pogt c expression in poplar results in a reduced glucuronoxylan content and an increased wood digestibility by cellulase the arabidopsis family gt glycosyltransferases form two functionally nonredundant groups essential for the elongation of glucuronoxylan backbone glycosylated neurotensin analogues exhibit sub-picomolar anticonvulsant potency in a pharmacoresistant model of epilepsy techniques and tactics used in determining the structure of the trimeric ebolavirus glycoprotein isolation and tandem mass fragmentations of an anti-inflammatory compound from aralia elata high-throughput quantitative analysis of plant nglycan using a dna sequencer synthesis of selenium nanowires morphologically directed by shinorhizobial oligosaccharides core o-glycan synthase suppresses tumor formation and metastasis of prostate carcinoma pc and lncap cells through down-regulation of a b integrin complex core o-glycan structure is essential for the cell surface expression of sucrase isomaltase and dipeptidyl peptidase-iv during intestinal cell differentiation synthesis and cellular uptake properties of guanidine-containing molecular transporters built on the sucrose scaffold tracing the development of structural elucidation of nglycans synthesis of undecaprenyl pyrophosphate-linked glycans as donor substrates for bacterial protein nglycosylation deficiency of dol-p-man synthase subunit dpm bridges the congenital disorders of glycosylation with the dystroglycanopathies n-glycosylation patterns of hemolymph glycoproteins from biomphalaria glabrata strains expressing different susceptibility to schistosoma mansoni infection multivalent binding of carbohydrates by the human a-defensin, hd structural analysis of sulfated glycans by sequential double-permethylation using methyl iodide and deuteromethyl iodide sequential enrichment of sulfated glycans by strong anion-exchange chromatography prior to mass spectrometric measurements a new allergen from ragweed (ambrosia artemisiifolia) with homology to art v from mugwort an unusual galactofuranose lipopolysaccharide that ensures the intracellular survival of toxin-producing bacteria in their fungal host human l-selectin preferentially binds synthetic glycosulfopeptides modeled after endoglycan and containing tyrosine sulfate residues and sialyl lewis x in core o-glycans efficient synthesis and protein conjugation of b-( - )-d-n-acetylglucosamine oligosaccharides from the polysaccharide intercellular adhesin steroid compounds from two pacific starfish of the genus evasterias n-glycosylation site analysis of human platelet proteins by hydrazide affinity capturing and lc-ms/ ms phosphoethanolamine substitution of lipid a and resistance of neisseria gonorrhoeae to cationic antimicrobial peptides and complement-mediated killing by normal human serum infrared multiphoton dissociation mass spectrometry for structural elucidation of oligosaccharides collision-induced dissociation tandem mass spectrometry for structural elucidation of glycans pancreatic cancer serum detection using a lectin/ glyco-antibody array method structure of pleiotrophin-and hepatocyte growth factor-binding sulfated hexasaccharide determined by biochemical and computational approaches overexpression and topology of bacterial oligosaccharyltransferase pglb effects of hapten density on the induced antibody repertoire advances in the glycosylation of milk protein light-switchable janus [ ] rotaxanes based on a-cyclodextrin derivatives bearing two recognition sites linked with oligo(ethylene glycol) maldi-tof-ms analysis of small molecules using modified mesoporous material sba- as assisted matrix preparation of homogenous oligosaccharide chains from glycosphingolipids mass spectrometry of fluorocarbon-labeled glycosphingolipids gold(i)-catalyzed glycosylation with glycosyl ortho-alkynylbenzoates as donors: general scope and application in the synthesis of a cyclic triterpene saponin immunologic glycosphingolipidomics and nkt cell development in mouse thymus comparison of sample pre-treatments for laser desorption ionization and secondary ion mass spectrometry imaging of miscanthus  giganteus glycan array: a powerful tool for glycomics studies switching of the core structures of glycosphingolipids from globo-and lacto-to ganglio-series upon human embryonic stem cell differentiation the effect of multivalent binding on the lateral phase separation of adhesive lipids -n, -o-carbonyl- , , -tri-o-chloroacetyl-protected sialyl donor for the stereoselective synthesis of a-( ! )-tetrasialic acid enhanced expression of b -galactosyltransferase activity is sufficient to induce in vivo synthesis of extended type chains on lactosylceramides of selected human colonic carcinoma cell lines a new naphthimidazole derivative for saccharide labeling with enhanced sensitivity in mass spectrometry detection i -catalyzed oxidative condensation of aldoses with diamines: synthesis of aldo-naphthimidazoles for carbohydrate analysis in vivo protection provided by a synthetic new alpha-galactosyl ceramide analog against bacterial and viral infections in murine models fabrication of oriented antibody-conjugated magnetic nanoprobes and their immunoaffinity application characterization of alginate-like exopolysaccharides isolated from aerobic granular sludge in pilot-plant synthesis of magnetic nanoparticles with immobilized aminophenylboronic acid for selective capture of glycoproteins the grignard reaction of cyclodextrin- -aldehydes revisited: a study of the stereoselectivity upon addition of organometallic reagents to aldehydes and ketones a bivalent glycopeptide to target two putative carbohydrate binding sites on fimh en route to photoaffinity labeling of the bacterial lectin fimh muropeptide rescue in bacillus subtilis involves sequential hydrolysis by b-n-acetylglucosaminidase and n-acetylmuramyl-lalanine amidase incoherent production reactions of positive and negative ions in matrix-assisted laser desorption/ionization initial ionization reaction in matrix-assisted laser desorption/ionization isolation and identification of two novel sds-resistant secreted chitinases from aeromonas schubertii norclerodane diterpenoids from rhizomes of dioscorea bulbifera an apple oligogalactan prevents against inflammation and carcinogenesis by targeting lps/tlr /nf-kb pathway in a mouse model of colitis-associated colon cancer phosphoryl moieties of lipid a from neisseria meningitidis and n. gonorrhoeae lipooligosaccharides play an important role in activation of both myd -and trif-dependent tlr -md- signaling pathways efficient synthesis of flaccidoside ii, a bioactive component of chinese folk medicine di wu concise synthesis of two natural triterpenoid saponins, oleanolic acid derivatives isolated from the roots of pulsatilla chinensis ionic liquids in sample preparation albizosides d and e, two new cytotoxic triterpene saponins from albizia chinensis cytotoxic oleanane triterpene saponins from albizia chinensis electrospray ionization mass spectrometry as a critical tool for revealing new properties of snake venom phospholipase a f-labeled galacto and pegylated rgd dimers for pet imaging of a v b integrin expression alteration of n-glycome in diethylnitrosamine-induced hepatocellular carcinoma mice: a noninvasive monitoring tool for liver cancer is permethylation strategy always applicable to protein n-glycosylation study? a case study on the o-acetylation of sialic acid in fish serum glycans microwave-assisted kochetkov amination followed by permanent charge derivatization: a facile strategy for glycomics methylamidation for sialoglycomics by maldi-ms: a facile derivatization strategy for both a , -and a , -linked sialic acids investigation of sample preparation artifacts formed during the enzymatic release of n-linked glycans prior to analysis by capillary electrophoresis supramolecular assembly of perylene bisimide with b-cyclodextrin grafts as a solid-state fluorescence sensor for vapor detection elevation of sulfatides in ovarian cancer: an integrated transcriptomic and lipidomic analysis including tissue-imaging mass spectrometry tandem o stable isotope labeling for quantification of n-glycoproteome plant-expressed recombinant mountain cedar allergen jun a is allergenic and has limited pectate lyase activity modular approach to triazole-linked , -a-d-oligomannosides to the discovery of inhibitors of mycobacterium tuberculosis cell wall synthetase glycosylation of b subunits regulates gaba a receptor biogenesis and channel gating lipidomic analysis of porcine olfactory epithelial membranes and cilia a versatile and scalable strategy for glycoprofiling bifidobacterial consumption of human milk oligosaccharides the egmur xyloglucan galactosyltransferase from eucalyptus grandis complements the mur cell wall phenotype in arabidopsis thaliana morphological, toxicological and molecular characterization of a benthic nodularia isolated from atlantic estuarine environments enthalpic studies of xyloglucan-cellulose interactions titania microparticles and nanoparticles as matrixes for in vitro and in situ analysis of small molecules by maldi-ms reactivity-based onepot synthesis of immunosuppressive glycolipids from the caribbean sponge plakortis simplex chemical synthesis of a hyaluronic acid decasaccharide a yeast glycoprotein shows highaffinity binding to the broadly neutralizing human immunodeficiency virus antibody g and inhibits gp interactions with g and dc-sign fatal outcome due to deficiency of subunit of the conserved oligomeric golgi complex leading to a new type of congenital disorders of glycosylation two kdo-heptose regions identified in hafnia alvei lipopolysaccharide: the complete core structure and serological screening of different hafnia o serotypes structural analysis of the lipid a isolated from hafnia alvei and pcm lipopolysaccharides functional l-lysine dendritic macromolecules as liver-imaging probes structural characterization of n-linked oligosaccharides of defibrase from agikistrodon acutus by sequential exoglycosidase digestion and maldi-tof mass spectrometry studying a cell division amidase using defined peptidoglycan substrates data mining the pdb for glyco-related data facile preparation of fluorescent neoglycoproteins using p-nitrophenyl anthranilate as a heterobifunctional linker synthesis of the pentasaccharide repeating unit of escherichia coli o antigen a b-d-allopyranoside-grafted ru(ii) complex: synthesis and acid-base and dna-binding properties new phosphane based on a b-cyclodextrin, exhibiting a solventtunable conformation, and its catalytic properties structural analysis of the o-specific polysaccharide isolated from plesiomonas shigelloides o lipopolysaccharide the conformational properties of the glc man unit suggest conformational biasing within the chaperone-assisted glycoprotein folding pathway euscaphinin, a new ellagitannin dimer from euscaphis japonica (thunb.) kanitz study of lysozyme glycation reaction by mass spectrometry and nmr spectroscopy fut -null mice display an altered glycosylation profile and impaired babamediated helicobacter pylori adhesion to gastric mucosa aglp is a s-adenosyl-l-methionine-dependent methyltransferase that participates in the n-glycosylation pathway of haloferax volcanii manipulation of electrostatic and saccharide linker interactions in the design of efficient glycopolypeptide-based cholera toxin inhibitors chemical synthesis and proinflammatory responses of monophosphoryl lipid a adjuvant candidates glucooligosaccharides production from glucan of leuconostoc mesenteroides nrrl b- by microwave assisted hydrolysis isorhizochalin: a minor unprecedented bipolar sphingolipid of stereodivergent biogenesis from the rhizochalina incrustata development of continuous flow type hydrothermal reactor for hemicellulose fraction recovery from corncob phenolic compounds from bursera simaruba sarg. bark: phytochemical investigation and quantitative analysis by tandem mass spectrometry iodine-hexamethyldisilane (hmds)-mediated anomerization of peracetylated , -trans-linked alkyl and aryl glycosides a simple, mild, and regioselective method for the benzylation of carbohydrate derivatives promoted by silver carbonate efficient synthesis of a fluorescent tripod detection system for pesticides by microwaveassisted click chemistry highly efficient deletion of fut in cho cell lines using zinc-finger nucleases yields cells that produce completely nonfucosylated antibodies rapid de-o-glycosylation concomitant with peptide labeling using microwave radiation and an alkyl amine base dissection of host cell signal transduction during acinetobacter baumannii-triggered inflammatory response consumption of human milk oligosaccharides by gutrelated microbes analysis of protein posttranslational modifications using mass spectrometry interactions of lipopolysaccharide and polymyxin studied by nmr spectroscopy amino-acetone-bridged cyclodextrins-artificial alcohol oxidases a systematic approach to protein glycosylation analysis: a path through the maze flavonoids with prolyl oligopeptidase inhibitory activity isolated from scutellaria racemosa pers glyconanoparticles: polyvalent tools to study carbohydrate-based interactions glycosylation protects proteins against free radicals generated from toxic xenobiotics gold manno-glyconanoparticles: multivalent systems to block hiv- gp binding to the lectin dc-sign antithrombin murcia (k e) causing antithrombin deficiency: a possible role for altered glycosylation a series of -o-trifluoromethylsulfonyl-dmannopyranosides as precursors for concomitant f-labeling and glycosylation by click chemistry synthesis and delivery activity of new cationic cholesteryl glucosides mutational deglycosylation of the fc portion of immunoglobulin g causes osulfation of tyrosine adjacently preceding the originally glycosylated site biochemical and immunological characterization of the plantderived candidate human immunodeficiency virus type mucosal vaccine ctb-mpr - supported molecular matrix electrophoresis: a new tool for characterization of glycoproteins development of an apparatus for rapid release of oligosaccharides at the glycosaminoglycan-protein linkage region in chondroitin sulfate-type proteoglycans synthesis of sialyllactosamine clusters using carbosilane as core scaffolds by means of chemical and enzymatic approaches novel synthesis of functional mucin glycopeptides containing both n-and o-glycans functional neoglycopeptides: synthesis and characterization of a new class of muc glycoprotein models having core -based o-glycan and complex-type n-glycan chains a tethering mechanism for length control in a processive carbohydrate polymerization structural analysis of arabinoxylans isolated from ball-milled switchgrass biomass extracellular monoenzyme deglycosylation system of -o-linked flavonoid brutinosides and its disaccharide transglycosylation activity from stilbella fimetaria biodistribution and excretion of monosaccharidealbumin conjugates measured with in vivo near-infrared fluorescence imaging glycoform and glycologue: two software applications for the rapid construction and display of n-glycans from mammalian sources site-directed mutagenesis to probe catalysis by a thermobifida fusca b- , -glucanase (lam a) the mass-mobility correlation redux: the conformational landscape of anhydrous biomolecules glycomic analysis by capillary electrophoresis-mass spectrometry solid-phase permethylation for glycomic analysis high-sensitivity analytical approaches to the analysis of n-glycans quantitative serum glycomics of esophageal adenocarcinoma and other esophageal disease onsets the effect of glycation on foam and structural properties of b-lactoglobulin structural characterization of glycans on omega- , a major schistosoma mansoni egg glycoprotein that drives th responses proteomic scale high-sensitivity analyses of gpi membrane anchors glycotyping of trypanosoma brucei variant surface glycoprotein mitat studies on the boronation of methyl-b-d-cellobioside-a cellulose model assessment of heat treatment of dairy products by synthesis of tetraglucosyl-and tetrapolyamine-tetrabenzoporphyrin conjugates for an application in pdt synthesis of an fmoc-asn-heptasaccharide building block and its application to chemoenzymatic glycopeptide synthesis fluorescent bodipylabelled gm gangliosides designed for exploring lipid membrane properties and specific membrane-target interactions helicobacter pylori binding to new glycans based on n-acetyllactosamine transcriptomic analysis of arabidopsis developing stems: a close-up on cell wall genes glycoforms of human endothelial cd that bind l-selectin carry sulfated sialyl lewis x capped o-and n-glycans chemical structure analysis of starch and cellulose derivatives genetic and structural characterization of l lipooligosaccharide from neisseria meningitidis serogroup a strains the n-glycolyl form of mouse sialyl lewis x is recognized by selectins but not by heca- and fh antibodies that were raised against human cells -(d-glucopyranosyl- -deoxy- -iminomethyl)- -hydroxybenzene as chemosensor for aromatic amino acids by switch-on fluorescence ultrahighly sensitive in situ metabolomic imaging for visualizing spatiotemporal metabolic behaviors rapid and simple solid-phase esterification of sialic acid residues for quantitative glycomics by mass spectrometry glycoblotting-assisted o-glycomics: ammonium carbamate allows for highly efficient o-glycan release from glycoproteins synthesis and absolute structures of mycoplasma pneumoniae bglyceroglycolipid antigens multivalent galacto-trehaloses: design, synthesis, and biological evaluation under the concept of carbohydrate modules promiscuous activity of er glucosidase ii discovered through donor specificity analysis of uggt novel rhamnosyltransferase involved in biosynthesis of serovar -specific glycopeptidolipid from mycobacterium avium complex systematic synthesis and inhibitory activity of haloacetamidyl oligosaccharide derivatives toward cytoplasmic peptide: n-glycanase structural and functional characterization of recombinant human serum transferrin secreted from pichia pastoris the cytotoxic activity of linum grandiflorum leaves new acylated flavone and cyanogenic glycosides from linum grandiflorum synthesis of novel gluco-and galacto-functionalized platinum complexes design of triazoletethered glycoclusters exhibiting three different spatial arrangements and comparative study of their affinities towards pa-il and rca by using a dna-based glycoarray capsule polysaccharide conjugate vaccine against diarrheal disease caused by campylobacter jejuni a-type crystals from dilute solutions of short amylose chains the n-glycosylation of classical swine fever virus e glycoprotein extracellular domain expressed in the milk of goat n-glycosylation pattern of e glycoprotein from classical swine fever virus glycation pattern of peptides condensed with maltose, lactose and glucose determined by ultraviolet matrix-assisted laser desorption/ionization tandem mass spectrometry regulated and aberrant glycosylation modulate cardiac electrical signaling synthesis of hyperbranched b-galceramide-containing dendritic polymers that bind hiv- rgp in vivo biocompatibility and biodegradability of dextrin-based hydrogels analysis of glycosylation and other post-translational modifications by mass spectrometry analysis of n-and olinked glycans from glycoproteins using maldi-tof mass spectrometry glycosylation pattern of brush borderassociated glycoproteins in enterocyte-like cells: involvement of complex-type n-glycans in apical trafficking anthocyanin components and mechanism for color development in blue veronica flowers biochemical characterization of plasma-derived tissue factor pathway inhibitor: post-translational modification of free, full-length form with particular reference to the sugar chain isolation of basidiomycetous yeast pseudozyma tsukubaensis and production of glycolipid biosurfactant, a diastereomer type of mannosylerythritol lipid-b production of glycolipid biosurfactants, mannosylerythritol lipids, by a smut fungus, nbrc production of a novel glycolipid biosurfactant, mannosylmannitol lipid, by pseudozyma parantarctica and its interfacial properties coupling of planar chromatography to mass spectrometry selective binding of rnase b glycoforms by polydopamine-immobilized concanavalin a glycosylated cu/zn-superoxide dismutase from kluyveromyces yeast, determined by mass spectrometry chemoenzymatic synthesis of conjugatable oligosialic acids analysis of recombinant cd glycans by maldi-tof-ms reveals prevalence of sialyl-t antigen separation and identification of oligosaccharides labeled with -amino- -ethylcarbazole using high performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry o-acetylation of peptidoglycan in gramnegative bacteria. identification and characterization of peptidoglycan o-acetyltransferase in neisseria gonorrhoeae identification of the n-glycosylation sites on recombinant bovine cd expressed in pichia pastoris: their impact on enzyme stability and catalytic activity efficient synthesis of -amino- -deoxy-l-arabinose and spacerequipped -amino- -deoxy-l-arabinopyranosides by transglycosylation reactions molecular weight determination of high molecular mass (glyco)proteins using cge-on-a-chip study of inclusion complexes of cycloamylose with surfactants by isothermal titration calorimetry analysis of n-and o-linked protein glycosylation in children with prader-willi syndrome supramolecular nanocycles comprising b-cyclodextrin-click-ferrocene units: rings of rings of rings unique cleavage of -acetamido- -deoxy-dglucose from the reducing end of biantennary complex type oligosaccharides efficient and systematic synthesis of a small glycoconjugate library having human complex type oligosaccharides imaging mass spectrometry: principle and application application of thin-layer chromatography/infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry to structural analysis of bacteria-binding glycosphingolipids selected by affinity detection chemoenzymatic synthesis of a new class of macrocyclic oligosaccharides advances on the compositional analysis of glycosphingolipids combining thin-layer chromatography with mass spectrometry shiga toxins, glycosphingolipid diversity, and endothelial cell injury heparin/heparan sulfate -o-sulfatase from flavobacterium heparinum. integrated structural and biochemical investigation of enzyme active site and substrate specificity heparin/heparan sulfate n-sulfamidase from flavobacterium heparinum. structural and biochemical investigation of catalytic nitrogen-sulfur bond cleavage utilizing ion-pairing hydrophilic interaction chromatography solid phase extraction for efficient glycopeptide enrichment in glycoproteomics anti-influenza virus activity and structure-activity relationship of aglycoristocetin derivatives with cyclobutenedione carrying hydrophobic chains structural and functional glycosphingolipidomics by glycoblotting with an aminooxy-functionalized gold nanoparticle synthesis of urea tethered glycosylated amino acids and glycopeptides mediated by dppa employing na-fmoc-asp/glu- -oxazolidinones toward fully synthetic homogeneous b-human follicle-stimulating hormone (b-hfsh) with a biantennary n-linked dodecasaccharide. synthesis of b-hfsh with chitobiose units at the natural linkage sites experiments on the synthesis of carotenoid glycosides glycomic analyses of glycoproteins in bile and serum during rat hepatocarcinogenesis transferrin receptordependent cytotoxicity of artemisinin-transferrin conjugates on prostate cancer cells and induction of apoptosis synthesis, structural analysis and application of novel acarbose fructoside using levansucrase extraction and characterization of native heteroxylans from delignified corn stover and aspen a strategy for discovery of cancer glyco-biomarkers in serum using newly developed technologies for glycoproteomics effect of glycosylation on cis/trans isomerization of prolines in iga -hinge peptide proteomic approaches to study structure, functions and toxicity of legume seeds lectins. perspectives for the assessment of food quality and safety engineering host cell lines to reduce terminal sialylation of secreted antibodies purification and partial characterization of cu/ zn superoxide dismutase from kluyveromyces marxianus yeast experimental evidence of chemical exchange over the b( ! ) glycosidic linkage and hydrogen bonding involving hydroxy protons in hyaluronan oligosaccharides by nmr spectroscopy synthesis of amino-bridged oligosaccharide mimetics production of chitin oligosaccharides with different molecular weights and their antioxidant effect in raw . cells genetic analysis of genes involved in synthesis of modified -amino- , -dideoxyglucose in flagellin of pseudomonas syringae pv. tabaci identification of a naturally-occurring -[a-d-glucopyranosyl-( - )-b-d-glucopyranosyl] daidzein from cultivated kudzu root novel rhamnolipid biosurfactants produced by a polycyclic aromatic hydrocarbon-degrading bacterium pseudomonas aeruginosa strain ny novel o-antigen of hafnia alvei pcm lipopolysaccharide with a teichoic acid-like structure structures of two novel, serologically nonrelated core oligosaccharides of yokenella regensburgei lipopolysaccharides differing only by a single hexose substitution syntheses and characterisation of novel cyclodextrin vinyl derivatives from cyclodextrin-nitrophenol-derivatives use of b-cyclodextrins to control the structure of water-soluble copolymers with hydrophobic parts facile synthesis of b-cyclodextrin-dextran polymers by "click" chemistry fullerene sugar balls gold nanoparticle arrangement on viral particles through carbohydrate recognition: a non-cross-linking approach to optical virus detection enrichment of glycopeptides for glycan structure and attachment site identification characterization of the quantitative relationship between signalto-noise (s/n) ratio and sample amount on-target by maldi-tof ms: determination of chondroitin sulfate subsequent to enzymatic digestion differently complex oligosaccharides can be easily identified by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry directly from a standard thin-layer chromatography plate identification of o-glycosylated decapeptides within the muc repeat domain as potential mhc class i (a ) binding epitopes mass spectrometric methods for analysis of oligosaccharides in human milk preparation and characterization of branched bcyclodextrins having a-l-fucopyranose and a study of their functions reverse thin layer method for enhanced ion yield of oligosaccharides in matrix-assisted laser desorption/ionization assessing the cluster glycoside effect during the binding of concanavalin a to mannosylated artificial lipid rafts systematic screens of a candida albicans homozygous deletion library decouple morphogenetic switching and pathogenicity onresin click-glycoconjugation of peptoids mass spectrometry in the analysis of n-linked and o-linked glycans glycomics profiling of chinese hamster ovary cell glycosylation mutants reveals n-glycans of a novel size and complexity mass spectrometric analysis of mutant mice characterization of the novel human age hn cell line for production of recombinant proteins production and characterization of monoclonal antibodies specific to lactotriaosylceramide repairing faulty genes by aminoglycosides: development of new derivatives of geneticin (g ) with enhanced suppression of diseases-causing nonsense mutations development of novel aminoglycoside (nb ) with reduced toxicity and enhanced suppression of disease-causing premature stop mutations enhanced detection and identification of glycopeptides in negative ion mode mass spectrometry structural characterization of an oligosaccharide made by neisseria sicca electrospray and maldi mass spectrometry: fundamentals, instrumentation, practicalities and biological applications microanalysis of plant cell wall polysaccharides molecular analysis of carbohydrate-antibody interactions: case study using a bacillus anthracis tetrasaccharide endo-b-n-acetylglucosaminidasecatalyzed polymerization of b-glcp-( - )-glcpnac oxazoline: a revisit to enzymatic transglycosylation syntheses and doxorubicininclusion abilities of b-cyclodextrin derivatives with a hydroquinone a-glycoside residue attached at the primary side synthesis of sialoglycopolypeptide for potentially blocking influenza virus infection using a rat a , -sialyltransferase expressed in bmnpv bacmid-injected silkworm larvae expression, purification, and analyses of glycosylation and disulfide bonds of stereum purpureum endopolygalacturonase i in pichia pastoris chemically responsive supramolecular assemblies of pyrene-b-cyclodextrin dimer matrix-assisted laser desorption/ionization mass spectrometric analysis of polysulfated-derived oligosaccharides using pyrenemethylguanidine structual analysis of neutral and acidic xylooligosaccharides from hardwood kraft pulp, and their utilization by intestinal bacteria in vitro structural study of the n-glycans of intercellular adhesion molecule- (telencephalin) an essential epitope of anti-muc monoclonal antibody kl- revealed by focused glycopeptide library simple and conveniently accessible bi-fluorescence-labeled substrates for amylases syntheses and biological evaluations of carbosilane dendrimers uniformly functionalized with sialyl a ( ! ) lactose moieties as inhibitors for human influenza viruses characterization of endoplasmic reticulum-localized udp-dgalactose: hydroxyproline o-galactosyltransferase using synthetic peptide substrates in arabidopsis structural analysis of three novel trisaccharides isolated from the fermented beverage of plant extracts novel fructopyranose oligosaccharides isolated from fermented beverage of plant extract convenient approach to access octa-glycosylated porphyrins via "click chemistry n-glycosylation engineering of lepidopteran insect cells by the introduction of the b , -n-acetylglucosaminyltransferase iii gene efficient substitution reaction from cysteine to the serine residue of glycosylated polypeptide: repetitive peptide segment ligation strategy and the synthesis of glycosylated tetracontapeptide having acid labile sialyl-tn antigens structural characterization and dynamics of globotetraosylceramide in vascular endothelial cells under tnf-a stimulation boronic acid lectin affinity chromatography (blac). . temperature dependence of glycoprotein isolation and enrichment physical mechanisms of bacterial survival revealed by combined grazing-incidence x-ray scattering and monte carlo simulation use of ftir and mass spectrometry for characterization of glycated caseins artefacts formed by addition of urea to n-linked glycans released with peptide-n-glycosidase f for analysis by mass spectrometry okicamelliaside, an extraordinarily potent antidegranulation glucoside isolated from leaves of camellia japonica quantification of fructooligosaccharides based on the evaluation of oligomer ratios using an artificial neural network quantitative glycomics results of the glycoprotein research group's (gprg) study on quantitative glycoprotein analysis click multivalent heterogeneous neoglycoconjugates-modular synthesis and evaluation of their binding affinities globular carbosilane dendrimers with mannose groups at the periphery: synthesis, characterization and toxicity in dendritic cells biosynthesis and structure of the burkholderia cenocepacia k - lipopolysaccharide core oligosaccharide. truncation of the core oligosaccharide leads to increased binding and sensitivity to polymyxin b switching of polymerization activity of cinnamoyl-a-cyclodextrin action of a-dglucosidase from aspergillus niger towards dextrin and starch contiguous ogalactosylation of (r)-hydroxy-l-proline residues forms very stable polyproline ii helices microarrays with varying carbohydrate density reveal distinct subpopulations of serum antibodies synthesis, in vitro and in vivo studies of gd-dtpa-xda-d -glc(oh) complex as a new potential mri contrast agent structural characterization and hypoglycemic effects of arabinogalactan-protein from the tuberous cortex of the white-skinned sweet potato (ipomoea batatas l.) comparison of fluorescent labels for oligosaccharides and introduction of a new postlabeling purification method characterization of minor n-linked glycans on antibodies using endo h release and maldi-mass spectrometry methods in molecular biology, glycomics: methods and protocols the caenorhabditis elegans bus- mutant reveals a new class of o-glycans affecting bacterial resistance surfactant-assisted lipopolysaccharide conjugation employing a cyanopyridinium agent and its application to a competitive assay multifaceted approaches including neoglycolipid oligosaccharide microarrays to ligand discovery for malectin glycoproteomic profile in wine: a 'sweet' molecular renaissance mass spectrometry based targeted protein quantification: methods and applications convergent synthesis of a common pentasaccharide-repeating unit corresponding to the o-specific polysaccharide of escherichia coli o : k , o : k , and o : k synthesis of penta-and hexasaccharide fragments corresponding to the o-antigen of escherichia coli o analysis of the human seminal plasma glycome reveals the presence of immunomodulatory carbohydrate functional groups fast access to robust c-sialoside multimers synthesis, conjugation, and immunological evaluation of the analysis of carbohydrates and glycoconjugates & serogroup pneumococcal oligosaccharides human igg expression in silkworm larval hemolymph using bmnpv bacmids and its n-linked glycan structure the synthesis and spectral properties of an encapsulated aminoazobenzene dye o-glcnacylation disrupts glyceraldehyde- -phosphate dehydrogenase homo-tetramer formation and mediates its nuclear translocation immunochemical studies of trehalose-containing major glycolipid from tsukamurella pulmonis structural characterization of the major glycolipids from arthrobacter globiformis and arthrobacter scleromae bioconjugation and detection of lactosamine moiety using a , -galactosyltransferase mutants that transfer c -modified galactose with a chemical handle muropeptides trigger distinct activation profiles in macrophages and dendritic cells rapid assembly of gp oligosaccharide moieties via one-pot glycosidation-deprotection sequences one-pot catalytic glycosidation/fmoc removal-an iterable sequence for straightforward assembly of oligosaccharides related to hiv gp analysis of the dispersity in carbohydrate loading of synthetic glycoproteins using maldi-tof mass spectrometry solvent-free synthesis of thioglycosides by ball milling synthesis of mycobacterial triacylated phosphatidylinositol dimannoside containing an acyl lipid chain at -o of inositol o-glycan inhibitors generate arylglycans, induce apoptosis and lead to growth inhibition in colorectal cancer cell lines detection of carbohydrates and steroids by cation-enhanced nanostructure-initiator mass spectrometry (nims) for biofluid analysis and tissue imaging protein glycosylation analysis with capillary-based electromigrative separation techniques glycoprotein analysis using protein microarrays and mass spectrometry synthesis of -deoxy cyclic and linear oligosaccharides by oligomerization of monomers maintaining product titer while replacing undefined components in a cho culture system model a-mannoside conjugates: immunogenicity and induction of candidacidal activity hydrodynamic properties of cyclodextrin molecules in dilute solutions koenigs-knorr glycosylation with neuraminic acid derivatives synthesis of the core structure of the lipoteichoic acid of streptococcus pneumoniae de novo glycan structure search with the cid ms/ms spectra of native n-glycopeptides identification of a novel human udp-galnac transferase with unique catalytic activity and expression profile molecular engineering of exocytic vesicle traffic enhances the productivity of chinese hamster ovary cells the vesicletrafficking protein munc b increases the secretory capacity of mammalian cells detection of pathogenic streptococcus suis bacteria using magnetic glycoparticles rapid screening of lectins for multivalency effects with a glycodendrimer microarray imaging ms methodology for more chemical information in less data acquisition time utilizing a hybrid linear ion trap-orbitrap mass spectrometer atmospheric pressure laser desorption/ionization of plant metabolites and plant tissue using colloidal graphite click multivalent homogeneous neoglycoconjugates-synthesis and evaluation of their binding affinities role of lipid a acylation in yersinia enterocolitica virulence glycopeptide-preferring polypeptide galnac transferase (ppgalnac t ), involved in mucin-type o-glycosylation, has a unique galnac-o-ser/thr-binding site in its catalytic domain not found in ppgalnac t or t steroidal glycosides from the leaves of ruscus colchicus: isolation and structural elucidation based on a preliminary liquid chromatography-electrospray ionization tandem mass spectrometry profiling off-line coupling of microcolumn separations to desorption mass spectrometry selective discrimination of cyclodextrin diols using cyclic sulfates d-hplc and maldi-tof/tof analysis of barley proteins glycated during brewing synthesis of cationic derivatives of quil a and the preparation of cationic immunestimulating complexes (iscoms) cardenolides from pergularia tomentosa display cytotoxic activity resulting from their potent inhibition of na þ /k þ -atpase structural determination of the o-chain polysaccharide from the haloalkaliphilic halomonas alkaliantarctica bacterium strain crss structure of the core region from the lipopolysaccharide of plesiomonas shigelloides strain - (serotype o ) structural characterization of the core region from the lipopolysaccharide of the haloalkaliphilic bacterium halomonas alkaliantarctica strain crss the complete structure of the core of the lps from plesiomonas shigelloides - and the identification of its o-antigen biological repeating unit enhancement of repair of radiation induced dna strand breaks in human cells by ganoderma mushroom polysaccharides structural investigation of an exopolysaccharide substituted with a lactyl ether group produced by raoultella terrigena ez- - isolated in the chernobyl exclusion zone a protein important for antimicrobial peptide resistance, ydei/omda, is in the periplasm and interacts with ompd/nmpc role of e-cadherin n-glycosylation profile in a mammary tumor model influence of glycosylation on the adsorption of thermomyces lanuginosus lipase to hydrophobic and hydrophilic surfaces diazo transfer-click reaction route to new, lipophilic teicoplanin and ristocetin aglycon derivatives with high antibacterial and anti-influenza virus activity: an aggregation and receptor binding study high-energy collision induced dissociation of biomolecules: maldi-tof/rtof mass spectrometry in comparison to tandem sector mass spectrometry polymer structure of commercial hydrolyzable tannins by matrix-assisted laser desorption/ ionization-time-of-flight mass spectrometry design, synthesis, and evaluation of novel fluoroquinolone-aminoglycoside hybrid antibiotics cycloartane-type glycosides from astragalus amblolepis triterpenoid saponins from astragalus wiedemannianus fischer mechanisms involved during the ultrasonically induced depolymerization of chitosan: characterization and control oligonucleotide carbohydrate-centered galactosyl cluster conjugates synthesized by click and phosphoramidite chemistries substrate recognition and hydrolysis by a fungal xyloglucan-specific family hydrolase the structural elucidation of glycosaminoglycans chemical modification and biological evaluation of new semisynthetic derivatives of , -didehydronystatin a (s hp), a genetically engineered antifungal polyene macrolide context-dependent effects of asparagine glycosylation on pin ww folding kinetics and thermodynamics maldi-tof mass spectrometry of naturally occurring mixtures of monorhamnolipids and dirhamnolipids functionalized c-glycoside ketohydrazones: carbohydrate derivatives that retain the ring integrity of the terminal reducing sugar mass spectrometric-based stable isotopic -aminobenzoic acid glycan mapping for rapid glycan screening of biotherapeutics desorption electrospray ionization mass spectrometry of glycosaminoglycans and their protein noncovalent complex haba-based ionic liquid matrices for uv-maldi-ms analysis of heparin and heparan sulfate oligosaccharides mannose-substituted conjugated polyelectrolyte and oligomer as an intelligent energy transfer pair for label-free visual detection of concanavalin a development of a ce-ms method to analyze components of the potential biomarker vascular endothelial growth factor lipopeptides produced by a soil bacillus megaterium strain mass spectrometric analysis of the s-layer proteins from clostridium difficile demonstrates the absence of glycosylation facile synthesis of mercaptophenylboronic acid-functionalized core-shell structure fe o @c@au magnetic microspheres for selective enrichment of glycopeptides and glycoproteins a smart glycol-directed nanodevice from rationally designed macroporous materials secondary disorders of glycosylation in inborn errors of fructose metabolism online coupling of thin layer chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: synthesis and application of a new material for the identification of carbohydrates by thin layer chromatography/matrix free material enhanced laser desorption/ionization mass spectrometry acylated triterpenoidal saponins and cytokinins from gleditsia aquatica click chemistry approach for the synthesis of water-soluble glycodendrimer with triazole as building unit synthesis and evaluation of n-acetyl- -amino- -deoxy-a-d-galactosyl- -thio- -oxaceramide, a new analogue of a-d-galactosyl ceramide mass spectrometry for pectin structure analysis multiple site-specific in vitro labeling of single-chain antibody the effect of ph on the production of chitinolytic enzymes of verticillium fungicola in submerged cultures glycome-db. org: a portal for querying across the digital world of carbohydrate sequences iodine-sodium cyanoborohydride-mediated reductive ring opening of , -o-benzylidene acetals of hexopyranosides glycosylation patterns of hiv- gp depend on the type of expressing cells and affect antibody recognition the glycosylation of myeloperoxidase the glycosylation and characterization of the candidate gc macrophage activating factor controlled synthesis of linear acyclodextrin oligomers using copper-catalyzed huisgen , -dipolar cycloaddition two-step synthesis of galactosylated human serum albumin as a targeted optical imaging agent for peritoneal carcinomatosis handbook of experimental pharmacology , doping in sports synthesis of multivalent glycoconjugates containing the immunoactive lelte peptide: effect of glycosylation on cellular activation and natural killing by human peripheral blood mononuclear cells golgi function and dysfunction in the first cog -deficient cdg type ii patient mycobacterium abscessus glycopeptidolipids mask underlying cell wall phosphatidyl-myo-inositol mannosides blocking induction of human macrophage tnf-a by preventing interaction with tlr capsule anchoring in bacillus anthracis occurs by a transpeptidation reaction that is inhibited by capsidin synthesis of acceptor substrate analogs for the study of glycosyltransferases involved in the second step of the biosynthesis of o-antigen repeating units identification of n-linked carbohydrates from severe acute respiratory syndrome (sars) spike glycoprotein identification of n-glycans from ebola virus glycoproteins by matrix-assisted laser desorption/ionisation time-of-flight and negative ion electrospray tandem mass spectrometry synthesis, characterization, and immunogenicity in mice of shigella sonnei o-specific oligosaccharide-core-protein conjugates -aminoquinoline acting as matrix and derivatizing agent for maldi ms analysis of oligosaccharides cross-linked membranes based on acrylated cyclodextrins and polyethylene glycol dimethacrylates for aromatic/aliphatic separation mycobacterium marinum lipooligosaccharides are unique caryophyllose-containing cell wall glycolipids that inhibit tumor necrosis factor-a secretion in macrophages structural analysis of an unusual bioactive n-acylated lipo-oligosaccharide los-iv in mycobacterium marinum exploiting the cross-metathesis reaction in the synthesis of pseudo-oligosaccharides isolation and characterization of rhamnolipid-producing bacterial strains from a biodiesel facility characterisation of haptoglobin in the blood plasma of harbour seals (phoca vitulina) n-glycan moieties of the crustacean egg yolk protein and their glycosylation sites synthesis of lewis x epitopes on plant n-glycans glycodynamers: dynamic polymers bearing oligosaccharides residues -generation, structure, physicochemical, component exchange, and lectin binding properties oligosaccharide analysis by graphitized carbon liquid chromatography-mass spectrometry -picoline-borane: a non-toxic reducing agent for oligosaccharide labeling by reductive amination glycan labeling strategies and their use in identification and quantification optimized workflow for preparation of apts-labeled n-glycans allowing high-throughput analysis of human plasma glycomes using -channel multiplexed cge-lif decreased levels of bisecting glcnac glycoforms of igg are associated with human longevity antibacterial activity of n-quaternary chitosan derivatives: synthesis, characterization and structure activity relationship (sar) investigations the presence of monoglucosylated n -glycan is important for the structural stability of storage protein, arylphorin molecular cloning and characterization of trehalose synthase from thermotoga maritima dsm : syntheses of trehalose disaccharide analogues and ndp-glucoses maldi tof-tof characterization of a light stabilizer polymer contaminant from polypropylene or polyethylene plastic test tubes quantification of soyasaponins i and bg in italian lentil seeds by solid-phase extraction (spe) and high-performance liquid chromatography-mass spectrometry (hplc-ms) nutrition analysis by nanoparticle-assisted laser desorption/ionisation mass spectrometry isolation and characterization of antibodies against three consecutive tn-antigen clusters from a phage library displaying human single-chain variable fragments o-glcnac modification of the extracellular domain of notch receptors systematic syntheses of influenza neuraminidase inhibitors: a series of carbosilane dendrimers uniformly functionalized with thioglycoside-type sialic acid moieties supramolecular assemblies of oligothiophene derivatives bearing b-cyclodextrin h- , , -triazol- -yl thiodigalactoside derivatives as high affinity galectin- inhibitors synthesis and structural characterization of sialic acid-glutamic acid hybrid foldamers as conformational surrogates of a- , -linked polysialic acid the remarkable stability of chimeric, sialic acid derived a/d-peptides in human blood plasma lack of a-xylosidase activity in arabidopsis alters xyloglucan composition and results in growth defects intact and top-down characterization of biomolecules and direct analysis using infrared matrix-assisted laser desorption electrospray ionization coupled to ft-icr mass spectrometry triumfettosterol id and triumfettosaponin, a new (fatty acyl)-substituted steroid and a triterpenoid 'dimer' bis(b-d-glucopyranosyl) ester from the leaves of wild triumfetta cordifolia a. rich. (tiliaceae) fluorescence turn-on sensing of lectins with mannose-substituted tetraphenylethenes based on aggregation-induced emission synthesis of tri-and pentasaccharide fragments corresponding to the o-antigen of shigella boydii type biorecognition of escherichia coli k adhesin for glycated porcine albumin synthesis of a single-molecule l-rhamnose-containing three-component vaccine and evaluation of antigenicity in the presence of anti-lrhamnose antibodies glycosylation of liver acute-phase proteins in pancreatic cancer and chronic pancreatitis support vector machine prediction of n-and o-glycosylation sites using whole sequence information and subcellular localization silkworm expression and sugar profiling of human immune cell surface receptor, kir dl high levels of e -pha-reactive oligosaccharides: potential as marker for cells with characteristics of hepatic progenitor cells highsensitivity analysis of naturally occurring sugar chains, using a novel fluorescent linker molecule di-tert-butylsilylenedirected a-selective synthesis of p-nitrophenyl t-antigen analogues analysis of the human cancer glycome identifies a novel group of tumor-associated n-acetylglucosamine glycan antigens the n-glycome of human embryonic stem cells inhibition of dc-signmediated hiv infection by a linear trimannoside mimic in a tetravalent presentation chemo-enzymatic synthesis of poly-n-acetyllactosamine (poly-lacnac) structures and their characterization for cgl -galectin-mediated binding of ecm glycoproteins to biomaterial surfaces aziridine ring opening as regio-and stereoselective access to o-glycosyl amino acids and their transformation into o-glycopeptide mimetics identification of a polyprenylphosphomannosyl synthase involved in the synthesis of mycobacterial mannosides reductive amination of the lysine n eamino group leads to a bivalent glyco-amino acid building block suited for spps utilizing staudinger ligation for the synthesis of glycoamino acid building blocks and other glycomimetics cysteinebased mannoside glycoclusters: synthetic routes and antiadhesive properties development of dictyostelium discoideum is associated with alteration of fucosylated n-glycan structures tlc/ hptlc with direct mass spectrometric detection: a review of the progress achieved in the last years o-glycosylation modulates proprotein convertase activation of angiopoietin-like protein . possible role of polypeptide galnac-transferase- in regulation of concentrations of plasma lipids inhibition binding studies of glycodendrimer/lectin interactions using surface plasmon resonance self-assembling carbohydrate-functionalized oligothiophenes chemical synthesis using enzymatically generated building units for construction of the human milk pentasaccharides sialyllacto-n-tetraose and sialyllacto-n-neotetraose epimer hydroxypropyl-substituted b-cyclodextrins: influence of degree of substitution on the thermodynamics of complexation with tauroconjugated and glycoconjugated bile salts site-specific analysis of nlinked oligosaccharides of recombinant lysosomal arylsulfatase a produced in different cell lines synthesis of a gpi anchor module suitable for protein post-translational modification a combined method for producing homogeneous glycoproteins with eukaryotic n-glycosylation structural basis of multivalent binding to wheat germ agglutinin mass spectrometric characterization of the surface-associated kda lipoprotein jlpa as a glycosylated antigen in strains of campylobacter jejuni probing the lactose gm carbohydrate-carbohydrate interaction with glycodendrimers identification of n-glycosylation in prolyl endoprotease from aspergillus niger and evaluation of the enzyme for its possible application in proteomics rapid and efficient glycoprotein identification through microwave-assisted enzymatic digestion assigning nglycosylation sites of glycoproteins using lc/msms in conjunction with endo-m/exoglycosidase mixture glycan analysis by mass spectrometry immunoglobulin g glycopeptide profiling by matrixassisted laser desorption ionization fourier transform ion cyclotron resonance mass spectrometry glycan bioengineering in immunogen design for tumor t antigen immunotargeting characterisation of cell wall polysaccharides from okra (abelmoschus esculentus (l.) moench) okra pectin contains an unusual substitution of its rhamnosyl residues with acetyl and alpha-linked galactosyl groups synthesis and characterization of hydroquinone glucoside using leuconostoc mesenteroides dextransucrase lipoprotein lipase and hydrofluoric acid deactivate both bacterial lipoproteins and lipoteichoic acids, but platelet-activating factor-acetylhydrolase degrades only lipoteichoic acids a functional carbohydrate chip platform for analysis of carbohydrate-protein interaction structural and immunological characterization of the n-glycans from the major yellow jacket allergen ves v : the n-glycan structures are needed for the human antibody recognition identification of glycosyltransferase family members as xylosyltransferases acting on o-glucosylated notch epidermal growth factor repeats developments and applications of mass microscopy characterization of a putative -deoxy-d-manno- -octulosonic acid (kdo) transferase gene from arabidopsis thaliana structural characterisation of the pectic polysaccharide rhamnogalacturonan ii using an acidic fingerprinting methodology maldi linear tof mass spectrometry of pegylated (glyco) proteins synthetic glycosides of ent-caurene series containing substituents with benzyl, phenoxyl, and uracyl fragments sno @poly(hema-co-st-co-vpba) core-shell nanoparticles designed for selectively enriching glycopeptides followed by maldi-ms analysis mass spectrometry based analysis of protein o-glycosylation synthesis and characterization of watersoluble conjugated glycopolymer for fluorescent sensing of concanavalin a laser desorption ionization mass spectrometry by using surface plasmon excitation on gold nanoparticle the n-linked oligosaccharide at fcgriiia asn- : an inhibitory element for high fcgriiia binding affinity to igg glycoforms lacking core fucosylation monitoring the hydrolysis and transglycosylation activity of aglucosidase from aspergillus niger by nuclear magnetic resonance spectroscopy and mass spectrometry n-linked glycan analysis of glycoproteins secreted from rice cell suspension cultures under sugar starvation characterization of receptor proteins using affinity cross-linking with biotinylated ligands proton sponge: a novel and versatile maldi matrix for the analysis of metabolites using mass spectrometry , -bis(dimethylamino)naphthalene: a novel superbasic matrix for matrix-assisted laser desorption/ionization timeof-flight mass spectrometric analysis of fatty acids acid-base-driven matrixassisted mass spectrometry for targeted metabolomics phenolic compounds in the leaves of populus ussuriensis and their antioxidant activities phenolic compounds from populus davidiana wood the structure of the carbohydrate backbone of the lipooligosaccharide from the halophilic bacterium arcobacter halophilus the structure of the carbohydrate backbone of the lipooligosaccharide from an alkaliphilic halomonas sp delineation of the roles of fadd , fadd and fadd in the biosynthesis of phthiocerol dimycocerosates and related compounds in mycobacterium tuberculosis biodegradable biocompatible xyloglucan films for various applications transparent xyloglucan-chitosan complex hydrogels for different applications physico chemical properties of aminated tamarind xyloglucan fractionation of sugar beet pulp by introducing ion-exchange groups the antimicrobial action of low-molar-mass chitosan, chitosan derivatives and chitooligosaccharides on bifidobacteria structural details and composition of trichomonas vaginalis lipophosphoglycan in relevance to the epithelial immune function determination of distinctive carbohydrate signatures obtained from the aeromonas hydrophila (chemotype ii) core oligosaccharide pinpointing the presence of the -o-phosphorylated -o-linked kdo reducing end group using electrospray ionization quadrupole orthogonal time-offlight mass spectrometry and tandem mass spectrometry biosynthetic origin of the galactosamine substituent of arabinogalactan in mycobacterium tuberculosis aftd, a novel essential arabinofuranosyltransferase from mycobacteria nmr structure determination of a segmentally labeled glycoprotein using in vitro glycosylation the role of peptides and proteins in melanoidin formation optimization of matrix conditions for the control of maldi in-source decay of permethylated glycans high-spatial resolution matrix-assisted laser desorption ionization imaging analysis of glucosylceramide in spleen sections from a mouse model of gaucher disease a simple cellulose column procedure for selective enrichment of glycopeptides and characterization by nano lc coupled with electron-transfer and high-energy collisional-dissociation tandem mass spectrometry structure of compositionally simple lipopolysaccharide from marine synechococcus synthesis of water-soluble phthalocyanines bearing four or eight d-galactose units discrimination of isobaric leucine and isoleucine residues and analysis of post-translational modifications in peptides by maldi in-source decay mass spectrometry combined with collisional cooling effect of gas pressure and gas type on the fragmentation of peptide and oligosaccharide ions generated in an elevated pressure uv/ir-maldi ion source coupled to an orthogonal time-of-flight mass spectrometer formation and structure elucidation of n-( , , -tri-o-acetyl-b-d-glucopyranosyl)-n -acetylthiourea disaccharide-modified liposomes and their in vitro intracellular uptake carbohydrate arrays: recent developments in fabrication and detection methods with applications discovery of the first series of small molecule h n entry inhibitors transglycosylation properties of maltodextrin glucosidase (malz) from escherichia coli and its application for synthesis of a nigerose-containing oligosaccharide fluorescent glycosylamides produced by microscale derivatization of free glycans for natural glycan microarrays novel fluorescent glycan microarray strategy reveals ligands for galectins generation of a natural glycan microarray using -fluorenylmethyl chloroformate (fmoccl) as a cleavable fluorescent tag glycan microarray analysis of ptype lectins reveals distinct phosphomannose glycan recognition development and application of mass spectrometric methods for the analysis of progranulin n-glycosylation francisella tularensis blue-gray phase variation involves structural modifications of lipopolysaccharide oantigen, core and lipid a and affects intramacrophage survival and vaccine efficacy glycosylation of gp and gp envelope proteins of yellow head virus of penaeus monodon shrimp structural profiling of individual glycosphingolipids in a single thin-layer chromatogram by multiple sequential immunodetection matched with direct ir-maldi-o-tof mass spectrometry software utilities for the interpretation of mass spectrometric data of glycoconjugates: application to glycosphingolipids of human serum ionic liquids in analytical chemistry isolation of b-mannanase from cocos nucifera linn haustorium and its application in the depolymerization of b-( , )-linked d-mannans n-glycan trimming by glucosidase ii is essential for arabidopsis development immuno-maldi-tof ms: new perspectives for clinical applications of mass spectrometry a computational approach for deciphering the organization of glycosaminoglycans synthesis of galactofuranose-based acceptor substrates for the study of the carbohydrate polymerase glft analysis of n-glycans in embryonated chicken egg chorioallantoic and amniotic cells responsible for binding and adaptation of human and avian influenza viruses the mechanism of boron mobility in wheat and canola phloem removal of the outer kdo from helicobacter pylori lipopolysaccharide and its impact on the bacterial surface galectin- -regulated delivery of glycoproteins to the brush border membrane of enterocyte-like cells characterization of an immunodominant cancer-specific o-glycopeptide epitope in murine podoplanin (ots ) formation of homooligosaccharides using base-promoted glycosylation of unprotected glycosyl fluorides glycosyltransferase function in core -type protein o-glycosylation xyloglucan endotransglycosylases (xets) from germinating nasturtium (tropaeolum majus) seeds: isolation and characterization of the major form botulinum neurotoxin serotype d attacks neurons via two carbohydrate-binding sites in a ganglioside-dependent manner the chain length of lignan macromolecule from flaxseed hulls is determined by the incorporation of coumaric acid glucosides and ferulic acid glucosides analysis of human plasma lipids and soybean lecithin by means of high-performance thin-layer chromatography and matrix-assisted laser desorption/ionization mass spectrometry chemoenzymatic synthesis of a glycolipid library and elucidation of the antigenic epitope for construction of a vaccine against lyme disease acylated cholesteryl galactosides are specific antigens of borrelia causing lyme disease and frequently induce antibodies in late stages of disease detailed n-glycan analysis of mannose receptor purified from murine spleen indicates tissue specific sialylation synthesis and acid catalysis of cellulose-derived carbon-based solid acid experimental studies on the ring opening polymerization of p-dioxanone using an al(o i pr) -monosaccharide initiator system glucuronyltransferase activity of kfic from escherichia coli strain k requires association of kfia. kfic and kfia are essential enzymes for production of k polysaccharide, n-acetylheparosan current imaging mass spectrometry for metabolite molecules imaging mass spectrometry for visualization of drug and endogenous metabolite distribution: toward in situ pharmacometabolomes imaging mass spectrometry protocols for mass microscopy structure and kinetic investigation of streptococcus pyogenes family gh a-mannosidase strategic glycan elution map for the production of human-type n-linked oligosaccharides: the case of hen egg yolk and white comprehensive analysis of n-linked oligosaccharides from eggs of the family phasianidae pectic oligosaccharide analysis by fluorophore-assisted carbohydrate electrophoresis ionic liquids in analytical chemistry dsa affinity glycoproteome of human liver tissue isolation of n-linked glycopeptides by hydrazine-functionalized magnetic particles further insight into the roles of the glycans attached to human blood protein c inhibitor synthesis of -petn-a-d-galpnac-( ! )-b-d-galp-( ! )-b-d-glcpnac-( ! )-b-d-galp-( ! )-b-d-glcp, a haemophilus influenzae lipopolysacharide structure, and biotin and protein conjugates thereof computationally and experimentally derived general rules for fragmentation of various glycosyl bonds in sodium adduct oligosaccharides molecular cloning of pigeon udp-galactose: b-d-galactoside a , -galactosyltransferase and udp-galactose: b-dgalactoside b , -galactosyltransferase, two novel enzymes catalyzing the formation of gal-a - gal-b - gal-b - glcnac sequence structural analysis of n-glycans from gull egg white glycoproteins and egg yolk igg synthesis of a glycosylphosphatidylinositol anchor bearing unsaturated lipid chains rapid release of n-linked glycans from glycoproteins by pressure-cycling technology laser desorption/ionization mass spectrometric analysis of small molecules using fullerene-derivatized silica as energy-absorbing material synthesis of three regioisomers of the pentasaccharide part of the skp glycoprotein of dictyostelium discoideum b-glycosidation of sterically hindered alcohols an aeromonas caviae genomic island is required for both oantigen lipopolysaccharide biosynthesis and flagellin glycosylation new triterpene glycoside from cyclamen adzharicum tubers mycobacterium leprae phenolglycolipid- expressed by engineered m. bovis bcg modulates early interaction with human phagocytes defects in flagellin glycosylation affect the virulence of pseudomonas syringae pv. tabaci mass spectrometric imaging of ginsenosides localization in panax ginseng root thermosensitive hydrogels composed of cyclodextrin pseudorotaxanes. role of [ ]pseudorotaxane in the gel formation hydrogels composed of organic amphiphiles and a-cyclodextrin: supramolecular networks of their pseudorotaxanes in aqueous media a plant class v chitinase from a cycad (cycas revoluta): biochemical characterization, cdna isolation, and posttranslational modification distinct features of matrix-assisted mm infrared laser desorption/ionization mass spectrometry in biomolecular analysis dissociation profile of protonated fucosyl glycopeptides and quantitation of fucosylation levels of glycoproteins by mass spectrometry loose-fit polyrotaxane composed of g-cyclodextrin and single poly(ethyelene glycol) chain: making room in g-cd cavity for additional inclusion complexation optically pure fullerodendron formed by diastereoselective dielse-alder reaction physiological and glycomic characterization of n-acetylglucosaminyltransferase-iva and -ivb double deficient mice synthesis and ring-opening polymerizations of novel s-glycooxazolines enrichment of amadori products derived from the nonenzymatic glycation of proteins using microscale boronate affinity chromatography alterations in receptor-binding properties of swine influenza viruses of the h subtype after isolation in embryonated chicken eggs tlc blot (far-eastern blot) and its applications epimeric and amino disaccharide analogs as probes of an a-( - )-mannosyltransferase involved in mycobacterial lipoarabinomannan biosynthesis solution conformation of c-linked antifreeze glycoprotein analogues and modulation of ice recrystallization application of proteomics in biomarker discovery: a primer for the clinician synthesis of the glycosaminoglycan-protein linkage tetraosyl peptide moieties of betaglycan, which serve as a hexosamine acceptor for enzymatic glycosyl transfer baca is indispensable for successful mesorhizobium-astragalus symbiosis synthesis of a sialic acid containing complex-type n-glycan on a solid support a combined p-azaelectrocyclization/ staudinger approach to protein and cell engineering: noninvasive tumor targeting by n-glycan-engineered lymphocytes noninvasive imaging of dendrimer-type n-glycan clusters: in vivo dynamics dependence on oligosaccharide structure synthesis of non-natural xyloglucans by polycondensation of , -dimethoxy- , , -triazin- -yl oligosaccharide monomers catalyzed by endo-b- , -glucanase novel dialkoxytriazine-type glycosyl donors for cellulase-catalysed lactosylation on-plate-selective enrichment of glycopeptides using boronic acid-modified gold nanoparticles for direct maldi-qit-tof ms analysis concanavalin a-immobilized magnetic nanoparticles for selective enrichment of glycoproteins and application to glycoproteomics in hepatocelluar carcinoma cell line biomaterials from sugars: ring-opening polymerization of a carbohydrate lactone synthesis of a monophosphoryl derivative of escherichia coli lipid a and its efficient coupling to a tumor-associated carbohydrate antigen identification of n-glycan serum markers associated with hepatocellular carcinoma from mass spectrometry data novel neogala-series glycosphingolipids with terminal mannose and glucose residues from hirsutella rhossiliensis, an aureobasidin aresistant ascomycete fungus functional characterization of tlmh in streptoalloteichus hindustanus e - atcc unveiling new insight into tallysomycin biosynthesis and affording a novel bleomycin analog the effect of temperature on the stability of compounds used as uv-maldi-ms matrix: , -dihydroxybenzoic acid, , , -trihydroxyacetophenone, a-cyano- -hydroxycinnamic acid, , -dimethoxy- -hydroxycinnamic acid, nor-harmane and harmane towards an integrated proteomic and glycomic approach to finding cancer biomarkers chemoenzymatic synthesis of multivalent neoglycoconjugates carrying the helminth glycan antigen ldnf fragment-based development of triazole-substituted o-galactosyl aldoximes with fragmentinduced affinity and selectivity for galectin- tempo oxidation of gelatinized potato starch results in acid resistant blocks of glucuronic acid moieties molecular sieves provoke multiple substitutions in the enzymatic synthesis of fructose oligosaccharide-lauryl esters synthesis of organic-soluble conjugated polyrotaxanes by polymerization of linked rotaxanes insulated molecular wire with highly conductive p-conjugated polymer core reaction of the antitumor antibiotic olivomycin i with aryl diazonium salts. synthesis, cytotoxic and antiretroviral potency of -aryldiazenyl- -odeglycosyl derivatives of olivomycin i highly site-selective stability increases by glycosylation of dihydrofolate reductase gold-catalyzed glycosidations: synthesis of , -anhydro saccharides site-specific glycoprofiling of n-linked glycopeptides using maldi-tof ms: strong correlation between signal strength and glycoform quantities purification and characterization of a new recombinant factor viii (n ) identification and quantification of n-linked oligosaccharides released from glycoproteins: an inter-laboratory study determination of nlinked sialyl-sugar chains in the lungs of domestic cats and dogs in thailand susceptible to the highly pathogenic avian influenza virus (h n ) cyclodextrin-based hyperbranched polymers: molecule design, synthesis and characterization glycoproteomics: past, present and future molecular basis for galactosylation of core fucose residues in invertebrates. identification of caenorhabditis elegans n-glycan core a , -fucoside b , -galactosyltransferase galt- as a member of a novel glycosyltransferase family glycoproteomic analysis of abnormal n-glycosylation on the kappa chain of cryocrystalglobulin in a patient of multiple myeloma solvolytic depolymerization of chondroitin and dermatan sulfates a chromatographic approach for elevating the antibody-dependent cellular cytotoxicity of antibody composites structural characterization and surface-active properties of a succinoyl trehalose lipid produced by rhodococcus sp. sd- chemo-enzymatic synthesis of glycosylated insulin using a glcnac tag acceptor specificity in the transglycosylation reaction using endo-m coxiella burnetii glycomics and proteomics-tools for linking structure to function social self-sorting: alternating supramolecular oligomer consisting of isomers humanization of recombinant glycoproteins expressed in insect cells inactivation of mycobacterium tuberculosis mannosyltransferase pimb reduces the cell wall lipoarabinomannan and lipomannan content and increases the rate of bacterial-induced human macrophage cell death the recognition motif of the glycoprotein-folding sensor enzyme udp-glc: glycoprotein glucosyltransferase enrichment method of sulfated glycopeptides by a sulfate emerging and ion exchange chromatography enzymatic solubilization of brewers' spent grain by combined action of carbohydrases and peptidases poly(n-vinylpyrrolidone) bearing covalently attached cyclodextrin via click-chemistry: synthesis, characterization, and complexation behavior with phenolphthalein chemical and enzymatic n-glycan release comparison for n-glycan profiling of monoclonal antibodies expressed in plants analysis of carbohydrates on proteins by offline normal-phase liquid chromatography maldi-tof/tof-ms/ ms carbohydrate structural analysis of wheat flour arabinogalactan protein the fine structure of neisseria meningitidis lipooligosaccharide from the m strain and three of its variants dihydrobenzoic acid modified nanoparticle as a maldi-tof ms matrix for soft ionization and structure determination of small molecules with diverse structures photoinduced isomerization of allyl alcohols to carbonyl compounds using dendrimer disulfide as catalyst miniaturizing sample spots for matrix-assisted laser desorption/ionization mass spectrometry glycomics profiling of heparan sulfate structure and activity alkali-hydroxide-doped matrices for structural characterization of neutral underivatized oligosaccharides by maldi time-of-flight mass spectrometry phosphocholinecontaining glycosyl inositol-phosphoceramides from trichoderma viride induce defense responses in cultured rice cells targeted serum glycoproteomics for the discovery of lung cancer-associated glycosylation disorders using lectin-coupled pro-teinchip arrays chemoenzymatic synthesis of glycosylated glucagon-like peptide : effect of glycosylation on proteolytic resistance and in vivo blood glucoselowering activity daughter cell separation is controlled by cytokinetic ring-activated cell wall hydrolysis solid-phase synthesis of glycopeptide carrying a tetra-n-acetyllactosamine-containing core decasaccharide glycosylation specific for adhesion molecules in epidermis and its receptor revealed by glycoform-focused reverse genomics isolation and identification of arabinose mycolates of cell wall skeleton (cws) derived from mycobacterium bovis bcg tokyo (smp- ) synthesis, cell-surface binding, and cellular uptake of fluorescently labeled glucose-dna conjugates with different carbohydrate presentation use of a novel ionic liquid matrix for maldi-ms analysis of glycopeptides and glycans out of total tryptic digests efficient transfer of sialo-oligosaccharide onto proteins by combined use of a glycosynthase-like mutant of mucor hiemalis endoglycosidase and synthetic sialo-complex-type sugar oxazoline milk oligosaccharides structural and functional characterization of a putative polysaccharide deacetylase of the human parasite encephalitozoon cuniculi glycosyl conjugates of biotinylated diaminopyridine applied for study of carbohydrate-to-carbohydrate interaction an oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides deciphering carbohydrate structures by ims-ms. applications to biological features related to carbohydrate chemistry and biology software platform for high-throughput glycomics preparation and properties of polyurethanes based on castor oil chemically modified with yucca starch glycoside synthesis and characterization of neurostatin-related compounds with high inhibitory activity of glioma growth o-fucosylation of an antibody light chain: characterization of a modification occurring on an igg molecule asparagine-linked oligosaccharides present on a non-consensus amino acid sequence in the c h domain of human antibodies biochemical characterization of bovine plasma thrombin-activatable fibrinolysis inhibitor (tafi) glycosylation pattern of mature dimeric leukocyte and recombinant monomeric myeloperoxidase. glycosylation is required for optimal enzymatic activity n-linked glycosylation is an important parameter for optimal selection of cell lines producing biopharmaceutical human igg immunoglobulin g galactosylation and sialylation are associated with pregnancyinduced improvement of rheumatoid arthritis and the postpartum flare: results from a large prospective cohort study structural and functional analysis of glycosphingolipids of schistosoma mansoni elucidation of molecular diversity and body distribution of saponins in the sea cucumber holothuria forskali (echinodermata) by mass spectrometry qualitative and quantitative saponin contents in five sea cucumbers from the indian ocean localization of secondary metabolites in marine invertebrates: contribution of maldi msi for the study of saponins in cuvierian tubules of h. forskali elevated sulfatide levels in neurons cause lethal audiogenic seizures in mice rhizobium leguminosarum biovar viciae , deficient in -hydroxyoctacosanoate-modified lipopolysaccharide, is impaired in desiccation tolerance, biofilm formation and motility glycome profiling using modern glycomics technology: technical aspects and applications symbol nomenclature for glycan representation synthesis of cyclic b-glucan using laminarinase a glycosynthase mutant from the basidiomycete phanerochaete chrysosporium purification and nglycosylation analysis of melanoma antigen dopachrome tautomerase rapana venosa hemocyanin with antiviral activity stable isotope-enhanced two-and threedimensional diffusion ordered c nmr spectroscopy (sie-dosy c nmr) modern maldi time-of-flight mass spectrometry orthogonal activation of propargyl and n-pentenyl glycosides and , -orthoesters laser desorption-ionization of lipid transfers: tissue mass spectrometry imaging without maldi matrix characterization of branched polysaccharides using multiple-detection size separation techniques multigram synthesis of a hyaluronic acid subunit and synthesis of fully protected oligomers finding new posttranslational modifications in salivary proline-rich proteins enzymatic glycosylations on arrays rapid assembly of oligosaccharides: a highly convergent strategy for the assembly of a glycosylated amino acid derived from psgl- databases and informatics for glycobiology and glycomics production of nonfucosylated antibodies by co-expression of heterologous gdp- -deoxy-d-lyxo- -hexulose reductase characterization of the acidic n-linked glycans of the zona pellucida of prepuberal pigs by a mass spectrometric approach pectin, a versatile polysaccharide present in plant cell walls expression of talaromyces emersonii cellobiohydrolase cel a in saccharomyces cerevisiae and rational mutagenesis to improve its thermostability and activity structural characterization of flavonoid glycosides by multi-stage mass spectrometry the absence of an identifiable single catalytic base residue in thermobifida fusca exocellulase cel b quantitation of saccharide compositions of o-glycans by mass spectrometry of glycopeptides and its application to rheumatoid arthritis comparison of methods for profiling o-glycosylation. human proteome organisation human disease glycomics/proteome initiative multi-institutional study of iga fluorinated glycosyl amino acids for mucin-like glycopeptide antigen analogues a nonprotein thermal hysteresis-producing xylomannan antifreeze in the freeze-tolerant alaskan beetle upis ceramboides glycans on influenza hemagglutinin affect receptor binding and immune response synthesis of a mannose-capped disaccharide with a thiol terminus steroidal saponins from the rhizomes of polygonatum odoratum oxidative stress-induced peptidoglycan deacetylase in helicobacter pylori convenient synthesis of an n-glycan octasaccharide of the bisecting type matrix-assisted laser desorption/ionization mass spectrometry (maldi-ms). application in carbohydrate analysis molecular aggregation behavior of perylene-bridged bis(b-cyclodextrin) and its electronic interactions upon selective binding with aromatic guests functional characterization of tlmk unveiling unstable carbinolamide intermediates in the tallysomycin biosynthetic pathway silver-coated gold nanoparticles as concentrating probes and matrices for surface-assisted laser desorption/ionization mass spectrometric analysis of aminoglycosides conversion of squid pen by pseudomonas aeruginosa k fermentation for the production of nacetyl chitooligosaccharides and biofertilizers n-terminal deletion of peptide: n-glycanase results in enhanced deglycosylation activity a new chalcone glycoside, a new tetrahydrofuranoid lignan, and antioxidative constituents from the stems and leaves of viburnum propinquum cosmc is an essential chaperone for correct protein o-glycosylation role of a cytoplasmic dual-function glycosyltransferase in o regulation of development in dictyostelium crystal structure and statistical coupling analysis of highly glycosylated peroxidase from royal palm tree (roystonea regia) two-enzyme systems for glycolipid and polyglycerolphosphate lipoteichoic acid synthesis in listeria monocytogenes n-glycan analysis of recombinant l-selectin reveals sulfated galnac and galnac-galnac motifs glycosylation of the phosphate binding protein, psts, in streptomyces coelicolor by a pathway that resembles protein o-mannosylation in eukaryotes immobilization of enzyme on detonation nanodiamond for highly efficient proteolysis comparative glycoproteomics: approaches and applications covalent modification of chitin with silk-derivatives acts as an amphiphilic self-organizing template in nacre biomineralisation expression of helix pomatia lectin binding glycoproteins in women with breast cancer in relationship to their blood group phenotypes analysis of site-specific glycosylation of renal and hepatic g-glutamyl transpeptidase from normal human tissue release and characterization of single side chains of white cabbage pectin and their complement-fixing activity characterization of wbpb, wbpe, and wbpd and reconstitution of a pathway for the biosynthesis of udp- , -diacetamido- , -dideoxy-d-mannuronic acid in pseudomonas aeruginosa maldi-tof ms and ce-lif fingerprinting of plant cell wall polysaccharide digests as a screening tool for arabidopsis cell wall mutants branched arabino-oligosaccharides isolated from sugar beet arabinan derivatization of sialic acids for stabilization in matrix-assisted laser desorption/ionization mass spectrometry and concomitant differentiation of a( - ) and a( - ) isomers glycomic characterization of prostate-specific antigen and prostatic acid phosphatase in prostate cancer and benign disease seminal plasma fluids synthesis of bifunctional peptide derivatives based on a b-cyclodextrin core with drug delivery potential glycoprotein production for structure analysis with stable, glycosylation mutant cho cell lines established by fluorescence-activated cell sorting triple recognition of b-dna by a neomycin-hoechst -pyrene conjugate introductory glycosylation analysis using sds-page and peptide mass fingerprinting synthesis and cytotoxic activity of g pamam-nh dendrimer-modified digoxin and proscillaridin a conjugates in breast cancer cells quantitative site-specific analysis of protein glycosylation by lc-ms using different glycopeptide-enrichment strategies profiling of n-glycosylation gene expression in cho cell fed-batch cultures an investigation of intracellular glycosylation activities in cho cells: effects of nucleotide sugar precursor feeding vitro bacterial polysaccharide biosynthesis: defining the functions of wzy and wzz ultrasound ionization of biomolecules new development of glycan arrays gold nanoparticles as assisted matrices for the detection of biomolecules in a high-salt solution through laser desorption/ionization mass spectrometry development of an annotated library of neutral human milk oligosaccharides synthesis of monomeric and dimeric repeating units of the zwitterionic type capsular polysaccharide from streptococcus pneumoniae chemoenzymatic synthesis of glycosylphosphatidylinositol-anchored glycopeptides synthesis of mangiferin, isomangiferin, and homomangiferin sortase a-catalyzed transpeptidation of glycosylphosphatidylinositol derivatives for chemoenzymatic synthesis of gpi-anchored proteins synthesis of talosin a and b, two bioactive isoflavonoid glycosides direct labelling of peptides with -[ f]fluoro- -deoxy-d-glucose ([ f]fdg) structural glycomics using hydrophilic interaction chromatography (hilic) with mass spectrometry hexose rearrangements upon fragmentation of n-glycopeptides and reductively aminated nglycans ligand identification of carbohydrate-binding proteins employing a biotinylated glycan binding assay and tandem mass spectrometry glycan reductive isotope labeling for quantitative glycomics core -derived o-glycans are essential for intestinal mucus barrier function rapid comparison of a candidate biosimilar to an innovator monoclonal antibody with advanced liquid chromatography and mass spectrometry technologies preparation of chitooligosaccharides by the enzymatic hydrolysis of chitosan potent angiogenic inhibition effects of deacetylated chitohexaose separated from chitooligosaccharides and its mechanism of action in vitro synthesis of well-defined -arm and -arm poly(nisopropylacrylamide) star polymers with b-cyclodextrin cores via click chemistry and their thermal phase transition behavior in aqueous solution chitooligosaccharides protect human embryonic hepatocytes against oxidative stress nduced by hydrogen peroxide on-plate enrichment of glycopeptides by using boronic acid functionalized gold-coated si wafer highly specific enrichment of glycopeptides using boronic acid-functionalized mesoporous silica n-glycosylation profiling of turtle egg yolk: expression of galabiose structure development and application of high performance liquid chromatography map of glucuronyl n-glycans comparative studies on the structural features of o-glycans between leukemia and epithelial cell lines hyphenated technique for releasing and maldi ms analysis of oglycans in mucin-type glycoprotein samples preparation and characterization of phospha sugar analogues, , -dibromo- -methyl- -phenylphospholane -oxide derivatives, as novel anticancer agents synthesis of b-d-fructopyranosyl-( ! )-d-glucopyranose from d-glucose and d-fructose by a thermal treatment switching from altro-a-cyclodextrin dimer to pseudo[ ]rotaxane dimer through tumbling a molecular reel: shuttling of a rotor by tumbling of a macrocycle selective enrichment of glycopeptides/phosphopeptides using porous titania microspheres synthesis of di-and tetrasaccharide containing -deoxytalose from the o-antigenic polysaccharide of b. pseudomallei strain b mechanism of mild acid hydrolysis of galactan polysaccharides with highly ordered disaccharide repeats leading to a complete series of exclusively oddnumbered oligosaccharides heterogeneous components of chitosans glucopyranoside-incorporated n-heterocyclic carbene complexes of silver(i) and palladium(ii): efficient water-soluble suzuki-miyaura coupling palladium(ii) catalysts detection of small neutral carbohydrates using various supporting materials in laser desorption/onization mass spectrometry synthesis of a novel class of glycocluster with a cyclic a-( ! )-octaglucoside as a scaffold and their binding abilities to concanavalin a isolation and structural characterization of a polysaccharide fcap from the fruit of cornus officinalis expression, glycoform characterization, and antibody-binding of hiv- v glycopeptide domain fused with human igg -fc synthesis of kaempferol -o-( , -di-o-e-p-coumaroyl)-b-d-glucopyranoside, efficient glycosylation of flavonol -oh with glycosyl odlkynylbenzoates as donors total synthesis and structural revision of tmgchitotriomycin, a specific inhibitor of insect and fungal b-n-acetylglucosaminidases chemoselective glycosylation of carboxylic acid with glycosyl ortho-hexynylbenzoates as donors homogeneous synthesis of grgdy grafted chitosan on hydroxyl groups by photochemical reaction for improved cell adhesion facile synthesis of -mercaptophenylboronic acid functionalized gold nanoparticles for selective enrichment of glycopeptides new triterpenoid saponins from the roots of gypsophila paniculata l the crystal structure of a xyloglucan-specific endo-b- , -glucanase from geotrichum sp. m xyloglucanase reveals a key amino acid residue for substrate specificity imidazolium cation supported solution-phase assembly of homolinear a( ! )-linked octamannoside: an efficient alternate approach for oligosaccharide synthesis growth phasedependent expression of proteins with decreased plant-specific nglycans and immunogenicity in tobacco by cells oxidation of the primary hydroxyl group of galactose of galactaosyl ceramide analogue by chemical method-precursors for the synthesis of labeled conjugates developmental changes in glycolipids and synchronized expression of nutrient transporters in the mouse small intestine differences in n-glycan structures found on recombinant iga and iga produced in murine myeloma and cho cell lines extraction of glycosaminoglycan from sea hare, aplysia kurodai, and its functional properties . structural properties of purified glycosaminoglycan synthesis of dopamine and l-dopaa-glycosides by reaction with cyclomaltohexaose catalyzed by cyclomaltodextrin glucanyltransferase n-glycosylation status of b-haptoglobin in sera of patients with prostate cancer vs. benign prostate diseases unexpected tolerance of glycosylation by udp-galnac:polypeptide a-n-acetylgalactosaminyltransferase revealed by electron capture dissociation mass spectrometry: carbohydrate as potential protective groups modification-specific proteomics in plant biology effect and limitation of excess ammonium on the release of o-glycans in reducing forms from glycoproteins under mild alkaline conditions for glycomic and functional analysis hydrophilic interaction chromatography based enrichment of glycopeptides by using click maltose: a matrix with high selectivity and glycosylation heterogeneity coverage construction and transmembrane dissociation behavior of supramolecular assembly of quinolinocyclodextrin with porphyrin metabolic labeling of glycoconjugates with photocrosslinking sugars identification of blood group a/ a-leb/y and b/b-leb/y active glycotopes co-expressed on the oglycans isolated from two distinct human ovarian cyst fluids a supramolecular bifunctional artificial enzyme with superoxide dismutase and glutathione peroxidase activities enabling techniques and strategic workflow for sulfoglycomics based on mass spectrometry mapping and sequencing of permethylated sulfated glycans synthesis and c nmr spectroscopy of model compounds for the microstructure analysis of poly(vinyl glycoside)s production of anti-carbohydrate antibodies by phage display technologies. potential impairment of cell growth as a result of endogenous expression precise structure of acidic polysaccharide present in salvia hydrogels n-glycosylation in archaea: on the coordinated actions of haloferax volcanii aglf and aglm on-line separations combined with ms for analysis of glycosaminoglycans mass spectrometry and glycomics matrix-assisted laser desorption/ionization imaging mass spectrometry effect of enzyme processivity on the efficacy of a competitive chitinase inhibitor catalytic properties of endo- , -b-d-glucanase from the vietnamese edible mussel perna viridis cerebrocostomandibular-like syndrome and a mutation in the conserved oligomeric golgi complex, subunit a new mutation in cog extends the spectrum of cog subunit deficiencies clickable lipids: azido and alkynyl fatty acids and triacylglycerols expression, purification, and characterization of recombinant human transferrin from rice structural characterization and anti-fatigue activity of polysaccharides from the roots of morinda officinalis a modified synthesis and serological evaluation of neoglycoproteins containing the natural disaccharide of pgl-i from mycobacterium leprae synthesis of novel aminoglycosides via allylic azide rearrangement for investigating the significance of -amino group development of a plate-based scintillation proximity assay for the mycobacterial aftb enzyme involved in cell wall arabinan biosynthesis transient expression and purification of chimeric heavy chain antibodies extracting both peptide sequence and glycan structural information by nm photodissociation of n-linked glycopeptides synthesis of neu ac-gal-functionalized gold glyconanoparticles total synthesis of apigenin- -yl- -o-(pcoumaroyl)-b-d-glucopyranoside recent developments of nanoparticle-based enrichment methods for mass spectrometric analysis in proteomics specific enrichment methods for glycoproteome research boronic acid functionalized core-satellite composite nanoparticles for advanced enrichment of glycopeptides and glycoproteins condensed tannins from mangrove species kandelia candel and rhizophora mangle and their antioxidant activity lew , encoding a putative a- , -mannosyltransferase (alg ) in n-linked glycoprotein, plays vital roles in cell-wall biosynthesis and the abiotic stress response in arabidopsis thaliana a perspective on the maillard reaction and the analysis of protein glycation by mass spectrometry: probing the pathogenesis of chronic disease a novel strategy for maldi-tof ms analysis of small molecules steroidal saponins from the rhizomes of paris delavayi acute kidney injury induced by proteinoverload nephropathy down-regulates gene expression of hepatic cerebroside sulfotransferase in mice, resulting in reduction of liver and serum sulfatides prediction of collision-induced dissociation spectra of common n-glycopeptides for glycoform identification mass spectrometry for structural characterization of therapeutic antibodies analysis of protein glycosylation and phosphorylation using liquid phase separation, protein microarray technology, and mass spectrometry preparation and characterization of galacto-mannan-oligosaccharides hydrolyzed from guar gum by bmannanase inclusion behavior of bcyclodextrin with bipyridine molecules: factors governing host-guest inclusion geometries combination of b-elimination and liquid chromatography/quadrupole time-of-flight mass spectrometry for the determination of o-glycosylation sites study of matrix additives for sensitive analysis of lipid a by matrix-assisted laser desorption ionization mass spectrometry microwaveassisted sample preparation for rapid and sensitive analysis of h. pylori lipid a applicable to a single colony bc , a duf -containing and golgi-located type ii membrane protein, is required for cell-wall biosynthesis in rice synthesis of ionic liquids functionalized b-cyclodextrin-bonded chiral stationary phases and their applications in high-performance liquid chromatography rapid identification of gallotannins from chinese galls by matrix-assisted laser desorption/ ionization time-of-flight quadrupole ion trap mass spectrometry engineered nanoparticle surfaces for improved mass spectrometric analysis convenient synthesis of sulfated oligofucosides synthesis of a mannose hexasaccharide related to the cell wall mannan of candida dubliniensis and trychophyton mentagrophytes synthesis of the -deoxytalose-containing tri-and hexasaccharides of the o-antigen polysaccharide from mesorhizobium huakuii ifo t highly efficient removal of allyloxycarbonyl (alloc) function provides a practical orthogonal protective strategy for carbohydrates dual reversible self-assembly of pnipam-based amphiphiles formed by inclusion complexation cytotoxic triterpenoid saponins acetylated with monoterpenoid acid from albizia julibrissin